No abstract

The goal of this study was to characterize the Yersinia pestis KIM OmpX protein. Yersinia spp. provide a model for studying several virulence processes including attachment to, and internalization by, host cells. For Yersinia enterocolitica and Yersinia pseudotuberculosis, Ail, YadA and Inv, have been implicated in these processes. In Y. pestis, YadA and Inv are inactivated. Genomic analysis of two Y. pestis strains revealed four loci with sequence homology to Ail. One of these genes, designated y1324 in the Y. pestis KIM database, encodes a protein designated OmpX. The mature protein has a predicted molecular mass of 17.47 kDa, shares approximately 70 % sequence identity with Y. enterocolitica Ail, and has an identical homologue, designated Ail, in the Y. pestis CO92 database. The present study compared the Y. pestis KIM6 + parental strain with a mutant derivative having an engineered disruption of the OmpX structural gene. The parental strain (and a merodiploid control strain) expressed OmpX at 28 and 37 6C, and the protein was detectable throughout all phases of growth. OmpX was required for efficient adherence to, and internalization by, cultured HEp-2 cell monolayers and conferred resistance to the bactericidal effect of human serum. Deletion of ompX resulted in a significantly reduced autoaggregation phenotype and loss of pellicle formation in vitro. These results suggest that Y. pestis OmpX shares functional homology with Y. enterocolitica Ail in adherence, internalization into epithelial cells and serum resistance.

Caulobacter crescentus contains a 25-and a 27-kDa flagellin, which are assembled into the flagellar filament, and a 29-kDa flagellin, which is related in sequence but is of unknown function. We have used DNA sequence analysis and nuclease S1 assays to map the in vivo transcription start sites of the three flagellin genes and to study their regulation. These experiments lead to several conclusions. First, copies of the 29-, 25-, and 27-kDa flagellin genes are organized in a tandem array in the flaEY gene cluster of C. crescentus. Second, flagellin genes are under transcriptional control and each gene is expressed with a characteristic periodicity in the cell cycle. Third, flagellin gene promoters contain conserved nucleotide sequence elements at -13, -24, and -100 that are homologous to thefla genes in the hook gene cluster. The -13 and -24 sequences conform to afla gene promoter consensus The well-defined nature of asymmetric cell division in the aquatic bacterium Caulobacter crescentus has provided an attractive model for the study ofmechanisms that regulate the temporal and spatial patterns of development. Unlike Bacillus and Myxococcus, in which differentiation is largely controlled by environmental conditions, morphogenesis in C. crescentus is driven by an internal clock that appears, from genetic results, to be linked to steps in DNA synthetic or cell division pathways (1-3). Flagellum biosynthesis has been of special interest in the study of cell-cycle timing because formation of this structure is stage-specific in the cell cycle, and the major flagellar proteins are synthesized periodically at the time of flagellum assembly (1, 2, 4). These proteins include the 70-kDa hook protein, the 27-kDa flagellin, which is assembled proximal to the hook, and the 25-kDa flagellin, which is assembled at the distal end of the filament. These two flagellins and a 29-kDa flagellin of undetermined function have extensive sequence homology (5-7).Most C. crescentus flagellar (fla) genes are organized into three genetically unlinked clusters. The best characterized of these are the hook gene cluster (8,9), which contains transcription units I-V, and theflaEY cluster (10, 11), which contains the 29-(10), the 25-(12), and as shown in this communication, the 27-kDa flagellin genes. Transcription units I, II, and III of the hook gene cluster (refs. 13 and 14; D. Mullin, L.-S. Chen, and A.N., unpublished work) and members of the flagellin gene family are transcriptionally regulated (15, 16). We also know that de novo RNA synthesis is required for the periodic synthesis of the hook protein and the flagellins (2), and it seems likely from these results thatfla genes are differentially transcribed in the C. crescentus cell cycle and that promoter recognition plays a role in timing their expression (13,14). To date, however, only the promoters of transcription units II and II

Yersinia pestis, the causative agent of plague, is one of the most virulent microorganisms known. The outer membrane protein X (OmpX) in Y. pestis KIM is required for efficient bacterial adherence to and internalization by cultured HEp-2 cells and confers resistance to human serum. Here, we tested the contribution of OmpX to disease progression in the fully virulent Y. pestis CO92 strain by engineering a deletion mutant and comparing its ability in mediating pneumonic plague to that of the wild type in two animal models. The deletion of OmpX delayed the time to death up to 48 h in a mouse model and completely attenuated virulence in a rat model of disease. All rats challenged with 1 ؋ 10 8 CFU of the ompX mutant survived, compared to the 50% lethal dose (LD 50 ) of 1.2 ؋ 10 3 CFU for the wild-type strain. Because murine serum is not bactericidal for the ompX mutant, the mechanism underlying the delay in time to death in mice was attributed to loss of adhesion/internalization properties but not serum resistance. The rat model, which is most similar to humans, highlighted the critical role of serum resistance in disease. To resolve conflicting evidence for the role of Y. pestis lipopolysaccharide (LPS) and OmpX in serum resistance, ompX was cloned into Escherichia coli D21 and three isogenic derivatives engineered to have progressively truncated LPS core saccharides. OmpX-mediated serum resistance, adhesiveness, and invasiveness, although dependent on LPS core length, displayed these functions in E. coli, independently of other Yersinia proteins and/or LPS. Also, autoaggregation was required for efficient OmpX-mediated adhesiveness and internalization but not serum resistance.

Yersinia enterocolitica is a facultative intracellular parasite, displaying the ability to grow saprophytically or invade and persist intracellularly in the mammalian reticuloendothelial system. The transition between such diverse environments requires the co-ordinated regulation of specific sets of genes on both the chromosome and virulence plasmid. Temperature has a profound pleiotropic effect on gene expression and phenotypically promotes alterations in cell morphology, outer-membrane protein synthesis, urease production, lipopolysaccharide synthesis, motility, and synthesis of genes involved in invasion of eukaryotic host cells. By examining thermoregulated flagella biosynthesis, we have determined that motility is repressed at 25 degrees C (permissive temperature) with subinhibitory concentrations of novobiocin. These conditions also induce virulence gene expression suggesting novobiocin addition simulates, at least partially, a high-temperature environment. Furthermore, temperature-shift experiments, using Y. enterocolitica containing pACYC184 as a reporter plasmid, indicate that thermo-induced alterations of DNA supercoiling coincide with temperature-induced phenotypic changes. A class of putative DNA gyrase mutant (novobiocin resistant) likewise demonstrates the 37 degrees C phenotype when cultured at 25 degrees C; it is non-motile, urease negative, calcium growth dependent, and positive for Yop expression. These results support a model implicating DNA topology as a contributing factor of Y. enterocolitica thermoregulation.