L. S. Baron

Cooperative effects of bacterial mutations affecting λ N gene expression: I. Isolation and characterization of a nusB mutant

Abstract We report the isolation and characterization of an Escherichia coli mutant which limits the growth of phage λ by inhibiting the expression of the N gene regulatory function. The mutation involved maps near minute 11 of the E. coli chromosome and dominance tests show that the mutant allele is recessive to the wild one. Therefore, we conclude that the locus involved normally codes for a function necessary for N expression. Another mutant which exhibits a similar phenotype has previously been reported and the mutation involved, in that case, maps at minute 61. This mutant is called Nus ( N u tilization s ubstance); we have named the locus at minute 61 nus A, and the locus at minute 11, nus B. Although the nus A allele is not found in Salmonella typhosa , our studies demonstrate that the nus A allele is found in this closely related enterobacteriaciae. A nus A-1 nus B-5 double mutant was constructed and exhibited a far more restrictive effect on λ growth than either of the single nus mutants. Further, we have constructed a λ variant which carries the nus B + allele. This phage plates on nus B-5 mutants under restrictive conditions, but not on the nus A-1 mutants.

Novel virulence properties of the Salmonella typhimurium virulence-associated plasmid: Immune suppression and stimulation of splenomegaly

Mice infected subcutaneously with wild-type Salmonella typhimurium, SR11, developed a significant splenomegaly when compared with mice infected with an equal number of a plasmid-cured strain. Further, the bacterial load in the spleen at 14 days after infection, measured as colony-forming units per gram tissue, was significantly higher in mice infected with the parent strain than in mice infected with the plasmid-cured strain. These data confirm the previously reported plasmid-associated ability of Salmonella to multiply within the spleen. In addition, lymph node cells (LNC) from mice infected with the parent strain had a significantly reduced ability to proliferate in response to concanavalin A, a T-cell mitogen, and to heat-killed S. typhimurium cells when compared with LNC isolated from mice infected with the plasmid-cured strain. Finally, reintroduction of a functional Tn5-tagged 90-kb plasmid into a plasmid-free strain restored its capacity to cause a marked splenomegaly and to suppress lymph node cell proliferation in BALB/c mice. These data demonstrate that the 90-kb plasmid of highly virulent S. typhimurium strains mediates several novel pathogenic properties in infected mice: (1) enhancement of the ability of Salmonella to multiply within the spleen; (2) stimulation of a splenic inflammatory response as displayed by marked splenomegaly; and (3) a general suppression of lymphocyte responsiveness to both T-cell mitogens and specific Salmonella antigens.

Strain differences in expression of virulence by the 90 kilobase pair virulence plasmid of Salmonella serovar Typhimurium

A number of plasmidless strains were obtained by curing the 90 kilobase pair (kb) virulence plasmid from six strains, C5, TML, W118, SR11, LT2 and Fisher, of Salmonella serovar Typhimurium. A number of transposon (Tn5) tagged 90 kb plasmids, also derived from these Typhimurium strains, were then transferred back into these plasmidless strains. Plasmid-cured strains, reconstituted strains, and the parental strains were tested for their virulence in BALB/c mice. There were two groups of Typhimurium strains: one required the 90 kb plasmid to express high virulence (LD50 less than 50 bacteria), and the other, regardless of the presence or absence of the 90 kb plasmid, maintained the same level of virulence at LD50 = 10 to 7 x 10(5) bacteria. Among the plasmidless strains, there were strains with a virulence level as low as LD50 = 10(7) bacteria, which was unaffected by the presence of the 90 kb plasmid.

Tn5 mutagenesis of the Salmonella typhimurium 100 kb plasmid: definition of new virulence regions

In this study, the 100 kb plasmid of Salmonella typhimurium, which is known to contribute to the pathogenicity of the organism, was tagged with the transposon Tn5 to define regions of the plasmid contributing to overall virulence. Eleven randomly selected vir::Tn5 plasmids carried by the plasmid-free S. typhimurium strain WS1321 were physically mapped and then examined in mice for subcutaneous LD50 value, ability to induce splenomegaly, and ability to grow to high numbers in the spleens of infected mice. Nine strains were found to be virulence-attenuated and showed varied levels of growth in the spleens of subcutaneously infected BALB/c mice. Eight of these nine strains carried Tn5 insertions which lie outside the previously defined virulence region. These studies corroborate the findings of other investigators as well as defining novel regions of the 100 kb virulence plasmid involved in the pathogenicity of this organism.

Somatic antigen addition in salmonella by bacteriophage

Abstract A bacteriophage that will lysogenize certain species of Salmonella has been isolated from Salmonella kentucky . This phage confers on the cells which it has lysogenized the ability to produce an added somatic antigenic component. The process of antigen addition, which appears to be dependent on the introduction and presence of the prophage, has resulted in the production of a novel somatic serotype, VI, VIII, XX.

Genetic studies of hybrids between coliphage ? and Salmonella phage P22: Genetic analysis of the P22-? hybrid class

SummaryP22-λ hybrids which retain the protein coat of P22 have been isolated and characterized into two types. Type 1 hybrids which have the c through O-P genes of λ are unable to grow lytically on Salmonella typhimurium. On the other hand, type 2 hybrids which contain only the c region of λ, plated on S. typhimurium. Both hybrid types retained the generalized transducing and antigenic conversion capabilities of P22.

