UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Molecular studies of the LysR-type regulator, mexT and its regulation of the outer membrane porin protein… McCusker, Matthew Patrick 1998

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1998-0536.pdf [ 6.3MB ]
Metadata
JSON: 831-1.0088644.json
JSON-LD: 831-1.0088644-ld.json
RDF/XML (Pretty): 831-1.0088644-rdf.xml
RDF/JSON: 831-1.0088644-rdf.json
Turtle: 831-1.0088644-turtle.txt
N-Triples: 831-1.0088644-rdf-ntriples.txt
Original Record: 831-1.0088644-source.json
Full Text
831-1.0088644-fulltext.txt
Citation
831-1.0088644.ris

Full Text

Molecular studies of the LysR-type regulator, mexT, and its regulation of the Pseudomonas aeruginosa outer membrane porin, OprD.  by Matthew Patrick Mc Cusker B.A.(Mod) in Microbiology ,1995. Trinity College Dublin, Ireland.  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (MICROBIOLOGY P R O G R A M )  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A September 1998 © Matthew Patrick M c Cusker, 1998  in  presenting  this  thesis in  partial  fulfilment  of  the  requirements  for  an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for department  or  by  his  scholarly purposes may be granted by the head of  or  her  representatives.  It  publication of this thesis for financial gain shall not  is  understood  that  MUcfo? g X  O LOQy  The University of British Columbia Vancouver, Canada Da,e  DE-6 (2/88)  ^ A / i ?  r^J,  or  be allowed without my written  permission.  Department of  copying  my  XM\ALLV<DL69  &V  ABSTRACT  Pseudomonas  a e r u g i n o s a OprD is a specific porin which facilitates the uptake of basic  amino acids and imipenem, a carbepenem antibiotic with high potency against P.aeruginosa.  Resistance to imipenem occurs frequently during antibiotic therapy of  patients with P. a e r u g i n o s a infections. This study was initiated to investigate the regulatory mechanisms in P . a e r u g i n o s a for the regulation of o p r D . A putative LysR-type regulator, designated mexT,  was cloned on a 1.2 kb PCR fragment. D N A sequencing  predicted a 304 amino acid mature MexT protein. It showed strong homology to other LysR-type regulators. When overexpressed it induced expression of the  mexEF-oprN  efflux operon while repressing transcription of o p r D . A second line of investigation was to try and identify conditions where this mexT  gene  might play a role in the regulation of its target genes in P . a e r u g i n o s a . Salicylate, an aromatic weak acid known to reduce porin expression and induce low levels of multiple antibiotic resistance in other bacteria, was found to reduce expression of oprD  via  transcription. No change in expression of any of the efflux operons was noted. Sodium benzoate, another aromatic weak acid, was shown to reduce the levels of OprD in the outermembranes of P . a e r u g i n o s a but the mechanism of reduction was found not to via transcription. Neither iron or zinc concentrations were found effect expression of o p r D or any of the efflux operons. Finally, expression of these genes during growth phase was examined. No changes were noticed.  ii  T A B L E OF CONTENTS ABSTRACT  ii  T A B L E OF CONTENTS  iii  LIST OF FIGURES  v  LIST OF T A B L E S  vi  LIST OF A B B R E V I A T I O N S  vii  ACKNOWLEDGEMENT 1  INTRODUCTION 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8  2  '  1  Pseudomonas aeruginosa Pseudomonas aeruginosa Outer Membrane Porins and their role in P. aeruginosa Antibiotic uptake across the outer membrane Imipenem Multiple-antibiotic Resistance in Pseudomonas aeruginosa OprD and its regulation Aims of this Study  METHODS A N D MATERIALS 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12  3  ix  1 4 8 9 15 18  11  20  Strains, plasmids and growth conditions Reagents Genetic manipulations D N A sequencing Transfer of D N A into P.aeruginosa Cloning strategy for the mexT gene Cloning and analysis of the mexT gene Overexpression of the mexT gene in P. aeruginosa Membrane isolation and Protein analysis Determination of Minimal Inhibitory Concentration (MIC) Assay X y l E assays : Growth phase experiments  RESULTS  20 20 21 21 23 23 24 24 25 27 29 29 30  3.1.1 Molecular cloning of the mexT Gene 3.1.2 Nucleotide sequence of the mexT gene 3.1.3 MexT shows similarity to NahR and other LysR-type transcriptional regulators 3.1.4 Overexpression of the me^rgene in P.aeruginosa 3.1.5 MexT reduces transcription of oprD  in  30 31 33 35 39  2  3.1.6 Summary 3.1.7 Introduction 3.1.8 Salicylate and sodium benzoate; effects on antibiotic susceptibilities and outer membrane protein production in P. aeruginosa 3.1.9 Salicylate reduces transcription of the oprD gene in P.aeruginosa 3.1.10 Effect of iron and zinc concentrations on the antibiotic susceptibilities of P. aeruginosa 3.1.11 Effect of growth phase on expression of the mex efflux operons of P.aeruginosa 3.1.12 Summary 4  DISCUSSION  40 41 42 47 48 52 54 55  REFERENCES  67  iv  LIST OF FIGURES Fig 1:  Schematic diagram of the cell envelope of Gram negative bacteria  Fig 2:  Structures of some antibiotics and amino acids that penetrate through the OprD channel  Fig 3:  :  5  7  Schematic representation of the organization of the mex efflux operons in 14  P. aeruginosa Fig 4:  Diagram of plasmid p M C l utilized for sequencing of the mexT gene  Fig 5:  Diagram of plasmid pMC2 utilized for the overexpression of the mexT gene  26  28  Fig 6:  Physical map of the mexT gene  30  Fig 7:  Nucleotide and deduced amino acid sequence of the mexTgene  32  Fig 8:  Similarity of MexT to NahR  34  Fig 9:  SDS-PAGE demonstrating repression of OprD by overexpression of the 37  mexT gene Fig 10: Western immunoblots of P.aeruginosa strains  38  Fig 11: Effect of salicylate and sodium benzoate on the outer membrane proteins oi P. aeruginosa P A O l Fig 12: Western immunoblots of outer membranes of P.aeruginosa grown in the presence of 32 m M salicylate and 16 m M sodium benzoate Fig 13: Outer membrane profiles of P.aeruginosa P A O l grown in MuellerHinton with different concentrations of zinc Fig 14: Outer membrane profiles of P.aeruginosa PAOlduring growth phases  v  45 46 51 53  LIST OF TABLES Table I: Bacterial strains  21  Table II: Plasmids used in study  22  Table III: Oligonucleotide primers used in study  27  Table IV:MICs for P.aeruginosa strains  36  Table V: Effect of MexT on an oprD::xylE chromosomal transcriptional fusion in 40  P.aeruginosa Table VI: Antibiotic susceptibilities of P.aeruginosa P A O l in Mueller-Hinton in the presence of salicylate and other structurally related compounds  44  Table VII: Effect of salicylate and sodium benzoate on an oprDr.xylE chromosomal transcriptional fusion in P.aeruginosa  48  Table VIII: Effect of iron and zinc concentrations on the antibiotic susceptibilities of P. aeruginosa  50  vi  LIST O F ABBREVIATIONS  MIC  minimum inhibitory concentration  OD  optical density  EDTA  ethylenediamine tetra acetate  MH  Mueller Hinton  LBNS  Luria broth, normal salt  LPS  lipopolysaccharide  Cm  chloramphenicol  Tet  tetracycline  Gent  gentamycin  Amp  ampicillin  Carb  carbenicillin  Im  imipenem  Cefp  cefpirome  Nor  norfloxacin  SDS  sodium dodecyl sulfate  SDS-PAGE  SDS polyacrylamide gel electrophoresis  mAb  monoclonal antibody  bp  base pair  kb  kilobase  kDa  kilodalton  PCR  polymerase chain reaction vii  ORF  open reading frame  LTTR  LysR-type transcriptional regulator  HTH  helix-turn-helix  MCS  multiple cloning site  Mr  molecular mass  His  histidine  Arg  arginine  Ala  alanine  Glu  glutamate  viii  ACKNOWLEDGEMENT  I am grateful to my supervisor, Dr. Robert E. W. Hancock, for his guidance and support during the time in his lab. A special thanks to Dr. Martina Ochs for her advice and patience over the last two years. I also thank Susan Farmer and Manjeet Bains as well as the rest of Hancock lab for their technical help and encouragement during this project. I also acknowledge the help and support of my supervising committee, Drs. Fernandez and Mohn. Last but not least a personal word of thanks to Dr. Tony Warren for his emotional support during the first and very difficult year in the department. The financial support of the Medical Research Council of Canada is gratefully acknowledged.  ix  1  1.1  INTRODUCTION  Pseudomonas aeruginosa  Pseudomonas aeruginosa is an opportunistic Gram negative pathogen which is a major cause of nosocomial (hospital-acquired) infections (Schimpff et al., 1970). These infections usually occur in immunocompromised hosts such as burn victims and cancer patients, or in people with cystic fibrosis. P.aeruginosa produces a variety of toxins, enzymes and pigments, some of which undoubtedly contribute to the pathogenic properties of the bacteria (Liu, 1974). P.aeruginosa is becoming a major clinical problem and is one of the most difficult infections to treat due to its high intrinsic resistance to the many of the most commonly used antibiotics including first and second generation penicillins and cephalosporins, tetracycline, chloramphenicol, quinolones and their derivatives (Bryan, 1979). This phenomenon has been partly attributed to the low outer membrane permeability of P. aeruginosa. It has been shown that the permeability of-the outer membrane of P. aeruginosa to (3-lactam antibiotics is from twelve to one hundred fold lower than the permeability of the E.coli membrane to the same compounds (Nicas and Hancock, 1983). This low outer membrane permeability, which springs from the properties of the pore-forming proteins, porins, in the outer membrane  P.aeruginosa,  acts only to slow down the movement of the antibiotics into the cell (Nikaido and Hancock, 1986). It does not itself cause resistance.  1  1.2  Pseudomonas aeruginosa Outer Membrane  The cell envelope of P.aeruginosa consists of two membranes separated by a layer of peptidoglycan and a cellular compartment called the periplasm (Fig. 1). The inner or cytoplasmic membrane is a phospholipid bilayer containing a large number of polypeptides. The peptidoglycan is located under the outer membrane and is primarily responsible for the mechanical strength of the cell wall (protecting the cell e.g. against osmotic shock) and for maintaining cell shape (Oliver, 1987). The periplasm lies between the inner and outer membranes. Proteins residing in the periplasmic space fulfill important functions in the detection and processing of essential nutrients and their transport into the cell (Oliver, 1987). The outer membrane consists of an asymmetric bilayer (Fig. 1), in which the inner monolayer is composed of phospholipid, while the outer monolayer  contains  lipopolysaccharide (LPS) (Nikaido and Nakae, 1979). LPS is a complex molecule consisting of three covalently linked regions; lipid A , a core oligosaccharide region and an O-specific chain (Rietschel et al., 1982). The lipid A region is conserved, consisting of substituted B-linked glucosamine disaccharide, usually (1—>6)-linked. The disaccharide is fully substituted with six or seven saturated or hydroxyl fatty acid residues, phosphate, and the core oligosaccharide (Karunaratne et al., 1992). The core oligosaccharide is a complex oligosaccharide usually linked to lipid A via 2-keto-3-deoxyoctonate, K D O . In P.aeruginosa the core contains glucose, heptose, K D O , rhamnose, galactosamine and alanine (Kropinski et al., 1979). The core may be capped with repeating tri- to pentasaccharide units termed the O-antigen. This is the immunodominant part of the LPS  2  molecule in the intact bacterial cell. It has been shown that the O-antigen of P.aeruginosa often contains such sugars as glucose, rhamnose, glucosamine, fucosamine and quinavosamine (Rropinski et al., 1985). The asymmetric distribution and chemical characteristics of LPS give the outer membrane many of its unique barrier properties. The presence of a large amount of phosphate in the core region of P.aeruginosa LPS results in the strong surface negative charge (Sherbert and Lakshmi, 1973). The non-covalent cross-bridging of adjacent LPS molecules with divalent cations (Mg  2+  or Ca ) (Rottem and Leive, 1977), and the 2+  hydrophobic interactions between the outer membrane proteins and Lipid A (Nikaido and Vaara, 1985), also contribute to the stability of the LPS in the outer membrane. The combination of these properties makes the P. aeruginosa highly resistant to hydrophobic antibiotics, bile salts, detergents, proteases, lipases and lysozyme (Nikaido and Vaara, 1985). The P.aeruginosa outer membrane also contains a number of "major" proteins (Fig. 1). These include the murein lipoproteins, the major outer membrane protein, OprF, and porins. The two lipoproteins of P.aeruginosa, OprL and OprI are inserted in the inner phospholipid monolayer and are non-covalently associated with peptidoglycan (Hancock et al., 1981). They have a structural role in stabilizing the architecture of the outer membrane-peptidoglycan complex. OprF is also strongly but not covalently associated with peptidoglycan and plays a role in outer membrane stability and cell shape determination (Woodruff and Hancock, 1989). Porins are a group of proteins forming trans-outer-membrane, water filled channels. In general, porins have monomer molecular 3  weights in the range of 28 kDa to 48 kDa, are present in the membrane as oligomers, usually trimers and have a high content of R-sheet structure. In P.aeruginosa, OprB, OprC, OprD, OprE, OprF, OprO and OprP have been identified as porins (for a review, see Hancock et al., 1990).  1.3  Porins and their role in P. aeruginosa  Porins are generally divided into two classes: non-specific or general porins and specific porins (Nikaido and Vaara, 1985). General porins form water-filled channels that permit the passive diffusion of hydrophilic molecules below a certain molecular size, and thus are responsible for the non-specific exclusion limit of the outer membrane. Specific porins also produce water-filled channels which contain stereospecific substrate-binding sites (Hancock, 1987). The diffusion of the specific substrate is accelerated when the solute concentration is low, but is slowed down when the concentration is high. General porins take up molecules based on size, electric charge and hydrophilicity. OprF, the major outer membrane porin of P.aeruginosa is a non-specific porin (Nikaido and Vaara, 1985). Its pore-forming properties have been shown in a model membrane system and in intact cells (Benz and Hancock, 1981). The pore diameter was estimated to be 20 A, about twice the width of the E.coli porin channels, and can allow passage of saccharides with molecular weights of approximately 3,000 (Nikaido and Hancock, 1986). However, only 400 of the estimated 200,000 OprF molecules per cell are proposed  4  Fig 1:  Schematic diagram of the cell envelope of Gram negative bacteria.  