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The role of adenylate cyclase in the regulation of competence development in haemophilus influenzae Dorocicz, Irene Renate 1992

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THE ROLE OF ADENYLATE CYCLASEIN THE REGULATION OFCOMPETENCE DEVELOPMENTIN HAEMOPHILUS INFLUENZAEbyIRENE RENATE DOROCICZB. Sc., The University of British Columbia, 1990A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Biochemistry)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIANovember 1992© Irene Renate Dorocicz, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of  BiochemistryThe University of British ColumbiaVancouver, CanadaDate 	Oct 1 3/92DE-6 (2/88)iiABSTRACTTo study the role of adenylate cyclase in competence development, a partialclone of the Haemophilus influenzae cya gene was isolated by complementation of aAcya Escherichia coli strain. Adenylate cyclase was believed to have a role incompetence development because it catalyzes production of adenosine 3':5'-cyclicmonophosphate (cAMP), a known regulator of competence. To prove thatadenylate cyclase was essential for competence development, transposonmutagenesis was used to form the cya- H. influenzae strain RR668, with aninsertion in the region of cya coding for the catalytic domain. Characterization ofthis mutant has shown that cya is an essential gene for spontaneous late logcompetence, and for competence induced by starvation conditions. The partialpreliminary sequence of the cloned gene had significant amino acid homology tothe cya genes from enteric bacteria and the more closely related bacteriumPasteurella multocida. Examination of the cya sequence also revealed a possibleCRP binding site (with 55% homology to the consensus Escherichia coli site) locatedupstream of the putative start codon GTG. The presence of the presumptive CRPsite indicated that H. influenzae, like other bacterial species, may regulate cAMPsynthesis by CRP mediated feedback repression of transcription. If the start codonwas correctly identified as GTG, then this is the first known bacterial cya gene to useGTG as a start codon instead of ATG or TTG.iiiTable of ContentsABSTRACT	 iiList of Tables 	 ivList of Figures 	List of Abbreviations	 viAcknowledgements	 viiINTRODUCTION 	 11.1 Competence development in various bacterial species	 11.11 Bacillus subtilis 	 21.12 Streptococcus pneumoniae	 21.13 Neisseria gonorrhoeae	21.2 Competence development in Haemophilus influenzae	 31.21 The process of transformation	 31.22 The function of competence development	 61.23 The role of cAMP in competence development	 71.3 The thesis project	 8MATERIAL AND METHODS	 91.1 Materials	 91.11 Bacterial strains, plasmids and phage	91.12 Media and antibiotics	 141.2 Methods	 151.21 Library construction	 151.22 Competence techniques	 151.23 cAMP assay 	 161.24 Sequencing 	 171.25 Analysis of DNA by Southern hybridization	 17RESULTS	 191.1 Cloning and mapping cya	 191.2 Competence studies	 291.3 Sequencing cya 	 341.4 Comparison of crp- mutant to cya- mutant	 40DISCUSSION 	 421.1 Significance of competence results	 421.2 Comparison of bacterial cya nucleotideand amino acid sequences	 431.3 Comparison of cya mutant to crp mutant 	 451.4 Future experiments	 45REFERENCES	 48-51ivLIST OF TABLESTable 1 	 List of bacterial strains, plasmids and phages	 10-11Table 2 	 Levels of cAMP production by various H. influenzaeand E. coli strains 	 21Table 3 	 Transformation frequencies for RR668, RR665 and KW20in MIV starvation medium 	 31Table 4 	 Analysis of codon usage for the first 20 amino acids of cya ..46-47VLIST OF FIGURESFigure 1 	 Details of the construction of cya containing plasmidsand cya- mutant strains from H. influenzae	 12-13Figure 2a 	 Partial restriction map of pID1 and the cya gene,including locations of the transposons Tn7-15and Tn1-6	 20Figure 2b The subclones of pID1	 22Figure 3 	 Mapping the cya gene to the H. influenzae genome	23-24Figure 4 	 Probing H. influenzae chromosomal DNA withpID1 subfragments 	 27-28Figure 5 	 Comparison of spontaneous competence levelsfrom RR668, RR665 and KW20	 30Figure 6 	 Comparison of competence levels for RR668 in theabsence or presence of cAMP	 32Figure 7 	 Comparison of competence levels for RR665 in theabsence or presence of cAMP	 33Figure 8 	 Nucleotide sequence of the H. influenzae cya geneand the deduced adenylate cyclase amino acidsequence 	 35-37Figure 9 	 Alignment of deduced H. influenzae adenylate cyclaseamino acid sequence to cya from four other bacteria	 38-39Figure 10 	 Cell survival on plates	 41viLIST OF ABBREVIATIONSBHI 	 brain heart infusion brothsBHI 	 brain heart infusion supplemented with NAD and hemincAMP 	 adenosine 3':5'-cyclic monophosphatecya- 	 defective cya gene: does not produce fully functional product.Other abbreviations used for cya- strains include Acya (= part ofcoding sequence deleted) or cya::Tn (= gene disrupted byminiTn10 transposon)MI V 	 starvation medium used for induction of maximumcompetence levels (formula taken from Herriot et al., 1970)viiACKNOWLEDGEMENTSAll of the work described in this thesis was carried out by the author, exceptfor the assays for cAMP concentration and protein content, which were performedby Rosemary Redfield. I would like to thank Pascale Williams for her excellenttechnical assistance and moral support, and the members of the Dennis lab,especially Lawrence Shimmin, Phalgun Joshi, Luc Bissonnette and Diedre deJong-Wong, for their superlative experimental advice. Last but not least, I wouldlike to give a very special thanks to Dr. Rosemary Redfield for the excellentsupervision, constant encouragement and support she gave me during the course ofthis study.1INTRODUCTIONTransformation is the process by which bacteria take up DNA from theexternal environment and recombine it into their chromosome. Althoughtransformation can be artificially induced in the lab by procedures such as CaC12treatment (Sambrook et al., 1989), there are several bacterial species that becomespontaneously competent to perform transformation (Kahn and Smith, 1984).These species include Bacillus subtilis, Neisseria gonorrhoeae, Streptococcuspneumoniae and Haemophilus influenzae. I am studying H. influenzae to learnhow transformation is regulated and to determine the role of competencedevelopment in this bacterium. Specifically, my thesis project involves studyingthe role of adenylate cyclase in the regulation of competence development in H.influenzae.1.1 Competence development in various bacterial species I would like to begin by first summarizing the known facts about spontaneouscompetence development in bacterial species other than H. influenzae. Thecomplete process of competence development and transformation is not yet fullyunderstood for any bacterium. Transformation is a multi-step process, involvingthe binding of DNA to the cell surface, translocation of the DNA into the cell, andthe recombination of the new DNA into the chromosome. All naturally competentbacteria use a similar recombination process, and only recombine a single strand ofDNA into the chromosome. However, different methods of DNA binding anduptake into the cell are used by gram-positive and gram-negative bacteria, whichmay be due to the differences in their cell wall composition.21.11 Bacillus subtilisThe competence system of this gram-positive bacterium has been extensivelystudied(Dubnau, 1991). In B. subtilis, competence development usually occurs instationary phase and involves a RecA-like protein. After the DNA binds to the cellsurface, single strand degradation occurs, and only a single strand of DNA enters thecell, where recombination takes place. The two gene products involved in theinitial step of competence development are the kinase ComP, and its target proteinComA. After ComA has been phosphorylated, it acts with two other proteins toactivate the srfA operon, which induces competence when it is overexpressed(Dubnau, 1991).1.12 Streptococcus pneumoniaeCultures of this gram-positive bacterium become spontaneously competentfor a brief period at high culture densities of approximately 5 x10 8 cfu/ml (Hui andMorrison, 1991). This abrupt development of competence is dependent on celldensity and occurs in response to a small competence factor protein released by thecells. When added to non-competent cultures, the competence factor inducescompetence. So far, S. pneumoniae is the only naturally competent species that usessecretion of a protein competence factor to induce competence. As is the case withB. subtilis, single strand degradation of the transforming DNA occurs at the cellsurface, before the DNA enters the cell.1.13 Neisseria gonorrhoeaeIt appears that competence development in this gram-negative bacterium isnot regulated, as piliated cells are fully competent throughout their life cycle, while3non-piliated cells never become competent. Unlike the gram-positive species, DNAtranslocation in N. gonorrhoeae involves using the short DNA recognitionsequence GCCGTCTGAA (Goodman and Scocca, 1988) to preferentially take uphomologous DNA. Further details about the process of transformation have not yetbeen studied in this bacterium.1.2 