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Neisseria meningitidis pili are filamentous protein structures that are essential adhesins in capsulate bacteria. Pili of adhesion variants of meningococcal strain C311 contain glycosyl residues on pilin (PilE), their major structural subunit. Despite the presence of three potential N-linked glycosylation sites, none appears to be occupied in these pilins. Instead, a novel O-linked trisaccharide substituent, not previously found as a constituent of glycoproteins, is present within a peptide spanning amino acid residues 45 to 73 of the PilE molecule. This structure contains a terminal 1-4-linked digalactose moiety covalently linked to a 2,4-diacetamido-2,4,6-trideoxyhexose sugar which is directly attached to pilin. Pilins derived from galactose epimerase (galE) mutants lack the digalactosyl moiety, but retain the diacetamidotrideoxyhexose substitution. Both parental (#3) pilins and those derived from a hyper-adherent variant (#16) contained identical sugar substitutions in this region of pilin, and galE mutants of #3 were similar to the parental phenotype in their adherence to host cells. These studies have confirmed our previous observations that meningococcal pili are glycosylated and provided the first structural evidence for the presence of covalently linked carbohydrate on pili. In addition, they have revealed a completely novel protein/saccharide linkage.

The presence of methanogenic bacteria was assessed in peat and soil cores taken from upland moors. The sampling area was largely covered by blanket bog peat together with small areas of red-brown limestone and peaty gley. A 30-cm-deep core of each soil type was taken, and DNA was extracted from 5-cm transverse sections. Purified DNA was subjected to PCR amplification with primers 1Af and 1100Ar, which specifically amplify 1.1 kb of the archaeal 16S rRNA gene, and ME1 and ME2, which were designed to amplify a 0.75-kb region of the ␣-subunit gene for methyl coenzyme M reductase (MCR). Amplification with both primer pairs was obtained only with DNA extracted from the two deepest sections of the blanket bog peat core. This is consistent with the notion that anaerobiosis is required for activity and survival of the methanogen population. PCR products from both amplifications were cloned, and the resulting transformants were screened with specific oligonucleotide probes internal to the MCR or archaeal 16S rRNA PCR product. Plasmid DNA was extracted from probe-positive clones of both types and the insert was sequenced. The DNA sequences of 8 MCR clones were identical, as were those of 16 of the 17 16S rRNA clones. One clone showed marked variation from the remainder in specific regions of the sequence. From a comparison of these two different 16S rRNA sequences, an oligonucleotide was synthesized that was 100% homologous to a sequence region of the first 16 clones but had six mismatches with the variant. This probe was used to screen primary populations of PCR clones, and all of those that were probe negative were checked for the presence of inserts, which were then sequenced. By using this strategy, further novel methanogen 16S rRNA variants were identified and analyzed. The sequences recovered from the peat formed two clusters on the end of long branches within the methanogen radiation that are distinct from each other. These cannot be placed directly with sequences from any cultured taxa for which sequence information is available.

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Adherence of capsulate Neisseria meningitidis to endothelial and epithelial cells is facilitated in variants that express pili. Whereas piliated variants of N. meningitidis strain C311 adhered to endothelial cells in large numbers (> 150 bacteria/cell), derivatives containing specific mutations that disrupt pilE encoding the pilin subunit were both non-piliated and failed to adhere to endothelial cells (< 1 bacterium/cell). In addition, meningococcal pili recognized human endothelial and epithelial cells but not cells originating from other animals. Variants of strain C311 were obtained that expressed pilins of reduced apparent M(r) and exhibited a marked increase in adherence to epithelial cells. Structural analysis of pilins from two hyper-adherent variants and the parent strain were carried out by DNA sequencing of their pilE genes. Deduced molecular weights of pilins were considerably lower compared with their apparent M(r) values on SDS-PAGE. Hyper-adherent pilins shared unique changes in sequence including substitution of Asn-113 for Asp-113 and changes from Asn-Asp-Thr-Asp to Thr-Asp-Ala-Lys at residues 127-130 in mature pilin. Asn residues 113 and 127 of 'parental' pilin both form part of the typical eukaryotic N-glycosylation motif Asn-X-Ser/Thr and could potentially be glycosylated post-translationally. The presence of carbohydrate on pilin was demonstrated and when pilins were deglycosylated, their migration on SDS-PAGE increased, supporting the notion that variable glycosylation accounts for discrepancies in apparent and deduced molecular weights. Functionally distinct pilins produced by two fully piliated variants of a second strain (MC58) differed only in that the putative glycosylation motif Asn-60-Asn-61-Thr-62 in an adherent variant was replaced with Asp-60-Asn-61-Ser-62 in a non-adherent variant. Fully adherent backswitchers obtained from the non-adherent variant always regained Asn-60 but retained Ser-62. We propose, therefore, that functional variations in N. meningitidis pili may be modulated in large part by primary amino acid sequence changes that ablate or create N-linked glycosylation sites on the pilin subunit.

