Lantibiotics are polycyclic peptides containing unusual amino acids, which have binding specificity for bacterial cells, targeting the bacterial cell wall component lipid II to form pores and thereby lyse the cells. Yet several members of these lipid II-targeted lantibiotics are too short to be able to span the lipid bilayer and cannot form pores, but somehow they maintain their antibacterial efficacy. We describe an alternative mechanism by which members of the lantibiotic family kill Gram-positive bacteria by removing lipid II from the cell division site (or septum) and thus block cell wall synthesis.

Many important virulence genes of pathogenic bacteria are preferentially expressed in vivo. We used the recently developed in vivo-induced antigen technology (IVIAT) to identify Vibrio vulnificus genes induced in vivo. An expression library of V. vulnificus was screened by colony blot analysis by using pooled convalescentphase serum that had been thoroughly adsorbed with in vitro-expressed V. vulnificus whole cells and lysates. Twelve clones were selected, and the sequences of the insert DNAs were analyzed. The DNA sequences showed homologies with genes encoding proteins of diverse functions: these functions included chemotaxis (a methylaccepting chemotaxis protein), signaling (a GGDEF-containing protein and a putative serine/threonine kinase), biosynthesis and metabolism (PyrH, PurH, and The expression of virulence determinants in bacteria is known to be regulated by various environmental and host factors (38). During host-parasite interactions, many novel genes that not expressed during in vitro growth have been demonstrated to be coordinately regulated or stimulated by host factors encountered in vivo (20). The usefulness of the information concerning virulence expression gained from in vitro studies is therefore incomplete in relation to in vivo bacterial pathogenesis.Vibrio vulnificus, an opportunistic pathogen, experiences a dramatic environmental change during its infection process. V. vulnificus is an estuarine bacterium that preferentially affects individuals who are heavy drinkers of alcohol and patients with underlying hepatic diseases and other immunocompromised conditions. The pathogen frequently causes fatal septicemia with a rapid progress, resulting in a mortality rate of more than 50% within a few days. The putative virulence factors of V. vulnificus reported so far include a hemolysin (15), a protease (29), phospholipase A2 (55), siderophores (53), and capsular polysaccharides (61a). We reported that the ToxRS system of V. vulnificus, a transmembrane signal-transducing transcription activator, regulated the expression of the hemolysin gene vvhA (32). The ToxRS system was reported to play an important role in regulating in vivo virulence gene expression during V. cholerae infection in a mouse model (33). However, whether the V. vulnificus ToxRS system plays an important role in regulating in vivo virulence gene expression during infection needs further study. V. vulnificus, while infecting the susceptible hosts, passes through gastric acidity, experiences an abrupt pH increase in the duodenum, receives bile secretion, invades into intestinal mucosa, and eventually enters the bloodstream where the pathogen multiplies. During this complicated infection process, V. vulnificus should be able to sense changes in the environmental parameters in the host milieu. The changing signals are likely relayed to specific genes by cognate signal transduction systems, resulting in the expression of specific virulence factors (33). Virulence factors required for in vivo survival and growth of V. vulnificus are ex...