Competence development in Haemophilus influenzaeMy research has been focussed on competence development in H. influenzae.Rd, a nonpathogenic derivative of a serotype d strain (Alexander and Leidy, 1951).This gram-negative bacterium is a member of the family Pasteurellaceae and livesin the human upper respiratory tract. There is medical interest in this bacteriumbecause one of its serotype b strains is the major cause of infant meningitis(Hammond et al., 1988). Studying transformation may be medically beneficial, sincetransformation could be involved in H. influenzae's pathogenicity and in theincreasing numbers of antibiotic resistant strains.1.21 The process of transformationIt is known that late log cultures of H. influenzae become spontaneouslycompetent to take up DNA and recombine it into the chromosome. Althoughtransformation is a tightly regulated process, the signal that induces competencedevelopment has not yet been identified. However, competent cells have alteredcellular membranes (Zoon et al., 1975), and have an increased capacity forrecombination. During transformation, H. influenzae preferentially takes uphomologous DNA (Scocca et al., 1974) and recognizes such DNA by means of the9-11 by recognition sequence AAGTGCGGT(CA) (Danner et al., 1980). This methodof DNA binding is similar to the method used by N. gonorrhoeae. After binding tothe cell surface, the DNA is rapidly transported into the cell. Unlike the situation in4gram-positive bacteria, DNA fragmentation and single strand degradation does notoccur outside of the cell. Immediately after crossing the outer membrane, the DNAis protected from both extracellular nucleases and intracellular restriction enzymes(Kahn et al., 1982). This data has been interpreted to mean that the DNA istemporarily stored between the outer membrane of the cell wall and the innercellular membrane. There is some disagreement as to the exact nature of thisstorage compartment, as some workers think that membrane blebs sometimes seenin electron micrographs are the DNA-containing 'transformasomes° (Kahn et al.,1983), while others think that the DNA is simply held in the periplasmic space.Although the preliminary binding and entry steps are not dependent on the DNAconformation, the DNA must have a free end in order to enter the cytoplasm(Barany et al., 1983). This limitation traps covalently closed circles, but permitslinearized DNA to enter the cytoplasm. After the DNA has passed through the cellmembrane, DNA degradation begins, with the leading 5' strand degraded morerapidly than the othr strand. The remains of the 3' strand are integrated intohomologous regions in the chromosome (Barany et al., 1983).The steps in transformation are studied by using labelled DNA to quantitateDNA uptake, and by measuring phage recombination to monitor the ability of thecell to perform recombination. By performing these tests on corn- H. influenzaemutants, it is possible to identify which step in transformation or competencedevelopment has been interfered with. So far, these studies have identified rec-1and rec-2 as genes coding for proteins involved in transformation, and the crp andtopA as regulatory competence genes.The rec-1 gene is similar to the Escherichia coli gene recA, as both genes havehomologous amino acid sequences, and both possess a copy of the E. coli consensus5LexA binding site in the upstream region (Setlow et al., 1988). Mutations in thesegenes cause similar cellular defects, such as UV sensitivity and a reduction in theability to perform DNA repair or prophage induction. H. influenzae strains withmutant rec-1 alleles can bind DNA and translocate it into the cytoplasm, butrecombination cannot occur (Barouki and Smith, 1985). The competence deficientrec2 mutants do not undergo transformation because they cannot translocate DNAinto the cell interior (McCarthy, 1989). Recent experiments have shown thatalthough rec2 is not essential for plasmid or chromosomal recombination, it isrequired for phage recombination (Kupfer et al., 1992).Sequencing of the defective genes in two of the 'JGcom-'series oftransformation mutants (Tomb et al., 1989) led to the identification of two essentialcompetence genes that were homologous to the E. coli crp and topA genes(Chandler, 1992; Chandler pers. comm.). The crp gene codes for the cAMP-bindingcatabolite regulatory protein (CRP). H. influenzae cells with disruptions in the crpgene not only fail to become competent, but they also differ from wild type cells inthat crp- cells have better growth rates and longterm plate survival (Redfield pers.comm.). The growth phenotype of H. influenzae crp- cells differs from thephenotype of E. coli crp- cells, since the E. coli mutants grow less well than theirwild type counterparts (D'Ari et al., 1988). In other bacteria, the cAMP/CRP complexis an important regulator of the expression of many catabolic genes, such as the lacoperon, and is also required for other functions such as flagellum synthesis andtoxin production (Botsford and Harman, 1992). The topA gene producestopoisomerase I, which acts in E. coli to relax negative DNA supercoils byintroducing single strand breaks in the DNA (Wang, 1971). These genes areidentified as regulators of competence because disruption of the coding region ofeither of these genes completely prevents DNA uptake and recombination. Thisinformation implies that the amount of DNA supercoiling influences competence6development; and it supports earlier results that indicate that cAMP is involved incompetence regulation (Wise et al., 1973). Further work needs to be done to isolateother competence and transformation genes, so that all the steps in the process canbe identified and fully understood.1.22 The function of competence developmentAs mentioned previously, competence levels in H. influenzae increasespontaneously in late log growth, with the transformation frequency rising from10-8 to 10-4 (Wise et al., 1973). In addition to these late log increases in competence,H. influenzae competence levels also rise when cells are treated with the starvationmedium MIV (Herriot et al., 1970), grown in the presence of cAMP (Wise et al.,1973), or are deprived of oxygen. The highest transformation frequencies of 10-2 areobserved in MIV medium, which lacks nucleotides and a carbon source and whichtherefore limits DNA synthesis and cell division.Several theories have been proposed to explain the function of competencedevelopment. It is commonly assumed that the primary function of competencedevelopment is as a method of gene transfer. By increasing the amount of geneticinformation available to the cell, pathogenic H. influenzae can alter their antigenicdeterminants to avoid the host immune system and antibiotic treatment. Thistheory explains the preference for homologous DNA. Some researchers think thatcompetence development acts during starvation situations to permit DNA to betaken up as a nutritional source of nucleotides (Stewart & Carlson, 1986; Redfield, inpress). It is also possible that H. influenzae specifically takes up homologous DNAto act as templates for DNA repair (Michod et al., 1988; Wojciechowski et al., 1989;Hoelzer and Michod, 1991), although recent work with DNA damaging agents such7as UV light and mitomycin C does not support this hypothesis (Redfield,submitted).1.23 The role of cAMP in competence developmentIn 1973, Wise et al. reported that the addition of cAMP to early log cellsinduces competence to the same moderate level as seen spontaneously in late logcultures, with transformation frequencies of 10 -4 . This was the first evidence thatcAMP might be an important molecular signal for competence development (Wiseet al., 1973). As mentioned earlier, the importance of cAMP was confirmed whenthe defective gene in the H. influenzae corn- mutant strain JG87 was identified ascrp, the gene coding for the cAMP-binding catabolite regulatory protein (Chandler,1992). Since exogenous cAMP increases H. influenzae's competence, intracellularcAMP may function with CRP to activate other competence genes. Control ofintracellular cAMP levels may therefore be important in regulating competence.Intracellular cAMP concentrations can be regulated in other bacteria bycontrolling cAMP synthesis, degradation and export (Botsford and Harman, 1992).The synthesis of cAMP by adenylate cyclase is regulated in E. colt by feedbackrepression of cya transcription (Aiba, 1985), use of rare tRNA amino acid codons tolimit translation (Aiba et al., 1984), and by phosphotransferase system activation ofadenylate cyclase activity (Meadow et al., 1990). Adenylate cyclase may be controlledby similar regulatory mechanisms in H. influenzae.81.3 The thesis projectSince cAMP induces competence development, and crp- H. influenzae cellscannot become competent, this led me to believe that controlling intracellularcAMP levels is important in competence development. Therefore, I decided tostudy the role of adenylate cyclase, the enzyme that catalyzes synthesis of cAMP, incompetence development. I planned on proving that adenylate cyclase is essentialfor competence development by showing that cya- H. influenzae cells cannotbecome competent unless