Differences between the anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferases of Salmonella typhimurium strains LT2 and LT7.

The anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferases (PRT), coded by the second structural gene (trpB) of the tryptophan (trp) operon in strains LT2 and LT7 of Salmonella typhimurium, differ from each other in a number of parameters. These include the apparent Km values for their substrates anthranilic acid and 5-phosphoribosylpyrophosphate, thermostability, sensitivity to substrate inhibition by anthranilic acid, as well as end-product inhibition by tryptophan and specific activity. The PRT of strain LT7 further differs from that of strain LT2 in that its apparent Km for 5-phosphoribosylpyrophosphate is three to seven times higher when associated with anthranilate synthase in the enzyme complex which catalyses the first two steps of tryptophan biosynthesis than in its free uncomplexed form, which the PRT of strain LT2 shows the same apparent Km for this substrate in both its free and complexed forms. These results confirm and extend the finding of Stuttard (1975) that strains LT2 and LT7 differ genetically form each other at a single site within region II of the trpB gene.     

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.     

Electrotransformation in Salmonella typhimurium LT2

Electroporation gives high efficiency of transformation in Salmonella typhimurium LT2, yielding 10(8)-10(9) electrotransformants per microgram of pBR322 DNA. Lipopolysaccharide (LPS) composition has little influence on electrotransformation efficiency by electroporation, unlike Ca2+ shock methods, which give ca. 10(6) transformants/microgram DNA with strains with Rc or Rd2 LPS, 10(4) transformants with most smooth and rough strains, and 10(2) transformants with strains with Re LPS. Thus cell envelope properties are less crucial in electrotransformation than in Ca2+ shock methods. The reciprocal restriction barrier between Escherichia coli K-12 and S. typhimurium LT2 reduces electrotransformation by ca. 100-fold, but host-restriction mutants reduce or eliminate the barrier.     

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.     

Spontaneous conversion between mutL and 6 bpDeltamutL in Salmonella typhimurium LT7: association with genome diversification and possible roles in bacterial adaptation

Previously, we reported the phenomenon of genome diversification in Salmonella typhimurium LT7, i.e., individual strains derived from LT7 kept changing the genome structure by inversions, translocations, duplications, and mutations. To elucidate the genetic basis, we sequenced selected genes of the mismatch repair (MMR) system for correlations between MMR defects and genome diversification. We chose S. typhimurium LT7 mutants 8111F2 and 9052D1 for mut gene sequence analyses and found that both mutants had a deletion of one of three tandem 6-bp repeats, GCTGGC GCTGGC GCTGGC, within mutL, which was designated 6 bpDeltamutL. mutS and mutH genes were unchanged in the mutants analyzed. Some sublines of 8111F2 and 9052D1 spontaneously stopped the genome diversification process at certain stages during single-colony restreaking passages, and in these strains the 6 bpDeltamutL genotype also became wild-type mutL. We conclude that conversion between mutL and 6 bpDeltamutL occurs spontaneously and that transient defects of mutL facilitate genome diversification without leading to the accumulation of multiple detrimental genetic changes. Spontaneous conversion between mutL and 6 bpDeltamutL may be an important mechanism used by bacteria to regulate genetic stability in adaptation to changing environments.     

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.     

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.     

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 nature of typhimuricin--a substance produced by Salmonella typhimurium LT2 and relation of its synthesis to the cryptic plasmid

The effect of typhimuricin on Escherichia coli K12 cells and the properties of this substance produced by Salmonella typhimurium LT2 have been studied. The obtained data permit one to conclude that typhimuricin is similar to the aminoacid L-valine or the chemical very similar to it in properties. The synthesis of typhimuricin is not controlled by the cryptic plasmid present in Salmonella typhimurium LT2 cells. The experiments aimed at isolation of enterobacterial strains producing low molecular mass antibiotics--microcines have been done. It was found that the capability to produce macrocines is not widespread among the isolates of enterobacteria.     

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.     

