- Volume 152, Issue 11, 2006

Yersinia pestis biofilm formation causes massive adsorption of haemin or Congo red in vitro as well as colonization and eventual blockage of the flea proventriculus in vivo. This blockage allows effective transmission of plague from some fleas, like the oriental rat flea, to mammals. Four Hms proteins, HmsH, HmsF, HmsR and HmsS, are essential for biofilm formation, with HmsT and HmsP acting as positive and negative regulators, respectively. HmsH has a β-barrel structure with a large periplasmic domain while HmsF possesses polysaccharide deacetylase and COG1649 domains. HmsR is a putative glycosyltransferase while HmsS has no recognized domains. In this study, specific amino acids within conserved domains or within regions of high similarity in HmsH, HmsF, HmsR and HmsS proteins were selected for site-directed mutagenesis. Some but not all of the substitutions in HmsS and within the periplasmic domain of HmsH were critical for protein function. Substitutions within the glycosyltransferase domain of HmsR and the deacetylase domain of HmsF abolished biofilm formation in Y. pestis. Surprisingly, substitution of highly conserved residues within COG1649 did not affect HmsF function.

Orthologues of the Salmonella enterica serovar Typhimurium (S. typhimurium) BarA/SirA two-component system are important for biofilm formation and virulence in many γ-Proteobacteria. In S. typhimurium, SirA activates the csrB and csrC carbon storage regulatory RNAs and the virulence gene regulators hilA and hilC. The regulatory RNAs antagonize the activity of the CsrA protein, allowing translation of those same virulence genes, and inhibiting the translation of flagellar genes. In this report, it was determined that SirA and the Csr system also control the fim operon that encodes type 1 fimbriae. sirA orthologues in other bacterial species, and the fim operon of S. typhimurium, are known to play a role in biofilm formation; therefore, all members of the S. typhimurium sirA regulon were tested for in vitro biofilm production. A sirA mutant, a csrB csrC double mutant, and a fimI mutant, were all defective in biofilm formation. Conversely, inactivation of flhDC increased biofilm formation. Therefore, SirA activates csrB, csrC and the fim operon to promote biofilm formation. In turn, csrB and csrC promote the translation of the fim operon, while at the same time inhibiting the translation of flagella, which are inhibitory to biofilm formation.

This paper is the first detailed description of the development and use of new genetic tools specifically for the safe manipulation of highly pathogenic Francisella tularensis subsp. tularensis. Most of these tools are also demonstrated to work with other F. tularensis subspecies. Kanamycin and hygromycin resistance determinants that function as genetic markers in F. tularensis subsp. tularensis strain Schu and sets of episomal shuttle vectors that are either unstable or stably maintained in the absence of selection were developed. In addition, the hyg gene, expressed from the F. tularensis groESL promoter, was successfully used as a marker for transposon mutagenesis. This work also includes the development of sacB-based suicide plasmids expressing kanamycin resistance that can be used for electroporation-mediated allelic exchange of unmarked mutations in Schu and the F. tularensis live vaccine strain (LVS). Using these plasmids, the two predicted β-lactamase genes, blaA and blaB, in Schu and LVS were deleted. Only the ΔblaB1 mutants had increased susceptibility to ampicillin, and this phenotype was complemented by a plasmid expressing blaB +. The results suggest that the β-lactam antibiotic resistance phenotype of Schu and LVS is likely due to only one of the two β-lactamase genes present and that ampicillin resistance can be used as an additional selectable marker in β-lactamase deletion mutants. The collection of tools presented in this report will be helpful for the genetic analyses of F. tularensis subsp. tularensis pathogenesis.

Heterologous expression of bacterial virulence factors in Saccharomyces cerevisiae is a feasible approach to study their molecular function. The authors have previously reported that the Salmonella typhimurium SigD protein, a phosphatidylinositol phosphatase involved in invasion of the host cell, inhibits yeast growth, presumably by depleting an essential pool of phosphatidylinositol 4,5-bisphosphate, and also that a catalytically inactive version, SigDR468A, was able to arrest growth by a different mechanism that involved disruption of the actin cytoskeleton. This paper describes marked differences between the phenotypes elicited by expression of SigD and SigDR468A in yeast. First, expression of SigDR468A caused accumulation of large unbudded cells and loss of septin organization, while SigD expression caused none of these effects. Second, growth inhibition by SigDR468A was mediated by a cell cycle arrest in G2 dependent on the Swe1 morphogenetic checkpoint, but SigD-induced growth inhibition was cell cycle independent. And third, SigD caused strong activation of the yeast MAP kinase Slt2, whereas SigDR468A rather inactivated another MAP kinase, Kss1. In a screen for suppressors of SigDR468A-induced growth arrest by overexpression of a yeast cDNA library, the Cdc42 GTPase was isolated. Furthermore, SigDR468A was co-purified with Cdc42 from yeast lysates. It is concluded that the Salmonella SigD protein deprived of its phosphatase activity is able to disrupt yeast morphogenesis by interfering with Cdc42 function, opening the possibility that the SigD N-terminal region might directly modulate small GTPases from the host during infection.