Invasive Bacterial Pathogens of the Intestine: Shigella Virulence Plasmids and Potential Vaccine Approaches

Bacterial diseases of the gastrointestinal tract usually occur by one of three overall mechanisms. The first mechanism, termed “intoxication,” occurs by bacterial secretion of an exotoxin that oftentimes is preformed in food prior to ingestion by the host. This process is exemplified by staphylococcal or clostridial food poisoning. In contrast, the remaining two processes require living and multiplying disease agents. In the “enterotoxigenic” mechanism, as discussed elsewhere in this volume, bacteria colonize the small intestine, usually in the jejunum or duodenum. These bacteria multiply on the intestinal surface and elaborate an enterotoxin that stimulates excessive fluid and electrolyte efflux resulting in a watery diarrhea. Enteropathogenic Escherichia coli and Vibrio cholera serve as typical examples. Finally, a third group of organisms, termed “invasive,” actually penetrate the epithelial mucosa of the large intestine. Subsequently, these organisms multiply intracellularly and disseminate within or through the mucosa. This latter mechanism, classically typified by Shigella and Salmonella, is now thought to be used by invasive strains of 15. coli, Yersinia, and, possibly, Campylobacter. In contrast to other invasive bacterial diseases like salmonellosis in which the invading bacteria are disseminated throughout the host, shigellosis is a disease normally confined to the intestinal lining. Whereas toxigenic organisms generally require a large dose of organisms to cause disease, previous studies have shown that as few as ten virulent cells of Shigella can cause disease in humans. Thus, these features distinquish the toxigenic from the invasive mechanism of intestinal disease (see reviewsl,2).

Role of IS1 in the conversion of virulence (Vi) antigen expression in Enterobacteriaceae

SummaryWhen Escherichia coli HB101 harbors pWR127, a plasmid comprising the viaB gene from Citrobacter freundii WR7004 and the ColEl-derived pACKC1, the strain produces the virulence (Vi) antigen. Vi antigen expression is abolished (Vi− phenotype), however, when an IS1 or IS1-like DNA element inserts into the viaB region. To determine the sites of IS1 insertion, pWR127 DNAs extracted from 95 independently isolated Vi− strains were analyzed by digestion with the restriction endonuclease PstI and agarose gel electrophoresis. Ten insertion sites were found distributed nonrandomly in an area of about 1.3 kb. Nine Vi+ strains (two Citrobacter, two E. coli, and five Salmonella strains), four of which contain pWR127, were then tested for the presence of IS1 by DNA-DNA hybridization. Of the nine strains, five were stable Vi+ and did not contain IS1. The other four which generated Vi− strains, contained IS1. When pRR134, a plasmid that contains IS1 was transferred into a stable Vi+Salmonella typhimurium strain carrying pWR127 (OU5140), Vi− strains were produced from which pWR127 derivatives carrying IS1 inserts could be isolated. It appears, therefore, that the presence of an IS1I or IS1-like element in a strain is required for conversion of the Vi+ expression state to the Vi− expression state.

Extensive segments of the Escherichia coli K12 chromosome in Proteus mirabilis diploids

SummaryVarious Escherichia coli K12 Hfr donors transfer at low frequency portions of the E. coli genome to Proteus mirabilis. By remating such Proteus hybrids with the same or a different E. coli Hfr strain, other genetic characters could be added to yield diploid Proteus hybrids which contained more than 30% of the E. coli genome. The extent of the E. coli genetic material in these unstable Proteus diploid hybrids included segments with the following selected markers: gal, lac, ara, mel, mtl, and malA. Unselected markers known to map throughout this region of the chromosome were also detected in these hybrids. Among the markers expressed in Proteus hybrids with the E. coli malA region was the receptor site for coliphage lambda. Although plaques were not seen, lambda was adsorbed by the Proteus hybrids. Examination of DNA from the various Proteus hybrids by CsCl density gradient centrifugation showed a satellite component of E. coli DNA with a size that corresponded to the extent of the E. coli genome present as determined by genetic analysis.

High Frequency Transduction by Phage Hybrids Between Coliphage ø80 and Salmonella Phage P22

phi 80immP22dis, a hybrid between phi 80 and P22, carries all the late genes of phi 80 and most of the P22 early region including the immC and immI bipartite immunity loci. The presence of the immI region allows this hybrid to grow on lysogens of phi 80immP22 hybrids which have the immC locus, but not the immI locus. In addition to these P22 immunity regions, phi 80immP22dis contains the P22 att marker so that the prophage can be inserted into the chromosomal P22 attachment site adjacent to the proA-proB region of the host. Unlike its phi 80 parent which performs specialized transduction of the trp region, phi 80immP22dis transduces markers located adjacent to its attachment site to Escherichia coli K12 recipients at high frequencies (0.3% for argF and 0.18% for proA). Induction of phi 80immP22dis lysogens yields new hybrid phage clones which have incorporated E. coli K12 chromosomal segments in place of the P22 immI to att segment. Having lost the immI region, the new hybrids no longer grow in phi 80immP22 lysogens. These new hybrids, termed phi 80immP22dis-, possess specialized transducing properties, transferring the argF and proA markers at higher frequencies (21% for argF and 12% for proA) than previously obtained with the phi 80immP22dis phage.