The cell envelope of Gram negative bacteria consists of two membranes, the cytoplasmic and outer membrane which are separated by the periplasm. The outer membrane is an asymmetric bilayer with lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. The periplasm separates the two membranes and contains the meshlike peptidoglycan layer. The inner membrane (cytoplasmic membrane) is a phospholipid bilayer. Proteins shown are: a three-component resistance-nodulation-division efflux system consisting of the integral outer membrane pore proteins, the periplasmic membrane fusion proteins (MF) and the integral cytoplasmic R N D pump protein; an outer membrane porin protein forming a diffusion channel and a peripheral periplasmic penicillin binding protein (PBP) which functions in the synthesis of the peptidoglycan layer.  5  to form such large channels. The rest appear to form small channels that are thought to be impermeable to antibiotics (Woodruff et al., 1986). Two other porins, namely OprC and OprE, are general porins with a small channel size and are anaerobically induced. It is generally accepted that the low overall permeability of the channels of the general porins, explains the low outer membrane permeability of P.aeruginosa compared to E.coli (Angus et al., 1982; Nicas and Hancock, 1983). This in turn, was proposed to contribute to the high intrinsic resistance of P.aeruginosa to antibiotics (Nikaido and Hancock, 1986). This low outer membrane permeability may be an advantage for excluding antibiotics but it means that the bacterium also excludes certain essential nutrients. To overcome this and allow effective uptake of essential nutrients available at low concentrations in the medium, several specific porins are present in the P.aeruginosa outer membrane. OprB is induced in the presence of glucose and is involved in the uptake of glucose and xylose (Trias et al, 1987). OprP is induced under phosphate limiting conditions (< 0.15 mM) (Hancock et al., 1982). It is part of the phosphate specific transport (Pst) system of P.aeruginosa (Poole and Hancock, 1986). OprO, another porin with high homology to OprP, forms pyrophosphate-specific channels (Hancock et al., 1992). OprD was discovered due to its role in the facilitated uptake of imipenem, a carbepenem which shows excellent activity against P.aeruginosa (Trias and Nikaido, 1990a). The natural substrate for OprD is not imipenem, but its structural analogues, basic amino acids and small peptides containing those amino acids (Fig. 2) (Trias and Nikaido, 1990b).  6  Imipenem  COOH  COOH  NH  CH — C H - C H - C H - N H  2  2  2  2  2  2  Lysine COOH  Arginine  NH .  .CH —CH  2  \  2  2  —NH—C-NH  ^ J  2  II NH  COOH  CH — N H \  II  Histidine  N  H  -  / C H  2  . C H , - C H - N H ^ COOH  Fig 2: Structures of some antibiotics and amino acids that penetrate through the OprD channel.  7  1.4  Antibiotic uptake across the outer membrane  There exist three general pathways for antibiotic uptake in Gram negative bacteria: the porin pathway, the hydrophobic pathway and the self promoted uptake pathway (Hancock, 1997). Small hydrophilic molecules enter the periplasm by diffusion through the porin channels that are present in the outer membrane. Uptake of small hydrophilic antibiotics usually occurs through the general porins. Antibiotic uptake through the specific porins is not well documented, but it has been shown that the basic amino acid-specific OprD porin of P.aeruginosa mediates the uptake of the B-lactam antibiotic imipenem (Sumita et al., 1993; Yoneyama et al, 1993). Lipophilic and amphipathic antibiotics enter the cell through the hydrophobic pathway by passive permeation through the outer membrane bilayer (Hancock & Bell, 1988). The mechanism of uptake of hydrophobic and amphipathic antibiotics is not well understood but probably involves the partitioning into the hydrocarbon core of the outer membrane and simple diffusion. It has become clear that the intrinsic and high level of resistance of bacteria to many hydrophobic antimicrobials is mediated by active efflux mechanisms rather than changes in outer-membrane permeability. Cationic antimicrobials such as the aminoglycosides and cationic antimicrobial peptides enter the cells by competing for divalent cation binding sites on the surface LPS in a process that has been termed self promoted uptake (Hancock, 1997). It is believed that the binding of these compounds to the outer membrane displaces the native divalent  8  cations and, due to their bulkiness, they destabilize (permeabilize) the outer membrane and thereby facilitate further uptake of themselves and other molecules.  1.5  Imipenem  Imipenem, or N-formimidoyl thienamycin, is derived from thienamycin, a natural product of the soil organism Streptomyces  cattleya  (Kahan et al., 1979). Thienamycin has unique  structural features that distinguish it from all natural and synthetic (3-lactam antibiotics previously described (Albers-Schonberg et al., 1978). It is the first representative of a new class of antibiotics, the carbepenems (Fig. 2). Imipenem is of particular interest because of it's high potency, broad spectrum and lack of microbial cross resistance to other (3-lactam antibiotics (Rohnson et al., 1986). Furthermore, imipenem had greater bactericidal activity  in vivo  and greater protective efficacy in experimental infections  against pathogenic species than other antibiotics (Kropp et al., 1980). The high potency and unusually broad spectrum of antimicrobial activity of imipenem is due to three aspects. Firstly, it is able to relatively easily penetrate the outer membranes of many Gram-negative bacteria. Imipenem has a compact structure with a molecular weight of 229 and is zwitterionic, and both these features facilitate its diffusion through the outer membranes of Gram-negative bacteria by specific porin channels (Lipman and Nea, 1988). In P.aeruginosa, imipenem can overcome the poor outer membrane permeability by penetrating through the specific porin OprD (Trias and Nikaido, 1990a). Secondly, imipenem has high affinity for its target penicillin binding proteins (PBPs), namely PBP1 and PBP-2 (Hashizame et al., 1984). Thirdly, imipemen is a poor substrate for a range  9  of P-lactamases. Despite being a strong inducer of chromosomal cephalosporinases, a class of P-lactamases induced in the presence of selected P-lactams and capable of hydrolyzing many P-lactams, imipenem is only weakly hydrolyzed by these P-lactamases (Livermore and Yang, 1987). These properties may account for the general lack of cross resistance of microbes to imipenem with other P-lactams. Resistance to imipenem can arise through three different mechanisms. Firstly, the constituants of the outer membrane may be modified to prevent the uptake of imipenem. Imipenem resistant mutants of P. aeruginosa have been isolated both in the laboratory and from clinical sources. Their resistance usually stems from the reduced or lack of expression of the outer membrane porin, OprD (Lynch et a l , 1987). Genetic analysis of these mutants has shown that the loss of OprD expression is due to rearrangements in the oprD coding region or the upstream promoter region (Yoneyana and Nakae, 1993). Secondly, imipenems target, the PBPs, may be altered to reduce its effect on the cell wall (Helinger and Brewer, 1991). Thirdly, P-lactamases may be expressed which are capable of hydrolyzing the P-lactam ring of imipenem thereby inactivating it. Pseudomonas maltophilia, which is resistant to imipenem, produces an imipenem-hydrolyzing Plactamase (Saino et al., 1982). This enzyme is part of a unique class of Zn -containing p2+  lactamases analogous to the metaloproteases. Interestingly, full resistance to imipenem in P.aeruginosa seems to require both reduced permeability due to the loss of OprD and slow hydrolysis due to production of the chromosomally encoded (Livermore, 1992).  10  P-lactamase  1.6  Multiple-antibiotic Resistance in Pseudomonas aeruginosa  Infections of low-virulence bacteria in immunocompromised patients are of major concern. Bacteria such as Staphylococcus aureus, Streptococcus spp., Enterococcus, and Pseudomonas aeruginosa, found commonly in hospitals, often have low or no virulence in healthy individuals but can cause potentially life threatening infections in people where the immune system is not working properly. These types of infections can usually be treated successfully with antibiotics. A major drawback of the use of antibiotics is the selection of strains that exhibit antibiotic resistance. Antibiotic resistance, as stated previously, can arise by different mechanisms. The most worrying of these mechanisms is active efflux, as this mechanism gives resistance to a wide range of structurally unrelated antibiotics (for a comprehensive review see Paulsen et al., 1996). Multiple-antibiotic resistance in P.aeruginosa has been shown to correlate with resistance to the quinolone class of antimicrobial agents (Hirai and Mitsuhashi, 1987). The fluoroquinolones in particular seem to select strains demonstrating cross-resistance, not only to other quinolones, but to structurally unrelated antibiotics (Nikaido, 1996). Three of these mutants, nalB, nfxB, and nfxC, which display a multiple-drug resistance phenotype (MDR) phenotype and overexpress outer membrane proteins in the range of 50kDa, have been described in P.aeruginosa (Rella and Hass, 1982; Hirai et al., 1987; Fukuda etal., 1990). NalB-type  mutants  chloramphenicol,  show increased  tetracycline,  resistance  |3-lactam  to meropenem, fluoroquinolones,  antibiotics  (including  penicillins  and  cephalosporins), as well as 2,2'-dipyridyl (Rella and Hass, 1982). These mutants also 11  overexpress an outer membrane protein with an apparent molecular weight of 50kDa. Poole et al., cloned an operon that overcame a growth deficiency in a mutant that lacked the ferri-pyoverdin receptor in the presence of the non-metabolizable iron-chelator, 2,2'dipyridyl. The operon contained three open reading frames, later designated mexA, mexB and oprM (Fig. 3) (Poole et al., 1993). These genes coded for proteins of 41, 112 and 50kDa respectively. MexB had amino acid similarity to other members of the RestrictionNodulation-Division (RND) family of cytoplasmic pump proteins. OprM has been shown to be in the outer membrane and to be the same protein seen in nalB-type mutants (Gotoh et al., 1994). MexA was predicted to be anchored in the cytoplasmic membrane and to form a bridge between, MexB in the cytoplasmic membrane, and OprM in the outer membrane. It has been proposed that these proteins, as for their homologues in E.coli, form a continuous channel from the cytoplasm to the external medium, and use the proton motive force to actively efflux their substrates (Fig. 1). A small ORF designated mexR was found upstream of the mexA/B-oprM operon and was shown to negatively regulate the efflux operon (Fig. 3) (Poole et al., 1996). As yet, the nalB gene has not been cloned. Mutants of the nfxB-type M D R phenotype show increased resistance to fluoroquinolones, chloramphenicol, tetracycline, erythromycin and cefpirome, and increased susceptibility to the classic P-lactams (Hirai et al., 1987). Outer membrane analysis of these mutants demonstrated the overexpression of a 54kDa protein designated OprJ. Poole et al. cloned the gene encoding OprJ and sequence analysis of the upstream region revealed the presence of two additional coding regions (Poole et al., 1996). These genes were designated mexC, which codes for a 112kDa membrane fusion protein, and mexD, which  12  encodes a 40kDa cytoplasmic membrane protein. These proteins showed significant homology to the MexA and MexB proteins respectively. The nfxB gene was located upstream of the mexCD-oprJ operon and coded for a putative D N A binding protein (Fig. 3). It was subsequently shown to negatively regulate itself and the efflux operon. The nfxB mutants produce a defective repressor that results in derepression of the mexC/DoprJ operon and, therefore, overproduction of the MexC, MexD, and OprJ proteins (Poole etal., 1996). NfxC-type mutants show increased resistance to chloramphenicol, quinolones and the carbepenem, imipenem (Fukuda et al., 1990). They show increased susceptibility towards most p-lactam antibiotics (except imipemen) and aminoglycoside antibiotics (Fukuda et al., 1990). Unlike the other types of mutations there are two changes in the outer membrane profile. There is a reduction in expression of the imipenem specific porin, OprD, and overexpression of a 51kDa protein designated OprN (Masuda et al., 1995). Kohler et al. recently cloned the operon responsible for the nfxC-type phenotype (Kohler et al., 1997). It consisted of three ORFs which coded for a cytoplasmic membrane protein MexF (115kDa), a membrane fusion protein MexE (45kDa), and an outer membrane protein OprN (51kDa). MexE MexF and OprN showed high similarity the MexA, MexB and OprM, respectively. Upstream of this operon was an ORF that encoded a putative LysR-type transcriptional activator (Fig. 3). Overexpression of this fragment resulted in overexpression of OprN and reduction of a protein with an apparent molecular mass of 45kDa. It has been suggested that this efflux machinery is involved in the excretion of intermediates for the biosynthesis of pyocyanain (Kohler et al., 1997).  13  mexA-mexB-oprM nalB  mexR  mexA • •'•  mexB  oprM  • • • • •  mexC-mexD-oprJ nfxB  23K  21K  mexC  mexD  oprJ  AA mexE-mexF-oprN oprD  exT  m  mexE  mexF  oprN  Fig 3: Schematic representation of the organization of the mex efflux operons in P.aeruginosa. Distances between genes and their sizes do not correspond to their physical sizes. Arrows above operons correspond to the direction of transcription of the genes in the operon. Arrows below correspond to regulatory proteins produced and their putative binding positions.  