exogenous cAMP is present. To form cya- H. influenzae, Ihad to clone the cya gene. Sequencing the cloned cya gene could also provideinformation about how H. influenzae regulates the activity of adenylate cyclase.9MATERIAL AND METHODS1.1 Materials 1.11 Bacterial strains, plasmids and phageThe bacterial strains, plasmids, and phages used in this study are described inTable 1. All H. influenzae strains were descendants of the original H. influenzae Rdof Alexander (Alexander and Leidy, 1951). The cya- mutant H. influenzae strainsRR665 and RR668 were constructed as follows. The cya+ plasmid pID1 wastransformed into the nonsuppressing E. coli strain NM554. The resulting strain wastransduced with the miniTnlOKan-containing phage X1316, following the procedureof Nancy Kleckner (N. Kleckner et al., 1991). Transposition of the miniTnlOkantransposon into pID1 resulted in approximately 14000 cya- plasmids, includingpID1::Tn7-15 and pID1::Tn1-6. These two plasmids were then digested with EcoRIand transformed into the wild type H. influenzae strain KW20. In vivorecombination occurred between the plasmid insert fragments and the KW20chromosome, yielding the kanamycin resistant cya- strains RR665 and RR668.The kanamycin resistant plasmids pID7T and pID6T, each containing adifferent part of the cya gene, were cloned from PstI libraries prepared from RR665and RR668, respectively. The miniTnlO transposon was removed from pID6T bytransforming the plasmid into wild-type KW20. After recombination between themutant plasmid gene and the wild type chromosomal copy had occurred, thekanamycin sensitive, transposon-free plasmid pID6 was isolated from the cell. Therelationship between the various plasmids and H. influenzae strains developedduring this project is shown in Figure 1.1 0Table 1: Bacterial strains, plasmids, and bacteriophagesStrain, etc 	 Genotype/Phenotype 	 Source or referenceE. coli strainsCAA8306Na1r 	 thi,dcya,g1p+,nal	 Kielyet al., 1983DH5a 	 supE44,4/acU169(f80/acZAM15)hsdR17 	 D. Hanahan,recA1,endA1,gyrA96,thiLrelA1	 1983NM554 	 araD139,A(ara-leu)7696,A(lac)174, 	 N. MurraygalU,galK,hsdR,strA,recA13H. influenzae strains KW20 	 wild type H. influenzae strain 	 Alexanderet al., 1951MAP7 	 strr na ir kan r navr s tvr six '. v ia l-	 J. SetlowRR665 	 cya::miniTnlOkan;	 this studycya- (regulatory), kanRRR668 	 cya::miniTn1Okan; 	 this studycya- (catalytic), kanRRR540 	 crp::miniTn1Okan; 	 Tomb et al.,crp-, kanR, strR 	 1989Plasmids pSU2718 	 chloramphenicol resistant shuttle vector 	 Martinezfor E. coli and H. influenzae (P15A origin) 	 et al., 1988pID1 	 7.3 kb insert (containing 	 this studyN-term —1.5kb cya gene) in pSU2718;cya+, cmRpID1::Tn1 -6	 pID1 with miniTnlOkan insertion in the	 this studyregulatory domain of cya (bp#-1800);cya-, cmRpID1::Tn7-15 	 pID1 with miniTnlOkan insertion in the 	 this studycatalytic domain of cya (bp#-900);cya-, cmR1 1Table 1 continuedStrain, etc 	 Genotype/Phenotype 	 Source or referencePlasmidspID4 	 3.7kb KpnI-HindIII pID1 fragment 	 this studysubcloned in pSU2718;cya-, cmRpID5 	 1.0kb XbaI-KpnI pID1 fragment 	 this studysubcloned in pSU2718;cya-, cmRpID6T 	 4.7kb PstI fragment from RR668 	 this studywith 1.8kb as cya::miniTn1Okan7-15;cya-, cmR, kanRpID6 	 2.9kb PstI fragment from pID6T 	 this studyin pSU2718 (miniTnlOkan removed);cya-, cmRpID7T	 9.3kb PstI fragment from RR665 	 this studywith 1.8kb as cya::miniTn1Okan1-6;cya-, cmR, kanRPhageX1316 	 X b522 cI857 Pam80 nin5::(miniTn10 	 N. Klecknerkan/ptac-ATS); 	 et al., 1991kanRLegend: For more details about plasmid inserts and construction, see Figure 1. Inthe central column the genotype information for some of the H. influenzae strainsand the plasmids is separated from the phenotypic details by ';'1 2Figure 1: 	 Details of the construction of cya containing plasmidsand cya- mutant H. influenzae strains Legend: DNA from the H. influenzae strain MAP 7 was used to prepare a library ofpartially digested Sau3A fragments ligated into the BamHI site of the plasmidpSU2718. By screening this library using a Acya E. colt complementation assay, asdescribed in the text, the cya+ plasmid pID1 was isolated. All other plasmids and cyamutant strains are derived from this plasmid, and their relationship to pID1 isshown in this figure. In this figure only part of the insert DNA for pID1::Tn1-6 andpID1::Tn7-15 is shown (solid lines) to indicate the position of the transposon inthese plasmids (arrow). The location of the cya gene in pID1, pID1::Tn1-6 andpID1::Tn7-15 is indicated by the arrow underneath the plasmid restriction maps.The dashed portion of this arrow indicated the section of the cya gene that may notbe present in these plasmids.Abbreviations: 	 kanR = kanamycin resistantkanS = kanamycin sensitivecmR = chloramphenicol resistantRestriction enzyme abbreviations: 	 B = BamHI 	 C = ClaIH = HindIII	 K = KpnIP = PstI	 Pv = PvuIIV = EcoRV	 X = XbaIStippled boxes in pID6T and pID7T diagram indicate the miniTnlOkan transposonpID1::Tn7-159.1 kb insertkanR cmR cya-X P 	 X 	 Pv 	 X P11 	 I 	 1 	 I pID1::Tn1-69.1 kb insertkanR cinR cya-PVVKB C 	 V 	 C 	 H1 111! 1 3Figure 1: Details of the construction of cya containing plasmids and cya- mutant H. influenzae strains H. influenzae chromosomal MAP7 DNASau3A partial digest; ligate into BamHI digestedpSU2718 => library in DI-I5atransform into Acya E. colifor complementation assay= dcya(pID1)	pID1 insert: X P 	 X 	 Pv 	 X PVVKB C 	 V 	 C 	 H7.3 kb 	I I I 	 I 	 I 	 I 	 Ill! 1 I 	 I 	 I 	 I 1cmR cya+=NM554(pID1)transposon mutagenesis with X1316--->- cyatransform into NM554)0- cyaTn7-15	 Tn1-61  EcoRI digest 	 1 EcoRI digesttransform into KW20 =>RR668: catalytic mutant	 transform into KW20 =>RR665cya- kanRcya- kanRiv Pstl digest	PstI digestligate into PstI digested pSU2718	 ligate into PstI digested pSU2718=>library	 =>libraryscreen for kanR 	 4 screen for kanR=pID6T 4.7 kb insert cya-	 =pID7T 9.3 kb insert cya- P 	 X 	 Pv 	 X 	 H C 	 p	 PVVK C H B CI	 	 I	WOLLEMEM I 	 I II I   K 	 p1     transform into KW20;screen for kanS cmR=pID6 2.9 kb insert cya-PI 	X 	 Pv 	 XI I I	 I)             141.12 Media and antibioticsGrowth media components were purchased from Difco and BDH. Allantibiotics were obtained from Sigma Chemical Co. Restriction enzymes, nucleases,and T4 ligase were purchased from Pharmacia, Inc. and Boehringer Mannheim.NYTRAN-brand nylon filters for Southern Blots were purchased from Schleicherand Schuell. 5'-[a 32P1-dCTP (3000Ci/mmol) for sequencing and probe labelling wasobtained from New England Nuclear.All H. influenzae strains were routinely grown at 37°C in Difco brain heartinfusion broth (BHI) supplemented with 21.tg/m1 NAD and 10µg/ml equine hemin(sBHI). Experiments for the maximal induction of competence development wereperformed using the starvation medium MIV (Herriott et al., 1970). Antibiotics inbroth and on 1.2% agar + sBHI plates were used at the following concentrations inH. influenzae experiments: novobiocin 2.51.1g/m1; kanamycin 7.0n/m1;chloramphenicol 2.04g/ml. Additional hemin (250111 of lmg/ml) was applied tosBHI plates more than 24 hours old.E. coli strains were grown in Luria Bertani broth and plates (Sambrook et al.,1989). The minimal medium M9 (J. Miller, 1972), supplemented with either 0.4%glycerol, or 0.4% glucose and 0.4% casamino acids, was used for the E. coli cAMPconcentration measurements. The Acya E. coli complementation assay wasperformed on MacConkey lactose plates or on MacConkey base agar plates with 1.0%maltose as the carbon source. Where necessary, antibiotics were added to plates orbroth cultures to the following final concentrations: ampicillin 50µg/ml;kanamycin 104g/m1; chloramphenicol 25µg/ml.151.2 Methods1.21 Library constructionH. influenzae chromosomal DNA was used to form libraries in thechloramphenicol resistant plasmid pSU2718 (Martinez et al., 1988). This plasmidcan be used as a shuttle vector between H. influenzae and E. coli (M. Chandler, 1991).DNA from the wild type strain KW20 was partially digested with Sau3A and ligatedto the BamHI site in pSU2718 with T4 ligase, following the procedure described inSambrook et al. (1989). Pstl libraries were also prepared from the cya- H. influenzaestrains RR665 and RR668. These libraries were screened for kanamycin resistantclones that were expected to contain part of the cya gene.1.22 Competence techniquesE. coli cells were made competent using the calcium chloride procedure fromSambrook et al. (1989). To study spontaneous competence development in H.influenzae, it was necessary to innoculate cultures with cells in early log growth(0D600 <0.1) instead of cells taken directly from colonies on a plate. Since cells fromplates are in stationary phase, they already have become spontaneous competent,which would have made it difficult to distinguish between the elevatedtransformation frequency of the initial innoculum and the increase intransformation frequency that results from competence development in the newculture. The early log cells were obtained from small cultures grown overnight(without vigorous aeration) at 30°C. After innoculating the new cultures to aninitial OD600 of 0.005, these cultures were grown at 37°C to an OD600 of 0.4. At thispoint, and at half hour intervals, the transformation frequency was measured byadding a saturating amount of MAP7 DNA to one milliltre culture samples.16To test cAMP induction of competence, cultures were grown to OD600 =—0.030 and cAMP was added to half of the culture to a final concentration of 1 mM.One ml samples were tested for competence after continued growth at 37°. Maximalinduction experiments with the starvation medium MIV (Herriott et al., 1970) wereperformed by growing cultures to approximately 10 9 cfu/ml, and transferring thecells to an equal volume of MIV medium, as per the procedure of Herriott et al.