Francis L. Macrina

A multiple plasmid-containing Escherichia coli strain: convenient source of size reference plasmid molecules.

Abstract A strain of Escherichia coli is described that contains eight plasmid species ranging in size from 1.36 × 106 to 35.8 × 106 daltons. This strain can be employed as a single source of covalently closed circular deoxyribonucleic acid molecules of different sizes for use as references in agarose gel electrophoretic analysis.

A cloning vector able to replicate in Escherichia coli and Streptococcus sanguis

A plasmid that is able to replicate in both Escherichia coli and Streptococcus sanguis has been constructed by the in vitro joining of the pACYC184 (Cmr Tcr) and pVA749 (Emr) replicons. This plasmid, designated pVA838, is 9.2 kb in size and expresses Emr in both E. coli and S. sanguis. Its Cmr marker is expressed only in E. coli and may be inactivated by addition of DNA inserts at its internal EcoRI or PvuII sites. The pVA838 molecule also contains unique SalI, SphI, BamHI, NruI and XbaI cleavage sites suitable for molecular cloning. pVA838 may be amplified in E. coli but not in S. sanguis. We have used the pVA838 plasmid as a shuttle vector to clone streptococcal plasmid fragments in E. coli. Such chimeras isolated from E. coli were readily introduced into S. sanguis by transformation.

Genome of the Opportunistic Pathogen Streptococcus sanguinis

The genome of Streptococcus sanguinis is a circular DNA molecule consisting of 2,388,435 bp and is 177 to 590 kb larger than the other 21 streptococcal genomes that have been sequenced. The G+C content of the S. sanguinis genome is 43.4%, which is considerably higher than the G+C contents of other streptococci. The genome encodes 2,274 predicted proteins, 61 tRNAs, and four rRNA operons. A 70-kb region encoding pathways for vitamin B(12) biosynthesis and degradation of ethanolamine and propanediol was apparently acquired by horizontal gene transfer. The gene complement suggests new hypotheses for the pathogenesis and virulence of S. sanguinis and differs from the gene complements of other pathogenic and nonpathogenic streptococci. In particular, S. sanguinis possesses a remarkable abundance of putative surface proteins, which may permit it to be a primary colonizer of the oral cavity and agent of streptococcal endocarditis and infection in neutropenic patients.

Genetic Characterization of a Streptococcus mutans LraI Family Operon and Role in Virulence

ABSTRACT Proteins belonging to the LraI (for “lipoprotein receptor antigen”) family function as adhesins in several streptococci, as a virulence factor for endocarditis in at least one of these species, and potentially as metal transporters in many bacteria. We have identified and characterized the chromosomal locus containing the LraI family gene (designated sloC) from Streptococcus mutans, an agent of dental caries and endocarditis in humans. Northern blot analysis indicated that sloC is cotranscribed with three other genes. As with other LraI operons, the sloA andsloB genes apparently encode components of an ATP-binding cassette transport system. The product of the fourth gene,sloR, has homology to the metal-dependent regulator fromCorynebacterium diphtheriae, DtxR. A potential binding site for SloR was identified upstream from the sloABCR operon and was conserved upstream from LraI operons in several other streptococci. Potential SloR homologs were identified in the unfinished genomic sequences from two of these, S. pneumoniae andS. pyogenes. Mutagenesis of sloC in S. mutans resulted in apparent loss of expression of the entire operon as assessed by Northern blot analysis. The sloCmutant was indistinguishable from its wild-type parent in a gnotobiotic rat model of caries but was significantly less virulent in a rat model of endocarditis. Virulence for endocarditis was restored by correction of the sloC mutation but not by provision of thesloC gene in trans, suggesting that virulence requires the expression of other genes in the sloC operon.

Sucrose-derived exopolysaccharides of Streptococcus mutans V403 contribute to infectivity in endocarditis

We used an isogenic mutant of Streptococcus mutans V403, which differs from the wild‐type V403 in genes involved in glucan and fructan production, to examine the importance of these exopolysaccharides as factors affecting infectivity in endocarditis. Rats inoculated with V403 developed endocarditis more frequently than animals inoculated with the mutant strain which produced neither glucan nor fructan (58% versus 12%, P<0.01). In phagocytosis assays, both strains were found to be associated with the human granulocytes but a greater number of live V403 than of mutant organisms could be recovered. Colony counts recovered from fibrin plates incubated with the mutant were lower than those incubated with V403. These experiments indicate that exopolysaccharides produced by Streptococcus mutans contribute to its infectivity in endocarditis.