14  1.7  OprD and its regulation  As stated previously, imipenem is highly active against P.aeruginosa. However, during antibiotic therapy, imipenem resistant isolates arise at a significant rate (Quinn et al., 1986), and usually the resistant strains are not cross resistant to other antibiotics, don't show any changes in their PBPs and cannot hydrolyze or modify imipenem. Instead, they lack the outer membrane protein, OprD (Quinn et al., 1986; Lynch et al., 1987). OprD has an apparent molecular weight of 46,000 in an SDS-polyacrylamide gel electrophoretogram when the protein is solubilized at 95°C (Yoshihara et al., 1991). This corresponds to its monomer form. OprD exists as trimers in the native outer membrane (Yoshihara et al., 1991). Native OprD shows a p-strand-rich conformation which is typical of many other porins (Huang et al., 1995). OprD has been proposed to function as a general porin, allowing the diffusion of monosaccharides, disacchrides and amino acids at a significant rate . Of more importance, OprD has been shown to act as a specific porin for the uptake of imipenem, basic amino acids and their structural analogues (Fig. 2) (Trias and Nikaido, 1990).  While a lot is known about the structure and function of OprD, little is known about the regulation of its expression . Huang et al. described the isolation of a Tn501 mutant of P.aeruginosa that was resistant to imipenem and lacking OprD (Huang et al., 1992). This mutant could be complemented both with relation to imipenem susceptibility and OprD expression with a 6.0-kb fragment of D N A from the region of the chromosome of the wild-type strain surrounding the site of Tn501 insertion. When sequenced this region contained three open reading frames (ORF), one of which was found to be interrupted by  15  the Tn501 transposon. The latter ORF was designated opdE (for putative regulator of oprD expression). It coded for a putative hydrophobic protein of molecular mass (Mr) 41,592. They hypothesized that the opdE gene, or one or two of the other ORFs, encodes a protein that influences the expression of OprD. Recent work done in this lab has cast doubt on the presumption that the insertion in the opdE gene is responsible for the reduction in OprD expression (Ochs, unpublished data). Work to elucidate this is in progress. More recently, Park et al. described the cloning and characterisation of a gene, argR, involved in the regulation of arginine biosynthesis and catabolism (Park et al., 1997). This gene was found to be the sixth and terminal gene of an operon for the transport of arginine. The argR gene was inactivated by gene replacement, using a gentamicin cassette. Inactivation of argR abolished arginine control of the biosynthetic enzymes encoded by the car and argF operons. Both the car operon, which encodes carbamoyltransferase (CPS), and the argF operon, which encodes the anabolic ornithine carbamoyltransferase (aOCT), are negatively regulated by arginine. The argR gene product, ArgR was also shown to regulate the aru operon. This operon is involved in the arginine succinyltransferase (AST) pathway, which converts arginine to glutamate, and is considered the major route for catabolism in P.aeruginosa under aerobic conditions (Hass et al., 1990). Enzymes of the A S T pathway catalyze the conversion of arginine to glutamate and are strongly induced by exogenous arginine. Arginine also induces the catabolic NAD-dependent glutamate dehydrogenase (NAD-GDH), which is required for  16  the utilization of glutamate and regeneration of succinyl coenzyme A (succinyl-CoA), through the citric acid cycle (Hass et al., 1990). ArgR was purified to homogeneity. Using DNase footprinting, it was shown that ArgR bound directly to the promoter regions of the car, argF, and aru operons (Park et al., 1997). Alignment of the ArgR binding sites revealed that the ArgR binding sites contained a consensus sequence. The consensus sequence is T G T C G C N A A N . They 8  5  also looked at the promoter sequence of oprD which, as previously stated, is involved in the uptake of basic amino acids. A n ArgR consensus sequence was found upstream of the oprD coding region. DNase footprinting experiments showed direct binding of ArgR to the oprD promoter region (Park et al., unpublished data). This is the first report of a regulatory protein directly binding to the oprD promoter region and thereby affecting OprD expression. Work done in this lab looked at the effect of different carbon and nitrogen sources on OprD expression. A l l amino acids tested (His, Arg, Ala, Glu), strongly induced OprD when used as sole carbon sources (Ochs, unpublished data). This induction could be abolished by succinate. Non-inducing carbon sources included succinate, glucose, gluconate and glycerol. The effect of different nitrogen sources was also looked at. Histidine and arginine slightly induced OprD expression, while alanine and glutamate resulted in strong induction when used as sole nitrogen sources (Ochs, unpublished data). Addition of ammonium sulphate abolished induction. Induction of OprD by using arginine as the sole nitrogen source was abolished in an argR mutant background. Interestingly in an argR mutant background, glutamate still induced OprD expression 17  (Ochs, unpublished data). This suggests that there is a separate mechanism for the regulation of OprD by glutamate. Recent analysis of the promoter region of oprD has found potential sigma70 and sigma54 consensus sequences. Investigations to see i f indeed oprD is regulated by these sigma factors is underway in the lab. Taken together, the above data suggest that oprD is a highly regulated gene with a complex network of regulatory signals and factors involved. This is not unexpected due to the observation that oprD is poorly expressed from its own promoter in E.coli (Huang et al., 1992). This observation is consistent with a gene that has a weak promoter and that is highly regulated.  1.8  Aims of this Study  OprD, a specific porin for imipenem and basic amino acids has been studied extensively as a model for the mechanisms of antibiotic and nutrient uptake through the specific porins. Despite all this research, little is known about the regulation of expression of the oprD gene itself. Recent work and other work performed during the investigations described here, suggest that oprD expression is highly regulated by multiple factors. One of the regulatory factors described was a putative transcriptional regulator belonging to the LysR-type family of transcriptional regulators. This regulatory ORF was found upstream of the mexE-mexF-oprN and was shown to positively regulate the operon and potentially negatively regulate oprD.  The aims of this study were, 1) to clone, sequence and analyze this putative transcriptional regulator; 2) show it directly regulates transcription of oprD; 3) attempt to  18  identify possible cofactors and 4) try to find conditions under which this gene regulates oprD expression.  19  2  2.1  METHODS AND MATERIALS  Strains, plasmids and growth conditions  Strains used in these studies are listed in Table I and all plasmids used are listed in Table II. E. coli strains were routinely grown in Luria Bertani (LB) broth (1.0% Tryptone, 0.5% yeast extract,0.5% NaCl) containing 0.5 % glucose; for solid media, agar was added to 1.5% (wt/vol). P.aeruginosa strains were routinely grown in Mueller Hinton broth; for solid media, agar was added to 1.5% (wt/vol). B M 2 minimal media was made as previously described (Poole et al., 1995). A l l media components were purchased from Difco Laboratories (Detroit, MI). Antibiotics were used for E. coli when required at the following concentrations:  ampicillin,  100  (J.g/ml; for P.aeruginosa: carbenicillin,  400|ag/ml; norfloxacin, l|j,g/ml; gentamycin, 10(ag/ml. 2.2  Reagents  Chemical reagents used in this study were purchased from Sigma Chemical (St. Louis, MO).  Restriction enzymes, polymerases and other molecular biology reagents were  purchased from Gibco B R L (Burlington, ON). A l l enzymes were used as described in the manufacturer's instructions Rabbit anti-OprD monoclonal antibody (YD11), mouse antiOprM monoclonal antibody (TM01), mouse anti-OprJ (HJ004) and mouse anti-OprN (TN009) monoclonal antibodies were all provided by Dr. Gotoh. Alkaline phosphatase conjugated anti-goat and anti-mouse antibodies were purchased from BioRad (Richmond, CA).  20  Table I:  Strain:  Bacterial strains  Relevant characteristics:  Source/reference:  supE44, /act/169 (<j) 80/acZM15), hsdRU, recAl, endAJ, gyrA96, thi-1, relAX  BRL  P A O l prototroph: wild type reference strain HI 03 containing a chromosomal oprD-xylE fusion Clinical isolate nfxC-type mutant of 1008  Hancock, et al., 1979 M . Ochs Masuda, et al., 1995 Masuda, et al., 1995  E. coli: DH5ct  P. aeruginosa H103 H103-1 1008 1008OIR01  2.3  Genetic manipulations  General molecular biology protocols were performed according to standard methods described in Sambrook et al. (1989). D N A fragments were isolated using the Gene Clean kit (BiolOl Inc., Vista, CA). Sticky end ligations were performed at RT for 30-60 min; blunt end ligations were done overnight in a thermocycler programmed to continuously cycle between 10°C and 30°C for 30 sec at each temperature. 2.4  D N A sequencing  D N A sequencing was done on an A B I 373 automated D N A sequencer (Applied Biosystems, Foster City, C A ) using the A B I Prism dye terminator sequencing kit according to the manufacturer's directions. Plasmid D N A was prepared for sequencing using Qiagen columns (Qiagen, Inc., Chatsworth, CA) and plasmid D N A was quantified using a Hoefer T K O 100 mini spectrofluorimeter. Oligonucleotide sequencing primers were synthesized as needed on a A B I 392 D N A / R N A oligonucleotide synthesizer according to the manufacturer's directions.  21  Table II:  Plasmids used in study  Plasmid:  Relevant characteristics:  pUCP18/19  pUC18/19-derived broad host range plasmid which can be maintained in both E.coli and P.aeruginosa pUCPl 8 containing a 1.2kb BamHIIEcoRl fragment encoding mexT cloned in the reverse orientation to the promoterless lacZgene pUCPl 9 containing a 1.2kb BamHIIEcoRl fragment encoding mexT cloned in the same orientation as the promoterless lacZ gene pMCl with a 500bp BamHIIBgUI de letion in the 5' end of the mexT gene pMCl with a 700bp Sail deletion in the 5' end of the wex^gene  pMCl pMC2 pMC3 pMC4  Source/re ferenee: Schweizer, 1991 This study This study This study This study  Oligonucleotide primers were purified as follows: following deprotection for 8 h at 55°C, the oligonucleotides were lyophilized and redissolved in 500 ul deionized water. The resuspended oligonucleotide was centrifuged for 5 min at maximum speed (-14,000 rpm) in a microcentrifuge to remove insoluble material and the supernatant was retained. The concentration of oligonucleotide was determined by measuring the absorbance at 260 nm. To completely sequence the cloned mexT D N A fragment two strategies were used. One was the building of custom oligonucleotide primers (Table III). The second was the construction of two plasmids containing deletions in the mexTUNA fragment (Table II). The first deletion was from the BamHI site of the pUCP18 M C S to the BgUI site oimexT (Fig. 6). The second deletion was from the Sail site of the pUCP18 M C S to the Sail site of mexT (Fig. 6). Nucleotide and deduced amino acid sequences were analyzed with the PC Gene software package (Intelligenetics Inc., Mountain View, CA). The D N A sequence was used for homology searches of the National Center for Biotechnology Investigation  (NCBI)  non-redundant  database  22  with  the  BLASTX  program  (vvrv^.ncbi.nlm.nih.gov/cgi-bin/BLAST/). The default settings for the B L A S T X program was used for all the searches. The D N A sequence was also used for homology searches of the contents of the Pseudomonas aeruginosa genome sequencing project with the B L A S T N program (www.ncbi.nlm.nih.gov/BLAST/pseudoabl.html). 2.5  Transfer of D N A into P. aeruginosa  Plasmids were transferred in P.aeruginosa by transformation (Olsen et al., 1982). Briefly, cells to be transformed were grown to an O D  600  of 0.6-0.8 in M H . Cells were pelleted and  resuspended in 150 m M M g C l , placed on ice for 5 min. This step was repeated except 2  for the cells were put on ice for 20 min. The cells were then pelleted and resuspended in 1/10 volume of ice cold 150 m M M g C l , and kept at 4°C for 16 hrs. D N A (0.1 to ljig) 2  was added to 200ul of competent cells and left on ice for 1 hr. Cells were heat shocked by placing them at 42°C for 5 min and then were returned to ice for a further 5 min. 1ml of M H broth was added and the mixture was incubated at 37°C for 2-3 hrs to allow expression of the plasmid antibiotic resistance gene. Aliquots (50-100 ul) of cells were plated on selective medium and grown for 24 to 48 hrs.  2.6  Cloning strategy for the mexT gene  According to Kohler et al., the mexT gene was directly upstream and in the same orientation as the mexE gene (Kohler et al., 1997). The sequence of mexE and its upstream region was retrieved from the EBI databank under accession number X99514. The 5' end of the sequence and all the available upstream sequence was used to search the contents of the P.aeruginosa genome sequencing project for a Contig containing the 5' end of mexE and as much upstream sequence as possible. Contig 1523 was retrieved and 23  when analysed was found to contain the 5' end of mexE and one other ORF upstream. The sequence of this ORF was used for homology searches of the National Center for Biotechnology Investigation (NCBI) non-redundant database with the B L A S T X program (www.ncbi.nlm.nih.gov/cgi-bin/BLAST/V It showed high homology to LysR-type regulators which was consistent with it being mexT. Using this sequence primers were designed to allow amplification of the mexT gene using P C R (Table. III). To allow easier cloning the primers contained convenient restriction sites. Primer mexTF89 contained a BamHI site while mexTRext contained an EcoRI site. 2.7  Cloning and analysis of the mexT gene  Amplification of the mexT gene from P.aeruginosa P A O l (HI03) was achieved using PCR. The reaction mixture (total volume lOOul) included: 2 units of Vent polymerase (New  England Biolab, Beverly, M A ) , l x Vent reaction buffer, 0.3  m M each  deoxynucleoside triphosphate, 0.5 u M of each primer (mexTF89 and mexTRext), and lOOng of HI03 genomic D N A . The mixture was treated for 5 min at 95°C, followed by one cycle of 5 min at 95°C, 2 min at 55-60°C and 1 min 45 sec at 72°C. This was followed with 30 more cycles of 2 min at 95°C, 2 min at 55-60°C and 1 min 45 sec at 72°C, before finishing with 10 min at 72°C. P C R products were examined on a 0.8% (wt/vol) agarose gel and purified with the QIAquick-spin PCR purification kit (Qiagen Inc., Chatsworth, CA). 