New antibiotic substance produced by Salmonella typhimurium LT2

Salmonella typhimurium LT2 excreted under certain conditions an antibiotic substance designated typhimuricin. It is suggested that the LT2 “cryptic” plasmid is involved in its production and in the immunity to it. Preliminary characterization of typhimuricin is presented.     

Stabilization of glucose-starved Escherichia coli K12 and Salmonella typhimurium LT2 by peptidase-deficient mutants

Escherichia coli K12 and Salmonella typhimurium LT2 cells were stabilized during carbon starvation in the presence of peptidase-deficient mutant strains. The rate of loss of viability of the wild-type S. typhimurium strain was decreased an average of 2-fold, and the rate for the wild-type E. coli strain was decreased about 2.3-fold, when either was starved in the presence of the multiply peptidase-deficient S. typhimurium strain TN852; other peptidase-deficient strains exhibited similar stabilizing effects. Starving wild-type S. typhimurium LT2 cells utilized peptides excreted by the starving peptidase-deficient cells for protein synthesis, and, to a lesser extent, as respiratory substrates. Provision of free amino acids in steady-state levels to starving E. coli K12 cells in a cell recycle apparatus had a stabilizing effect similar to that of mixing with peptidase-deficient cells.     

Silent genes in bacteria: the previously designated 'cryptic'ilvHI locus of 'Salmonella typhimurium LT2' is active in natural isolates

Abstract Gene ilvG in Escherichia coli K-12 and ilvl in ' Salmonella typhimurium LT2' ( S. enterica serotype Typhimurium, strain LT2) are inactive due to frameshift or nonsense mutations, respectively. These inactive genes have been suggested to be part of 'cryptic' genetic systems which are defined as being of long-term regulatory and evolutionary significance. We have shown that the nonsense mutation in ilvI is present only in derivatives of the laboratory strain ' S. typhimurium LT2'. All natural isolates of Salmonella examined have an arginine codon at the corresponding location of their ilvl sequences. Further, two randomly selected natural isolates of serotype Typhimurium are shown to each have an active ALS III isozyme. Our findings strongly suggest that the only Salmonella strains which lack a functional ilvHI locus are LT2 isolates. We suggest that the mutations leading to inactivation of both ilvI in ' S. typhimurium LT2' and ilvG in E. coli K-12 are more likely to have been acquired during laboratory storage and/or cultivation, rather than representing cryptic systems of gene regulation.     

The rfaS gene, which is involved in production of a rough form of lipopolysaccharide core in Escherichia coli K-12, is not present in the rfa cluster of Salmonella typhimurium LT2.

Partial sequencing of the rfa cluster of Salmonella typhimurium LT2 indicated a region of 336 bp between rfaP and rfaB in the site occupied by the rfaS gene in Escherichia coli K-12. This region does not contain a functional rfaS gene, although DNA analysis suggests that the region may have contained an ancestral gene. This conclusion that S. typhimurium LT2 lacks rfaS is supported by its lipopolysaccharide (LPS) gel phenotype, since LT2 does not make the lipooligosaccharide band characteristic of LPS from smooth strains of E. coli K-12.     

Salmonella typhimurium LT7 and LT2 strains carrying the imp operon on colIa.

The imp operon is carried on a transmissible plasmid, ColIa, in original isolates of Salmonella typhimurium LT7. LT2 strain recipients of F' factors from LT7 strains harboring ColIa can acquire ColIa and imp under nonselective conditions. Thus, S. typhimurium LT2 strains that have received plasmids by conjugal transfer from LT7 strains might be inadvertently harboring ColI factors.     

Tryptophan biosynthesis in Salmonella typhimurium: location in trpB of a genetic difference between strains LT2 and LT7.