Salmonella typhimurium LT2 possesses three distinct 23S rRNA intervening sequences.

The rrl genes for 23S rRNA of Salmonella typhimurium LT2 are known to carry intervening sequences (IVSs) at two sites, helix-25 and helix-45, which are excised by RNase III during rRNA maturation, resulting in rRNA which is fragmented but nevertheless functional. We isolated DNA fragments containing the seven rrl genes from BlnI, I-CeuI, and SpeI genomic digests following pulsed-field gel electrophoresis and used these DNA fragments as templates for PCRs utilizing primers upstream and downstream of helix-25 and helix-45. Variance in amplicon length and cycle sequencing indicated that rrlG and rrlH have IVSs in helix-25 of approximately 110 bp which are only 56% identical. rrnA, rrnB, rrnC, rrnD, rrnE, and rrnH have IVSs of approximately 90 bp in helix-45, and all have the same nucleotide sequence. Twenty-one independent wild-type strains of S. typhimurium from Salmonella Reference Collection A were analyzed for IVSs by using PCRs with genomic DNAs and by denaturing agarose electrophoresis of RNAs. Many strains resemble LT2, but some have no IVSs in helix-25 and others have IVSs in helix-45 in all seven rrl genes. However, the IVSs in individual wild-type lines are relatively stable, for several LT2 isolates separated over many years by many single-colony isolations are indistinguishable from one another, with the exception of line LB5010, which differs by one helix-25 IVS. We postulate that IVSs have entered strain LT2 by three independent lateral-transfer events and that the IVS in helix-45 was dispersed to and maintained in the same sequence in six of the seven rrl genes by the mechanism of gene conversion.     

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.     

Identification and characterization of the mutL and mutS gene products of Salmonella typhimurium LT2.

The gene products of the mutL and mutS loci play essential roles in the dam-directed mismatch repair in both Salmonella typhimurium LT2 and Escherichia coli K-12. Mutations in these genes result in a spontaneous mutator phenotype. We have cloned the mutL and mutS genes from S. typhimurium by generating mutL- and mutS-specific probes from an S. typhimurium mutL::Tn10 and an mutS::Tn10 strain and using these to screen an S. typhimurium library. Both the mutL and mutS genes from S. typhimurium were able to complement E. coli mutL and mutS strains, respectively. By a combination of Tn1000 insertion mutagenesis and the maxicell technique, the products of the mutL and mutS genes were shown to have molecular weights of 70,000 and 98,000 respectively. A phi (mutL'-lacZ+) gene fusion was constructed; no change in the expression of the fusion could be detected by treatment with DNA-damaging agents. In crude extracts, the MutS protein binds single-stranded DNA, but not double-stranded DNA, with high affinity.     

An inducible pathway is required for mutagenesis in Salmonella typhimurium LT2.

UV mutability of Salmonella typhimurium LT2 was eliminated in the presence of a multicopy plasmid carrying the Escherichia coli lexA+ gene. This result suggests that inducible, SOS-like functions are required for UV mutagenesis in S. typhimurium. S. typhimurium strains carrying either point or deletion mutations in topA had previously been shown to lose their mutability by UV or methyl methanesulfonate (K. Overbye and P. Margolin, J. Bacteriol. 146:170-178, 1981; K. Overbye, S. M. Basu, and P. Margolin, Cold Spring Harbor Symp. Quant. Biol. 47:785-791, 1983). Mitomycin C induction of the phi(mucB'-lacZ') fusion (a DNA damage-inducible locus carried on plasmid pSE205) in S. typhimurium topA was normal, suggesting that RecA is activated in topA mutants. These observations lead us to deduce that S. typhimurium has at least one DNA damage-inducible locus in addition to recA that is required for UV mutability.     

Genetic mapping of a mutation conferring sensitivity to bacteriophage Mu in Salmonella typhimurium LT2.