(1970). After 100 minutes of aerated growth at 37°, a saturating amount of MAP 7chromosomal DNA (1µg/m1) was added to one milliltre of cells.In all H. influenzae competence experiments, after cells + DNA were grownat 37° for 15 minutes to permit DNA uptake to occur, excess DNA was degraded withDNase over a period of 5 minutes, and the cells were then plated to select fornovobiocin resistant transformants. To transform intact plasmid DNA into MIVcompetent H. influenzae, glycerol was added to a final concentration of 30% after the15 minute DNA incubation step. After 10 minutes at room temperature, DNase wasadded and the cells were plated as described above.1.23 cAMP assayFor the E. colt strains, the cAMP determinations were made on cells grown inM9 medium supplemented with 0.4% glucose and 0.4% casamino acids. The H.influenzae strains were tested after growth in sBHI medium. The cAMP present inthe cells of these cultures was measured by filtering samples and rinsing the boundcells to remove the growth medium. The resulting filters with the bound cells weresealed into bags containing one millilitre of the cAMP assay buffer (provided in theNEN assay kit) and were boiled. After boiling, the protein content of these sampleswas also measured using the DC protein assay kit from Biorad. This kit measured1 7protein concentrations by using a modified Lowry reaction. The cellular debris fromthe boiled cells was then removed by centrifugation before the cAMP assay wasperformed.The cAMP assay was performed with the cAMP 125I-RIA kit from NEN.Using this kit, the cAMP present in the sample and standards was acetylated and itsconcentration was measured by using a competition reaction between unlabelledcAMP and a radioactively labelled [ 125112*-0-succinyl-cAMP tyrosine methyl estertracer for binding to a cAMP-specific antibody.1.24 SequencingDouble stranded plasmid DNA was isolated using the alkaline lysis prep fromSambrook et al. (1989), and was prepared for sequencing using the Magic Miniprepkit from Promega. Sanger dideoxy-chain termination sequencing (Sambrook et al.,1989) was performed using the T7 sequencing kit, the deazo G/A mixes, and thereverse and universal sequencing primers from Pharmacia.1.25 Analysis of DNA by Southern HybridiationTo map the cya gene in the H. influenzae genome, pulsed field gel nylonfilters (prepared by Dr. Redfield) carrying H. influenzae DNA digested by Apal, Smal,Eagl, and Nael were probed at 65°C with the 1.0 kb cya-containing insert from pID5.Since the Apal and Smal fragments have been physically mapped (Redfield and Lee,1990), by identifying the Apal and Smal fragments in the filter that the cya probehybridizes to, the location of cya can be determined.Other Southern blots were prepared from restriction enzyme digestedgenomic and plasmid DNA that were separated by electrophoresis in regular agarose1 8gels. Transfer of this DNA to nylon filters was performed using the method ofSouthern (Sambrook et al., 1989). These filters were probed with DNA labelled with5'-[a 32P]-dCTP. DNA hybridization reactions were performed at temperaturesranging between 40° and 65° C.19RESULTS1.1 Cloning and mapping cya Several attempts were made to isolate the H. influenzae cya gene. Initially, Itried probing filters of restriction-enzyme digested genomic H. influenzae DNAwith cya genes cloned from E. coli (Koop et al., 1984)and Bradyrhizobium japonicum(Guerinot and Chelm, 1984), in the hope that there would be sufficient sequencesimilarity between the cya genes that I could use the heterologous cya to isolate theH. influenzae cya gene from a plasmid library. Unfortunately, hybridizationconditions could not be found that permitted specific binding to the cya gene. I alsotried to use screens for cya- cells that have been originally developed for E. coli andSalmonella typhimurium (Alper and Ames, 1975; Brickman et al., 1973; S. Kumar,1976), but unfortunately these methods could not be used to isolate cya- H.influenzae cells.Finally, I decided to isolate the adenylate cyclase gene of H. influenzae byusing the E. coli Acya strain CAA8306 to screen a Sau3A H. influenzae plasmidlibrary for the H. influenzae cya gene. This screen selects for cells containing thecya+ plasmid by utilizing the fact that Acya E. coli cannot metabolize cataboliterepressed sugars (Kiely and O'Gara, 1983). E. coli with defective cya are thereforewhite on MacConkey lactose plates, while strains with the cya+ plasmid are red. Byusing this screen, the cya+ plasmid pID1 was isolated from the above mentionedlibrary (see Figure 2a). This plasmid was initially believed to contain the entire cyagene.2 0Figure 2a:  Partial restriction map of pID1 and cya gene, including locations of the transposons Tn7-15 and Tn1-6 -0. cyaE	 X P	 X	 Pv	 X PVVKB C	 V	 C	 H	 EL 	 II 	 I 	 I 	 I 	 1 111 	 1 1I 	 I 	I 	 I 	 I ]Tn 7-15 Tn 1-6Legend: F---I = 1kBRE abbreviations: B =BamHIE = EcoRIP = PstlV = EcoRVC = ClalH = HindIIIPv = PvuIIX = XbalHatched boxes in figure indicate the plasmid pSU2718Dashed line indicate region of cya that may not be present in pID1pSU2718 carries the multiple cloning site (MCS) of pUC18(Yanisch-Perron et al., 1985); the sites of the MCS are notpresented in this figure.The presence of cya in pID1 was confirmed by growth of the pID1-containingAcya strain on maltose, another catabolite repressed sugar, and by measuring levelsof cAMP production. The radioimmunoassay kit used to measure the amount ofcAMP produced by the cells did not give very satisfactory results, as it appeared thatthe kit was unable to detect cAMP concentrations below a certain background level.These limitations made it very difficult to determine the cAMP production levels.It was also difficult to compare the results from different strains since the cAMPvalues were not all taken at the same stage of culture growth. Therefore, the specificvalues for the cAMP results are not necessarily correct. Nevertheless, the generaltrend of these results indicated that the presence of pID1 in the Acya strain CAA8306increased the cAMP production compared to the Acya strain alone. These resultsindicate that the 7.3kB insert of pID1 codes for a protein with adenylate cyclaseactivity.21Table 2: Levels of cAMP production by various H. influenzaeand E. coli strains StrainE. coliCAA8306CAA8306(pID1)CAA8306(pID1::Tn7-15)H. influenzae:KW20 in sBHIRR668 in sBHIAccumulation14.7 ± 0.9 pmol/mg protein36.1 ± 0.3 pmol/mg protein13.6 ± 0.7 pmol/mg protein12.7 - 21.2 pmol/mg protein7.9 - 11.0 pmol/mg proteinLegend: The level of cAMP production by cultures of various E. coli and H.influenzae strains was measured using the RIA kit from NEN. The E. coli valueseach represent a single time point from a culture at OD600 0.15, grown in M9 +0.4%glucose and 0.4% casamino acids. The H. influenzae results are a range of resultsseen during culture growth. To standardize the cAMP results to the amount of cellspresent, the protein content of these cultures were measured using the Biorad DCprotein assay kit. For further details, see the Materials and Methods section.Several subclones of pID1 were prepared (see Figure 2b). Preliminaryrestriction enzyme digestions had shown that the cya gene must span the cluster of 6restriction enzyme sites located in the centre of pID1. To locate the cya gene in pID1,the subclone pID4, containing the 3.2 kB Kpnl-HindIII fragment of the pID1 insert,was formed. By sequencing outward from the Kpnl site in the pID4 insert, theposition of the cya gene was determined (see figure 2a).22Figure 2b: The subclones of pID1	  * cya	SXP	 X	 Pv	 X PVVK B C	 V	 C	 H	11	 I 	 I 	 I 	 111111 	 I 	 I 	 I KN, 1 K	 H pID4 insert X	 KI---I pID5 insertx 	 xI 	  pID8 insert Legend:S	 B C	 CI 	 I	SmaI - BamHI subfragment 	 2.1 kb C/aI subfragment= 1kB  RE abbreviations: 	 B =BamHI	 C = ClaIE = EcoRI 	 H = HindIIIP = PstI 	 Pv = PvuIIV = EcoRV 	 S = SmaIX = XbaIHatched boxes in figure indicate the plasmid pSU2718Dashed line indicate region of cya that may not be present in pID1Sma I site is located in pSU2718 multiple cloning site, not in insert DNAThe subclones pID4 and pID5 were partially sequenced. The subclonepID5 and the two sub fragments were used to probe chromosomal DNA.The plasmid pID5, carrying a 1.0 kb Xbal-Kpnl insert subcloned from pID1,was used as a probe in Southern blot hybridization to map the cya gene in the H.influenzae genome (see Figure 3). By comparing the banding pattern, it can beshown that the pID5 probe hybridizes to a single band in each of the 4 genomiclanes. As seen in the figure, these bands are the 32kb bands ApaI 0/P, 18 kb bandEagI U, 27 kb band Nal 0, and 30kb band SmaI K. Using the physical map of the H.influenzae genome (Redfield and Lee, 1990) it was concluded that the cya gene islocated within the 30 kb region bounded by the ApaI 0/ SmaI K fragments.2 3Figure 3: Mapping the cya gene to the H. influenzae genomeLegend: To map the H. influenzae cya gene to the genome, the insert of the plasmidpID5 was used to probe filters prepared from digested H. influenzae chromosomalDNA. One of these filters, probed with labelled total MAP7 genomic DNA and the Aconcatemer size standard DNA is shown at the top left. The bands in the sizestandard lanes are 48.5 kb, 97 kb, and 145.5kb in size. The same filter, probed withpID5, is shown at the top right of the figure. Letters at the top of the filter indicate: A= A DNA molecular weight standard; A = ApaI genomic digest; E = EagI genomicdigest; N = Nad genomic digest; S = Smal genomic digest. The physical map of theH. influenzae genome (Redfield and Lee, (1990), shown at the bottom of the figure,was used to determine where the cya-containing ApaI and Smal fragments map inthe genome (see arrow).