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A polymerase chain reaction (PCR)-based procedure without any cloning step was developed for a rapid mutagenesis/deletion of chromosomal target genes in Yersinia. For this purpose, a PCR fragment carrying an antibiotic resistance gene flanked by regions homologous to the target locus is electroporated into a recipient strain expressing the highly proficient homologous recombination system encoded by plasmid pKOBEG-sacB. Two PCR procedures were tested to generate an amplification product formed of an antibiotic resistance gene flanked by short (55 bp) or long (500 bp) homology extensions. Using this method, three chromosomal loci were successfully disrupted in Yersinia pseudotuberculosis. The use of this technique allows rapid and efficient large-scale mutagenesis of Yersinia target chromosomal genes.

The bacA gene product of Escherichia coli was recently purified to near homogeneity and identified as an undecaprenyl pyrophosphate phosphatase activity (El Ghachi, M., Bouhss, A., Blanot, D., and Mengin-Lecreulx, D. (2004) J. Biol. Chem. 279, 30106 -30113). The enzyme function is to synthesize the carrier lipid undecaprenyl phosphate that is essential for the biosynthesis of peptidoglycan and other cell wall components. The inactivation of the chromosomal bacA gene was not lethal but led to a significant, but not total, depletion of undecaprenyl pyrophosphate phosphatase activity in E. coli membranes, suggesting that other(s) protein(s) should exist and account for the residual activity and viability of the mutant strain. Here we report that inactivation of two additional genes, ybjG and pgpB, is required to abolish growth of the bacA mutant strain. Overexpression of either of these genes, or of a fourth identified one, yeiU, is shown to result in bacitracin resistance and increased levels of undecaprenyl pyrophosphate phosphatase activity, as previously observed for bacA. A thermosensitive conditional triple mutant ⌬bacA,⌬ybjG,⌬pgpB in which the expression of bacA is impaired at 42°C was constructed. This strain was shown to accumulate soluble peptidoglycan nucleotide precursors and to lyse when grown at the restrictive temperature, due to the depletion of the pool of undecaprenyl phosphate and consequent arrest of cell wall synthesis. This work provides evidence that two different classes of proteins exhibit undecaprenyl pyrophosphate phosphatase activity in E. coli and probably other bacterial species; they are the BacA enzyme and several members from a superfamily of phosphatases that, different from BacA, share in common a characteristic phosphatase sequence motif.An essential carrier lipid, undecaprenyl phosphate (C 55 -P), 1 is required for the synthesis of various bacterial cell wall polymers such as peptidoglycan, lipopolysaccharides, and teichoic acids (1-5) (Scheme 1). It is synthesized as a pyrophosphate precursor (C 55 -PP) by the addition of eight isoprene units to farnesyl pyrophosphate, a reaction catalyzed by the well characterized cis-prenyl-pyrophosphate synthase UppS (6 -10). However, genes and enzymes involved in subsequent steps of C 55 -P synthesis and recycling still remained to be identified. Bacitracin is a dodecapeptide antibiotic known to specifically block this metabolism by forming a specific complex with C 55 -PP. As a result, cell wall biosynthesis is inhibited and cell lysis finally occurs (11)(12)(13)(14). Bacillus licheniformis strains that produce bacitracin are resistant to this antibiotic due to the presence of an appropriate ABC transporter efflux system (15, 16). Several mutations leading to bacitracin resistance were identified in Escherichia coli and other Gram-negative bacteria. Interestingly, all these mutations were shown to block the synthesis of non-essential cell envelope polymers such as osmoregulated periplasmic glycans and capsule polysaccharides that also requ...