Two strains of Salmonella typhimurium LT2, SA1475 and MA411, were fortuitously found to be sensitive to bacteriophage Mu. The Mu-sensitivity allele of SA1475 was called musA1 and shown to be linked to the histidine operon both in conjugation and transduction experiments. The Mus allele of MA411 was unlinked to the his region and was tentatively designated musB2. Strains carrying large deletions of the his operon were also tested for Mu sensitivity; those of which the his-rib region is deleted were also sensitive to Mu. Transduction data led to the order zee-2 hisOGDCBAHFIE gnd musA. An Hfr injecting the his operon early (HfrK9) an carrying hisG9424::Tn10 delta 4 delta 11 and musA1 was isolated; this Hfr made it possible to introduce the Mus character into most derivatives of S. typhimurium LT2. Since strain SA1475 is resistant to bacteriophage P1, it could be used to select a new P1-Mu hybrid which has the host range of Mu and the transduction properties of P1.     

Salmonella typhimurium LT2 strains which are r- m+ for all three chromosomally located systems of DNA restriction and modification.

We describe the derivation of two strains of Salmonella typhimurium LT2 which are r- m+ for all three of the known chromosomal genes for the restriction and modification of DNA, hsdLT, hsdSA, and hsdSB; the strains were designated LB5000 and LB5010. LB5000 is a smooth derivative sensitive to phage P22; LB5010 is a galE strain sensitive to phage P1.     

β-Lactamases of R Factors Derived from Shigella and Salmonella Strains

R. factors conferring resistance to penicillins and cephalosporins were transferred by conjugation from six strains of Shigella and from four strains of Salmonella typhimurium to a standard strain of S. typhimurium LT2. The beta-lactamases produced were then characterized by using cell-free extracts. The enzymes were of two types, I and II, with respect to specific activity against benzyl penicillin, substrate profile, K(m), pH optimum, temperature optimum, inhibition by chloride and nitrate ions, and heat inactivation. The six type I enzymes were associated with R factors from Shigella strains; five of these were R(f) factors. The four type II enzymes were associated with R factors from S. typhimurium; all these were R(i) factors.     

Spontaneous mutators of salmonella typhimurium LT2 generated by insertion of transposable elements.

Spontaneous mutators of Salmonella typhimurium LT2 were generated by inserting the transposable element Tn5 or Tn10 into the bacterial chromosome. Two mutators mapped at the position of the mutH and mutL loci of S. typhimurium, and two other mutators mapped at positions corresponding to the mutS and uvrD loci of Escherichia coli. A fifth mutator, mutB, did not map at a position corresponding to any of the known mutators of S. typhimurium or E. coli. The mutH,L,S and uvrD alleles increased the frequency of both spontaneous base substitution and frameshift mutations, whereas the mutB allele increased the frequency only of spontaneous base substitution mutations. The increased frequency of base substitution mutations was recA+ independent in the mutH, mutL, and uvrD strains and partially recA+ independent in the mutS strain. The uvrD mutation decreased the resistance of the cells to killing by ultraviolet irradiation. The mutH,L,S and uvrD strains showed an increased sensitivity to mutagenesis by the alkylating agents methyl methane sulfonate and ethyl methane sulfonate, but not to mutagenesis by 4-nitroquinoline-1-oxide.     

Transport and metabolism of vitamin B6 in Salmonella typhimurium LT2.

Salmonella typhimurium LT2 concentrates radioactivity intracellularly from [3H]pyridoxal or [3H]pyridoxine up to 25 times the external concentration. After 1 min of uptake intracellular radioactivity is found as phosphorylated vitamin B6. The process is sensitive to temperature and is maximally active at pH 8.1, but under the conditions tested it is insensitive to monovalent cations or metabolic inhibitors, and does not require an exogenous energy source. The Km values for uptake of pyridoxine and pyridoxal are 2.0 x 10(-7) M and 1.2 x 10(-7) M, respectively; [3H]pyridoxamine is not transported. Evidence is presented for an uptake mechanism involving facilitated diffusion followed by trapping by pyridoxal kinase. S. typhimurium also appears to lack a periplasmic binding protein for vitamin B6.