•a le•OP AOAir 4. 	 OW411011°' *."4110 tOPop 410ompPl, tit,atthiPl, 	 hinc110.4ompP2 	 Rsrll4C4oP24Figure 3: Mapping the cya gene to the H. influenzae genomeR 	 ENSMOP	 INWR	 E N 5alpPiePhysical and genetic map of H. influenza° Rd2 5Transposon mutagenesis was also performed on pID1, using the phage-borneminiTnlOkan transposon as described in the Materials and Methods. The procedureyielded the two cya- pID1 derivatives pID1::Tn1-6 and pID1::Tn7-15. As seen inFigure 2a, the transposon from pIDI::Tnl-6 was located in the 3' half of the cya gene,while pID1::Tn7-15's transposon was in the 5' half of cya. The defective cya gene inpID1::Tn1-6 was recombined into the wild type chromosome of KW20 to form thecya- mutant RR665, as described in the Materials and Methods. I later usedpID1::Tn7-15 to form RR668, in order to study a cya- H. influenzae strain with acatalytically defective cya gene. To prove that inserting the transposon 7-15 intopID1 rendered the plasmid's partial cya gene catalytically inactive, the cAMP levelfor the Acya strain CAA8306 carrying pID1::Tn7-15 was compared to the level ofcAMP produced by CAA8306 alone, and by CAA8306 carrying the cya+ plasmid pID1(see Table 2). The strain with the transposon-bearing plasmid had a cAMP levelsimilar to that of CAA8306, and substantially lower than that of CAA8306 with pID1,indicating that the transposon insertion prevented the H. influenzae cya fromfunctioning in E. coli. This result, as well as the cAMP levels for RR668 shown inTable 2, supported my belief that RR668 was incapable of synthesizing cAMP.Southern blot analysis using the 4.0 kb Smal - BamHI subfragment and the2.1kb Clal subfragment of pID1 as probes also revealed that the restriction enzymepattern of the pID1 insert did not match the pattern from chromosomal DNA. Inparticular, while the Smal - BamHI subfragment probe hybridized to the predictednumber and sizes of bands, the 2.1 kb Clal fragment from the pID1 insert did nothybridize to a 2.1 kb band in the chromosomal digest (see Figure 4). This indicatedthat the 5' end of cya is present in pID1, but pID1 may be missing up to 25% of the 3'end of the cya gene. An attempt was made to isolate the complete gene in twopieces, one each from libraries prepared from the two cya- strains RR665 and RR668.The two libraries each yielded one clone carrying part of the cya gene (pID6T, pID7T).2 6I had planned to remove the transposons from the plasmids, and then ligate theinserts together. Unfortunately, there is a problem with pID7T, as I was not able tosuccesfully remove its transposon. Attempts to reisolate the 3' end of cya from theRR665 library yielded 15 more kanamycin-resistant plasmids, all with differentrestriction maps. I interpreted these results as evidence that extensiverearrangement has occured, either during the formation of RR665 or during thecloning of the plasmids from the library. As a result of this rearrangement, I onlysequenced the unrearranged section of pID7T (i.e. the region that is common to bothpID7T and pID1). The transposon was successfully removed from pID6T, to formpID1. Preliminary partial sequence from one strand of the pID6 insert, whichcontains the 5' untranslated region and the N-terminal catalytic domain, has beendetermined.2 7Figure 4: Probing H. influenzae chromosomal DNA with pID1 subfra gments Legend: Two subfragments of the cya+ plasmid pID1 were used to probe filters ofdigested H. influenzae KW20 chromosomal DNA. Letters at the top of the filtersindicate: X = HindIII digested X DNA molecular weight standard; X = XbaI genomicdigest; C = Clal genomic digest; E = EcoRI genomic digest. The dots in the X laneindicate the 23.1 kb, 9.4kb, 6.6kb, 4.4kb, 2.3kb, and 2.0kb size standard bands . Thesehybridization experiments were performed at 65°C to compare the restrictionenzyme pattern of the pID1 insert and the genomic DNA. The 4.0 kb Smai-BamHIsubfragment of pID1 was used to probe filter 105-2, and the 2.1 kb ClaI subfragmentof pID1 was used to probe filter 105-1 (for further details of relationship of thesubfragments to pID1, see figure 2b). Note that the 2.1kb ClaI subfragment did nothybridize to a band of corresponding size in the genomic filter.Go.1 428Figure 4: Probing H. influenzae chromosomal DNA with pID1 subfragments105-2XXCE X105-1XXCE	 X291.2 Competence StudiesI was primarily interested in determining how adenylate cyclase wasinvolved in competence development. Therefore, I constructed the H. influenzaecya- strain RR665. This strain was tested for development of spontaneous late logcompetence and induction of competence by MIV medium. When spontaneouscompetence development for RR665 was measured, it was found that competencelevels rose throughout log growth, but were consistently 100 fold lower than thecorresponding wild type values (see Figure 5). The transformation frequenciesmeasured for this strain in MIV were 10 fold below the levels for KW20 (see Table3).At first, the results I had obtained for RR665 indicated that cya was notessential for competence development. However, when the location of the cya genein pID1 was determined, I realized that the transposon in RR665 was located in the3' half of the gene. Work in E. coli has shown that the analogous region of the E.coli cya gene can be deleted without completely eliminating the catalytic activity ofcya (Roy et al., 1983). It was therefore possible that the cya gene in RR665 was stillcatalytically active. I then formed the cya- strain RR668 from KW20 and the plasmidpID1::Tn7-15. Since the transposon in pID1::Tn7-15 is in a region of cya that aligns tothe catalytic domain of other bacterial cya, RR668 should not be able to synthesizecAMP.Spontaneous competence development in RR668 was measured throughoutall phases of growth, from early log to stationary phase. As seen in Figure 5, thetransformation frequency of RR668 did not rise during the growth of the culture.This result is quite different from the increase in competence seen in wild type H.influenzae and in RR665, and shows that RR668 cannot become spontaneously3 0competent. RR668 also failed to develop competence in the MIV starvationmedium, and its MIV transformation frequency was 10 4 fold below the wildtypevalue (see Table 3). The adenylate cyclase gene is therefore an essential competencegene.Figure 5: Comparison of spontaneous competence levels in sBHI from RR668, RR665 and KW20 10 10 7	cfu/m1KW20-a- cfu/ml RR668cfu/ml RR665cfloni 109108	10-3 	•10-410-5 .TF 10- 610-710- 8 .;	10-9 		4 	 5 	 6time (hr)-0- av TF KW20-a- av TF RR668-M- av TF RR6657 8Legend: The data for RR668 and RR665 were the average results of six experiments.The KW20 results were averaged from three experiments. Growth data waspresented to correlate the transformation frequencies to the state that the cultureswere in. Spontaneous competence was measured for these cultures from 0D600 0.4to OD600 1.3. The same time scale was used for both graphs.3 1Table 3: Transformation frequencies for RR668, RR665 and KW20 in MIV starvation medium no cAMP +5001.tM cAMPaKW 20 (3.3 ± 2.4) x10-3 11 (4.9 ± 1.4) x10-3 5RR665 (2.9 ± 2.2) x10-4 6 (7.1 ± 5.4) x10-4 4RR668 <(1.1± 1.0) x10-7 8 (1.1 ± 0.9) x10-3 6Legend: 	 a=minimum cAMP concentration needed to induce competence inRR668Underlined numbers refer to the number of samples used to calculate the averageresults. The less-than sign used in the RR668 result refers to the fact that four of theexperiments did not produce any transformants.I also measured competence development for RR668 and RR665 during logphase growth in sBHI and in MIV medium in the presence of added cAMP. I foundthat addition of cAMP to either of the mutant strains restored competencedevelopment in early and mid log phase growth in sBHI to the levels seen for wildtype cells (see Figures 6 and 7). The addition of cAMP to MIV medium inducedcompetence in RR668 to levels close to those seen for MIV-competence wild typecells. These results agreed with my initial assumption that RR668 cannot becomecompetent because it possesses