Salmonella typhimurium prototrophs carrying a trpR mutation synthesize tryptophan biosynthetic enzymes constitutively. When feedback inhibition of anthranilate synthetase but not 5'-phosphoribosylpyrophosphate phosphoribosyltransferase activity was by-passed by growing cells on media supplemented with anthranilic acid, all trpR prototrophs overproduced and excreted tryptophan. However, the rate of tryptophan production depended on both the ancestry of the trpR strain and the integrity of its trpA gene. Prototrophs with trp genes derived from S. typhimurium strain LT2 produced tryptophan more efficiently than those with trp genes derived from strain LT7. This strain difference was cryptic insofar as it did not affect the growth rate; it was revealed only as a rate-limiting step in the constitutive biosynthesis of tryptophan in the presence of anthranilic acid, and was due to a lesion in the LT7-derived trpB gene. Strains with LT7-derived trp genes bearing a deletion in trpA produced tryptophan as readily as LT2 trpR prototrophs. This indicated that LT7-specific 5-phosphoribosylpyrophosphate phosphoribosyltransferase must be aggregated with the trpA gene produce to give an observable reduction of constitutive tryptophan production. The discovery of this strain difference has particular implications for studies involving the activities of trpA and B genes and their products in S. typhimurium and may have general significance for other studies involving different strains of Salmonella.     

Cloning, sequencing and distribution of the Salmonella typhimurlum LT2 siaiidase gene, nanH, provides evidence for interspecies gene transfer

The Salmonella typhimurium LT2 sialidase (neuraminidase, EC 3.2.1.18) structural gene, nanH, has been cloned and sialidase overproduced from multicopy plasmids in Escherichia coli. Sialidase expression was regulated positively by cAMP. In contrast, certain Tn1000 insertions located upstream of nanH coding sequences reduced sialidase activity. A nanH chromosomal insertion mutation constructed by marker exchange demonstrated a single sialidase gene copy in S. typhimurium LT2. The complete nucleotide sequence of nanH, encoding a 41,300 dalton polypeptide, was determined and the derived primary structure was similar to sialidases from Clostridium perfringens, Clostridium sordellii, Bacteroides fragilis, and Trypanosoma cruzi. Comparative sequence analysis, including codon usage and secondary structure predictions, indicated that the S. typhimurium and clostridial sialidases are homologous, strongly suggestive of an interspecies gene transfer event. At least two primary sequence motifs of the bacterial enzymes were detected in influenza A virus sialidases. The predicted secondary structure of the bacterial enzymes was strikingly similar to viral sialidase. From the population distribution of nanH detected within a collection of salmonellae, it was apparent that S. typhimurium obtained its nanH copy most recently from Salmonella arizonae. S. typhimurium LT2 is thus a genetic mosaic that differs from other strains of even the same serotype by nanH plus potentially additional characters linked to nanH. These results have relevance to the evolution and function of sialidases in pathogenic microbes, and to the origin of the sialic acids.     

Identification of a umuDC locus in Salmonella typhimurium LT2.

The umuDC operon of Escherichia coli is required for efficient mutagenesis by UV light and many other DNA-damaging agents. The existence of a umuDC analog in Salmonella typhimurium has been questioned. With DNA probes to the E. coli umuD and umuC genes, we detected, by Southern blot hybridization, sequences similar to both of these genes in S. typhimurium LT2. We also confirmed that the presence of cloned E. coli umuD enhances the UV mutability and resistance of S. typhimurium. Our data strongly suggest that S. typhimurium contains a functional umuDC operon.     

Structural characterization of the Salmonella typhimurium LT2 umu operon.

The umuDC operon of Escherichia coli encodes functions required for mutagenesis induced by radiation and a wide variety of chemicals. The closely related organism Salmonella typhimurium is markedly less mutable than E. coli, but a umu homolog has recently been identified and cloned from the LT2 subline. In this study the nucleotide sequence and structure of the S. typhimurium LT2 umu operon have been determined and its gene products have been identified so that the molecular basis of umu activity might be understood more fully. S. typhimurium LT2 umu consists of a smaller 417-base-pair (bp) umuD gene ending 2 bp upstream of a larger 1,266-bp umuC gene. The only apparent structural difference between the two operons is the lack of gene overlap. An SOS box identical to that found in E. coli is present in the promoter region upstream of umuD. The calculated molecular masses of the umuD and umuC gene products were 15.3 and 47.8 kilodaltons, respectively, which agree with figures determined by transpositional disruption and maxicell analysis. The S. typhimurium and E. coli umuD sequences were 68% homologous and encoded products with 71% amino acid identity; the umuC sequences were 71% homologous and encoded products with 83% amino acid identity. Furthermore, the potential UmuD cleavage site and associated catalytic sites could be identified. Thus the very different mutagenic responses of S. typhimurium LT2 and E. coli cannot be accounted for by gross differences in operon structure or gene products. Rather, the ability of the cloned S. typhimurium umuD gene to give stronger complementation of E. coli umuD77 mutants in the absence of a functional umuC gene suggests that Salmonella UmuC protein normally constrains UmuD protein activity.     

