A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.

This report describes a set of Escherichia coli and Salmonella typhimurium strains that permits the reversible transfer of lac fusions between a plasmid and either bacterial chromosome. The system relies on homologous recombination in an E. coli recD host for transfer from plasmid to chromosome. This E. coli strain carries the S. typhimurium put operon inserted into trp, and the resulting fusions are of the form trp::put::[Kanr-X-lac], where X is the promoter or gene fragment under study. The put homology flanks the lac fusion segment, so that fusions can be transduced into S. typhimurium, replacing the resident put operon. Subsequent transduction into an S. typhimurium strain with a large chromosomal deletion covering put allows selection for recombinants that inherit the fusion on a plasmid. A transposable version of the put operon was constructed and used to direct lac fusions to novel locations, including the F plasmid and the ara locus. Transductional crosses between strains with fusions bearing different segments of the hemA-prfA operon were used to determine the contribution of the hemA promoter region to expression of the prfA gene and other genes downstream of hemA in S. typhimurium.     

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.     

Locations of the opp and supX genes of Salmonella typhimurium and Escherichia coli.

The chromosomal locations of the supX and opp loci of Salmonella typhimurium LT2 and Escherichia coli K-12 were identified and found to result in the same gene sequence in both species, namely, pyrF-cysB-supX-trpPOLEDCBA-tonB(chr)-opp. These results differ from a previously reported location of the opp gene on the E. coli chromosome. Evidence indicates that the opp gene lies between chr(tonB) and galU in S. typhimurium.     

Influence of Chromosome Structure on the Frequency of tonB trp Deletions in Escherichia coli

The frequency of tonB trp deletions varies in different strains and substrains of Escherichia coli. Studies with chromosomal hybrids constructed by transducing various segments of the cysB-trp-suIII region from K-12(Ymel) into K-12(W3110) indicate that the characteristic low deletion frequency of K-12(Ymel) is determined largely by the (genetic) structure of the trp-suIII region of the chromosome. Transduction of the trp region from K-12(W3110) or K-12(Ymel) into strain B has little effect on the frequency of tonB trp deletions in that strain. When tonB trp deletions occur at 42 C rather than at 37 C, there is a significant reduction in the frequency of deletions in all strains examined except K-12(Ymel) and hybrids exhibiting a Ymel deletion pattern. The magnitude of this temperature effect in different K-12 strains increases proportionally with the frequency of tonB trp deletions at 37 C. At 42 C the frequency of tonB trp deletions in all K-12 strains approaches the low frequency observed for Ymel at 37 or 42 C. In contrast, spontaneous deletions in another region of the genome which simultaneously result in resistance to phages T7 and lambda and in proline auxotrophy (tfrA pro deletions) occur at a constant frequency regardless of growth temperature or the structure of the chromosome in the trp region. Two mutants of strain KB30 obtained after treatment with nitrosoguanidine show very low tonB trp deletion frequencies. The alterations in both mutants map in the trp region of the chromosome. These studies indicate that the structure of the cysB-trp-suIII region is responsible for many of the characteristic deletion frequencies observed.     

Gene ilvY of Salmonella typhimurium.

Evidence is presented for the existence in Salmonella typhimurium LT2 of the regulatory gene ilv Y. The Escherichia coli K-12 ilvY gene product is shown to complement a S. typhimurium ilvY mutation in vivo.     

Properties of cysK mutants of Escherichia coli K12.

Triazole and azaserine resistant mutants of E. coli K12 affecting cysK gene coding for O-acetylserine sulphydrylase were isolated. The cysK gene in E. coli is located in the same region of chromosome as the cycK gene in Salmonella typhimurium. All azaserine and some triazole resistant mutants require cysteine for growth at a normal rate. The cysK mutants have reduced sulphate uptake. Stability and transfer by conjugation of triazole resistant phenotype were checked. Differences in sulphate metabolism between closely related organisms E. coli and S. typhimurium are discussed.     