a catalytically defective cya gene.10-5 1TF3 2Figure 6: Comparison of competence levels for RR668 in sBHI in the absenceor presence of cAMP 10 10 F-13- KW20 no cAMP-0- KW20 + cAMP-0- RR668 no cAMP-U- RR668 + cAMP   1 09 7cfu/ml  108 7101 o-4Tatip1-a- RR668-11- RR668 +cAMP-0- KW20-•- KW20 +cAMP1 0-90 2	 3 	 4time (hr)Legend: The data in this figure was taken from sBHI cultures grown with orwithout 1mM cAMP as described in Materials and Methods. For time values t=0refers to time when the cultures were divided and cAMP was added to one of them.These cultures were studied from OD600 0.050 to OD600 0.9. Growth data waspresented to correlate the transformation frequencies to the state that the cultureswere in.i1 	 13 3Figure 7: Comparison of competence levels for RR665 in sBHI in the absenceor presence of cAMP10 1010 9cfu/ml1081 071 0-31 o-410- 5TF 10-610-10--41- RR665 no cAMP"ar RR665 + cAMP-0" KW20 no cAMP-410- KW20 +cAMP4' - RR665ner- RR665 +cAMP-0- KW20-4•- KW20 +cAMP10- 9 	 10 	 1 	 2 	 3 	 4time (hr)Legend: The data in this figure was taken from sBHI cultures grown with orwithout 1mM cAMP as described in Materials and Methods. For time values t=0refers to time when the cultures were divided and cAMP was added to one of them.These cultures were studied from OD600 0.050 to OD600 0.4. Growth data waspresented to correlate the transformation frequencies to the state that the cultureswere in.341.3 Sequencing cyaSections of the H. influenzae cya gene were sequenced from one strand of the2.9kB insert of pID6, the 3.2 kb insert of pID4, the 1.0 kb insert of pID5, the 1.4 kbinsert of pID8 and the 9.7 kb insert of pID7T. The resulting preliminary cya sequenceand the deduced amino acid sequence is shown in Figure 8. The deduced H.influenzae amino acid sequence was aligned to the N-terminal cya sequences fromfour other bacteria (see Figure 9). This alignment has identified 69/399 identical and43/399 conservative amino acids common to all five proteins. Also, this alignmenthas shown that start codons of the other bacterial cya genes align with the unusualputative start codon GTG in the H. influenzae cya sequence. Analysis of the H.influenzae DNA sequence located a poor Shine Delgarno site (AGGGA) at -11 to -7and a CRP binding site with 12/22 matches to the consensus sequenceAAATGTGATCTAGATCACATTT (Botsford et al., 1992)) at -80 to -58, whichsupported my belief that the GTG codon at +1 was the putative start codon. Asexpected, the PasteureHa multocida gene has the highest amino acid identity to theH. influenzae gene (55%), while the three enteric bacteria all have lower identities of—30%. The H. influenzae cya gene is the first bacterial cya gene found to use GTG asa start codon (Aiba et al., 1984; Beuve et al., 1990; Danchin and Lenzen, 1988; Mock etal., 1991; Thorner et al., 1990).Figure 8: Nucleotide sequence of the H. influenzae cya gene and deduced amino acid sequence m000pocxxx mooc0000cx xxxxxx)ocxx xxxxx>oocxx xxmocoocx xttttttgtc attttgattgaaatttatag tattttnnaa aatt=qat ttatcrtcact ttttttcrcat ttatactccc gtaagtggtaV E C N L A Q A K E Wtaatttttgt tgttctggta crgcracaccac GIG GAA TOT MT CTA GCA CAA GCA MA GAA MG+1FE R ALQGSGDAF QGCC TIG GAT CAA CGC CGT '1 11 1 ' GAG CGT GCA '1'1'A CAA GGT TCA GGC GAT GCA TIC CAAttgataggtaaattgtt tgg3 S(31'1 1 AGC39H VCAC GTT102LAIVPLLLHLNHPQLPGYVIHTTA GCG Ail' Oft CCC' '1`1A C1 11 1 TEA CAC Cl'A PAC CAT CCA CPA (21'1 1 CCC GGC TAT (31'1' A1'1' CAC165APSGIASF LASDYQK K W L TNEGCC CCC 'ICA GGT Ali GCA AC31 1 TIC MC GCA MT GAT TAT CAA MA PAG MG CTC ACC MT CAA228YGIHYADHK PS T L K SAVNFHETAC GGT Ai'i' CAT TAT GCC GAT CAT AAA CCA `ICA ACA CTC AAA AGT GM CIA MT TIT CAC GAA2913 FPPILGVYVMGSFGSISQTSGIT 1T2 CCA CCA Ar.i.".Li.LA GGT G.LT TAT (3.1.A MG GGT AGC 'PIT CCC 'ICA ATA ACT CAA ACC ICT354S SDLDTWICVRDGLSLDEY TLTCT 'ICA GAT Cr!' GAT ACT MG Al'!' a= arc car GAT GGC TIG AGE CTA GAT GM TAT ACA Carr417L T Q K AK R ISEWAMQFNVEINFCIT ACT CAA AAA GCC AAG CGG AlT ACT GAA TGG GCT ATG CAA 1 '1T MT GIG GM A1'1' MT '1' 1'1'480YLMDQQRFRNEHYADPL TIENTAT TIG NIG GAT CAA CM CGT TIC CGC AAT GM CAT TAT GCT GAC CCA CIG ACC' Al'I' GM AAT543 *DPYIAIL AK V Tx Y L TAL S E F KGAT Cell TAC ALL' GCC Al'i' (21T GCA AAA CIA ACC CAA TAT TTA ACC GCA Cl'i' 'ICI' GAA '1T1' AAA873? 	 936RLDFVHRCF Y V K A TEDF AR Y QCGT TIC GAT TiT GIG CAC COT rIGT TIC TAT GIG AAA GCA ACG GM GAT T1'1' GCA CGT TAC CAA999ANNWR I R YME IL A Q E W G W S A EGCC AAT AAT TOG COT A'l'l' CGT TAT MG GAA ATC Cl'll GCG CAA GAA 'IGG GGA TOG TOT GCA1062TVKHLNK R P F WK IK A 	 ENV K H DACn G'1A AM CAT CIC MT AAG ail' CCA Tr1' TOG AAA Al'!' MC GCA GTA AAA GAA AAC CAC GAT1125NIMKFLML 	 FAR K H HAAC ATA ATG AAA Ti'1"1 1 A ATG TIG AGT TAT CGT AAT TTA GIG GM T1'1' GCT CGA AAG CAT CACcn 	 1188IHSSVVPQDINILSRKLY TAFAl ' !'CAC TOG AGC GIC GIG CCA CAA GAT MC MC Al'!' C1'1' TOT CGT AAA Ci'1' TAT ACC' GCT 1111251EEL PGK VSLLNTQISHNL SEAGAA   CCC GGC AAA GIG TCC TTA 	 AAC ACA CAG ATA TCT CAC AAT 'l'IA TCA GAA GCA1314HL T F VEVRGNK H F K DGWY x x xCAC CIG AC1"1T1' GIG GAA C1T CGC GGC AAT AAA CAT '1'1'1' AAG GAT GGT TUG TAC Cnn nnn nnn1377x x x xxxxx SK ERVIE YGE S L Nnnn nnn nnn nnn nnn nnn nnn nnC TCC AAA GAA CGG GIG ATC GAA TAC CGA GAA ACC CIG AAT1440KLVSWAYFNHLL T E K TEL SIFAAA TTA 	 TOT 'IGG GCT TAT TIC MT CAT '1 1 A CIG ACT GM AAA ACC GM TTG TCA Al'l' IT11503SKNVTL S T L Q R F V TNLRQS..:L_tzL__ 124 RP 13-20 RPN 	 1 	 1 cyaen -200 	 -100 	 +1 	 100 	 200 	 300 	 400 	 500 	 600Legend: 	 n = region not yet sequenced 	 x = unknown amino acid * = —330 by gapOther lower case letters in nucleotide sequence indicate untranslated regionsUnderlined bases in 5' region indicate a putative CRP binding site (bases #-80 to -59), -35 and -10promotor regions (at #-85 to -79 and #-59 to -54) and a possible Shine Delgarno site (at #-11 to -7).DNA numbering begins from +1 at first base of putative start codonApproximately 1.0 kB of cya has not yet been sequencedRegions of pID4, pID5, pID6, pID8 and pID7T that have been sequenced:Preliminary sequence from the 5' end of the H. influenzae cya gene	....	-...(1) 	ID8 UP (2) If. pID5 UP(1) 	12_19 Rc,(2(2)(3)-41 6-49(1RI, -.1-- 	 13-)16 RP	 13-1 RPAGC AAG AAT GIG AC:1"1 1 A AGC ACA '1'1A CAA CGC Tirf GIC ACT AAC TIG CGT CAG rICT1566-40	Preliminary sequence from the centre of the H. influenzae cya gene(3) 	 1.. 	 pID7T UP 	 (1)(4)(1)-.41 	pID5 SP6pID6, pID6T RP110, pID4 RPcya700 	 800	 9001 inch =100 by1000 	 1100 	 1200 	 1300 	 1400 	 1500— Sequence from coding strand-411— = Sequence from noncoding strand12 -1, 13-20, 6-49, 13-16, 13-1, and12-19 are ExoIII deletion productsof pID6Bracketed number indicates number oftimes that region has been sequenced1600GYV-IHAPSGGYV-ADAPVGGYLEGNVPKGGYLITIKVPHGGYLEGNVPSGIASF--LASD 67IADFVISPYQICLY--TPDEICLF- -SPDEICFY- - TPDESSSDLDIWIC VRDGLSLDEY 136PKSDLDIWVC HRDDLSTKEKCSSDT nIWVC HQSWLDSEERCSSDLDIWVC HQSWLDNEERCSSDLDIWVC HQSWLDGEERXXXXXXXXXX xxxxx>000cx 206MLLLDEFYRS AIRLAC1KPT  T IT T DEFYRT AVRLA=LIT T DEFyRT AVFMACYRILIT TT DEFYRT AVRLACYRILFigureH.iP.mE.cEr.cS .tH.iP.mE.cEr.cS .tH.iP.mE.cEr.cS .tH.iP.mE.cEr.cS .tH.iP.mE.cEr.c9: Alignment of deduced H. influenzae adenylate cyclase amino acidbacteria sequence to cya from four otherMECNLAQAKQ WVSALIDQRRF ERALCGSGDA FQHVLAIVPL LLELNHPQLPMNYDLFSAQK KVEYLDKLRI ERALSGSSGE FQHVFQLLTL LLHINEPNLPMYLYIETLKQ RLDAINQLRV DRALAANEPA FQQVVSLLPT LLHYHHPLMPMYFYIETLKQ RLDAINQLRV DRALEAMKPA FQQVYSLLPV LLHHHEPLMPMYLYIETLKQ RLDAINQLRV DRALAANEPA FQQVYSLLPT LLHYHHPLMPYQ}aWLTNEY GE-IYADKHPS TLKSAVNFHE VFPPIL-VYV IvESFGSISCT2KQYLLTIVPS LEANQSLLPS FSYRSTN-- - - - -AILGVYV IvESIASISQrTQRHYLNELE LYRCMSVQDP PKGEL 	  --PITGVYT MGSTSSVGQSKQQHYLDSVE LRWGELSAPD RKGEL 	  --PITGVYS NESTSSIGQSTUHYLNELE LYRGMTPQDP PKGEL 	  --PITGVYT NGSTSSVGQSTLLTQKAKRI SEWAM)FNVE INFYLMDQQR FRNalYADPL TiM00000(EALQRKTHLL KNWAKQFNIE INFYLMCQKR. FRCFRYAEPL TAENCGSAQYQLLQRKCSLL ENv\IAASLGVEVSVFLIDENR FRHNESGS-L CGEDCGSTQHQ-IJQQKCSLL EKAAACQGVD VSFFLMDENR FRHNESGS-L GGEDCGSTQHQLLQRKCSLL ESWAASI_GVE VSFFLI= FRHNESGS-L OGEDCGSTQHxxxxxm000c xxmaococxx xxxxxx>cxxx xxx>0000cxx xxxxxxxxxx x)oocxxmcxWLHLLIEQEE NYESEVERLV RIQQICIDDW VDEGGLOQLS	 QLYKGIDAPYWNMVPCDEEE HYDDYVMILY AQGGLTPNEW LDLGGLSSLS a	 W QLYKSIDSPYWNMVPVEEEA HY=VLSLY A i• A • 111, 	 LDLGGLSALS A a 	 QLYKSIDSPYWSMVPCDEEE HYDDYVMILY AQGVLTPNEW LDLOGLSSLS AEEYFGATLW QLYKSIDSPYxxxmcc000c. xxxxxxNRDL LAGNrNPDI-11 FUPYIAILAK VTxYLTALSE FKRLDFVHRCTYSSEYPNrY LIARQFKEEL LTGKLNPSHH 1.1.)PYLAMLQR ATRYLTK-INE LKRLGFVRRSAYSWEYPNPR LLAKDIKQRL HEGEIV-SeG T nPYCMMLER VTEYLTAIED FIRLDLVRRCAYSWEYPNIR T T SSEIKARL I-EGEIV-SM LDPYCI4vILER VTQYLDAIND QTRLDLVRRCxxxxxxxxxx 276KSVIKIT iF,KAKAKAFYVKATEDFA 346VYLKATEGMCFYLKVCEMLSFYLKVCEKLSRY---QANNT01 RIRYMEILAQ EW3WSAETVK HLNERPFWKIWQDPNATNLIAI RLQHICKIJIQ EWEWSDALIE ELNQRAWKIRE- -RACVGW RRAVLSQLVS EWagDEARLA MLDNRANtAIKIRE--RACTAW RRQLLTC,MCQ AW3WSDERLV MLDNRANAIKIRE--RACVGW RREVLSQLVS EAEWDDARLT MLDNRANWKIHSSVVPQDIN ILSRKLYTAFNSSIMPQDIS VLTRKLYTAFSVSASPQDIG VLTRKLYAAFSVSASPQDIG VLTRKLYAAFSVSASPEELPGKVSLL NIQISFELSEEELPGKITLL NPQISPDLSEEALPGKVTLV NPQISPDLSEEALPGKVTLV NPQISPDLSES.tH.iP.mE.cEr.cS.tH.iP.mE.cEr.cS.tH.iP.mE.cEr.cAYSWEYPNPR LLAKDIKQRL HDGEIV-SFG T DPYCMMLER VTEYLTAIED PTGLDLVRRC