Oligonucleotide sequences selected from the 163 rRNA genes of various species of ammonia-oxidizing bacteria were evaluated as specif ic PCR amplification primers and probes. The spedficities of primer pairs for eubacterial, Nitmsospira and Nibrosomones rRNA genes were established with sequence databases, and the primer pairs were used to amplify DNA from laboratory cultures and environmental samples. Eubacterial rRNA genes amplified from samples of soil and activated sludge hybridized with an oligonucleotide probe specific for Nifmsospifa Spp.8 but not with a Nitmsomonas-specif ic probe. Lakewater and sediment samples were analysed using a nested PCR technique in which eubacterial rRNA genes were subjected to a secondary amplification with Nifmsomnas or Nitrosospira specific primers. Again, the presence of Nifmsospira DNA, but not Nitmsomonas DNA, was detected and this was confirmed by hybridization of the amplified DNA with an internal oligonucleotide probe. Enrichments of lakewater and sediment samples, incubated for two weeks in the presence of ammonium, produced nitrite and were found to contain DNA from both Nitmsospira and Nitmsomonas as determined by nested PCR amplification and probing of 163 rRNA genes. This demonstrates that Nifmsospira spp. are widespread in the environment. The implications of the detection of Nitmsomnas DNA only after enrichment culture are discussed.

A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt 2 ), was used to infect Enterobacteriaceae strains. A number of Shigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.Verocytotoxigenic Escherichia coli (VTEC) is a serious pathogen of considerable public health concern worldwide. Infection is usually characterized by bloody diarrhea and can be life threatening due to the subsequent development of hemolytic-uremic syndrome mediated by verocytotoxins (VTs), of which there are two forms, VT1 and VT2. In almost all cases, the VT genes are carried on temperate bacteriophages (VT phages). Although E. coli O157 is the most commonly isolated VTEC serogroup in the United Kingdom, North America, and Japan, more than 30 disease-causing non-0157 VTECs have been described (1) and over 100 serotypes are capable of producing VT (6). VT production has been observed in other members of the Enterobacteriaceae, including Enterobacter cloacae (8) and Citrobacter freundii (12), but was first described in Shigella dysenteriae as Shiga toxin (3). The localization of vt genes on a bacteriophage was first described by Smith et al. (13), but their acquisition by pathogenic E. coli strains remained anomalous because only nonpathogenic (rough) E. coli strains could apparently be infected with VT phage. Previously, the vt 2 gene of a bacteriophage (24 B ), isolated from an E. coli O157 strain, had been inactivated by insertion of a selectable marker (kanamycin resistance) (10). This provided an ideal opportunity to investigate the host range of a lysogenic VT bacteriophage and thus its potential to transfer the ability to produce VT between E. coli and related gram-negative bacteria.The host range of this recombinant VT2 phage (24 B ::Kan) was determined by infection of pathogenic and commensal strains of E. coli and other Enterobacteriaceae strains from human and animal sources. Lysogens were detected by spreading phage-infected cultures of the host bacteria (100 l) onto Luria-Bertani Miller (LB) agar (Difco) plates containing kanamycin (50 g ml Ϫ1 ). As it is clear that some phage infections create lysogens and do not result in a lytic infection, plaque assays may not necessarily detect all infectious phage particles. Induction of the VT phage lytic cycle is RecA dependent (7). RecA plays a central role in the SOS response of E. coli, during which phage-mediated lysis is induced. The recA441 mutant E. coli K-12 strain...