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Comparison of S-layer secretion genes in freshwater caulobacters Iuga, Mihai

Abstract

The Smit laboratory strain collection contains numerous freshwater caulobacter strains (FWC) isolated from locales throughout North America. 40 of these strains are morphologically similar to Caulobacter crescentus and secrete a surface (S-) layer. Their S-layer proteins have an estimated molecular weight (MW) ranging from 100 kDa to 193 kDa, and react positively to an antibody raised against the C. crescentus S-layer protein, RsaA. They can be divided in three size groups based on their MW: the small, medium and large S-layer groups. This thesis investigated whether all FWC strains, independent of the size of their S-layer, secreted the S-layer protein to the surface by a type I secretion system. The transporter genes composing the type I apparatus and the S-layer secretion signal were cloned, sequenced and compared for a subset of FWC strains, two from each S-layer size group. The protein cross-expression ability of the FWC strains and the arrangement of the S-layer secreting genes were also examined. The transporter genes and the regions encoding the C-termini of S-layer proteins were isolated by screening libraries or by PCR amplification of genomic DNA from a subset of FWC strains. The sequence data showed that all FWC strains studied contained adjacent genes encoding ABC transporters and membrane fusion proteins (MFP) that were highly similar to those of C. crescentus. Comparison of the available S-layer Ctermini showed low similarity for the small and medium size groups, with the presence of conserved residues and predicted secondary structure features, typical for type I secretion signals. Protein cross-expression studies showed that FWC strains.were capable of recognizing secretion signals from C. crescentus and other type I systems (Pseudomonas aeruginosa alkaline protease AprA), and that C. crescentus secreted the C-terminus of one FWC strain, FWC19. This suggested the presence of functional type I transporters in FWC strains, and that the S-layer subunits are secreted by a type I mechanism. The successful creation, in FWC strains from all size groups, of ABC transporter-inactivated mutants that no longer secrete an S-layer demonstrated that the identified type I transport system was the one that secreted the S-layer subunit. The arrangement of S-layer secretion genes was also analyzed. The ABC transporter and MFP genes were adjacent in all strains examined, the same as in C. crescentus. The S-layer gene was adjacent to the transporter genes in the small S-layer group, located further upstream in the medium S-layer group, and elsewhere on the genome in the large S-layer group. In all FWC strains studied, the OMP gene had the same disposition as in C. crescentus, further downstream of the OMP gene. Interestingly, the S-layer gene location is correlated with the sequence divergence of transporter proteins among size groups, dividing the S-layer secreting FWC strains into at least two groups. The small S-layer group has the same arrangement of S-layer secretion genes as C. crescentus, almost identical transporter genes and low similarity of secretion signals. The medium-large S-layer group has highly similar transporter genes and low similarity of secretion signals. The S-layer gene location further divides this group into subgroups: the medium S-layer group (S-layer gene located further upstream of the transporter genes) and the large S-layer group (S-layer gene located elsewhere in the genome). Overall, these results show that the FWC strains studied secrete their S-layer by a type I secretion system proprietary to the S-layer protein.

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