New vectors were constructed for efficient transposon Tn917-mediated mutagenesis of poorly transformable strains of Streptococcus mutans(pTV1-OK) and subsequent recovery of interrupted genes in Escherichia coli(pTV21⌬2TetM). In this report, we demonstrate the utility of Tn917 mutagenesis of a poorly transformable strain of S. mutans (JH1005) by showing (i) the conditional replication of pTV1-OK, a repA(Ts) derivative of the broad-host-range plasmid pWVO1 harboring Tn917, in JH1005 at the permissive temperature (30؇C) versus that at the nonpermissive temperature (45؇C); (ii) transposition frequencies similar to those reported for Bacillus subtilis (10 ؊5 to 10 ؊4) with efficient plasmid curing in 90 to 97% of the erythromycin-resistant survivors following a temperature shift to 42 to 45؇C; and (iii) the apparent randomness of Tn917 insertion as determined by Southern hybridization analysis and the ability to isolate nutritional mutants, mutants in acid tolerance, and mutants in bacteriocin production, at frequencies ranging from 0.1 to 0.7%. Recovery of transposon-interrupted genes was achieved by two methods: (i) marker rescue in E. coli with the recovery vector pTV21⌬2TetM, a tetracycline-resistant and ampicillin-sensitive Tn917-pBR322 hybrid, and (ii) "shotgun" cloning of genomic libraries of Tn917 mutants into pUC19. Sequence analyses revealed insertions at five different genetic loci in sequences displaying homologies to Clostridium spp. fhs (66% identity), E. coli dfp (43% identity), and B. subtilis ylxM-ffh (58% identity), icd (citC [69% identity]), and argD (61% identity). Insertions in icd and argD caused nutritional requirements; the one in ylxM-ffh caused acid sensitivity, while those in fhs and dfp caused both acid sensitivity and nutritional requirements. This paper describes the construction of pTV1-OK and demonstrates that it can be efficiently employed to deliver Tn917 into S. mutans for genetic analyses with some degree of randomness and that insertions in the chromosome can be easily recovered for subsequent characterization. This represents the first published report of successful Tn917 mutagenesis in the genus Streptococcus.

Samples of subgingival plaque were taken from 275 active and inactive sites in 35 subjects with destructive periodontal diseases. The predominant cultivable microbiota was determined in each of the samples by characterizing 50 randomly selected isolates recovered on anaerobically incubated Trypticase soy agar supplemented with 5% sheep blood. Microbial associations between species were determined by computing the odds ratio of a site being infected by a “target” species in the presence of an “effector” species. Data are presented for 22 numerically dominant “effector” species and 8 “target” species which consisted of suspected periodontal pathogens. These included Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Bacteroides gingivalis, Bacteroides intermedicus, Bacteroides melaninogenicus, Peptostreptococcus micros, Streptococcus intermedius and Wolinella recta. Positive associations were considered to be relationships in which the odds ratio of detecting a “target” species was >2:1 in the presence of the “effector” species, while negative associations were defined as those in which the odds ratio of detecting the “target” species was < 0.5:1 in the presence of the “effector” species. In general, species of streptococci, an unnamed Actinomyces sp. and Propionibacterium acnes showed a negative association with the 8 suspected pathogens, while many Gram‐negative species tended to show a positive association with these suspected pathogens. There appeared to be specificity in the associations observed. For example, S. intermedius was positively associated with Streptococcus mitis and Streptococcus sanguis I, but negatively associated with Bacteroides forsythus. In contrast, W. recta was negatively associated with S. mitis and Streptococcus sanguis I, but positively associated with Bacteroides forsythus. S. sanguis II, Streptococcus uberis, P. acnes, Capnocytophaga ochracea and an unnamed Actinomyces sp. showed negative associations with 4 of the suspected periodontal pathogens, while B. gingivalis, Fusobacterium nucleatum and the Eubacterium sp. showed only positive or “neutral” associations with the “target” species. Data from this and other investigations suggest that microbial associations might play an important role in controlling the composition of subgingival plaque and, thus, be a major determinant of health or destructive periodontal diseases.

No abstract

An effector strain has been constructed for use in the replacement therapy of dental caries. Recombinant DNA methods were used to make the Streptococcus mutans supercolonizing strain, JH1140, lactate dehydrogenase deficient by deleting virtually all of the ldh open reading frame (ORF). To compensate for the resulting metabolic imbalance, a supplemental alcohol dehydrogenase activity was introduced by substituting the adhB ORF from Zymomonas mobilis in place of the deleted ldh ORF. The resulting clone, BCS3-L1, was found to produce no detectable lactic acid during growth on a variety of carbon sources, and it produced significantly less total acid due to its increased production of ethanol and acetoin. BCS3-L1 was significantly less cariogenic than JH1140 in both gnotobiotic-and conventional-rodent models. It colonized the teeth of conventional rats as well as JH1140 in both aggressive-displacement and preemptive-colonization models. No gross or microscopic abnormalities of major organs were associated with oral colonization of rats with BCS3-L1 for 6 months. Acid-producing revertants of BCS3-L1 were not observed in samples taken from infected animals (reversion frequency, <10 ؊3 ) or by screening cultures grown in vitro, where no revertants were observed among 10 5 colonies examined on pH indicator medium. The reduced pathogenic potential of BCS3-L1, its strong colonization potential, and its genetic stability suggest that this strain is well suited to serve as an effector strain in the replacement therapy of dental caries in humans.