Streptococcal surface enolase (SEN) is a major plasminogen-binding protein of group A streptococci. Our earlier biochemical studies have suggested that the region responsible for this property is likely located at the C-terminal end of the SEN molecule. In the present study, the gene encoding SEN was cloned from group A streptococci M6 isolate D471. A series of mutations in the sen gene corresponding to the C-terminal region ( 428 KSFYNLKK 435 ) of the SEN molecule were created by either deleting one or more terminal lysine residues or replacing them with leucine. All purified recombinant SEN proteins with altered C-terminal ends were found to be enzymatically active and were analyzed for their Glu-and Lys-plasminogen-binding activities. Wild-type SEN bound to Lys-plasminogen with almost three times more affinity than to Glu-plasminogen. However, the recombinant mutant SEN proteins with a deletion of Lys434-435 or with K435L and K434-435L replacements showed a significant decrease in Glu-and Lys-plasminogen-binding activities. Accordingly, a streptococcal mutant expressing SEN-K434-435L showed a significant decrease in Glu-and Lys-plasminogen-binding activities. Biochemical and functional analyses of the isogenic mutant strain revealed a significant decrease in its abilities to cleave a chromogenic tripeptide substrate, acquire plasminogen from human plasma, and penetrate the extracellular matrix. Together, these data indicate that the last two C-terminal lysine residues of surfaceexposed SEN contribute significantly to the plasminogen-binding activity of intact group A streptococci and hence to their ability to exploit host properties to their own advantage in tissue invasion.

SummaryYersinia pestis, the plague bacillus, has an exceptional pathogenicity but the factors responsible for its extreme virulence are still unknown. A genome comparison with its less virulent ancestor Yersinia pseudotuberculosis identified a few Y. pestis-specific regions acquired after their divergence. One of them potentially encodes a prophage (YpfF), similar to filamentous phages associated with virulence in other pathogens. We show here that YpfF forms filamentous phage particles infectious for other Y. pestis isolates. Although it was previously suggested that YpfF is restricted to the Orientalis branch, our results indicate that it was acquired by the Y. pestis ancestor. In Antiqua and Medievalis strains, YpfF genome forms an unstable episome whereas in Orientalis isolates it is stably integrated as tandem repeats. Deletion of the YpfF genome does not affect Y. pestis ability to colonize and block the flea proventriculus, but results in an alteration of Y. pestis pathogenicity in mice. Our results show that transformation of Y. pestis from a classical enteropathogen to the highly virulent plague bacillus was accompanied by the acquisition of an unstable filamentous phage. Continued maintenance of YpfF despite its high in vitro instability suggests that it confers selective advantages to Y. pestis under natural conditions.

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BackgroundPlague is still a public health problem in the world and is re-emerging, but no efficient vaccine is available. We previously reported that oral inoculation of a live attenuated Yersinia pseudotuberculosis, the recent ancestor of Yersinia pestis, provided protection against bubonic plague. However, the strain poorly protected against pneumonic plague, the most deadly and contagious form of the disease, and was not genetically defined.Methodology and Principal FindingsThe sequenced Y. pseudotuberculosis IP32953 has been irreversibly attenuated by deletion of genes encoding three essential virulence factors. An encapsulated Y. pseudotuberculosis was generated by cloning the Y. pestis F1-encoding caf operon and expressing it in the attenuated strain. The new V674pF1 strain produced the F1 capsule in vitro and in vivo. Oral inoculation of V674pF1 allowed the colonization of the gut without lesions to Peyer's patches and the spleen. Vaccination induced both humoral and cellular components of immunity, at the systemic (IgG and Th1 cells) and the mucosal levels (IgA and Th17 cells). A single oral dose conferred 100% protection against a lethal pneumonic plague challenge (33×LD50 of the fully virulent Y. pestis CO92 strain) and 94% against a high challenge dose (3,300×LD50). Both F1 and other Yersinia antigens were recognized and V674pF1 efficiently protected against a F1-negative Y. pestis.Conclusions and SignificanceThe encapsulated Y. pseudotuberculosis V674pF1 is an efficient live oral vaccine against pneumonic plague, and could be developed for mass vaccination in tropical endemic areas to control pneumonic plague transmission and mortality.