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Virulent Yersinia species harbor a common plasmid that encodes essential virulence determinants (Yersinia outer proteins [Yops]), which are regulated by the extracellular stimuli Ca2+ and temperature. The V-antigen-encoding operon has been shown to be involved in the Ca(2+)-regulated negative pathway. The genetic organization of the V-antigen operon and the sequence of the lcrGVH genes were recently presented. The V-antigen operon was shown to be a polycistronic operon having the gene order lcrGVH-yopBD (T. Bergman, S. Håkansson, A. Forsberg, L. Norlander, A. Macellaro, A. Bäckman, I. Bölin, and H. Wolf-Watz, J. Bacteriol. 173:1607-1616, 1991; S. B. Price, K. Y. Leung, S. S. Barve, and S. C. Straley, J. Bacteriol. 171:5646-5653, 1989). We present here the sequence of the distal part of the V-antigen operons of Yersinia pseudotuberculosis and Yersinia enterocolitica. The sequence information encompasses the yopB and yopD genes and a downstream region in both species. We conclude that the V-antigen operon ends with the yopD gene. This conclusion is strengthened by the observation of an insertion-like element downstream of the yopD gene. The translational start codons of YopB and YopD have been identified by N-terminal amino acid sequencing. By computer analysis, the yopB and yopD gene products were found to be possible transmembrane proteins, and YopD was shown to contain an amphipathic alpha-helix in its carboxy terminus. These findings contrast with the general globular pattern observed for other Yops. Homology between Yersinia LcrH and Shigella flexneri IppI and between Yersinia YopB and S. flexneri IpaB was found, suggesting conservation of this locus between these two genera. YopB was also found to have a moderate level of homology, especially within the hydrophobic regions, to members of the RTX protein family of alpha-hemolysins and leukotoxins, indicating that YopB might exhibit a similar function.

Virulent Yersinia species possess a common plasmid that encodes essential virulence determinants (Yops) which are regulated by the extracellular stimuli Ca2+ and temperature. The V antigen operon was recently shown to be involved in the Ca2(+)-regulated negative pathway (A. Forsberg and H. Wolf-Watz, Mol. Microbiol. 2:121-133, 1988). We show here that the V antigen-containing operon of Yersinia pseudotuberculosis is a polycistronic operon having the gene order lcrGVH-yopBD. DNA sequencing analysis of lcrGVH revealed a high homology to the corresponding genes of Yersinia pestis. LcrG was conserved and LcrH showed only one amino acid difference, while LcrV showed only 96.6% identity. The amino acid substitutions of LcrV occurred in the central domain of the protein, while the two ends of the protein were conserved. Northern (RNA) blotting experiments showed that the operon is regulated at the transcriptional level by the extracellular stimuli temperature and calcium. One 4.6-kb transcriptional product of the operon was identified. This mRNA is rapidly processed at its 5' end, resulting in different mRNA species of variable stability. By genetic analysis, the lcrV and lcrH gene products were found to be regulatory proteins having important roles in the Ca2(+)-controlled regulation of Yop expression. The activity of LcrH is modulated by a gene product of the operon that inhibits the negative action of LcrH on yop transcription in the absence of Ca2+.

The three virulent members of the genus Yersinia harbour related virulence Plasmids with a molecular weight of about 60-70 kb (11). When exponential phase cultures of these organisms growing in a Ca2+ free medium are shifted from 26C to 37C, growth ceases over a period of about 2 generations (10). If however 2.5 mM Ca2+ is present in the medium growth continues normally. These bacteria are referred to as being Ca2+ dependent (CD). Plasmid cured strains, however, do not show this dependency on Ca2+ and are thus, Ca2+ independent (CI). Such bacteria are always avirulent. By transposon insertion mutagenesis a 20 kb region of the virulence plasmid has been identified which is involved in this low calcium response (1cr). Such CI-mutants do not require Ca2+ for prolonged growth at 37C and they are not virulent. Although the plasmids of Y. enterocolitica. Y. pestis and Y. pseudotuberculosis have been subjected to rearrengements, the Ca2+ region, however, of the different plasmids is conserved (11).

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

During infection of cultured epithelial cells, surface-located Yersinia pseudotuberculosis deliver Yop (Yersinia outer protein) virulence factors into the cytoplasm of the target cell. A non-polar yopB mutant strain displays a wild-type phenotype with respect to in vitro Yop regulation and secretion but fails to elicit a cytotoxic response in cultured HeLa cells and is unable to inhibit phagocytosis by macrophage-like J774 cells. Additionally, the yopB mutant strain was avirulent in the mouse model. No YopE or YopH protein were observed within HeLa cells infected with the yopB mutant strain, suggesting that the loss of virulence of the mutant strain was due to its inability to translocate Yop effector proteins through the target cell plasma membrane. Expression of YopB is necessary for Yersinia-induced lysis of sheep erythrocytes. Purified YopB was shown to have membrane disruptive activity in vitro. YopB-dependent haemolytic activity required cell contact between the bacteria and the erythrocytes and could be inhibited by high, but not low, molecular weight carbohydrates. Similarly, expression of YopE reduced haemolytic activity. Therefore, we propose that YopB is essential for the formation of a pore in the target cell membrane that is required for the cell-to-cell transfer of Yop effector proteins.