In vivo-induced antigen technology is a method to identify proteins expressed by pathogenic bacteria during human infection. Sera from 10 patients convalescing from cholera infection in Bangladesh were pooled, adsorbed against in vitro-grown El Tor Vibrio cholerae O1, and used to probe a genomic expression library in Escherichia coli constructed from El Tor V. cholerae O1 strain N16961. We identified 38 positive clones in the screen, encoding pili (PilA and TcpA), cell membrane proteins (PilQ, MshO, MshP, and CapK), methyl-accepting chemotaxis proteins, chemotaxis and motility proteins (CheA and CheR), a quorum-sensing protein (LuxP), and four hypothetical proteins. Analysis of immune responses to purified PilA and TcpA in individual patients demonstrated that the majority seroconverted to these proteins, confirming results with pooled sera. These results suggest that PilA and its outer membrane secretin, PilQ, are expressed during human infection and may be involved in colonization of the gastrointestinal tract. These results also demonstrate substantial immune responses to TcpA in patients infected with El Tor V. cholerae O1. In vivo-induced antigen technology provides a simple method for identifying microbial proteins expressed during human infection, but not during in vitro growth. Vibrio cholerae is a Gram-negative bacillus that causes a severe, dehydrating diarrhea in humans (1). V. cholerae can be differentiated by the lipopolysaccharide in the outer membrane; strains of V. cholerae that produce cholera belong to serogroup O1 or O139. V. cholerae O1 is divided into two biotypes, classical and El Tor; the current global pandemic of V. cholerae O1 infection is caused by El Tor strains.A major virulence factor for pathogenic strains of V. cholerae is cholera toxin, a protein exotoxin that consists of a single A subunit noncovalently associated with five B subunits (2). A second major virulence factor of V. cholerae is the toxin coregulated pilus (TCP; ref. 3). TCP is essential for colonization and virulence in both mouse models of cholera (3) and human volunteer studies (4). TcpA, the 20.5-kDa major structural subunit of TCP, has homology to the type IV pili of several other bacterial pathogens (5). TcpA from El Tor and classical strains of V. cholerae show Ϸ80% protein homology; monoclonal antibodies demonstrate epitope differences between these proteins in the two biotypes (6, 7).In addition to TcpA, the V. cholerae genome encodes two other type IV pili, the mannose-sensitive hemagglutinin (MSHA) and PilA (8). MSHA is a thin, flexible pilus composed of a 17-kDa subunit (9). A strain of V. cholerae deleted in mshA showed no defect in colonization of human volunteers (10). Recently, Fullner et al. (11) described a four-gene cluster, pilABCD, encoding a third type IV pilus in V. cholerae. A deletion of pilA had no effect on colonization in infant mice. The role of PilA in human infection has not been previously examined.

Streptococcus mutans strain JH1005 is a mutant that produces levels of bacteriocin activity three-fold-elevated than those produced by its parent, JH1001. A single infection regimen with JH1005 was found to result in persistent colonization of the teeth of all three adult subjects tested. This is a significant improvement over JH1001, which required multiple exposures in order to colonize the teeth of humans reliably. The levels of total cultivable bacteria and indigenous S. sanguis were not affected by JH1005 colonization. In two of the three subjects, total (indigenous plus JH1005) S. mutans levels were significantly decreased. The results provide additional support for the role of bacteriocin production as an ecological determinant in colonization by S. mutans. They also indicate that a practical regimen for infection by an effector strain might be achieved for use in the replacement therapy of dental caries.