2.8  Overexpression of the mexT gene in P. aeruginosa  To overexpress MexT, the 1.2 kb BamHIIEcoRl fragment from p M C l containing the mexT gene was cloned into pUCP19 to form the plasmid pMC2, so that the direction of 24  expression of the mexT gene was in the same orientation as the lac promoter (Fig. 5). It was then transformed into P.aeruginosa HI03 and HI03-1. 2.9  Membrane isolation and Protein analysis  Outer membranes were prepared by sucrose gradient density centrifugation (Hancock and Nikaido, 1978) or by differential Sarcosyl solubilisation (Poxton et al., 1985). A l l samples were taken from cultures at an O D  600  of 1.0. Analysis of protein profiles was  done by electrophoresis through SDS 12%- polyacrylamide gels (PAGE) (Mutharia & Hancock, 1983). Proteins were solubilized in sample buffer (0.5 M Tris-HCl buffer, pH 6.8, containing 2% SDS, 10% glycerol, 5% P-mercaptoethanol and bromophenol blue) for 10 min at 98°C before being applied to the gel. Following electrophoresis proteins were visualized by staining with Coomassie Brilliant Blue R250 (Bio-Rad, Richmond, CA). Immunoblotting procedures were performed as described previously (Mutharia & Hancock, 1983). Antibodies for Western immunoblotting were used at the following dilutions: OprD, 1:5000; OprM, 1:3000; OprJ, 1:714; OprN, 1:1250.  25  F i g 4:  Diagram of plasmid pMCl utilized for sequencing of the mexT gene.  The coding region of the mexT gene is shown. The orientations of the ampicillin resistance gene, origin of replication and the lac promoter are indicated. The 1.8 kb stabilizing fragment is for maintenance of the plasmid in P. aeruginosa.  26  Oligonucleotide primers used in study  Table III:  Primer:  D N A Sequence from 5' to 3':  mexTF89  TTACATG<JATCCTGGCTATGCCTGTCAGTG  mexTRext  TTCTACGAATTCCGTTTTCTGTAACAGTCG  UNI  TGTAAAACGACGGCCAGT  REV  CAGGAAACAGCTATGACC  mexT17AFwd  CTGCTGATCGTGTTCGAGACC  mexT17BRev  CATGGCGGTGGAGATGGAACT  mexT13ARev  GAGGATCTTCGGCTTGCTGCG  mexT12ARev  CAAGCCATCGACAGTTCGAAG  mexT21AFwd  CTTCGAACTGTCGATGGCTTG  2.10  Determination of Minimal Inhibitory Concentration (MIC) Assay  The MIC of various antimicrobial agents for P. aeruginosa strains was determined by the broth dilution method (Amsterdam., 1991). Briefly, cells were grown overnight at 37°C in Mueller-Hinton and diluted 1 in 10 000 in the same medium to give concentrations of about 10 -10 colony-forming units/ml. Serial dilutions of each antibiotic were made in 4  5  Mueller-Hinton medium in 96-well microtitre plates (Costar).  Each well was then  inoculated with 10u.l of the test organism. Samples of the bacterial inoculum were plated to ensure they were within the proper range. The MIC was determined after overnight incubation of the plates at 37°C. The microtitre plates were scored for growth in the wells and the M I C was taken as the lowest antibiotic concentration at which growth was inhibited. The influence of iron and zinc on the antibiotic susceptibilities of P. aeruginosa strains was determined using B M 2 minimal media with succinate as a carbon source and varying concentrations of each ion.  27  Fig 5:  Diagram of plasmid pMC2 utilized for the overexpression of the mexT gene.  The coding region of the mexT gene is shown. The orientations of the ampicillin resistance gene, origin of replication and the lac promoter are indicated. The 1.8 kb stabilizing fragment is for maintenance of the plasmid in P. aeruginosa.  28  2.11  X y l E assays  P.aeruginosa strains containing a chromosomal oprD::xylE transcriptional fusion and either no plasmid, pUCP19, or pMC2, were assayed for X y l E activity as described previously (Deretic and Konyecsni, 1988). Briefly, cells were grown with appropriate antibiotic selection to and O D  600  of -0.5  and then harvested. Cell pellets were  resuspended in 750ul of 50mM K phosphate buffer, pH 7.5, and 10% acetone. Cells were broken by 1 min sonication on ice. Unbroken cells and cell debris were removed by centrifugation for 20 min at 5000 rpm, at 4°C. Supernatant was removed to a clean tube. Protein concentration was determined as described previously. Two volumes of supernatant were assayed from each sample in a total volume of 1 ml containing 50mM K phosphate buffer, p H 7.5 and 0.3 m M catechol. Reaction rates were recorded in a PerkinElmer (Lambda3) dual-beam spectrophotometer coupled to a Perkin-Elmer 561 chart recorder, by following the change of absorbance at 375 nm. Results are given in pmol of product produced per min per \xg of extract. The molar extinction coefficient of the product, 2-hydroxymuconic semialdehyde, is 4.4 x 10 . A l l experiments were done in 4  triplicate.  2.12  Growth phase experiments  For Growth phase experiments, P.aeruginosa P A O l (HI03) was grown in M H broth with shaking at 37°C. Samples for analysis of the outer membrane profiles were taken in midlog, late-log and stationary phases of growth. Stage of growth was determined by monitoring O D  600  and plotting the growth curve.  29  3  RESULTS  Chapter One: Analysis of a Gene Involved in the Regulation of Expression of OprD  3.1.1  Molecular cloning of the mexT Gene  Using the primers designed on the basis of the data from the P.aeruginosa genome sequencing project, a P C R reaction was done. A fragment of approximately 1.2 kb was obtained. This was consistent with the size predicted from analysis of the sequence in the P.aeruginosa genome sequencing project. The 1.2 kb fragment was cut with BamHI and EcoRI and cloned into pUCP18 to yield plasmid p M C l (reverse orientation compared to the lac promoter) (Fig. 4), and into pUCP19 to yield plasmid pMC2 (forward orientation compared to the lac promoter) (Fig. 5).  BgH(34) BamHI(l)  BgUI(564)  , /  AraI(5S6) tadl(38)  B«I  /  BgJI(869)  s»Hf7«l P™n(673) t ! S ! d I  7 4 3  C M (1076)  \EnII(904) \ B M I\  Piral(1080) , EcoRI(1151)  1151  mexT lOObp  Fig 6:  Physical map of the mexT gene.  Some of the endonuclease restriction sites used for the cloning and subcloning of mexT are shown. The BamHI and EcoRI restriction sites were engineered into the primers used to P C R up the mexT gene and are not part of the sequence.  30  3.1.2  Nucleotide sequence of the mexT gene  Both strands of the 1.2 kb BamHI/'EcoRI P C R fragment were sequenced. When this sequence was compared to the sequence from the P.aeruginosa genome sequencing project, a number of discrepancies were noticed. There was an 8bp duplication, a lbp deletion and 4bps that were different to the sequence obtained. From analysis of the chromatograms obtained during the sequencing of the P C R product, it was determined that the sequence shown in Fig. 7 is the correct sequence. The errors in the genome project sequence probably resulted from the fact that this sequence from the sequencing project has not been fully checked and edited yet. The fragment contains a single open reading frame (ORF) of 912 nucleotides (Fig. 7). It encodes a 304 amino acid protein with a calculated molecular weight of 33,422. Consistent with this, overexpression of the gene, designated mexT, using the pET-based expression system (Invitrogen, Ca.), yielded a major protein band with an M r of approximately 33kDa (data not shown). The GC content of the whole gene is 68%, and at the third codon position it reaches 82.2%. Both of these sequence features are typical of a P.aeruginosa gene. Putative -35 and -10 promoter regions were identified upstream of mexT (Fig. 7). The 6 nucleotides, A A G G A G , which constitute a consensus bacterial ribosome binding site (SD) are not present upstream of the mexT start site. However a putative SD sequence, A A C G A G , does exist (Fig. 7). There are at least two potential A T G start codons at the predicted 5' end of the mexT gene, although the largest ORF is likely to be correct in the light of the similarity between the largest deduced MexT product and other bacterial proteins (see below).  31  CCTGTCAGTGATCCTATGCCCCTCCGGCACCTCGCCAGGCCCCGCCCCGTCTCGCACGCA -35  -10  SD  60  +l->  AGGCTTGACGGCGAGCCCCCGCGGTTGCAGCCTCTAGCCCCTGGAAACGAGGAACGCCAT M  120 1  GAACCGAAACGACCTGCGCCGCGTCGATCTGAACCTGCTGATCGTGTTCGAGACCCTGAT N R N D L R R V D L N L L I V F E T L M 2  1  180  GCACGAACGCAGCGTGACCCGCGCCGCAGAGAAACTGTTCCTCGGCCAGCCGGCCATCAG H E R S V T R A A E K L F L G Q P A I S 4  1  CGCCGCGCTGTCGCGCCTGCGCACGCTGTTCGACGACCCGCTGTTCGTCCGTACCGGACG A A L S R L R T L F D D P L F V R T G R 6  1  CAGCATGGAGCCCACCGCGCGAGCCCAGGAAATCTTCGCCCACCTGTCGCCGGCGCTGGA S M E P T A R A Q E I F A H L S P A L D 8  1  240  300  3 60  TTCCATCTCCACCGCCATGAGTCGCGCCAGCGAGTTCGATCCGGCGACCAGCACCGCGGT S I S T A M S R A S E F D P A T S T A V  4 20 101  GTTCCGCATCGGCCTTTCCGACGACGTCGAGTTCGGCCTGTTGCCGCCCATGCTCCGCCG F R I G L S D D V E F G L L P P M L R R  480 121  CCTGCGCGCGGAGGCGCCGGGGATCGTCCTCGTCGTGCGCCGCGCCAACTATCTATTGAT L R A E A P G I V L V V R R A N Y L L M  540 141  GCCGAACCTGCTGGCCTCGGGGGAGATCTCGGTGGGCGTCAGCTACACCGACGAACTGCC P N L L A S G E I S V G V S Y T D E L P  600 161  GGCCAACGCCAAGCGCAAGACCGTGCGCCGCAGCAAGCCGAAGATCCTCCGCGCCGACTC A N A K R K T V R R S K P K I L R A D S  660 181  CGCGCCCGGCCAGCTGACCCTCGACGACTACTGCGCGCGACCGCACGCGCTGGTGTCCTT A P G Q L T L D D Y C A R P H A L V S F  7 20 201  CGCCGGCGACCTCAGCGGCTTCGTCGACGAGGAGCTGGAAAAATTCGGCCGCAAACGCAA A G D L S G F V D E E L E K F G R K R K  7 80 221  GGTGGTCCTGGCGGTGCCGCAGTTCAACGGCCTCGGCACCCTCCTGGCCGGCACCGACAT V V L A V P Q F N G L G T L L A G T D I  840 241  CATCGCCACCGTGCCCGACTACGCCGCCCAGGCGCTGATCGCCGCCGGCGGCCTACGCGC I A T V P D Y A A Q A L I A A G G L R A  900 261  CGAGGACCCACCGTTCGAGACCCGCGCCTTCGAACTGTCGATGGCTTGGCGCGGCGCCCA E D P P F E T R A F E L S M A W R G A Q  960 281  GGACAACGATCCGGCCGAACGCTGGCTGCGCTCGCGGATCAGCATGTTCATCGGCGATCC D N D P A E R W L R S R I S M F I G D P  1020 301  GGACAGTCTCTGAGCCCTCCGGCAGCTACCCGCACGAGGCGTCGCAACGGGAAAATCGAT D S L *  1080 304  CGCGCGCCGCGGGTGTGCGGCTTATTCCATCCGAAAGCACTGTCCATAACCATCGACTGT  1040  TACAGAAAACG  1051  Fig 7:  Nucleotide and deduced amino acid sequence o f the mexTgene.  A potential Shine-Dalgarno (SD) sequence is shown (dashed line). The start site o f the mexTgene is indicted along with its putative - 1 0 and - 3 5 promoter regions.  32  3.1.3  MexT shows similarity to NahR and other LysR-type transcriptional regulators  The deduced MexT protein sequence was compared with sequences in the Genbank database using the B L A S T algorithm. The search for amino acid sequences similar to MexT identified 14 proteins with similariity scores greater than 115. Thirteen out of the first 14 were NodD proteins from various species of Rhizobium. The protein with the highest similarity was NahR. NahR is a LysR-type transcriptional regulator (LTTR) that regulates production of enzymes for the metabolism of naphthalene and salicylate as sole carbon sources in Pseudomonas putida (You., et al. 1988). Alignment of the two sequences (MexT, 304 aa; NahR, 300 aa) revealed that there were 33% identical residues and 14.5% conserved residue changes (Fig. 8). The regions of similarity were concentrated somewhat in the N-terminal portion of the proteins. This region of NahR contains the D N A binding helix-turn-helix - (HTH) domain (Fig. 8. residues 26-56) of the protein. A number of very highly conserved residues in NahR essential for D N A binding (Ala 27, Pro 35, Thr 56) were also conserved in MexT (Fig. 8. bold). It is likely therefore that the N-terminal region of MexT contained a D N A binding H T H domain. Analysis of the region of NahR which binds salicylate as a coninducer (Fig. 8. aa 95-170), and the residues important for binding, showed little similarity and no conservation of residues in the same region of MexT. This was consistent with other LTTRs which show little similarity in this region. The C-terminal portion of the two proteins also showed limited similarity.  33  MEXT  MNRNDLRRVDLNLLIVFETLMHERSVTRAAEKLFLGQPAISAALSRLRTL * _** _*****_** * * * ** * * *** * ** ****  NAHR  M  MEXT  FDDPLFVRTGRSMEPTARAQEIFAHLSPALDSISTAMSRASEFDPATSTA ******* **** * _ .. *. . * *** **  100  NAHR  LQDPLFVRTHQGMEPTPYAAHLAEPVTSAMHALRNALQHHESFDPLTSER  97  MEXT  VFRIGLSDDVEFGLLPPMLRRLRAEAPGIVLVVRRANYLLMPNLLASGEI  150  ELRDLDLNLLVVFNQLLVDRRVSITAENLGLTQPAVSNALKRLRTS  *  *  *  *  *  **  *  *  *  *  NAHR  TFTLAMTDIGEIYFMPRLMDVLAHQAPNCVISTVRDSSMSLMQALQNGTV  MEXT  SVGVSYTDELPANAKRKTVRRSKPKILRADSAPGQ  *  * _  *  LTLDDYCARPHA  *  * * * * *  50 4 7  14 7 197  NAHR  DLAVGLLPNLQTGFFQRRLLQNHYVCLCRKDHPVTREPLTLERFCSYGHV  197  MEXT  LVSFAGDLSGFVDEELEKFGRKRKVVLAVPQFNGLGTLLAGTDIIATVPD  2 47  *  **  ***  ^ ^ *  _*  _ * * * *  _ *  _ *  **__****  NAHR  RVIAAGTGHGEVDTYMTRVGIRRDIRLEVPHFAAVGHILQRTDLLATVPI  2 47  MEXT  YAAQALIAAGGLRAEDPPFETRAFELSMAWRGAQDNDPAERWLRSRISMF * _ ** * * * * * * ***  2 97  NAHR  RLADCCVEPFGLSALPHPVVLPEIAINMFWHAKYHKDLANIWLRQLMFDL  2 97  MEXT  IGDPDSL *  304  NAHR  FTD  300  Fig 8.  Similarity of MexT to NahR.  