Derepression of F factor function in Salmonella typhimurium

In Salmonella typhimurium LT2 the F factor of Escherichia coli K-12 replicates normally but is repressed; Flac+ cells give no visible lysis on solid media with male-specific phages, low frequency transfer of Flac+ (0.001-0.007 per donor cell), few f2 infective centers (0.002-0.006 per cell), and they propagate male-specific phages to low titers. Thus they display a Fin+ (fertility inhibition) phenotype. This repression, owing to pSLT, a 60 Mdal plasmid normally resident in S. typhimurium, was circumvented by the following materials: (i) Flac+ plasmids from E. coli with mutations in finP or traO; (ii) a S. typhimurium line which had been cured of pSLT; (iii) pKZl, a KmR plasmid in the same Inc group as pSLT, which caused expulsion of pSLT and made Fin- lines; (iv) F-Fin- mutants which originated spontaneously and which are present in most Hfr strains of S. typhimurium. Strains which are derepressed for F function by the above methods give visible lysis on solid media with male-specific phages, ca. 1.0 Lac+ recombinants per donor cell in conjugal transfer, ca. 0.82 f2 infective centers per cell, over 80% of cells with visible F pili, and propagation of male-specific phages to high titer. These data confirm earlier observations that pSLT represses F by the FinOP system. In addition, it shows that there is no other mechanism which represses F function in S. typhimurium. If donor function is derepressed by one of the above methods, and if rough recipient strains are used, F-mediated conjugation in S. typhimurium LT2 is as efficient as in E. coli K-12.     

crp genes of Shigella flexneri, Salmonella typhimurium, and Escherichia coli.

The complete nucleotide sequences of the Salmonella typhimurium LT2 and Shigella flexneri 2B crp genes were determined and compared with those of the Escherichia coli K-12 crp gene. The Shigella flexneri gene was almost like the E. coli crp gene, with only four silent base pair changes. The S. typhimurium and E. coli crp genes presented a higher degree of divergence in their nucleotide sequence with 77 changes, but the corresponding amino acid sequences presented only one amino acid difference. The nucleotide sequences of the crp genes diverged to the same extent as in the other genes, trp, ompA, metJ, and araC, which are structural or regulatory genes. An analysis of the amino acid divergence, however, revealed that the catabolite gene activator protein, the crp gene product, is the most conserved protein observed so far. Comparison of codon usage in S. typhimurium and E. coli for all genes sequenced in both organisms showed that their patterns were similar. Comparison of the regulatory regions of the S. typhimurium and E. coli crp genes showed that the most conserved sequences were those known to be essential for the expression of E. coli crp.     

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.     

Genetic and biochemical analyses of pantothenate biosynthesis in Escherichia coli and Salmonella typhimurium.

Pantothenate (pan) auxotrophs of Escherichia coli K-12 and Salmonella typhimurium LT2 were characterized by enzymatic and genetic analyses. The panB mutants of both organisms and the pan-6 ("panA") mutant of S. typhimurium are deficient in ketopantoate hydroxymethyltransferase, whereas the panC mutants lack pantothenate synthetase. panD mutants of E. coli K-12 were previously shown to be deficient in aspartate 1-decarboxylase. All mutants showed only a single enzyme defect. The finding that the pan-6 mutant was deficient in ketopantoate hydroxymethyltransferase indicates that the genetic lesion is a panB allele. The pan-6 mutant therefore is deficient in the utilization of alpha-ketoisovalerate rather than the synthesis of alpha-ketoisovalerate, as originally proposed. The order of the pan genes of E. coli K-12 was determined by phage P1-mediated three-factor crosses. The clockwise order was found to be aceF panB panD panC tonA on the genetic map of E. coli K-12. The three-factor crosses were greatly facilitated by use of a closely linked Tn10 transposon as the outside marker. We also found that supplementation of E. coli K-12 auxotrophs with a high concentration of pantothenate or beta-alanine increased the intracellular coenzyme A level two- to threefold above the normal level. Supplementation with pantoate or ketopantoate resulted in smaller increases.     

