- Volume 146, Issue 6, 2000
Volume 146, Issue 6, 2000
- Genetics And Molecular Biology
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Characterization of transposon Tn1549, conferring VanB-type resistance in Enterococcus spp.
More LessThe GenBank accession number for the 33803 bp sequence of Tn1549 is AJ192329.
Transfer of VanB-type resistance to glycopeptides among enterococci has been reported to be associated with the movement of large chromosomal genetic elements or of plasmids. The authors report the characterization of the 34 kb transposon Tn1549 borne by a plasmid related to pAD1 and conferring vancomycin resistance in clinical isolates of Enterococcus spp. Tn1549 contained 30 ORFs and appeared to be organized like the Tn916 family of conjugative transposons into three functional regions: (i) the right end, implicated in the excision–integration process; (ii) the central part, in which the vanB2 operon replaces the tet(M) gene; and (iii) the left extremity, in which eight of the 18 ORFs could be implicated in the conjugative transfer.
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- Pathogenicity And Medical Microbiology
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Intimin from enteropathogenic Escherichia coli mediates remodelling of the eukaryotic cell surface
More LessAdhesion to cultured epithelial cells by enteropathogenic Escherichia coli (EPEC) is associated with extensive rearrangement of the host cell cytoskeleton. Evidence has been presented that EPEC adhesion is associated with activation of signal transduction pathways leading to production of a characteristic histopathological feature known as the attaching and effacing (A/E) lesion. A/E lesion formation requires intimin, an EPEC adhesion molecule and several EPEC secreted proteins (EspA, B, D and Tir) involved in cell signalling and protein translocation. In this study it is shown that HEp-2 cells respond during the early stages of infection with two wild-type EPEC strains (B171 and E2348/69) by producing microvillus-like processes (MLP) at the site of initial bacterial adherence. Intimin appears to play a key role in MLP elongation. At later stages of infection with these wild-type EPEC strains, when A/E lesions have formed, the MLP were reduced in number and length to appear as at time zero, and the cell surface in the vicinity of bacterial clusters appeared unaffected. In contrast, infection with EspA- or EspB-negative, but intimin-positive, EPEC strains (UMD872 and UMD864, respectively) resulted in enhanced MLP proliferation and formation of ‘cage-like’ structures engulfing the bacteria. Inoculating HEp-2 cells with intimin-coated latex spheres induced similar ‘cage-like’ structures. Caco-2 cells did not show intimin-induced microvillus elongation in response to EPEC infection, although microvillus effacement and reduction in number occurred. Similar phenomena appeared on B171 and E2348/69 infection of paediatric intestine using in vitro organ culture, i.e. elongated microvilli were seen in association with small colonies and at the periphery of large localized colonies, along with evidence of microvillus breakdown and debris in the colony centre. These results show that intimin activates signal transduction pathways involved in the remodelling of the eukaryotic cell surface, probably via binding to a receptor encoded by the host cell.
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Identification of novel loci involved in entry by Legionella pneumophila
More LessThe GenBank accession numbers for the enh1 and enh2 loci reported in this paper are AF057703 and AF057704, respectively.
Legionella pneumophila is primarily an intracellular pathogen during infection; thus, the mechanisms of entry into host cells are likely to be important for pathogenesis. Several L. pneumophila mutants that display an enhanced-entry (Enh) phenotype were isolated by selecting for bacteria that enter host cells at a higher frequency than wild-type. In the course of characterizing the genetic basis of one of these mutants, C3, a strategy was developed for the isolation of laboratory-media-repressed virulence determinants from L. pneumophila. Screens for dominant mutations using a genomic DNA library from C3 resulted in the isolation of three cosmids that confer an Enh phenotype to wild-type L. pneumophila. Transposon mutagenesis of these cosmids allowed identification of three loci that affect entry. Analysis of the putative proteins encoded by these loci, designated rtxA and enhC, demonstrated similarity to repeats in the structural toxin protein and the secreted Sel-1 protein from Caenorhabditis elegans, respectively. L. pneumophila rtxA and enhC mutants display significantly reduced entry into host cells, compared to wild-type bacteria. The phenotype that the cosmids containing these loci confer is most likely due to elevated expression resulting from their presence on multicopy vectors. The use of increased gene copy number to overexpress genes that are normally repressed under laboratory growth conditions is generally applicable to the isolation of virulence determinants from L. pneumophila and other bacterial pathogens.
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Streptococcus equi with truncated M-proteins isolated from outwardly healthy horses
More LessThe EMBL accession numbers of the M-protein gene partial sequences of S. equi isolates A1–A3 and B–E are AJ249868–AJ249874, respectively.