Amino acid sequences of MexT and NahR were aligned with the P A L I G N program of the PCGene software package (Intelligenetics Inc.). The character to show that two aligned residues are identical was '*'. The character to show that two aligned residues are similar was '.' Amino acids considered to be 'similar' are: A,S,T; D,E; N,Q; R,K; I,L,M,V; F,Y,W  34  3.1.4  Overexpression of the mexT gene in P.aeruginosa  To determine the effect of overexpressing mexT in P.aeruginosa, plasmid pMC2 was transformed into a P.aeruginosa wild-type strain, HI03. mexT is in the forward orientation compared to the lac promoter, allowing overexpression of mexT, as the lac promoter is constitutive in P.aeruginosa. The antimicrobial susceptibility and outer membrane profiles of this strain were examined. The MICs for the various strains are shown in Table IV. Overexpression of mexT resulted in a 32 fold increase in resistance to chloramphenicol, an 8-fold increase in resistance to norfloxacin and a 2-4-fold increase in resistance to imipenem. There was a 2-fold increase in susceptibility to gentamicin while there was no change in the M I C of tetracycline. This was in comparison to the wild-type strain with and without the vector. The high M I C for carbenicillin in the strains containing pUCP19 and pMC2 was due to the plasmid encoded antibiotic marker. The antibiotic susceptibility profile when mexT was overexpressed was consistent with that of the nfxC-type mutant 1008OIR01 (Table IV). The characteristic increase in susceptibility to carbenicillin seen in nfxC-type mutants could not be confirmed due the use of carbenicillin to maintain the plasmids.  Analysis by SDS-PAGE of the outer membrane proteins of the strains in Table IV showed reduced expression of a 45kDa protein (Fig. 9, lane 4) in the mexT overexpressing strain compared to the control strains (Fig. 9, lanes 2 and 3). This was confirmed to be OprD by Western blot analysis using an OprD specific mAb (Fig. 10A, lanes 2, 3 and 4).  35  Table IV:  MICs for P. a e r u g i n o s a strains.  Strains  H103 H103 + p U C P 1 9 H103 + pMC2 1008 1008OIR01  MICs ( ucj/ml) CARB  CEFP  IM  GENT  NOR  CM  TET  25 >400 >400 25 6.25  0.5 1 1 0.5 0.5  1 0.5 2 1 4  0.39 0.39 .0.195 0.39 0.195  0.195 0.195 1.56 0.195 3.13  12.5 12.5 400 25 >800  3.13 1.56 3.13 3.13 3.13  •Abbreviations: CARB, carbenicillin; CEFP, cefpirome; IM, imipenem; GENT, gentamicin; NOR, norfloxacin; CM, chloamphenicol; TET, tetracycline.  This reduction of OprD in the outer membrane was consistent with the nficC mutant (Fig. 9, lane 6 and Fig. 10A, lane 6). Outer membrane preparations were further analysed by Western blotting using anti OprM, OprJ and OprN mAbs. In the mexT  overexpressing  and nfxC mutant, a protein of 50kDa hybridised with the OprN mAb (Fig. 10B, lanes 4 and 6). No OprN expression was detected in the parent and control strains (Fig. 10B, lanes 2, 3 and 5 ). No cross hybridization bands were seen with the OprJ antibody (data not shown), while the OprM mAb revealed only wild-type levels of OprM in all strains (Fig. 10C, lanes 2-6). No other changes in the outer membrane profiles were observed. These results suggest that overexpression of mexT n/xC-type phenotype.  36  is itself sufficient to reproduce an  OprD (45kDa)  1  Fig 9: gene.  2  3  4  5  6  SDS-PAGE demonstrating repression of OprD by overexpression of the mexT  The banding position of OprD is indicated by the arrow on the right. Lanes: 1, molecular markers; 2, H103; 3, H103(pUCP19); 4, H103(pMC2); 5, 1008; 6, 1008OIR01. For each lane, 20pg of outer membrane protein was applied.  37  .  4fc  —«  4  OprD(46kDa)  4  OprN (50kDa)  B  1 Fig 10:  2  3  4  5  6  Western immunoblots of P. aeruginosa strains.  Lanes: 1, molecular markers; 2, H103; 3, H103(pUCP19); 4, H103(pMC2); 5, 1008; 6, 1008OIR01. For each lane, 20ug of outer membrane protein was applied. A , B, C were blotted with anti-OprD m A B , anti-OprN mAb and anti-OprM mAB respectively.  38  3.1.5  MexT reduces transcription of oprD  Given the fact that MexT had high homology to the LysR-type transcriptional regulator NahR, and that overexpression of mexT resulted in reduction in the level of OprD in the outer membrane, it was believed likely that MexT exerted its effect at the transcriptional level. To test this directly, plasmid pMC2 containing the mexT gene in the forward orientation relative to the lac promoter, was transformed into the P. aeruginosa strain, HI03-1,  which had an oprDr.xylE  transcriptional  fusion in the chromosome.  Overexpression of mexT in this strain resulted in a 2.7-fold reduction in X y l E activity (Table V). To rule out the possible involvement of other genes present on the pM2 plasmid in the observed repression, vector alone (pUCP19) was transformed in HI03-1. In this instance only a 10% reduction in X y l E activity was detected (Table V). The effect of overexpressing mexT'm E.coli on an oprDr.xylE plasmid encoded fusion could not be determined due to the instability of the mexT overexpressing construct in E.coli. This would have allowed us to test whether mexT regulates oprD via a third gene.  39  Table V: Effect of MexT on an oprDr.xylE chromosomal transcriptional fusion in P.aeruginosa. i  !  Strain H103-1  Vector  (  none  | XylE activity (values given in I pmol of product per min per ug extract)* | 61 +/- 5  pUCP19  I  55+/-12  pMC2 22 +/- 5 *Data are reported as the means of three separate experiments. Data from each experiment was in turn the mean of two different measurements using different protein concentrations  3.1.6  Summary  The mexT structural gene was obtained by using sequence data from the P. aeruginosa genome sequencing project to design primers that allowed amplification of the gene from the genome of P.aeruginosa P A O l using PCR. D N A sequencing of the obtained PCR product revealed an ORF of 912 bp, that encoded a 304 aa mature MexT protein. The sequence showed homology to the LysR-type transcriptional regulator (LTTR) family of proteins. It showed highest homology to the LTTR, NahR from P.putida. Overexpression of the mexT gene in P.aeruginosa increased resistance to norfloxacin, chloramphenicol and imipenem. Outer membrane profiles were also changed by mexT overexpression, with reduction of OprD and expression of the outer membrane component of the mexE/FoprN efflux operon, OprN. MexT was shown to reduce transcription of the oprD gene using a chromosomal oprDr.xylE transcriptional fusion.  40  Chapter Two: The search for factors affecting mexT expression in P.aeruginosa. 3.1.7  Introduction  In E.coli, salicylate, a plant-derived compound, has been shown to reduce the expression of outer membrane porins as well as induce low-level resistance to some antibiotics. Salicylate does this by antagonizing the repressor activity of the MarR protein which negatively regulates another regulatory locus, marAB. By reducing binding of MarR to the promoter region of the marAB genes, salicylate relieves the repression of MarA which in turn results in changes in the expression of several unlinked target genes. Among these, two have clear relevance to antibiotic resistance. The levels of the major outer membrane porin OmpF are substantially reduced, and expression of the multi-drug efflux pump, AcrAB is induced (for review see Miller and Sulavik, 1996). In P.aeruginosa, it has been shown that salicylate reduces expression of an outer membrane protein with a molecular mass of 45 kDa (Sumita and Fukasawa, 1993). Salicylate has also been suggested to induce expression of a 50-kDa protein thought to be OprN (Masuda et al., 1995). With these results in mind, as well as the fact that MexT was found to be similar to NahR, we were interested in investigating the possibility that salicylate or other structurally related compounds may be an inducer of the mexE/F-oprN operon by acting as a cofactor of the MexT protein. This was done by examining the effects of these compounds on the antimicrobial susceptibilities and outer membrane profiles of P. aeruginosa.  41  A number of other factors suspected to play a role in the expression of the known efflux operons as well as expression of the imipenem specific porin, OprD, in P.aeruginosa were investigated. These included iron and zinc concentrations as well as the growth phase of the bacterium. This was done by looking at either MICs, outer membrane profiles or a combination of both. 3.1.8  Salicylate and sodium benzoate; effects on antibiotic susceptibilities and outer membrane protein production in P.aeruginosa.  Shown in Table VI are the antibiotic susceptibilities of P.aeruginosa P A O l in the presence of salicylate and a number of other compounds known to induce multiple antibiotic resistance in E.coli. Salicylate induced an 8 fold increase in resistance to imipenem, a 2 fold increase in resistance to chloramphenicol, norfloxacin and cefpirome and a 4 fold increase in susceptibility to carbenicillin (Table VI). The only significant change in M I C with sodium benzoate was a 4 fold increase in resistance to imipenem (Table VI). Dinitrophenol, naphthalene, and catechol induced no significant changes in susceptibility to any of the antibiotics tested, while naphthaquinone showed only a 2 fold increase in resistance to carbenicillin, cefpirome, norfloxacin and tetracycline (Table VI).  Interestingly, the antimicrobial susceptibility profile of P.aeruginosa in the presence of salicylate was similar to that of an nfxC-type mutant;  i.e. increased resistance to  norfloxacin, chloramphenico, imipenem and increased sensitivity to carbenicillin (Table IV). To determine whether these changes were due to expression of one of the efflux operons, we examined the outer membrane profiles of P.aeruginosa when grown in the presence of 32 m M salicylate. We also looked at the outer membrane profiles of 42  P.aeruginosa when grown in the presence of 16 m M sodium benzoate to see whether the increased resistance to imipenem might be due to reduced OprD.  43  Table V I :  Antibiotic susceptibilities of P.aeruginosa P A O l in Mueller-Hinton in the  presence of salicylate and other structurally related compounds.  I'" ' Compound none | Salicylate I Sodium benzoate Dinitrophenol Naphthalene Naphlhaquinone Catechol  " "~  |MIC (ug/ml)*  CARB 25  '  25 12.5 2 5 50 50  ' —  CEFP 0.5 1 "~ 1 0.5 "1 1 1 _ 1  IM | 1 ! 8 l~ 4 I 1 ! 1 " ' 1 | 1  _j_  GENT 0.39 1.56 0.78 0.39 0.39 0.39 o : 3 g  NOR CM IET 0.195 12.5 3.13 " 0.39 25 3.13 0.195 12.5 " " 3 . 1 3 0.195 | 12.5 0.195 " 1 2 . 5 3.13 0.39 " 6.25 " 0.195 ^ 6.25 ~ 3.13 3  1 3  "  [  "Abbreviations: CARB , carbenicil in; CEFP, cefpirome; IM, imipenem; GENT, gentamicin; NOR, norfloxacin; CM, chloramphenicol; TET, tetracycline. |  Salicylate and sodium benzoate both reduced expression of a 45 kDa protein when included in the media at the appropriate concentrations (Sal, 32 m M ; SB, 16 mM) (Fig. 11, lanes 3 and 4), compared to media alone (Fig. 11, lane 2). This was confirmed to be OprD using Western blotting with anti-OprD mAb (Fig 12A, lanes 2-4). Wild type levels of OprM were detected in all samples using anti-OprM mAb (Fig. 12B, lanes 2-4). No cross-reacting bands were revealed with the OprJ antibody (data not shown), or the OprN antibody (Fig. 12C, lanes 4-6).  44  OprD(46kDa)  1  Fig 11:  2  3  4  Effect of salicylate and sodium benzoate on the outer membrane proteins of  P.aeruginosa P A O l .  Lanes: 1, molecular markers; 2, H103 grown in M H ; 3, H103 grown in M H plus salicylate (32mM); 4, H103 grown in M H plus sodium benzoate (16mM). For each lane, 20u.g of outer membrane protein was applied.  45  •sin  «•*  Hjjjj  '  g <—  <«— O p r M (50kDa)  OprD (46kDa)  1  2  3  4  OprN(50kDa)  F i g 12: Western immunoblots o f outer membranes o f P. aeruginosa grown in the presence o f 32 m M salicylate and 16 m M sodium benzoate. A and B Lanes: 1, molecular markers; 2, H I 0 3 grown in M H ; 3, H I 0 3 grown in M H plus salicylate (32mM); 4, H103 grown in M H plus sodium benzoate (16mM). C Lanes: 1, molecular markers; 2, 1008; 3, 1008OIR01; 4, H103 grown in M H ; 5, H103 grown in M H plus salicylate (32mM); 6, H I 0 3 grown in M H plus sodium benzoate (16mM). For each lane, 20ug o f outer membrane protein was applied. A , B, C were blotted with antiOprD m A B , anti-OprM m A b and anti-OprN m A b respectively.  46  3.1.9  Salicylate reduces transcription of the oprD gene in P.aeruginosa.  To further examine the mechanism through which salicylate and sodium benzoate reduce OprD levels in the outer membrane, we looked at the effect the compounds had on the oprDr.xylE chromosomal transcriptional fusion in P.aeruginosa P A O l . When grown in M H supplemented with 32 m M salicylate, X y l E activity was reduced by 3.3 fold compared to M H alone (Table VII). This indicated that salicylate reduced OprD expression by reducing transcription of the oprD gene. When grown in M H with 16 m M sodium benzoate X y l E activity increased by 1.3 fold compared to M H alone. This suggests that  sodium benzoate  exerts its  transcriptionally.  47  effects  on OprD  expression  post-  Table VII:  Effect of salicylate and sodium benzoate on an oprDr.xylE chromosomal  transcriptional fusion in P.aeruginosa.  pmol of product per min per ug extract)* H103-1  none  [ 82 +/-11  \  Ii  32mM Sal  j 25+/-9 i M i l +/-14" TBrnWI S& Data are reported as the means of three separate experiments. Data from each experiment is in turn the mean of two different measurements using fwo different "protein concentrations I | I " S a l , salicylate (32m M); SB, sodium benzoate (TBmM).  3.1.10 Effect of iron and zinc concentrations on the antibiotic susceptibilities of P.aeruginosa. Table VIII shows the antimicrobial susceptibilities of P.aeruginosa P A O l to various antibiotics grown in M H and B M 2 minimal medium with varying concentrations of iron and zinc. The addition of excess iron to M H resulted in no changes in the M I C of the antibiotics tested (Table VIII). Addition of supplementary zinc to the M H media resulted in a four fold increase in resistance to imipenem and a two fold increase in resistance to cefpirome (Table VIII). When B M 2 minimal medium supplemented with 20mM iron was used instead of M H , there was a two fold increase in susceptibility to tetracycline, chloramphenicol, imipenem, and cefpirome, no change for carbenicillin and norfloxacin, and a two fold increase in resistance to gentamicin (Table VIII). When no iron was added to the B M 2 minimal media making it iron deficient, there was a consistent two to four  48  fold increase in the susceptibility to all the antibiotics tested compared to iron sufficient medium, except for gentamicin which showed a two fold increase in its MIC (Table VIII). Interestingly, when iron deficient B M 2 was supplemented with 0.1 mM zinc, the MIC of any of the antibiotics could not be determined due to no growth in any of the wells.  