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Listeria monocytogenes and other pathogenic bacteria modify their peptidoglycan to protect it against enzymatic attack through the host innate immune system, such as the cell wall hydrolase lysozyme. During our studies on GpsB, a late cell division protein that controls activity of the bi-functional penicillin binding protein PBP A1, we discovered that GpsB influences lysozyme resistance of L. monocytogenes as mutant strains lacking gpsB showed an increased lysozyme resistance. Deletion of pbpA1 corrected this effect, demonstrating that PBP A1 is also involved in this. Susceptibility to lysozyme mainly depends on two peptidoglycan modifying enzymes: The peptidoglycan N-deacetylase PgdA and the peptidoglycan O-acetyltransferase OatA. Genetic and biochemical experiments consistently demonstrated that the increased lysozyme resistance of the ΔgpsB mutant was PgdA-dependent and OatA-independent. Protein-protein interaction studies supported the idea that GpsB, PBP A1 and PgdA form a complex in L. monocytogenes and identified the regions in PBP A1 and PgdA required for complex formation. These results establish a physiological connection between GpsB, PBP A1 and the peptidoglycan modifying enzyme PgdA. To our knowledge, this is the first reported link between a GpsB-like cell division protein and factors important for escape from the host immune system.

FliZ is an activator of class 2 flagellar gene expression in Salmonella enterica . To understand its role in flagellar assembly, we investigated how FliZ affects gene expression dynamics. We demonstrate that FliZ participates in a positive-feedback loop that induces a kinetic switch in class 2 gene expression.

Each Salmonella enterica serovar Typhimurium cell produces a discrete number of complete flagella. Flagellar assembly responds to changes in growth rates through FlhD(4) C(2) activity. FlhD(4) C(2) activity is negatively regulated by the type 3 secretion chaperone FliT. FliT is known to interact with the flagellar filament cap protein FliD as well as components of the flagellar type 3 secretion apparatus. FliD is proposed to act as an anti-regulator, in a manner similar to FlgM inhibition of σ(28) activity. We have found that efficient growth-dependent regulation of FlhD(4) C(2) requires FliT regulation. In turn, FliD regulation of FliT modulates the response. We also show that, unlike other flagellar-specific regulatory circuits, deletion of fliT or fliD did not lead to an all-or-nothing response in FlhD(4) C(2) activity. To investigate why, we characterized the biochemical interactions in the FliT : FliD : FlhD(4) C(2) circuit. When FlhD(4) C(2) was not bound to DNA, FliT disrupted the FlhD(4) C(2) complex. Interestingly, when FlhD(4) C(2) was bound to DNA it was insensitive to FliT regulation. This suggests that the FliT circuit regulates FlhD(4) C(2) activity by preventing the formation of the FlhD(4) C(2) :DNA complex. Our data would suggest that this level of endogenous regulation of FlhD(4) C(2) activity allows the flagellar system to efficiently respond to external signals.

Background: BA0330 and BA0331 are the only two lipoproteins among 11 known or putative polysaccharide deacetylases from Bacillus anthracis. Results: Both proteins lack deacetylase activity and are important for cell shape maintenance (BA0331) or high salt stress adaptation of the bacterium (BA0330). Conclusion: BA0330 and BA0331 stabilize the cell wall of B. anthracis. Significance: Understanding the mechanisms by which lipoproteins maintain cell wall integrity., Membrane-anchored lipoproteins have a broad range of functions and play key roles in several cellular processes in Gram-positive bacteria. BA0330 and BA0331 are the only lipoproteins among the 11 known or putative polysaccharide deacetylases of Bacillus anthracis. We found that both lipoproteins exhibit unique characteristics. BA0330 and BA0331 interact with peptidoglycan, and BA0330 is important for the adaptation of the bacterium to grow in the presence of a high concentration of salt, whereas BA0331 contributes to the maintenance of a uniform cell shape. They appear not to alter the peptidoglycan structure and do not contribute to lysozyme resistance. The high resolution x-ray structure of BA0330 revealed a C-terminal domain with the typical fold of a carbohydrate esterase 4 and an N-terminal domain unique for this family, composed of a two-layered (4 + 3) β-sandwich with structural similarity to fibronectin type 3 domains. Our data suggest that BA0330 and BA0331 have a structural role in stabilizing the cell wall of B. anthracis.