The M-protein genes of Streptococcus equi isolated from 17 outwardly healthy horses after 4 strangles outbreaks had ended, including a quarantined animal, were compared with those of S. equi isolates from 167 active cases of strangles across 4 countries. The healthy horses included 16 persistent S. equi carriers, at least one from each of the four outbreaks. These carriers, despite being outwardly healthy, had empyema of the guttural pouch(es), an enlargement of the equine Eustachian tube. A persistent carrier from two of these outbreaks, the quarantined animal and a healthy animal with normal guttural pouches, from which S. equi was isolated only once, were colonized by variant S. equi with truncated M-protein genes (24% of outwardly healthy animals with S. equi). The truncated M-protein genes had in-frame deletions in slightly different positions between the signal sequence and the central repeat region, equivalent to approximately 20% of the mature expressed protein. Immunoblotting with antibody to recombinant M-protein confirmed that the variants expressed a truncated form of the M-protein. In contrast to the outwardly healthy S. equi carriers, only 1/167 of S. equi isolates from strangles cases possessed a truncated M-protein gene (<1%; Fisher’s exact test, P=0·0002). Compared with isolates from healthy horses with a truncated M-protein, much more of the N terminus of the truncated M-protein was retained in the variant S. equi from a strangles case. Variant S. equi from outwardly healthy animals were more susceptible to phagocytosis by neutrophils in vitro than typical isolates. This is the first report of detection of S. equi with a truncated M-protein. The distribution of the variants between strangles cases and carriers suggests that the 80% of the M-protein retained in the variants may contribute to colonization whilst the deleted portion of the gene may be needed for full virulence.
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Tripartite haemolysin BL: isolation and characterization of two distinct homologous sets of components from a single Bacillus cereus isolate
More LessHaemolysin BL (HBL), a three-component enterotoxic/necrotizing/vascular permeability toxin, is a likely virulence factor of Bacillus cereus diarrhoeal food poisoning and necrotic infections. This paper describes the isolation of two distinct homologous sets of all three HBL components from a single B. cereus isolate, MGBC 145. The proteins of one set (designated HBL, consisting of B, L1 and L2), were about 87–100% identical in N-terminal amino acid sequences to their respective prototype components from strain F837/76, and the proteins of the homologous set (HBLa, consisting of Ba, L1a and L2a) were all about 62–65% identical. Only the latter homologues differed immunochemically and physicochemically from the prototypes. HBL and HBLa exhibited similar haemolytic and vascular permeability potencies, and the homologues could be interchanged freely. There were no notable differences in activity between the L component homologues. However, components B and Ba were significantly different. Both were secreted as monomers, but unlike B, Ba was isolated as a relatively inactive complex that could be reactivated with urea. When Ba was substituted for B in gel-diffusion assays the distinct discontinuous haemolysis pattern typical of the presence of B did not occur. In suspension assays, excess B inhibited the haemolysis of B-primed cells by L1 (as previously described), but not that of Ba-primed cells. Excess Ba had the opposite effect and enhanced lysis of Ba-primed cells, but not that of B-primed cells. These differences reveal details about how the toxin components interact on target cell membranes. The authors’ observations indicate that HBL represents a new family of multicomponent toxins that was generated by a process of gene and operon duplication that occurred either intracellularly or by horizontal transfer, and raise the possibility of the existence of other related toxins in the genetically diverse B. cereus taxonomic group.
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- Systematics And Evolution
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Comparison of conserved structural and regulatory domains within divergent 16S rRNA–23S rRNA spacer sequences of cyanobacteria
More LessThe GenBank accession numbers for the sequences reported in this paper are AF180968 and AF180969 for ITS-L and ITS-S, respectively.
PCR amplification of the internal transcribed spacer (ITS) between the 16S rRNA and 23S rRNA genes of the cyanobacterium Nostoc PCC 7120 gave three products. Two represented true ITS regions of different sizes, while the third was a heteroduplex. The longer spacer (ITS-L) contained 512 nucleotides and carried tRNAIle and tRNAAla genes, separated by a large stem–loop structure (V2) composed of short tandemly repeated repetitive sequences. Both tRNA genes, and the 5′ half of the intervening stem, were absent from the shorter spacer (ITS-S), of length 283 nucleotides, which was otherwise almost completely identical to ITS-L. The two spacer regions of Nostoc PCC 7120 were aligned to published ITS sequences of cyanobacteria, the cyanelle of Cyanophora paradoxa and Escherichia coli. Although the ITS regions of cyanobacteria vary in length from 283 to 545 nucleotides and contain either both tRNAIle and tRNAAla genes, only the tRNAIle gene, or neither, there is no correlation between ITS size and coding capacity for tRNAs. Putative secondary structures were determined for the deduced transcripts of the rrn operons of several cyanobacteria and were compared to that of E. coli. Highly conserved motifs important for folding and for maturation of the rRNA transcripts were identified, and regions homologous to bacterial antiterminators (box B–box A) were located. The conserved and variable regions of the cyanobacterial ITS are potential targets of PCR primers and oligonucleotide probes for detection and identification of cyanobacteria at different taxonomic levels.
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