FYLKVCEKLSSKERVIEYGEVQKRYTEYSEISHQPLEYNRISHQPLEYNRSLNKLVSWAYSLNKLVAWAYYLNKLVAVAWYLNKLVAWAYFI\ELLTEKTEFNRILTANIDFNGLLTSR1hFNGLLTSSMLSIFSKN-VTLHIISPN-VSLYIKGNGIVDLHIKGHELCDKAVKINHDNIKQVKKAHt'ELCQVREAHNELGQVREAHNELDQVREAHNELAHLTFVEVRGKNLLFFEVKGPNLTI.IYVPPTNILLVIYVPALSTLQRFVTNLTILRHFVTDLPICLOEMVADIARLQELVSDIKELMLSYRNIA II	 •LDAMIADSYRNLDAMMQSYRNLVEFARKHHI 413LVAFARKHKTLIRFARRNNLLIRF'ARRNNLLNRFARRNNLNICHFKIDGWYx xxxxxxx= 483VNUIPSVAGFGRANRSGWYL YNRAPNIESIGRANRSGWYL YNQAPSMDAILRQSFPSxxx xx)c<xxxxxx 553LRLSFPVTVS SVTNEDLTHA*VSHHFPLRLP APTPKALYSP*VSSHFPLRVA APTPKALYSP*Legend:P.m=P. multocida; E.c=E. coli; Er.c=Erwinia chrysanthemi; S.t-S. typhimurium	 influenzaeP. multocida alignment to enteric bacteria sequence taken from Mock et al., 1991 Alignment gap indicated by'-'. Unknown amino acids indicated by 'x'. The '*' symbol indicates that -300 C-terminal amino acids are notpresented in this figure. Numbering refers to H. influenzae amino acids. Only the first 419 deduced aminoacids of the S. typhimurium cya are presented here, as the remaining 1.2kb of thegene has notr yet beensequenced.4 01.4: Comparison of crp- mutant to cya- mutant:As mentioned previously, the phenotype of the H. influenzae crp- mutantdiffers from the E. coli crp- and cya- mutants in that H. influenzae crp- strains havebetter growth rates and longterm plate survival than KW20 (Redfield, pers. comm.).In E. coli, mutations in the crp or cya gene result in strains that do not grow as wellas the wild type cells. (D'Ari et al., 1988). I wanted to determine if cya- H. influenzaebehaved like crp- H. influenzae, or if it was more similar to E. coli cya- mutants.Since cAMP acts with CRP in bacteria, then the H. influenzae crp- mutant and cya-mutants should have similar phenotypes. I have monitored cell death for RR668and KW20 on plates kept at room temperature for a period of 8 days. As seen inFigure 10, the results were similar for all strains, with no significant cell deathoccuring until the fourth day. Comparison of the log phase growth rate of the cya-mutants to the wild type KW20 has shown that the average doubling times of 31 ± 9min for RR668 and 34 ± 7 min for RR665 are higher than the 28 ± 5 min averagedoubling time of KW20, indicating that the mutant strains have a longer generationtime than KW20.—Et— KW20—a-- RR6680 241Figure 10: Cell survival on platesLegend: Single colonies of KW20 and RR668 were used to innoculate sBHI andsBHI/kan plates. After overnight growth at 37°C, the plates were left at roomtemperature for the remainder of the experiment. For each of the time points, asingle colony was removed from the plates, and resuspended in one ml BHI. Theresuspended colony was diluted and samples were plated to determine the numberof viable cells in the colony.42DISCUSSIONThe role of spontaneous competence development in H. influenzae can bedetermined by identifying essential regulatory genes. Once these genes are known,data from other bacteria can be used to identify possible target genes and hint at thefunction of these genes in competence. Since it was already known that crp wasessential for competence development, this made us suspect that cya was alsoinvolved in competence development.1.1: Significance of competence resultsI have shown that the cya- mutant RR668 cannot become competent duringlog growth in sBHI medium or in MIV starvation medium, unless exogenouscAMP is present. The similarity of the competence levels for the wild type strainand RR668 in MIV in the presence of cAMP proves that the defect in RR668 is in thecya gene. These results prove that the cya gene is an essential competence regulatorygene.The competence phenotype of the cya mutant strain RR665 was also tested.Unlike RR668, this strain was capable of limited spontaneous and MIV-inducedcompetence development. The transposon insertion in RR665 is located in a sectionof the cya gene that aligns to the region of the P. multocida cya gene coding for thepostulated hinge joining the regulatory and catalytic domains of adenylate cyclase. Ifthe H. influenzae cya gene is organized into similar domains then the transposon inRR665 may not completely eliminate adenylate cyclase activity in this strain. Itherefore interpret the competent results for RR665 to mean that reduced cAMPproduction causes decreased competence development. Since it is likely that the cya4 3gene in RR665 still produces a catalytically active product, these results do notindicate that adenylate cyclase is unimportant for competence development.The transformation frequency for RR665 in MIV rises when exogenous cAMPis present, indicating that this mutant has not reached its maximum competencelevel. Since RR665 may have limited cAMP production, this result shows thatcAMP is required for maximum competence induction.1.2: Comparison of bacterial cya nucleotide and amino acid sequences Cloning cya proved to be more difficult than originally expected, since asmentioned previously, up to 60% from the 3' end of cya can be deleted withoutcompletely eliminating cya catalytic activity (Roy et al., 1983). The data fromSouthern blots has indicated that it is possible that the cya+ plasmid pID1 may bemissing up to 25% of the cya gene. Since the potentially rearranged insert of pID7Tshould contain the part of the cya gene missing in pID1, it is possible that this regionof the H. influenzae genome is particularily prone to recombination events.I have succeeded in sequencing one strand of —1.2 kb of the cya gene, a regioncoding for 412 amino acids. I was not able to obtain any sequence for 1.0 kb of the 3'end of the gene. It was not necessary to clone the entire gene to study adenylatecyclase's role in competence development, since this could be determined bystudying cya- mutants. The partial clone contained enough of the cya gene so thattransposon mutagenesis could be performed on it to form the cya- strains RR668 andRR665. The partial sequence was also sufficient to map cya in the genome andprovide information about possible transcriptional and translational regulation ofcya activity.4 4It was not surprising to find that the H. influenzae cya has the highesthomology to the gene from P. multocida, since P. multocida is a member of thesame family as H. influenzae. Despite this high homology, the H. influenzae cyagene differs from all known bacterial cya in that the H. influenzae cya gene usesGTG as its start codon instead of TTG or ATG (Aiba et al., 1984; Beuve et al., 1990;Danchin and Lenzen, 1988; Mock et al., 1991; Thorner et al., 1990). I think that thisGTG codon is the true start codon because it aligned with the start codons from fourother bacterial cya genes. This start codon also has a weak ribosome binding site(TAGGGA) located 8 basepairs upstream. Of the other possible start codons for cya,using the TTG codon at +42 or the ATG codon at +321 as alternative start codonswould remove a significant portion of the cya gene that would otherwise aligns tothe other bacterial cya genes.Work in E. coli has shown that while GTG and TTG both act to decreasetranslation efficiency, the TTG codon is one sixth as efficient than ATG while GTG isonly half as efficient as ATG (Botsford et al., 1992). If H. influenzae regulates cya bylimiting cya translation, then why isn't the TTG codon used? Is there a reason forhigher cAMP production in H. influenzae or does H. influenzae rely on anothersystem to control intracellular cAMP levels?Although only part of the gene has been sequenced, we have enoughsequence information to form tentative conclusions about the regulation of cya atthe transcriptional and translational level. The CRP binding site found upstream ofthe start codon has been seen in other bacterial cya genes. It has been proposed thatthis binding site is used as a method of feedback inhibition to decrease cyatranscription when intracellular cAMP concentrations are too high (J.P Fandl eta!.,1990). Based on H. influenzae codon usage data from the SGcom locus genes (J.Tomb pers. comm., see Table 4), there is no evidence that the first 20 codon of the H.4 5influenzae cya gene contains rare tRNA codons that would inhibit translationduring exponential growth, as has been proposed for E. coli (Aiba et al., 1984).However, as mentioned previously, the unusual start codon GTG is believed to playthe same role of limiting translation as does the TTG start codon of the other cyagenes (Botsford et al., 1992). H. influenzae is the first bacteria capable of spontaneouscompetence development to have its cya gene sequenced. It is possible that the GTGcodon is a feature shared by all spontaneously competent bacteria. To test this ideacya from other naturally competent bacteria such as N. gonorrhoeae should besequenced.1.3: Comparison of cya- mutant to crp- mutantFrom the plate survival and growth rate data, it appears that RR668 growsmore poorly than KW20. In this way it is more like the crp- and cya- mutants in E.coli than the H. influenzae crp- strain. This may mean that cAMP has a role in H.influenzae that is independent of the CRP protein.1.4: Future experimentsIt is essential that the cya gene be completely sequenced and that an intactversion of the gene be isolated. I also believe that the other genes involved