49  Table VIII:  Effect of iron and zinc concentrations on the antibiotic susceptibilities of  P.aeruginosa  Media MH MH + 20mM Fe** MH + 0.1 mM Zn** BM2 + 20mM Fe BM2 - Fe BM2 - Fe + 0.1 mM Zn  CARB 25 25 25 25 12.5 NG  CEFP 1 1 2 0.5 0.25 NG  IM 1 1 4 0.5 0.25 NG  MIC (ug/ml)* GENT 0.39 0.39 0.39 0.78 0.78 NG  NOR 0.195 0.195 0.195 0.195 0.097 NG  CM 12.5 12.5 12.5 6.25 3.13 NG  TET 3.13 3.13 3.13 1.56 0.78 NG  "Abbreviations: CARB, carbenicillin; CEFP, cefpirome; IM, imipenem; GENT, gentamicin; NOR, norfloxacin; CM, chloramphenicol; TET, tetracycline. NG, no growth **lron and Zinc added as Fe and Zn sulphate.  The increase in resistance of P.aeruginosa to imipenem in the presence of zinc was investigated further to try to identify the mechanism of resistance. Expression of the imipenem specific porin, OprD, was examined in the presence of varying concentrations of zinc (Fig. 13). There was no change in OprD expression as observed by using SDSP A G E (Fig. 13A), and Western blotting with an OprD mAb (Fig. 13 B). Expression of OprM, OprJ and OprN were also checked but no expression of OprJ or OprN was detected by Western blotting with the respective mAbs, while only wild type levels of OprM were found (data not shown).  50  OprD (46kDa)  OprD(46kDa)  1  2  3  4  Fig 13: Outer membrane profiles o f P.aeruginosa P A O l grown i n Mueller-Hinton with different concentrations of zinc. (A) S D S - P A G E and (B) Western-immunoblot with anti-OprD m A b . Lanes: 1, molecular standards; 2 , M H only; 3, M H with zinc (6ug/ml); 4, M H with zinc (12ug/ml). For each lane, 20ug o f outer membrane protein was applied.  51  3.1.11 Effect of growth phase on expression of the mex efflux operons of P.aeruginosa. Outer membrane profiles of P.aeruginosa P A O l during the various growth phases were examined using SDS-PAGE and Western blotting. Samples were taken during mid-log phase (Fig. 14 A , Lane 2), late log phase (Fig. 14 A , Lane 3), and late stationary phase (Fig. 14, Lane 4). No visible changes in OprD, OprE or OprF levels were observed during the growth. The level of OprG seemed slightly elevated during log phase compared to late log and stationary phase (Fig 14 A , lane2). OprH was highly produced during log phase (Fig. 14 A , lane 2), while, OprL showed increased levels in stationary phase (Fig. 14 A , lane 4). Analysis by Western blotting using monoclonal antibodies against OprM, OprJ and OprN, was also done. The OprM antibody detected only wild-type levels of OprM throughout growth (Fig. 14 B, lanes 2-4). No cross reacting bands were detected with either the OprJ or OprN antibodies in any of the samples (data not shown).  52  + <  OprD(46kDa) ' OprE(44kDa) OprF(35kDa)  OprG(25kDa) OprH(21kDa) OprL(20kDa)  B  Fig 14:  OprM(50kDa)  Outer membrane profiles of P.aeruginosa P A O l during growth phases.  (A) SDS-PAGE and (B) Western-immunoblot with anti-OprM mAb. Lanes: 1, molecular standards; 2, HI03 mid-log phase; 3, HI03 late-log phase; 4, late stationary phase. For each lane, 20ug of outer membrane protein was applied.  53  3.1.12 Summary When grown in the presence of 32mM salicylate, P.aeruginosa had increased resistance to imipenem, chloramphenicol, norfloxacin and gentamicin while becoming more sensitive to carbenicillin. Sodium benzoate gave increased resistance to imipenem while none of the other compounds tested, naphthalene, naphthaquinone, dinitrophenol or catechol, resulted in any significant increase in resistance to any of the antibiotics tested. Salicylate and sodium benzoate reduced the amount of the outer membrane porin, OprD. No other changes in the outer membrane profiles were noticed. Iron concentrations had no effect on the antibiotic susceptibilities oi P. aeruginosa while zinc increased its resistance to imipenem. This increase was not due to the loss of the imipenem specific porin OprD as seen by the normal levels of OprD seen in the outer membrane when grown in media containing high levels of zinc. The outer membrane profiles of P.aeruginosa were examined during the various growth phases for the presence of the 3 efflux operons known in P.aeruginosa, mexA/B-oprM, mexCD-oprJ and mexEF-oprN. There were no changes in the amounts, with only wildtype levels of OprM and no presence of either OprJ or OprN detected in the outer membrane during any of the growth phases.  54  4  DISCUSSION  The P.aeruginosa outer membrane porin, OprD, is an interesting protein as it facilitates the diffusion of basic amino acids and imipenem, a potent antibiotic that has been used for the treatment of P.aeruginosa. While much has been discovered about OprD's structure function relationships little has been uncovered about its regulation until recently. In this study the cloning, sequencing and characterization of a gene, mexT, involved not only in the regulation of oprD, but also in the regulation of an efflux operon, mexEF-oprN, is described. These and other tests set the ground for the detailed analysis of the mechanism of regulation of these genes by the MexT protein.  MexT; a new member of the LysR-type transcriptional regulator family. D N A sequencing of the mexT gene detected a 33kDa ORF which probably represents the amino acid sequence of the MexT protein (Fig. 7). This amino acid sequence has extensive similarity with the derived sequence of the NahR protein of Pseudomonas putida (Fig. 8). It also has lower but significant similarity with the derived sequences of the NodD proteins of Rhizobium species (data not shown). A l l three of these proteins are nearly identically sized transcriptional activators and it is likely that that the regions of greatest similarity between them represent protein domains involved in common functions (e.g., D N A binding or transcriptional activation or both). The amino acid sequence of the domain of MexT containing the region of greatest similarity between the three (common domain; residues 24-60) (Fig. 8), had a protein sequence which was  55  similar to the amino acid sequence of the helix-turn-helix (HTH) DNA-binding motif of the lambda Cro protein (Dodd and Egan, 1987). Henikoff et al. reported the discovery of a family of prokaryotic transcriptional activators evolutionary related to LysR (Henikoff et a l , 1988). This family included the NodD proteins and a number of other proteins, all of which were approximately 300 residues in length and most, if not all, of which are transcriptional activators. More recently, NahR has also been shown to belong to this family of transcriptional regulators (Schell and Sukordhaman, 1989). Comparison of the MexT protein sequence with the Henikoff consensus sequence for this protein family clearly showed that MexT is a member of this family (41% similarity over the first 150 residues; 33% similarity overall) (Henikoff et al., 1988). Schell and Sukordhaman (Schell and Sukordhaman, 1989) noticed that NahR and the NodD proteins had much greater similarity to each other than any other members of the family. They suggested that these two proteins might represent a subfamily within the LysR-type family of transcriptional activators. MexT may represent another member of this proposed subfamily based on its much greater similarity to NahR and the NodD proteins than any other members of the LysR-type family of transcriptional activators. Additional evidence that the more conserved N-terminal region of MexT is indeed as proposed here, a H T H domain, comes from the comparison of the LysR-type regulators consensus H T H sequence (Henikoff et a l , 1988), with the same region from MexT. This region showed 50% identity and 20% conserved residues compared to the consensus sequence. 56  MexT; an atypical LysR-type transcriptional regulator. Most members of the LysR-type transcriptional regulator (LTTR) family share four common characteristics: (a) They encode coinducer-responsive transcriptional activator proteins, (b) Independent of the presence of coinducer they bind at regulated targets to D N A sequences, (c) Each is divergently transcribed from a promoter that is very close to and often overlaps a promoter of a regulated target gene, (d) Because the overlapping divergent promoter structure  allows for simultaneous bi-directional control  of  transcription, most LTTRs repress their own transcription. The mexT gene and the protein which it encodes, MexT, show some interesting differences to the 'typical' members of the L T T R family. As judged from analysis of the sequence from the P.aeruginosa genome sequencing project, the mexT gene lacks the divergent promoter structure so characteristic of members of this family of proteins. Transcription occurs in the same direction as that of the mexEF-oprN operon (sequence data not shown) (Fig. 3 shows a schematic representation). Another exception to this rule is the phcA L T T R of Pseudomonas solanacearum which is transcribed in the same direction as its target genes (Brumbley et al., 1993) When overexpressed from a multi-copy plasmid, MexT not only resulted in expression of the mexEF-oprN operon, as judged by the presence of OprN in the outer membranes (Fig. 10B, Lane 4), but also resulted in reduced levels of the outer membrane porin, OprD (Fig. 10A, Lane 4). These result would suggest that MexT can not only act as an activator of  57  target genes, as in the case of the mexEF-oprN operon, but also as a repressor, as in the case of oprD. Most LTTRs described to date positively regulate their target genes while negatively regulating their own expression. They do this by binding to the same target D N A sequence in both cases, but the positioning of this sequence is very different. In activated genes the target sequence is upstream of the -35 region of the promoter, while in cases where the gene is repressed the target sequence overlaps the transcriptional start site. It seems likely that MexT would act in a similar manner with relation to the activation of the mexEF-oprN operon, but as discussed below, the mechanism of repression is unclear. While unusual, this would not be the first example of an L T T R acting both as a repressor and an activator. The TfdS protein of Agrobacterium eutrophus is reportedly a repressoractivator (Kaphammer and Olsen, 1990). Interestingly, this protein's repressor activity is low for a repressor, with a 40%  maximal repression of transcription. MexT  overproduction resulted in a 64%  repression of an oprDr.xylE chromosomal  transcriptional fusion in P. aeruginosa (Table V). This result is consistent with the level of OprD in the outer membrane in both the mexT overexpressing strain and the nfxC mutant (Fig. 10A, Lanes 4 and 6). This would suggest that the nfxC mutation results in overexpression of the mexT gene rather than by making the protein independent of any coinducer. It is be possible that MexT does not need a coinducer to function, as is the of AmpR protein of Enterobacter cloacae (Honore, et al., 1986) but it is more than likely that MexT has at least one coinducer that it responds to.  58  The mechanism by which MexT represses oprD expression is an interesting question. Normally, L T T R proteins repress transcription by occluding the promoter region by binding to and overlapping the transcription start site. However, in the case described here, one still gets some expression of the oprD gene. Once the exact binding site of the MexT protein in the promoter region is identified, it will allow us examine possible repression mechanisms more closely. Recent unpublished data (Kohler et al., 1998) has suggested that there is no mutation in the coding region of the mexT gene in the nfxC-type mutants. This would tend to rule out the possibility of a change in the binding properties and hence the normal function of the MexT protein. So how might the mexT gene get overexpressed in nfxC-type mutants as suggested here? It is likely that the mexT gene negatively autoregulates. A n explanation for this suspected mexT overexpression in nfxC-type mutants, would be that there is a mutation in the MexT target D N A sequence in the promoter region of the mexT gene itself. A mutation in the promoter region of the mexT gene that would eliminate this negative autoregulation would allow for overexpression of the mexT gene resulting in expression of the mexEF-oprN operon and repression of the oprD gene. Once the sequence to which MexT binds has been determined, the sequence in the promoter region oimexT in nfxC-type mutants can be examined for potential mutations. Work is ongoing in the lab to purify the mature MexT protein from the wild-type P. aeruginosa P A O l strain, and to test the binding of this protein to the promoter regions of mexT, mexE, and oprD. To assist this I have cloned the mexT gene into a His-Tag vector. Possible cofactors of MexT, as discussed below, will also be examined for their 59  affect on the binding of this protein to its target D N A sequences. These experiments will help reveal the mechanism by which MexT exerts its effects on its target genes.  