The human enteropathogen Yersinia enterocolitica survives and replicates in the lymphoid tissues of its host. Previous in vivo analyses of gene expression revealed that various chromosomal genes are expressed at this stage of infection, but not in vitro. One of these, termed hreP, encodes a protease that is necessary for full virulence of Y. enterocolitica. Using transposon mutagenesis, we identified three genes, pypA, pypB, and pypC, as positive regulators of hreP transcription. PypA is an inner membrane protein with no significant similarity to any known proteins; PypB is a ToxR-like transmembrane transcriptional regulator; and PypC is a cytoplasmic transcriptional regulator with an OmpR-like winged helix-turn-helix DNA binding motif. We show that all Pyp proteins are able to activate hreP independently of each other and that PypB and PypC interact directly with the hreP promoter region. Furthermore, pypB and pypC are autoregulated and regulate each other. Additional data indicate that transcription of hreP is repressed by the histone-like nucleoid-structuring protein H-NS in a temperature-dependent manner. Our data reveal a new regulatory network that might have implications for the controlled expression of further virulence-associated functions in Yersinia.

The DNA adenine methyltransferase (Dam) of gammaproteobacteria catalyzes the methylation of adenine residues at the N 6 position in GATC sequences. Methylation occurs directly after DNA replication with a delay, thereby leaving the newly synthesized daughter strand nonmethylated for a short period of time. Depending on their presence and their affinity, methylation-sensitive regulatory proteins either bind to the hemimethylated DNA during this time and prevent the subsequent methylation of GATC sequences or bind preferentially to fully methylated DNA. Therefore, the methylation status of GATC sequences has an impact on the binding of regulatory proteins and consequently is involved in the regulation of several basic processes of the bacterial cell, such as mismatch repair, chromosome replication, transposition, or transcription of genes (10,56 (11,20,23,25,26,30,34,42,46,48,55 (4,5,20,41).The food-borne human pathogen Yersinia enterocolitica is able to cause different gastrointestinal syndromes, ranging from self-limiting enteritis to mesenteric lymphadenitis. In rare cases, Y. enterocolitica is able to disseminate to deeper tissues and cause systemic infections (9, 16). We used a Dam OP strain as a tool to identify and analyze methylation-sensitive processes implicated in virulence of this pathogen. By using Dam OP strains as well as dam mutant strains, it is possible to alter methylation patterns in regulatory regions of genes, whereby the affinity of transcription factors for these regions can be altered, thereby mimicking a situation that can also be found in vivo (26,56). In previous studies, we could demonstrate that overproduction of the Dam enzyme in Y. enterocolitica leads to a relaxed Ca 2ϩ regulation of the Yop/Ysc type III secretion system. This effect depends at least in part on a ClpP-mediated degradation of the regulatory protein LcrG (21). Furthermore, we found that the expression of several in vivo-expressed genes is not changed in Dam OP strains. However, a Dam OP strain cured of the pYV virulence plasmid

Type IV pili are virulence factors in various bacteria and mediate, among other functions, the colonization of diverse surfaces. Various subclasses of type IV pili have been identified, but information on pilus expression, biogenesis, and the associated phenotypes is sparse for the genus Yersinia. We recently described the identification of PypB as a transcriptional regulator in Yersinia enterocolitica. Here we show that the pypB gene is associated with the tad locus, a genomic island that is widespread among bacterial and archaeal species. The genetic linkage of pypB with the tad locus is conserved throughout the yersiniae but is not found among other bacteria carrying the tad locus. We show that the genes of the tad locus form an operon in Y. enterocolitica that is controlled by PypB and that pypB is part of this operon. The tad genes encode functions necessary for the biogenesis of the Flp subfamily of type IVb pili initially described for Aggregatibacter actinomycetemcomitans to mediate a tight-adherence phenotype. In Y. enterocolitica, the Flp pilin protein shows some peculiarities in its amino acid sequence that imply similarities as well as differences compared to typical motifs found in the Flp subtype of type IVb pili. Flp is expressed and processed after PypB overproduction, resulting in microcolony formation but not in increased adherence to biotic or abiotic surfaces. Our data describe the transcriptional regulation of the tad type IVb pilus operon by PypB in Y. enterocolitica but fail to show most previously described phenotypes associated with this type of pilus in other bacteria.

Horizontal gene transfer plays an important role in bacterial evolution. DNA acquired by horizontal gene transfer has to be incorporated into existing regulatory networks. The histone-like nucleoid structuring protein H-NS acts as a silencer of horizontally acquired genes to avoid potential damage. However, specific regulators can overcome H-NS repression, resulting in the integration of newly acquired genes into existing regulatory networks. Here, we analyzed the influence of H-NS on the transcription of the Yersinia enterocolitica hreP gene and its regulators pypA, pypB, and pypC by establishing a dominant-negative H-NS version. Using transcriptional fusions and electrophoretic mobility shift assays, we show that H-NS silences hreP, pypA, pypB, and pypC by direct interactions. While the H-NS antagonist RovA activates pypC, it has no effect on pypA and pypB. Furthermore, H-NS affects biofilm formation in Y. enterocolitica.