Protective immunities induced by porins from mutant strains of Salmonella typhimurium

The protective immunity against Salmonella typhimurium-infection in mice immunized with porins from mutant strains of S. typhimurium was studied. A high level of protection against S. typhimurium infection was achieved in mice immunized with native porins from S. typhimurium LT2 (wild-type strain) but not from S. typhimurium SH6017, SH6260, or SH5551 (mutant strains), which produce 34K, 35K, or 36K porin, respectively. Moreover, when mice were immunized with mixtures of 34K, 35K, and 36K porins (34K + 35K, 35K + 36K, 34K + 36K, or 34K + 35K + 36K porin) or LT2 porin heated at 100 C for 2 min in 2% SDS (heat-denatured LT2 porin), the degree of protective immunities in the mice was very much lower than that in the mice immunized with the native LT2 porin. However, antisera raised against these porins showed no significant differences of the antibody titer against LT2 porin or LT2 whole cells. On the other hand, mice immunized with the native LT2 porin--but not 34K, 35K, 36K, 34K + 35K + 36K, and the heat-denatured LT2 porins--exhibited significant levels of delayed-type hypersensitivity reaction and interleukin-2 production when they were elicited with whole cells of S. typhimurium LT2. These observations suggested that the high level of protection induced by the native LT2 porin immunization was dependent on the induction of cell-mediated immunity.     

Protein sequences of linked genes are highly conserved in two bacterial species

It has been shown that proteins encoded by linked genes have similar rates of evolution and that clusters of essential genes are found in regions with low recombination rates. We show here that proteins encoded by linked genes in two closely related bacterial species, namely Escherichia coli K12 and Salmonella typhimurium LT2, evolve more slowly when compared with proteins encoded by genes that are not linked as assessed by protein sequence similarity. The proteins encoded by the identified linked genes share an average sequence identity of 82.5% compared with a 46.5% identity of proteins encoded by genes that are not linked.     

Location of a mutator gene in Salmonella typhimurium by cotransduction

Kirchner, Carl E. J. (Suffolk County Community College, Selden, N.Y.), and Matthew J. Rudden. Location of a mutator gene in Salmonella typhimurium by cotransduction. J. Bacteriol. 92:1453-1456. 1966.-The LT7 strain of Salmonella typhimurium has been shown to possess a mutator gene which is responsible for an increase in mutation frequency for most loci tested. Preliminary results suggested the gene might be responsible for the production of an abnormal purine or pyrimidine base. Phage prepared on the mutator strain were used to transduce selected purine and pyrimidine LT2 mutants that do not possess this gene. A high frequency (60%) of cotransduction was observed with mutants from only one locus, purA. Transduction of additional mutants from this region gave similar results, except for one mutant (purA1) which showed no transduction of the mutator gene or the purA1 region. The results show that the mutator gene is very closely linked to the purA locus and suggest that it might be part of it.     

Molecular Analysis of a Salmonella Enterica Group E1 Rfb Gene Cluster: O Antigen and the Genetic Basis of the Major Polymorphism

Salmonella enterica is highly polymorphic for the O antigen, a surface polysaccharide that is subject to intense selection by the host immune system. This polymorphism is used for serotyping Salmonella isolates. The genes encoding O antigen biosynthesis are located in the rfb gene cluster. We report here the cloning and sequence of the 19-kb rfb region from strain M32 (serovar anatum, group E1) and compare it with that of strain LT2 (serovar typhimurium, group B). Genes for biosynthetic pathways common to both strains are conserved and have very similar sequences. In contrast, the five genes for CDP-abequose synthesis, present in strain LT2, are absent in strain M32; three open reading frames (ORFs) of strain LT2, thought to include genes for transferases, are not present in strain M32 but are replaced by three different ORFs with little or low level of similarity. Both rfb gene clusters are low in G + C content, indicating that they were transferred from a common ancestral species with low G + C content to S. enterica relatively recently (in the evolutionary sense). We discuss the recombination and lateral transfer events which may have been involved in the evolution of the polymorphism.     