Salicylate; a repressor of oprD transcription and possible cofactor of the LysR-type transcriptional regulator protein, MexT. Given the observed apparent homology between the transcriptional regulators, MexT and NahR, and the previous reports that salicylate, one of NahR's natural cofactors, reduced expression of a 45kDa protein in P.aeruginosa, we were intrigued with the possibility that salicylate reduced oprD expression via the MexT protein. Salicylate is a membrane-permeable weak acid and is known to suppress the synthesis of some outer membrane porins in a number of Gram negative bacteria (Burns et al., 1992; Sawai, et al., 1987). The mechanism by which this occurs has not been elucidated thus far. In the presence of 32 m M salicylate, the levels of a 45kDa protein in the outer membrane and the susceptibility of P.aeruginosa to the carbepenem, imipenem, were reduced. This was consistent with previous reports (Sumita and Fukasawa, 1993 and Masuda et al., 1995). However, this thesis is the first report confirming that the 45kDa protein with reduced expression was indeed OprD (Fig. 12A). Using an oprDr.xylE chromosomal fusion, I determined that the mechanism by which salicylate reduces oprD expression was at the level of transcription. When grown in 32 m M salicylate, oprD expression was reduced by 3.3 fold compared to normal (Table VII). This level is similar to the level of repression seen when mexT was overexpressed (Table V). While the  60  involvement of another regulator cannot be ruled out, I hypothesize here that salicylate does indeed repress oprD expression via the MexT protein. Unfortunately, a mexT knock out, which would have allowed the direct testing of this hypothesis, could not be obtained during the course of this study. Its construction is ongoing in the lab and will hopefully allow us to conclusively show whether or not MexT represses oprD expression in response to salicylate. Masuda et al. (1995), reported that increased resistance to ofloxacin and the induction of a protein of approximately 50kDa occurred in the presence of 32 m M salicylate. This protein was not heat modifiable and was therefore thought to be OprN and not OprM. In the paper by Fukasawa and Sumita (Fukasawa and Sumita, 1993) however, no change in the M I C to quinolones was seen and no expression of a heat unmodifiable protein was noted in the presence of 32 m M salicylate. Our results showed an M I C profile consistent with that of an nfxC-type mutant but, as stated in the results, there was only a two fold difference in any of the M I C values (Table VI). These changes are not considered significant enough to suspect meaningful induction of an efflux operon. The outer membrane profiles showed that only OprD expression was changed and that there were wild-type levels of expression of OprM and the usual lack of expression of either OprJ or OprN (Fig. 12). These data seem to suggest that salicylate alone does not induce expression of the mexEF-oprN operon. This does not contradict the hypothesis that MexT down-regulates oprD in response to salicylate. LysR-type regulators often regulate different genes in response to different signals. MexT may repress oprD in response to salicylate while not affecting expression of the mexEF-oprN operon. A s stated  61  previously, work to purify the MexT protein is underway in the lab. Promoter binding studies in the presence and absence of salicylate along with the knockout of the mexT gene, will hopefully shed some light on whether salicylate indeed affects MexT interactions with its target D N A promoter sequences. Sodium benzoate reduced the levels of OprD in the outer membranes of P.aeruginosa almost to the same level as seen with salicylate (Fig . 12 and 13, lane 4). Similarly, no change in production of any of the efflux operon was observed (Fig. 13 B and C, lanes 4 and 6). Unlike salicylate though, sodium benzoate was found not to reduce transcription of the oprD gene. No conclusions can be drawn from this except to say that the mechanism of repression of oprD expression would be presumed to be posttranscriptional.  Iron and zinc as possible signals for efflux operon expression. While the role of the three mex operons of P.aeruginosa in antibiotic efflux has been widely documented (for a review see Nakae, 1998), their real function and natural substrates, i f any, await identification. It was originally proposed that the mexAB-oprM operon is involved in the excretion of the siderophore pyoverdine (Poole et al., 1993). More recently this theory has come into question (Poole, 1994). To try and elucidate whether or not iron concentrations in the growth medium were actually involved in the expression of any of the efflux operons, I looked at the effect of varying concentrations of iron on the MICs of P.aeruginosa to various antibiotics. Apart from a general increase in  62  susceptibility when the iron concentrations were limited (Table VIII), there were no noticeable changes in the pattern of susceptibility to any of the antibiotics tested. These increases in susceptibility can be simply explained by the reduced growth rate in minimal media, like B M 2 , compared to rich media, like M H . Another ion suspected in the induction of the efflux operons in P. aeruginosa is zinc. Two independent reports have shown that the high concentrations of zinc in the media can give rise to levels of imipenem resistance that are considered clinically significant (Cooper et al., 1993; Daly et al., 1997). Concentrations of 6 p.g/ml zinc were shown to significantly increase resistance to imipenem. This was consistent with the results obtained here, in which the M I C for imipenem rose from 1 u,g/ml to 4 ug/ml in the presence of supplementary zinc (Table VIII). This did still not represent a rise that would be considered 'resistant' but is a significant increase in M I C for imipenem. Previous studies have shown that cations such as zinc, calcium and magnesium affect aminoglycoside activity against P.aeruginosa (Riller et al., 1974; Casillas et a l , 1981). However, in general, cation influence on P-lactam activity has not been frequently described. Zinc-dependent P-lactamases have been isolated from only a few bacteria including from one P.aeruginosa isolate (Watanabe et al., 1991). Nonetheless, it is highly unlikely that zinc induction of P-lactamase production is a possible explanation for the increased resistance to imipenem in the presence of high zinc concentrations. Such a Plactamase would have to have limited activity against antipseudomonal cephalosporins, as seen from the only two fold increase in resistance to cefpirome (TableVIII), and would be able to specifically inactivate imipenem. Only one P-lactamase is present in the 63  P.aeruginosa P A O genome and it has been well described as having properties that could not be used to explain this zinc effect. A n enzyme which inactivates carbepenems but not cephalosporins has been found in Serratia marcescens (Yang and Livermore, 1990), but not in P.aeruginosa. Interestingly, it is activated by zinc. A n alternative explanation to the increased resistance to imipenem in the presence of high zinc concentrations, is that zinc may effect the binding of imipenem to their target PBPs. This has been shown in other bacteria but as yet not in P.aeruginosa (Sabath et al., 1990). Could zinc be acting by altering the uptake of imipenem into the cell? A n obvious candidate for this was the imipenem specific porin, OprD. Even when grown in high levels of zinc (12u.g/ml), there was no change in OprD expression (Fig. 13). Additionally, there was no change in expression of any of the outer membrane components of the mex efflux operon, namely, OprM, OprJ and OprN (data not shown). This was consistent with the MIC data obtained (Table VIII), where no increase in MIC was observed in any of the antibiotics known to be substrates of these efflux pumps. A final alternative for the observed increase in resistance to imipenem in the presence of zinc, is that zinc may be altering the chemical properties of imipenem prior to uptake into the cell reducing its activity. Poole et al. isolated a mutant that was deficient in the production of the siderophore, pyochelin, and the ferripyochelin receptor (Poole et al., 1993). This mutant when grown in iron-deficient B M 2 media supplemented with 0.1 m M ZnS0 , induced expression of a 4  protein of -50 kDa that was not heat modifiable, later thought to be OprN. The outer membrane profile also showed reduction in a protein of approximately 45 kDa, probably  x  64  OprD. Another independent report also suggested that zinc induced OprN expression in media containing high zinc concentrations (Hofte et al., 1991). As shown in Table VIII, MICs done under these very low iron and high zinc conditions resulted in no growth, although growth was observed in the preculture before the start of the assay. Interestingly, i f the preculture was grown under the same conditions except for the presence of zinc, an M I C could be determined. This would indicate that in the presence of zinc, iron limitation is so severe that the susceptibility of P. aeruginosa to the antibiotics tested was high enough to result in killing even at low antibiotic concentrations. Unfortunately, the question of whether or not the mexEF-oprN operon is indeed expressed under these conditions is unresolved. If expression of this efflux operon can be shown under these conditions, it would shed a lot of light onto possible natural roles of these systems.  Expression and role of the mex efflux operons in P.aeruginosa. The prevalence of PMF-dependent multidrug systems in a diversity of organisms raises several questions. What is the normal physiological role of such efflux systems? Is their primary role to protect the cell from toxic compounds in the environment? Or do they play roles other than detoxification, such as the transport of a particular substrate? In which cases are their abilities to efflux a wide range of structurally unrelated compounds only fortuitous rather than by design?  65  As yet it is not possible to answer these questions definitively, although, light is being shed slowly on the natural roles of these systems. The broad substrate specificity of these systems suggests that their natural role is to 'detoxify' the cells by excreting either toxic compounds from the environment that enter the cell, or getting rid of toxic by products produced by the cells metabolic processes. As stated previously the P.aeruginosa mexABoprM is highly expressed under conditions of iron starvation but is also expressed constitutively at low levels even in the presence of excess iron (Poole et al., 1993). On the other hand, nothing is know about the expression of the mexCD-oprJ and mexEF-oprN efflux operons. The most likely stage for the expression of these efflux operons is in the conversion stage from late log to stationary phase, when there is a build up of metabolic byproducts. Our experiments show that there is no change in expression of any of the three known efflux operons in P.aeruginosa under the growth conditions tested (Fig. 14). This seems to suggest that these efflux operons, especially the mexCD-oprJ and mexEFoprN efflux operons are tightly regulated and are not expressed under 'normal' lab growth conditions. A possible role for the mexEF-oprN efflux operon might be to excrete toxic byproducts from the metabolism of basic amino acids. Since OprD is known to be involved in the uptake of basic amino acids in P.aeruginosa and that it is linked via a common transcriptional regulator, MexT, it is conceivable that when the cellular levels of toxic compounds reach a certain level, it turns on the efflux pump to 'detoxify' the cell while turning off any further OprD production. Recent data from the lab using the P.aeruginosa strain harboring a oprDr.xylE chromosomal fusion and grown in medium where the basic amino acid arginine was used as the sole carbon source, suggest that in stationary phase, oprD expression is reduced (Ochs, unpublished data). 66  REFERENCES Albers-SchOnberg, G. B. H . Arison, O. D. Hensens, J. Hirschfield, K . Hoogsteen, E. A . Kaczka, R. E. Rhodes, J. S. Kahan, F. M . Kahan, R. W. Ratcliffe, E. Walton, L . J. Ruswinkle, R. B. Morin, and B. G. Christensen. 1978. Structure and absolute configuration of thienamycin. J. American Chemical Society. 100:6491-6499. Amsterdam. 1991. Antibiotics in laboratory medicine, (Lorian, V . , ed) Williams and Wilkins, Baltimore. 72-78. Angus, B. L., A . M . Carey, D. A . Caron, A . M . B. Kropinski, and R. E. W. Hancock. 1982. Outer membrane permeability in Pseudomonas aeruginosa: comparison of a wildtype with an antibiotic-supersusceptible mutant. Animicrob. Agents Chemother. 12:299309. Benz, R., and R. E. W. Hancock. 1981. Properties of the large ion-permeable pores formed from protein F oi Pseudomonas aeruginosa in lipid bilayer membranes. Biochem. Biophys. Acta. 646:298-308. Brumbley, S., B. Carney, and T. Denny. 1993. Phenotype conversion in Pseudomonas solanacearum due to spontaneous inactivation of PhcA, a putative LysR transcriptional regulator. J. Bacterid. 175:5477-5487. Bryan, L . E . 1979. Resistance to antimicrobial agents: The general nature of the problem and the basis of resistance. In "Pseudomonas aeruginosa: Clinical Manifestations of Infection and Current Therapy", R.G. Doggett (Ed.), Academic Press, New York, 219270. Burns, J. L . and D. K . Clark. 1992. Salicylate-inducible antibiotic resistance in Pseudomonas cepacia associated with absence of a pore-forming outer membrane protein. Antimicrob. Agents Chemother. 36:2280-2285. Casillas, E., M . A . Kenny, B. H . Minshew, and F. D. Schoenknecht. 1981. the effect of ionized calcium and soluble magnesium on the predictability of the performance of Mueller-Hinton agar susceptibility testing of Pseudomonas aeruginosa with gentamicin. Antimicrob. Agents Chemother. 19:987-992. Cebolla, A . , C. Sousa, and V . de Lorenzo. 1997. Effector specificity mutants of the transcriptional activator NahR of naphthalene degrading Pseudomonas define protein sites involved in binding of aromatic inducers. J. Biol. Chem. 272:3986-3992. Cooper, S. L., A . Louie, A . L. Baltch, A . C. Chu, R. P. Smith, W. J. Ritz, and P. Michelsen. 1993. Influence of zinc on Pseudomonas aeruginosa susceptibilities to imipenem. J. Clin. Micro. 31: 2366-2370.  67  Daly, J. S., R. A . Dodge, R. H . Glew, D. T. Soja, B. A . DeLuca, and S, Herbert. 1997. Effect of zinc concentration in Mueller-Hinton agar on susceptibility of Pseudomonas aeruginosa to imipenem. J. Clin. Micro. 35: 1027-1029. Dodd, I. B., and J. B. Egan. 1987. Systematic method for the detection of potential X Crolike DNA-binding regions in proteins. J. Mol. Biol. 194: 557-564. Fukuda, H . , M . Hosaka, K . Hirai, and S. Iyobe. 1990. New norfloxacin resistance gene in Pseudomonas aeruginosa PAO. 34:1757-1761. Geothals, K., M . Van Montagu, and M . Holsters. 1992. Conserved motifs in a divergent nodbox of Azorhizobium caulinodans ORS571 reveal a common structure in promoters regulated by LysR-type proteins. 89:1646-1650. Gotoh, N . , N . Itoh, H . Tsujimoto, J. L. Yamagishi, Y . Oyamada, and T. Nishino. 1994. Isolation of OprM-deficient mutants of Pseudomonas aeruginosa by transposon mutagenesis: evidence of involvement in multiple antibiotic resistance. F E M S Microbiol. Lett. 122:267-274. Haas, D., Galimands, M . Gamper, and A. Zimmermann. 