Extrachromosomal systems and gene transmission in anaerobic bacteria

Obligately anaerobic bacteria are important in terms of their role as medical pathogens as well as their degradative capacities in a variety of natural ecosystems. Two major anaerobic genera, Bacteroides and Clostridium, are examined in this review. Plasmid elements in both genera are reviewed within the context of conjugal transfer and drug resistance. Genetic systems that facilitate the study of these anaerobic bacteria have emerged during the past several years. In large part, these developments have been linked to work centered on extrachromosomal genetic systems in these organisms. Conjugal transfer of antibiotic resistance has been a central focus in this regard. Transposable genetic elements in the Bacteroides are discussed and the evolution and spread of resistance to lincosamide antibiotics are considered at the molecular level. Recombinant DNA systems that employ shuttle vectors which are mobilized by conjugative plasmids have been developed for use in Bacteroides and Clostridium. The application of transmission and recombinant DNA genetic systems to study these anaerobes is under way and is likely to lead to an increased understanding of this important group of procaryotes.

Vaccination with FimA from Streptococcus parasanguis Protects Rats from Endocarditis Caused by Other Viridans Streptococci

ABSTRACT The FimA protein of Streptococcus parasanguis is a virulence factor in the rat model of endocarditis, and immunization with FimA protects rats against homologous bacterial challenge. Because FimA-like proteins are widespread among the oral streptococci, the leading cause of native valve endocarditis, we evaluated the ability of this vaccinogen to protect rats when challenged by other streptococcal species. Here we report that FimA vaccination produced antibodies that cross-reacted with and protected against challenge by the oral streptococci S. mitis, S. mutans, and S. salivarius. FimA thus has promise as a vaccinogen to control infective endocarditis caused by oral streptococci.

Conjugation and Broad Host Range Plasmids in Streptococci and Staphylococci

Interest in conjugal genetic exchange in gram-positive bacteria has centered on two major themes. First, such gene transfer systems offer comparative models for the study of the mechanisms and consequences of genetic exchange in this group of organisms. Second, the role of conjugative gene exchange in the dissemination of antimicrobial resistance has prompted investigation in order to understand such systems and, eventually, to prevent or control the spread of resistance phenotypes.

Molecular characterization of unique deletion mutants of the streptococcal plasmid, pAMβ1

Abstract pAMβ1 is a 17 × 10 6 dalton plasmid originally isolated in a strain of Streptococcus faecalis . This plasmid confers constitutively expressed macrolide-lincosamide-streptogramin resistance. Following its introduction in Streptococcus sanguis (Challis) by transformation we have detected a class of pAMβ1 derivatives which carry site-specific deletions. Each of these independently obtained, smaller plasmids has been found to be missing an identical 60% of the pAMβ1 molecule when probed by restriction endonuclease digestion. A typical specific deletion derivative, designated pVA1, is present to the extent of ~10 copies per chromosomal equivalent. It is more stably inherited than pAMβ1 ( S. sanguis grown at 37 °C. However, both pVA1 and pAMβ1 appear to be rapidly segregated from S. sanguis cells grown at 42 °C. pVA1 should provide a useful replicon for genetic studies including those aimed at elucidating R plasmid organization, expression, and molecular cloning vector development in the streptococci.

Molecular Cloning in the Streptococci

The genus Streptococcus contains a large number of species able to cause infection in humans and in animals. Our research efforts have revolved around the study of streptococci that normally reside in the human oral cavity. In this regard, Streptococcus mutans is thought to play an important role in the etiology of dental caries (tooth decay) in humans (5). Its virulence has been well established in animal model systems (see 5) and studies with mutants indicate that S. mutans’ ability to synthesize water-insoluble glucans from sucrose is closely linked to its pathogenicity.