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The YscC secretin is a major component of the type III protein secretion system of Yersinia enterocolitica and forms an oligomeric structure in the outer membrane. In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin. To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in trans in a Y. enterocolitica strain lacking all other components of the secretion machinery. YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein. Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW. Pulse-chase experiments revealed that ~30 min were required before YscC oligomerization was completed. In the absence of YscW, oligomerization was delayed and the yield of YscC oligomers was strongly reduced. An unlipidated form of the YscW protein was not functional, although it still interacted with the secretin and caused mislocalization of YscC even in the presence of wild-type YscW. Hence, YscW interacts with the unassembled YscC protein and facilitates efficient oligomerization, likely at the outer membrane.

The influence of the master regulatory operon of the flagellum, flhDC, on the yop regulon in Yersinia enterocolitica is evaluated. Data show that in a mutant, the yop regulon is up-regulated and temperature independent, suggesting that in wild-type bacteria, flhDC represses the yop regulon.

Research reveals that Yersinia cells adhering to animal cells deliver a cocktail of effector Yops molecules through a type III secretion-translocation system. Data indicate that the binding domain residues of SycE mediate transfer into the target cells.

A complete deletion of lcrV in the pYV plasmid of Yersinia enterocolitica E40 was genetically engineered and the phenotype of the mutant was characterized to elucidate the participation of LcrV in the Yop virulon. Results of the complementation studies provided evidence that the mutation was not polar with respect to yopB and yopD. However, the mutation eliminated the secretion of YopB and YopD, whereas the secretion of the other Yops was not influenced and even showed an increase.

The nucleotide sequence of the fourth quadrant of the pYV 70-kb virulence plasmid of Yersinia enterolitica was analyzed to determine the biochemical function of the region. Sequence analysis of the fourth quadrant of low virulence Yersinia eneterolitica pYV plasmid indicated the presence of a defective transposon that induced microbial resistance to arsenite and arsenate. Furthermore, the pVY plasmid contained ars genes that produced an arsenite-inducible transcriptional repressor and a transmembrane channel.

A novel Yersinia enterocolitica gene, virG, most likely encodes a lipoprotein and is involved in virulence protein synthesis or secretion. The protein, VirG, has strong similarities to Pseudomonas aeruginosa's ExsB protein and most likely serves the same function. The virG mutant has interesting characteristics because it does not require Ca2+ for growth and yet still secretes Yops, a protein produced when Ca2+ is absent in the wildtype.

The fliA gene encoding the sigma28 homolog in Yersinia enterocolitica was cloned, sequenced, and mutagenized to show that fliA was needed for motility in mutagenesis but took no part in fibrilla synthesis and Yop secretion. Motility for Y. enterocolitica pathogenesis was not needed in the mouse model. Y. enterocolitica influences the formation of gastrointestinal syndromes.

Temperature and pH modulate the expression of the Yersinia enterocolitica surface antigen Myf at the transcriptional level. The myfA gene that codes for the antigen's 21-kDa major subunit is transcribed in an acidic medium at 37 degrees Celsius and requires the myfF and myfE genes that are upstream of myfA. The 18.5-kDa myfF product has a distinct hydrophobic region in the NH2-terminal end but does not exhibit any characteristic helix-turn-helix pattern. Osmotic shock, T7 expression and fractionation experiments reveals that MyfF bends to the inner membrane through its hydrophobic domain while the hydrophilic portion projects in the periplasm.

Experimental analyses of Yersinia enterocolitica reveal that the last virB locus gene encodes an inner membrane protein, yscU, which is similar to the Spa40 protein of Salmonella flexneri and SpaS of S. typhimurium, and mediates secretion of antihost Yop proteins. Structural analysis shows that the YscU product is of 40.3-kDa and consists of four transmembrane fragments that link the cytoplasmic carboxyl terminal domain to the inner membrane. Mutations in YsuC result in the inhibition of Yop secretion.

VirF belongs to the AraC family of transcriptional regulators and binds to multiple sites in the promoter regions of yopH, yopE, virC and lerG in Yersinia. There are strong and weak VirF-binding sites within the yop promoters and the affinity for binding sites of VirF is thermo-induced. Like AraC, the transcriptional control of VirF-regulated genes requires the binding of VirF to several sites.

The first gene of the virB locus is yscN, which is a part of the three type III secretion pathway, along with yop secretion. There are two nucleotide binding motifs, boxes A and B in the yscN. A modified form of the yscN protein without box A, namely pYV mutant, is synthesized. The yscN is a yop secretion component and is a part of the secretion machinery or the energies.

The Yersinia outer membrane protein (YOP) and the type III virulence system of the pathogenic bacteria were described. This system involved the transfer of bacterial proteins by the extracellular bacteria to the cytosol of the eukaryotic cell. The three species of Yersinia considered as pathogens in mammals are Y. pestis, Y. pseudotuberculosis and Y. enterocolitica. Genetic analysis revealed that majority of Yop proteins were necessary for virulence and cytotoxicity activity. The Yops were classified as secreted proteins. The pYV plasmid and the ysc genes encoded the Yop secretion apparatus.

A method for monitoring genetically engineered bacteria in the murine and bovine digestive tract was developed. Theprocedure involved the use of luxAB genes, which encodes bacterial luciferase, integrated in the chromosome of Yersinia enterocolitica. The survival of the recombinant bacteria in the digestive tracts of mice and cows was monitored. The results showed that the method is a rapid and reliable system for monitoring genetically engineered bacteria, were easily discriminated from indigenous bacterial populations.

The tools required for manipulating Capnocytophaga spp. are described. The genetic tools have paved the way not only for molecular studies of the pathogenesis of Capnocytophaga canimorsus, but also for studying other oral Capnocytophaga species.

The Yersinia Yop virulon, the common weapon of Y pseudotuberculosis, Y enterocolitica and Y pestis, enables the three pathogens to protect themselves from nonspecific immune response. An investigation of basic aspects such as control, secretion and translocation emphasizes its bacteriology rather than its eukaryotic cell biology. Pathological observations of infected animals suggest that yersiniae are largely extracellular.

Capnocytophaga canimorsus, a commensal bacterium of dog's mouth flora causing severe infections in humans after dog bites or scratches, has a lipopolysaccharide (LPS) (endotoxin) with low-inflammatory lipid A. In particular, it contains a phosphoethanolamine (P-Etn) instead of a free phosphate group at the C-1 position of the lipid A backbone, usually present in highly toxic enterobacterial Gram-negative lipid A. Here we show that the C. canimorsus genome comprises a single operon encoding a lipid A 1-phosphatase (LpxE) and a lipid A 1 P-Etn transferase (EptA). This suggests that lipid A is modified during biosynthesis after completing acylation of the backbone by removal of the 1-phosphate and subsequent addition of an P-Etn group. As endotoxicity of lipid A is known to depend largely on the degree of unsubstituted or unmodified phosphate residues, deletion of lpxE or eptA led to mutants lacking the P-Etn group, with consequently increased endotoxicity and decreased resistance to cationic antimicrobial peptides (CAMP). Consistent with the proposed sequential biosynthetic mechanism, the endotoxicity and CAMP resistance of a double deletion mutant of lpxE-eptA was similar to that of a single lpxE mutant. Finally, the proposed enzymatic activities of LpxE and EptA based on sequence similarity could be successfully validated by mass spectrometry (MS)-based analysis of lipid A isolated from the corresponding deletion mutant strains., (Copyright © 2016 Renzi et al.)