1990. Arginine network of Pseudomonas aeruginosa: specific and global controls, p303-316. In S. Silver, A . - M . Chakrabarty, B. Iglewski, and S. Kaplan (ed.), Pseudomonas . Biotransformations, pathogenesis, and evolving biotechnology. American Society of Microbiology, Washington, D. C. Hancock, R. E. W., and A . M . Carey. 1979. Outer membrane of Pseudomonas aeruginosa. Heat- and 2-mercaptoethanol-modifiable proteins. J. Bacterid. 140:902-910. Hancock, R. E. W. 1997. The bacterial outer membrane as a drug barrier. Trends in Microbiology 5: 37-42. Hancock, R. E. W., and A . Bell. 1988. Antibiotic uptake into gram-negative bacteria. European Journal of Clinical Microbiology and Infectious Disease 7:713-720. Hancock, R. E. W., C. Egli, R. Benz, and R. J. Siehnel. 1992. Overexpression in Escherichia coli and functional analysis of a novel Ppi-selective porin, OprO, from Pseudomonas aeruginosa. J. Bacteriol. 174:471-476. Hancock, R. E. W., R. T. Irvin, J. W. Costerton, and A. M . Carey. 1981. Pseudomonas aeruginosa outer membrane: peptidoglycan-associated proteins. J. Bacteriol. 145:628631. Hancock, R. E. W., R. Siehnel, and N . Martin. 1990. Outer membrane proteins of Pseudomonas. Mol. Microbiol. 4:1069-1075.  68  Hancock, R. E. W., K . Poole, and R. Benz. 1982. Outer membrane protein P of Pseudomonas aeruginosa: regulation by phosphate deficiency and formation of small anion-specific channels in lipid bilayer membranes. J. Bacteriol. 1 5 0 : 730-738. Hancock, R. E. W. 1987. Role of porins in outer membrane permeability. J. Bacteriol. 169:929-933.  Hancock, R. E. W., and H . Nikaido. 1978. Outer membrane of gram-negative bacteria. X I X . Isolation from Pseudomonas aeruginosa and use in reconstitution and definition of the permeability barrier. J. Bacteriol. 136:381-390. Hashizume, T., F. Ishino, J. I. Nagagawa, S. Tamaki, and M . Matsuhashi. 1984. Studies on the mechanism of action of imipemen (N-formimidoyl thienamycin) in vitro: binding to the penicillin-binding proteins (PBPs) in Escherichia coli and Pseudomonas aeruginosa, and inhibition of enzyme activities due to the PBPs in Escherichia coli. J. Antibiotics. 37:394-400. Helinger, W. C , andN. C. Brewer. 1991. Imipenem. Mayo Clin. Proc. 66:1074-1081. Henikoff, S., G. Haughn, J. Calvo and J. C. Wallace. 1988. A large family of bacterial activator proteins. Proc. Natl. Acad. Sci. 84: 4460-4464. Hirai, K., and S. Mitsuhashi. 1987. Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 31:582-586. Honore, N . , M . H . Nicolas, and S. Cole. 1986. Inducible cephalosporinase production in clinical isolates of Enterobacter cloacae is controlled by a regulatory gene that has been deleted from Escherichia coli. E M B O J. 5 : 3709-3714. Huang, H . and Hancock, R.E.W. 1993. Genetic definition of the substrate selectivity of outer membrane protein OprD of Pseudomonas aeruginosa. J. Bacteriol. 175:7793-7800. Huang, H . D. Jeanteur, F. Partus, and R. E. W. Hancock. 1995. Membrane topology and site-specific mutagenesis of Pseudomonas aeruginosa porin OprD. M o l . Microbiol. 16:931-941. Huang, H . and Hancock, R.E.W. 1996. The role of specific surface loop regions in determining the function of the imipenem-specific pore protein OprD oi Pseudomonas aeruginosa. J. Bacteriol. 178:3085-3090. Huang, H , R. Siehnel, F. Bellido, E. Rawling, and R. E. W. Hancock. 1992. Analysis of two gene regions involved in the expression of the imipenem-specific outer membrane porin protein OprD of Pseudomonas aeruginosa. F E M S Microbiol. Lett. 97:267-27'4.  69  Kahan, J. S., F. M . Kahan, R. Goegelman, S. A . Currie, M . Jackson, E. O. Stapley, T. W. miller, A . K . Miller, D. Hendlin, S. Mochales, S. Hernandez, H . B. Woodruff, and J. Birnbaum. 1979. Thienamycin, anew P-lactam antibiotic. Discovery, taxonomy, isolation and physical properties. J. Antibiot. 32:1-12. Kaphammer, B. K., and R. H . Olsen. 1990. Cloning and characterization of the tfdS, the repressor-activator gene of tfdB, from the 2,4-dicholorphenoxyacetic acid and catabolic plasmid pJP4. J. Bacteriol. 172: 5856-5862. Karunaratne, D.N. J.C. Richards, and R.E.W. Hancock. 1992. Characterization of lipid A from Pseudomonas aeruginosa O-antigen B band lipopolysaccharide by ID and 2D N M R and mass spectral analysis. Arch. Biochem. Biophy. 299: 368-376. Kohler, T., M . Michea-Hamzehpour, U . Henze, Naomasa Gotoh, L. K. Cuirty and J. C. Pechere. 1997. Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Mol. Microbiol. 23:345-354. Kohler, et al. 1998. unpublished data. Konyecsni, W. M . , and V . Deretic. 1988. Broad-host-range plasmid and M13 bacteriophage-derived vectors for promoter analysis in Escherichia coli and Pseudomonas aeruginosa. Gene. 74: 375-386. Kropinski, A . M . , L.C. Chan, and F.H. Milazzo. 1979. The extraction and analysis of lipopolysaccharide from Pseudomonas aeruginosa strain PAO, and three rough mutants. Can. J. Microbiol. 25: 390-398. Kropinski, A . M . , B Jewell, J.Kuzio, F.H. Milazzo, and D. Berry. 1985. Structure and functions Pseudomonas aeruginosa lipopolysaccharide. In Pseudomonas aeruginosa: New Therapeutic Approaches from Basic Research", D.P. Speert and R.E.W. Hancock (Eds.) Karger, Basel, Switzerland, 58-73. Kropp, H . , J. G. Sundelof, J. S. Kahan, F. M . Kahan, and J. Birnbaum. 1980. M K 0 7 8 7 (N-formimodoyl thienamycin): evaluation of in vivo and in vitro activities. Antimicrob. Agents Chemother. 17:993-1000. Lipman, B., and H . C. Neu. 1988. Imipenem: a new carbepenem antibiotic. Med. Clin. North. Am. 72:567579. Liu, P.V. 1974. Extracellular toxins of Pseudomonas aeruginosa. J. Infect. Dis. 130 (suppl): S94-S99. Livermore, D. M . and Y . J. Yang. 1987. ^-lactamase lability and inducer power of newerp-lactam antibiotics in relation to their activity against P-lactamase-inducibility mutants of Pseudomonas aeruginosa. J. Infect. Dis. 155:775-782.  70  Livermore, D. M . , 1992. Interplay of impermeability and chromosomal P-lactamase activity in imipenem-resistant Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 31:1892-1896. Lynch, M . J. G. L . Drusano, and H . L. T. Mobley. 1987. Emergence of resistance to imipenem in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 31:1892-1896. Masuda, N . , E. Sakagawa, and S. Ohya. 1995. Outer membrane proteins responsible for multiple drug resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 39:645-649. Miller, P. F., and M . C. Sulavik. 1996. Overlaps and parallels in the regulation of intrinsic multiple-antibiotic resistance in Escherichia coli. 21:441-448. Mutharia, L . M . and R. E. W. Hancock. 1983. Surface localization of Pseudomonas aeruginosa outer membrane protein F using monoclonal antibodies. Infect. Immun. 42:1027-1033. Nicas, T.I., and R.E.W. Hancock. 1983. Pseudomonas aeruginosa outer membrane permeability: isolation of porin protein F-deficient mutant. J. Bacteriol. 153: 281-285. Nikaido, H and T. Nakae. 1979. The outer membrane of Gram-negative bacteria. Adv. Microbiol. Physiol. 20: 163-250. Nikaido, H and R.E.W. Hancock. 1986. Outer membrane permeability of Pseudomonas aeruginosa. In "The Bacterial", J.R. Sokatch (ed.). Academic Press, New York, V o l X : 145-193. Nikaido, H . 1996. Multidrug efflux pumps make a major contribution to drug resistance in pseudomonads. P.353-362. In T.Nakazowa, K.Furukawa, D.Haas and S.Silver (ed.), Molecular biology of pseudomonads. A S M Press, Washington, D.C. Nikaido, H . , and M . Vaara. 1985. Molecular basis of bacterial outer membrane permeability. Microbiol. Rev. 49:1-32. Ochs, M . unpublished data. Oliver, D.B. 1987. Periplasm and protein secretion. In "Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology'" F.C. Neidhardt (ed.) A S M Publications, Washington, D . C , 56-69. Olsen, R. H , W. De Busscher, and W. R. M c Combie. 1982. Development of broad host range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa P A O chromosome. J. Bacteriol. 150:60-69.  71  Park, S.-M. C-D, L u , and A . T Abdelal. 1997. Cloning and characterization of argR, a gene that participates in regulation of arginine biosynthesis and catabolism in Pseudomonas aeruginosa P A O l . J.Bacteriol. 179:5300-5308. Park, S.-M. C-D, L u , and A . T Abdelal. 1997. Purification and characterization of an arginine regulatory protein, ArgR, from Pseudomonas aeruginosa and its interactions with the control regions for the car, argF, and aru operons. 179: 5309-5317. Park, S. unpublished data. Paulsen, I.T. M . Brown, and R. A . Skurray. 1996. Proton-dependent multidrug efflux systems. Microbiol. Rev. 60:575-608. Poole, K., and R. E. W. Hancock. 1986. Isolation of a Tn501 insertion mutant lacking porin protein P of Pseudomonas aeruginosa. Mol. Gen. Genet. 202:403-409. Poole, K., K. Krebes, C. McNally, and S. Neshat. 1993. Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon. J. Bacteriol. 175:7363-7372. Poole, K . 1994. Bacterial multidrug resistance-emphasis on efflux mechanisms in Pseudomonas aeruginosa. J. Antimicro. Chemother. 34:453-456. Poole, K., K . Tetro, Q. X . Zhao, S. Neshat, D. E. Heinrichs, and N . Bianco. 1996. Expression of the multidrug resistance operon mexA-mexB-oprM in Pseudomonas aeruginosa: mexR encodes a regulator of operon expression. Antimicrob. Agents Chemother. 40: 2021-2028. Poole, K . , N . Gotoh, H . Tsujimoto, Q. Zhao, A . Wada, T. Yamasaki, S. Neshat, J. Yamagishi, X - Z . L i , and T. Nishino. 1996. Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-type multi-drug-resistant strains of Pseudomonas aeruginosa. M o l . Micro. 21:713-724. Poxton, J. 1985. . F E M S Microbiol. Lett. 27:247-251. Quinn, J.P. et al. 1986. Emergence of resistance to imipenem during therapy for Pseudomonas aeruginosa infections. J.Infect.Des. 154:289-294. Rella, M . , and D. Hass. 1982. Resistance of Pseudomonas aeruginosa P A O l to nalidixic acid and low levels of (3-lactam antibiotics: Mapping of chromosomal genes. Antimicrob. Agents Chemother. 22:242-249. Reller, L. B., F. D. Schoenkncht, M . A . Kenny, and J. C. Sherris. 1974. Antibiotic susceptibility testing of Pseudomonas aeruginosa: selection of a control strain and criteria for magnesium and calcium content in media. J. Infect. Dis. 130:454-463.  72  Rohnson, G. N . 1986. (3-lactam antibiotics. J. Antimicrob. Chemother. 17:5-36. Rottem, S., and L. Leive. 1977. Effect of variations in lipopolysaccharides on the fluidity of the outer membrane of Escherichia coli. J. Antimicrob. Chemother. 17:5-36. Sabath, L . D., C. Liebeler, and J. Handley. 1990. Program Abstr. 30 Intersci. Conf. Antimicrob. Agents Chemother., abstr. 175, p. 116. th  Saino, Y., F. Kobayashi, M . Inoue, and S. Mitsuhashi. 1982. Purification and properties of inducible penicillin P-lactamase isolatedfromPseudomonas maltophilia. Antimicrob. Agents. Chemother. 22:564-570. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N . Y . Sawai, T., S. Hirano, and A. Yamaguchi. 1987. Repression of porin synthesis by salicylate in Escherichia coli, Klebsiella pneumonia and Serratia marcescens. F E M S Microbiol. Lett. 40: 233-237. Schell, M . A . , and E. Poser. 1989. Demonstration , characterisation, and mutational analysis of NahR protein binding to the nah and sal promoters. J. Bacteriol. 171: 837846. Schell, M . A . , and M . Sukordhaman. 1989. Evidence that the transcription activator encoded by the Pseudomonas putida nahR gene is evolutionally related to the transcription activators encoded by the Rhizobium nodD genes. J. Bacteriol. 171: 19521959. Schimpff, S.C., M . Moody, and V . M . Young. 1970. Relationship of colonization with Pseudomonas aeruginosa to develop of Pseudomonas bacteremia in cancer patients. Antimicrob. Agents Chemother. 10: 240-244. Schweizer, H . P. 1991. Escherichia-Pseudomonas shuttle vectors derived from pUC18/19. Gene. 97: 109-112. Sherbert, G. V . , and M . S. Lakshmi. 1973. Characterization oi Escherichia coli cell surface by isoelectric equilibrium analysis. 5:11-13. Sumita, Y . and M . Fukasawa. 1993. Transient carbapenem resistance induced by salicylate in Pseudomonas aeruginosa associated with suppression of outer membrane protein D2 synthesis. Antimicrob. Agents Chemother. 37:2743-2746. Trias, J. and . H . Nikaido 1990a. Protein D2 channel of the Pseudomonas aeruginosa outer membrane has a binding site for basic amino acids and peptides. J.Biol.Chem. 265:15680-15684.  73  Trias, J. and H . Nikaido. 1990b. Outer membrane protein D2 catalyses facilitated diffusion of carbepenems and penems through the outer membrane of Pseudomonas aeruginosa. Biochim. Biophys. Acta. 938:493-496. Trias, J., E. Y . Rosenberg, and H . Nikaido. 1987. Specificity of the glucose channel formed by the protein DI of Pseudomonas aeruginosa. Biochim. Biophys. Acta. 938:439-496. Watanabe, M . , S. Iyobe, M . Inoue, and S. Mitsuhashi. 1991. Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 35:147-151. Woodruff, W. A . , T. R. Parr, R. E. W. Hancock, L. F. Hanne, T. I. Nicas, and B. H . Iglewski. 1986. Expression in Escherichia coli and function of Pseudomonas aeruginosa outer membrane protein OprF. Mol. Microbiol. 10:283-292. Woodruff, W. A . , and R. E. W. Hancock. 1989. Pseudomonas aeruginosa outer membrane protein F: structural role and relationship to the Escherichia coli OmpA protein. J. Bacteriol. 171:3304-3309. Yang, Y . , P. Wu, and D. M . Livermore. 1990. Biochemical characterization of a Plactamase that hydrolyzes penems from two Serratia marcescens isolates. Antimicrob. Agents Chemother. 34:755-758. Yoneyama, H , and T. Nakea. 1993. Mechanism of efficient elimination of protein D2 in outer membrane of imipenem-resistant Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy. 37: 2385-2390. Yoshihara, E., H . Yoneyama, and T Nakae. 1991. In vitro assembly of the functional porin trimer from dissociated monomers in Pseudomonas aeruginosa. J. Biol. Chem. 266:636-642. Yoshimura, F. and Nikaido, H . 1982. Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic solutes. J. Bacteriol. 152:636-642. You, I. S., D. Ghosal, and I. C. Gunsalus. 1988. Nucleotide sequence of plasmid N A H 7 gene nahR and D N A binding of the nahR product. J. Bacteriol. 170:5409-5415.  74  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0088644/manifest

Comment

Related Items