incontrolling cAMP levels should be cloned so their influence on competence can bedetermined. It should be possible to use the cya- strain RR668 to search for thecompetence genes that cya regulates, by using mutagenesis to obtain cya-pseudorevertants, with mutations in the competence genes targetted by cya.4 6Table 4: Analysis of codon usage for the first 20 amino acids of cya#12,10cya AA cya Codon codon usage in other loci conclusionaValGluGTGGAAGTG 37% GTT 27%GTC 12%GAG 17%CCGTA 23%GAA 83%3 Cys TGT TGT 69% TGC 31% C4 Asn AAT AAT 74% AAC 26% C5 Leu CTA TTA 43% TTG 24% RCTT 15% CTC 6%CTA 6% CTG 6%6, 8 Ala GCA GCA 36% GCT 26% CGCG 25% GCC 13%7,17 Gln CAA CAA 77% CAG 23% C9 Lys AAA AAA 86% AAG 14% C11 Trp TGGb12 Val GTT GTG 37% GTT 27% AGTA 23% GTC 12%13 Ser AGC TCT 24% AGT 24% ATCA 20% AGC 17%TCG 8% TCC 7%14 Ala GCC GCA 36% GCT 26% RGCG 25% GCC 13%15 Leu TTG TTA 43% TTG 24% ACTT 15% CTC 6%CTA 6% CTG 6%16 Asp GAT GAT 80% GAC 20% C18 Arg CGC CGT 56% CGC 20% ACGA 15% CGG 4%AGA 4% AGG 1%47Table 4 continued# cya AA cya Codon codon usage in other loci conclusiona19 Arg CGT CGT 56% CGC 20% CCGA 15% CGG 4%AGA 4% 	 AGG 1%20 Phe TTT TTT 	 87% 	 TTC 13% CLegend: The codons used for the first 20 amino acids from the H. influenzae cyagene were compared to the codon usage patterns seen for 10 H. influenzae corn locigenes (data supplied by J-F. Tomb).Notes:-a) 	 'C' = 'most frequently used codon', 'R' = 'least frequently used codon' and'A' = 'moderately frequent codon'-b) 	 I could not determine if this was a rare or common codon since this is theonly tryptophan codon in existence4 8REFERENCESAiba, H., et al. (1984): The complete nucleotide sequence of the adenylate cyclasegene of Escherichia coli . Nucleic Acids Res. 12:9427-9439Aiba, H. (1985): Transcription of the Escherichia coli adenylate cyclase gene isnegatively regulated by cAMP-cAMP receptor protein. J. Biol. Chem. 260:3063-70Alexander, H., and G. Leidy (1951): Determination of inherited traits of H.influenzae by desoxyribonucleic acid fractions isolated from type-specific cells. J.Exp. Med. 93:345-59Alper, M.D., and B. N. Ames (1975): Transport of antibiotics and metabolite analogsby systems under cyclic AMP control: positive selection of Salmonellatyphimurium cya and crp mutants. J. Bacteriol. 133:149-57Barany, F., et al. (1983): Directional transport and integration of donor DNA inHaemophilus influenzae transformation. Proc. Natl. Acad. Sci. USA 80:7274-8Barouki, R. and H. 0. Smith (1985): Reexamination of phenotypic effects in rec-1and rec-2 mutants of Haemophilus influenzae Rd. J. Bacteriol. 163:629-34Beuve, A., et al. (1990): Rhizobium meliloti adenylate cyclase is related to eucaryoticadenylate and guanylate cyclases. J. Bacteriol. 172:2614-21Botsford, J., and (1992): Cyclic AMP in Prokaryotes. Microbiological reviews 56:100-22Brickman, E., et al. (1973): Genetic characterization of mutations which affectcatabolite-sensitive operons in Escherichia coli, including deletions of the gene foradenylate cyclase. J. Bacteriol. 116:582-7Chandler, M. S. (1991): New shuttle vectors for Haemophilus influenzae andEscherichia coli: P15A derived plasmids replicate in H. influenzae Rd. Plasmid25:221-4Chandler, M. S. (1992): The gene encoding cyclic AMP receptor protein is requiredfor competence development in Haemophilus influenzae Rd. Proc. Natl. Acad. Sci.USA 89:1626-30Danchin, A., and G. Lenzen. (1988): Structure and evolution of bacterial adenylatecyclase: comparison between Escherichia coli and Erwinia chrysanthemi. SecondMessengers and Phosphoproteins 12:7-28Danner, D.B., et al. (1980): An eleven-base-pair sequence determines the specificityof DNA uptake in Haemophilus transformation. Gene 11:311-84 9D'Ari, R., et al. (1988): Cyclic AMP and cell division in Escherichia coli. J. Bacteriol.170:65-70Dubnau, D. (1991): Genetic competence in Bacillus subtilis. Microbiol. Rev. 55:395-424Fandl, J.P., et al. (1990): Mutations that affect transcription and cyclic AMP-CRPregulation of the adenylate cyclase gene (cya) of Salmonella typhimurium. Genetics125:719-27Goodman, S.D., and J.J. Scocca (1988): Identification and arrangement of the DNAsequence recognized in specific transformation of Neisseria gonorrhoeae. Proc.Natl. Acad. Sci. USA 85:6982-6Guerinot, M. L., and Chelm, B. K. (1984): Isolation and expression of theBradyrhizobium japonicum adenylate cyclase (cya) in Escherichia coli. J. Bacteriol.159:1068-71Hammond, G. W., et al. (1988): Haemophilus influenzae meningitis in Manitobaand the Keewatin District, NWT: potential for mass vaccination. C.M.A.J. 139:743-7Herriott, R. M., et al. (1970): Defined Nongrowth Media for Stage II Development ofCompetence in Haemophilus influenzae. J. Bacteriol. 101:517-24Hoelzer, M. and R. Michod (1991): DNA repair and the evolution of transformationin Bacillus subtilis: III Sex with damaged DNA. Genetics 128:215-23Hui, F. M., and D. A. Morrison (1991): Genetic transformation in Streptococcuspneumoniae: Nucleotide sequence analysis shows comA, a gene required forcompetence induction, to be a member of the bacterial ATP-depedant transportprotein family. J. Bacteriol. 173:372-81Kahn, M. E., et al. (1982): Possible mechanism for donor DNA binding and transportin Haemophilus. Proc Natl Acad Sci USA 79:6370-4Kahn, M. E., et al. (1983): Transformasomes: specialized membranous structuresthat protect DNA during Haemophilus transformation. Proc Natl Acad Sci USA80:6927-31Kahn, M. E., and H. 0. Smith (1984): Transformation in Haemophilus: a problemin membrane biology. J. Membrane Biol 81:89-103Kiely, B. and F. O'Gara (1983): 3'5'-Adenosine monophosphate synthesis inRhizobium: Identification of a cloned sequence from Rhizobium meliloti codingfor adenyl cyclase. Mol. Gen. Genet. 192:230-4Kleckner N., et al. (1991): Uses of transposons with emphasis on Tn/ O. in Methodsin Enzymology. Miller, J. H. (Academic Press Inc, New York), Vol 204 p139-805 0Koop, A. H., et al. (1984): Analysis of the cya locus of Escherichia coli. Gene 28:133-46Kumar, S. (1976): Properties of adenyl cyclase and cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli. J.Bacteriol. 125:545-55Kupfer, D. M., and D. McCarthy. (1992): rec-2-dependent phage recombination inHaemophilus influenzae J. Bacteriol. 174:4960-6Martinez et al. (1988): pACYC184-derived cloning vectors containing the multiplecloning site and lacZa reporter gene of pUC8/9 and pUC18/19 plasmids Gene 68:159-62McCarthy, D. (1989): Cloning of the rec-2 locus of Haemophilus influenzae. Gene75:135-43Meadow, N., et al. (1990): The bacterial phosphoenolpyruvate glycosephosphotransferase system. Ann. Rev. Biochem. 59:497-542Michod, R. E., et al. (1988): DNA repair and the evolution of transformation in thebacterium Bacillus subtilis. Genetics 118:31-9Miller, J. H., (1972): Experiments in molecular genetics. (Cold Spring HarborLaboratory Press, Cold Spring Harbor)Mock, M., et al. (1991): Structural and Functional Relationships between Pasteurellamultocida and Enterobacterial Adenylate cyclases. J. Bacteriol. 173:6265-9Redfield, R. J., and J. J. Lee (1990): Haemophilus influenzae Rd. in Genetic Maps, 5thed. O'Brien, S. J., (Cold Spring Harbor Laboratory Press, Cold Spring Harbor)Redfield, R. J. (1991): sxy1, a Haemophilus influenzae mutation causing greatlyenhanced spontaneous competence. J. Bacteriol. 173:5612-8Redfield, R. J. (1992): Genes for Breakfast: the have-your-cake-and-eat-it-too ofbacterial transformation. To appear in the Journal of HeredityRoy, A., et al. (1983): Two functional domains in adenylate cyclase of Escherichiacoli. J. Mol. Biol. 165:197-202Sambrook, J., et al. (1989): Molecular cloning: a labortory manual, 2nd ed (ColdSpring Harbor Laboratory Press, Cold Spring Harbor)Scocca, J.J., et al. (1974): Specificity in deoxyribonucleic acid uptake by transformableHaemophilus influenzae. J. Bacteriol. 118:369-735 1Setlow, J. K., et al. (1988): Characterization of the rec-1 gene of Haemophilusinfluenzae and behavior of the gene in Escherichia co/i. J. Bacteriol. 170:3876-81Stewart, G.J., and C.A. Carlson (1986): The biology of natural transformation. Annu.Rev. Microbiol. 40:211-35Thorner, L. K., et al. (1990): Analysis of sequence elements important for expressionand regulation of the adenylate cyclase gene (cya) of Salmonella typhimurium.Genetics 125:709-17Tomb, J-F., et al. (1989): Transposon mutagenesis, characterization, and cloning oftransformation genes of Haemophilus influenzae Rd. J. Bacteriol. 171:3796-802Wang, J. C. (1971): Interaction between DNA and an E. coli protein co. J.Molec Bi.55:523-33Wise, E. M., et al. (1973): Adenosine 3'5'-cyclic monophosphate as a regulator ofbacterial transformation. Proc. Natl. Acad. Sci. USA 70:471-4Wojciechowski, M., et al. (1989): DNA repair and the evolution of transformationin Bacillus subtilis. II Role of inducible repair. Genetics 121:411-22Yanisch-Perron, C., et al. (1985): Improved M13 phage cloning vectors and hoststrains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103Zoon, K. C., et al. (1975): Multiple regulatory events in the development ofcompetence for genetic transformation in Haemophilus influenzae. J. Bacteriol.124:1607-9

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