Survival of dehydrated cells of Salmonella typhimurium LT2 at high temperatures

Cells of Salmonella typhimurium LT2 were dehydrated on hydrophobic membranes (Millipore FGLP2500) placed in a controlled atmosphere chamber held at 57% equilibrium relative humidity (ERH) and 37°C. Dehydration for 48 h under the above conditions increased the heat resistance of Salm. typhimurium LT2 when measured as the surviving fraction after a heat challenge of 135°C for 30 min. Results also showed that little or no death occurred during heat challenges of 1 h at temperatures of up to 100°C. The survival of Salm. typhimurium LT2 was measured as the ability to form colonies on solid media tryptone soy broth plus 1.2% agar (TSBA) after 24 h at 37°C. Incorporation of sodium pyruvate, at a concentration of 0.2% into the recovery medium, did not enhance the recovery of heated Salm. typhimurium LT2. Dehydrated cells of S. typhimurium LT2 showed a triphasic death curve. Increasing the period of dehydration from 48 h to 34 d, reduced initial numbers due to die off but did not alter the shape of the subsequent survival curve. and accepted 22 June 1989     

Survival of dehydrated cells of Salmonella typhimurium LT2 at high temperatures

Cells of Salmonella typhimurium LT2 were dehydrated on hydrophobic membranes (Millipore FGLP2500) placed in a controlled atmosphere chamber held at 57% equilibrium relative humidity (ERH) and 37 degrees C. Dehydration for 48 h under the above conditions increased the heat resistance of Salm. typhimurium LT2 when measured as the surviving fraction after a heat challenge of 135 degrees C for 30 min. Results also showed that little or no death occurred during heat challenges of 1 h at temperatures of up to 100 degrees C. The survival of Salm. typhimurium LT2 was measured as the ability to form colonies on solid media tryptone soy broth plus 1.2% agar (TSBA) after 24 h at 37 degrees C. Incorporation of sodium pyruvate, at a concentration of (TSBA) after 24 h at 37 degrees C. Incorporation of sodium pyruvate, at a concentration of 0.2% into the recovery medium, did not enhance the recovery of heated Salm. typhimurium LT2. Dehydrated cells of S. typhimurium LT2 showed a triphasic death curve. Increasing the period of dehydration from 48 h to 34 d, reduced initial numbers due to die off but did not alter the shape of the subsequent survival curve.     

Hemin-Deficient Mutants of Salmonella typhimurium

Nine hemin-deficient mutants of Salmonella typhimurium LT2 were isolated as neomycin-resistant colonies. Five of these mutants could be stimulated by Delta-aminolevulinic acid (Delta-ALA), thus representing hemA mutants. Since S. typhimurium LT2 is not able to incorporate hemin, the identification of the mutants not stimulated by Delta-ALA was made on the basis of the simultaneous loss of catalase activity and cytochromes. The hemA gene was mapped by conjugation in the trp region, probably in the order purB-pyrD-hemA-trp; the episome FT(71)trp does not carry the hemA gene. Transductional intercrosses by phage P22 indicate that hemA 11, 12, 13, and 37 are at very closely linked sites, whereas hemA14 is at a more distant site in the same or an adjacent gene. No joint transduction was detected between hemA and trp or pyrF. The loci affected in the other hemin-deficient mutants were linked in conjugation to the pro(+) marker (frequency of linkage, 88 to 97%), but cotransduction of the two markers could not be obtained. The episome F lac hem purE, which originates from Escherichia coli K-12, could complement these hemin-deficient mutants of S. typhimurium LT2. As a result, the sequence of the markers on the chromosome of S. typhimurium LT2 is probably pro heme purE, analogous to the sequence found in E. coli K-12. Thus, the chromosome of S. typhimurium also possesses two hem regions, with a location similar to that described in E. coli K-12.     

Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites

Abstract The susceptibility of Salmonella typhimurium LT2 and of S. typhi 1079 to oxygen metabolites were compared. S. typhimurium LT2 and S. typhi 1079 were killed to an equal extent (about 40%) by the xanthine-xanthine oxidase (200 mU/ml) system. Among the various scavengers of oxygen metabolites, catalase alone inhibited the killing of S. typhimurium LT2 and S. typhi 1079 by the xanthine-xanthine oxidase system, indicating that hydrogen peroxide contributed to the killing of Salmonellae . The respiratory burst of murine macrophages was efficiently triggered by the ingestion of S. typhimurium LT2, S. typhimurium SL1102, and S. typhi 1079 and all to the same extent. However, in the range of the concentration of hydrogen peroxide produced by murine macrophages, neither S. typhimurium LT2 nor S. typhi 1079 were killed. Only S. typhimurium SL1102, a rough mutant of S. typhimurium LT2, was markedly susceptible under these conditions. The findings suggest that both S. typhimurium LT2 and S. typhi 1079 are resistant to oxygen-dependent killing mechanisms.     

Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites

The susceptibility of Salmonella typhimurium LT2 and S. typhi 1079 to oxygen metabolites were compared. S. typhimurium LT2 and S. typhi 1079 were killed to an equal extent (about 40%) by the xanthine-xanthine oxidase (200 mU/ml) system. Among the various scavengers of oxygen metabolites, catalase alone inhibited the killing of S. typhimurium LT2 and S. typhi 1079 by the xanthine-xanthine oxidase system, indicating that hydrogen peroxide contributed to the killing of Salmonellae. The respiratory burst of murine macrophages was efficiently triggered by the ingestion of S. typhimurium LT2, S. typhimurium SL1102, and S. typhi 1079 and all to the same extent. However, in the range of the concentration of hydrogen peroxide produced by murine macrophages, neither S. typhimurium LT2 nor S. typhi 1079 were killed. Only S. typhimurium SL1102, a rough mutant of S. typhimurium LT2, was markedly susceptible under these conditions. The findings suggest that both S. typhimurium LT2 and S. typhi 1079 are resistant to oxygen-dependent killing mechanisms.     

Salmonella typhimurium HfrA, a mutant in which adenosine triphosphate can drive amino acid transport but not energy-dependent nicotinamide nucleotide transhydrogenation.

In contrast with wild-type Salmonella typhimurium LT2, strain HfrA did not have ATP-driven energy-dependent transhydrogenase activity, although ATP-dependent quenching of atebrin fluorescence was normal. Respiration-dependent and energy-independent transhydrogenase, and Ca2+-activated ATPase (adenosine triphosphatase) activities were similar in both strains. Purified ATPases from the two strains had similar specific activities, similar subunit polypeptides, and were equally effective in restoring energy-dependent transhydrogenase activities to membrane particles of strain LT2 from which the ATPase had been stripped. The purified ATPases from both strains could restore respiration-dependent but not ATP-dependent transhydrogenation to stripped particles of strain HfrA. Both strains grew aerobically equally well on salts media containing glucose, malate, succinate, citrate, acetate, pyruvate, fumarate, lactate or aspartate as substrates. Growth on glucose under anaerobic conditions was similar. Strains LT2 and HfrA were equally effective in the accumulation under both aerobic and anaerobic conditions of the amino acids proline, phenylalanine, histidine, lysine, isoleucine and aspartic acid. Inhibition of amino acid accumulation by KCN and dicyclohexylcarbodi-imide occurred to the same extent in both strains. The complete inhibition by dicyclohexylcarbodi-imide of amino acid uptake under anaerobic conditions suggested that ATP could drive amino acid uptake in both strains. The ability of strain HfrA to carry out ATP-dependent transport or quenching of atebrin fluorescence but not ATP-dependent transhydrogenation is different from the wild-type strain and from any previously described energy-coupling mutant. It is difficult to reconcile the properties of this mutant with the chemiosmotic hypothesis.     

Exclusion of high-molecular-weight maltosaccharides by lipopolysaccharide O-antigen of Escherichia coli and Salmonella typhimurium.

The barrier properties of lipopolysaccharide were studied by testing the influence of O-antigen on the binding of ligand to maltoporin in the outer membranes of Escherichia coli and Salmonella typhimurium. Maltoporin (LamB protein) of Escherichia coli K-12 was capable of interacting with macromolecular starch polysaccharides, as was previously shown by the binding of intact bacteria to fluorescein-labeled amylopectin or to starch-Sepharose columns. In contrast, strains with complete O-antigenic lipopolysaccharide showed reduced binding to these substrates. A similar result was obtained with Salmonella typhimurium LT2, which did not bind to starch unless rfa mutations removed noncore polysaccharide. The exclusion limit of the lipopolysaccharide permeability barrier to alpha-glucans was tested by measuring the maltoporin-dependent transport of maltose and its inhibition by maltodextrins of various sizes. Only amylopectin (molecular weight, greater than 25,000) was excluded in transport experiments, whereas maltodextrins with molecular weights of up to 2,000 were not excluded by the presence of an O-polysaccharide layer.