Bacterial flagella play key roles in surface attachment and host-bacterial interactions as well as driving motility. Here, we have investigated the ability of Caulobacter crescentus to assemble its flagellar filament from six flagellins: FljJ, FljK, FljL, FljM, FljN, and FljO. Flagellin gene deletion combinations exhibited a range of phenotypes from no motility or impaired motility to full motility. Characterization of the mutant collection showed the following: (i) that there is no strict requirement for any one of the six flagellins to assemble a filament; (ii) that there is a correlation between slower swimming speeds and shorter filament lengths in Δ fljK Δ fljM mutants; (iii) that the flagellins FljM to FljO are less stable than FljJ to FljL; and (iv) that the flagellins FljK, FljL, FljM, FljN, and FljO alone are able to assemble a filament.

Flagellar gene expression is temporally regulated in response to the assembly state of the growing flagellum. The key mechanism for enforcing this temporal hierarchy in Salmonella enterica serovar Typhimurium is the sigma(28)-FlgM checkpoint, which couples the expression of the late flagellar (P(class3)) genes to the completion of the hook-basal body. This checkpoint is triggered when FlgM is secreted from the cell. In addition to the sigma(28)-FlgM checkpoint, a number of other regulatory mechanisms respond to the secretion of late proteins. In this work, we examined how middle (P(class2)) and late (P(class3)) gene expression is affected by late protein secretion. Dynamic analysis of flagellar gene expression identified a novel mechanism where induction of P(class2) activity is delayed either when late protein secretion is abolished or when late protein secretion is increased. Using a number of different approaches, we were able to show that this mechanism did not involve any known flagellar regulator. Furthermore, the changes in P(class2) activity were not correlated with the associated changes in P(class3) activity, which was found to be proportional to late protein secretion rates. Our data indicate that both P(class2) and P(class3) promoters are continuously regulated in response to assembly and late protein secretion rates. These results suggest that flagellar regulation is more complex than previously thought.

The σ28 protein is a member of the bacterial σ70-family of transcription factors that directs RNA polymerase to flagellar late (class 3) promoters. The σ28 protein is regulated in response to flagellar assembly by the anti-σ28 factor FlgM. FlgM inhibits σ28-dependent transcription of genes whose products are needed late in assembly until the flagellar basal motor structure, the hook-basal body (HBB), is constructed. A second function for the σ28 transcription factor has been discovered: σ28 facilitates the secretion of FlgM through the HBB, acting as the FlgM Type III secretion chaperone. Transcription-specific mutants in σ28 were isolated that remained competent for FlgM-facilitated secretion separating the transcription and secretion-facilitation activities of σ 28. Conversely, we also describe the isolation of mutants in σ28 that are specific for FlgM-facilitated secretion. The data demonstrate that σ28 is the Type III secretion chaperone for its own anti-sigma factor FlgM. Thus, a novel role for a σ70-family transcription factor is described.

Signaling-protein mRNAs tend to have long untranslated regions (UTRs) containing binding sites for RNA-binding proteins regulating gene expression. Here we show that a PUF-family RNA-binding protein, Mpt5, represses the yeast MAP-kinase pathway controlling differentiation to the filamentous form. Mpt5 represses the protein levels of two pathway components, the Ste7 MAP-kinase kinase and the Tec1 transcriptional activator, and negatively regulates the kinase activity of the Kss1 MAP kinase. Moreover, Mpt5 specifically inhibits the output of the pathway in the absence of stimuli, and thereby prevents inappropriate cell differentiation. The results provide an example of what may be a genome-scale level of regulation at the interface of signaling networks and protein-RNA binding networks.