Genetics of Sensitivity of Salmonella Species to Colicin M and Bacteriophages T5, T1, and ES18

Nearly all of 62 strains of Salmonella paratyphi B were sensitive to colicin M and phage T5 but resistant to phages T1 and ES18 and to colicin B. All tested S. typhimurium strains were resistant to colicin M and phage T5, and many were sensitive to phage ES18. A rough S. typhimurium LT2 strain given the tonA region of Escherichia coli or S. paratyphi B became sensitive to colicin M and phage T5. We infer that the tonA allele of S. paratyphi B, like that of E. coli, determines an outer membrane protein that adsorbs T5 and colicin M but not phage ES18, whereas the S. typhimurium allele determines a protein able to adsorb only ES18. The partial T1 sensitivity of a rough LT2 strain with a tonA allele from E. coli or S. paratyphi B and also the tonB(+) phentotype of an E. coli B trp-tonB Delta mutant carrying an F' trp of LT2 origin showed that S. typhimurium LT2 has a tonB allele like that of E. coli with respect to determination of sensitivity to colicins and phage T1. Rough S. paratyphi B, although T5 sensitive, remained resistant to T1 even when given F' tonB(+) of E. coli origin. Classes of Salmonella mutants selected as resistant to colicin M included: T5-resistant mutants, probably tonA(-); mutants unchanged except for M resistance, perhaps tolerant; and Exb(+) mutants, producing a colicin inhibitor (presumably enterochelin). Some Exb(+) mutants were resistant to a bacteriocin inactive on E. coli but active on all tested S. paratyphi B and S. typhimurium strains (and on nearly all other tested Salmonella). A survey showed sensitivity to colicin M in several other species of Salmonella.     

Molecular analysis of the rfaD gene, for heptose synthesis, and the rfaF gene, for heptose transfer, in lipopolysaccharide synthesis in Salmonella typhimurium.

We report the analysis of three open reading frames of Salmonella typhimurium LT2 which we identified as rfaF, the structural gene for ADP-heptose:LPS heptosyltransferase II; rfaD, the structural gene for ADP-L-glycero-D-manno-heptose-6-epimerase; and part of kbl, the structural gene for 2-amino-3-ketobutyrate CoA ligase. A plasmid carrying rfaF complements an rfaF mutant of S. typhimurium; rfaD and kbl are homologous to and in the same location as the equivalent genes in Escherichia coli K-12. The RfaF (heptosyl transferase II) protein shares regions of amino acid homology with RfaC (heptosyltransferase I), RfaQ (postulated to be heptosyltransferase III), and KdtA (ketodeoxyoctonate transferase), suggesting that these regions function in heptose binding. E. coli contains a block of DNA of about 1,200 bp between kbl and rfaD which is missing from S. typhimurium. This DNA includes yibB, which is an open reading frame of unknown function, and two promoters upstream of rfaD (P3, a heat-shock promoter, and P2). Both S. typhimurium and E. coli rfaD genes share a normal consensus promoter (P1). We postulate that the yibB segment is an insertion into the line leading to E. coli from the common ancestor of the two genera, though it could be a deletion from the line leading to S. typhimurium. The G+C content of the rfaLKZYJI genes of both S. typhimurium LT2 and E. coli K-12 is about 35%, much lower than the average of enteric bacteria; if this low G+C content is due to lateral transfer from a source of low G+C content, it must have occurred prior to evolutionary divergence of the two genera.     

Identification of the enzymatic basis for delta-aminolevulinic acid auxotrophy in a hemA mutant of Escherichia coli.

The hemA mutation of Escherichia coli K-12 confers a requirement for delta-aminolevulinic acid (ALA). Cell extract prepared from the hemA strain SASX41B was incapable of producing ALA from either glutamate or glutamyl-tRNA, whereas extract of the hem+ strain HB101 formed colorimetrically detectable amounts of ALA and transferred label from 1-[14C]glutamate and 3,4-[3H]glutamyl-tRNA to ALA. Extracts of both strains converted glutamate-1-semialdehyde to ALA and were capable of aminoacylating tRNAGlu. Glutamyl-tRNA formed by extracts of both strains could be converted to ALA by the extract of hem+ cells. The extract of hemA cells did not convert glutamyl-tRNA formed by either strain to ALA. However, the hemA cell extract, when supplemented in vitro with glutamyl-tRNA dehydrogenase isolated from Chlorella vulgaris cells, formed about as much ALA as did the unsupplemented hem+ cell extract. We conclude from these observations that the enzyme activity that is lacking in the ALA auxotrophic strain carrying the hemA mutation is that of glutamyl-tRNA dehydrogenase.     

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.     

Initiator (DnaA) protein concentration as a function of growth rate in Escherichia coli and Salmonella typhimurium.

The DnaA protein concentration was determined in five different Escherichia coli strains and in Salmonella typhimurium LT2 growing at different growth rates. The DnaA protein concentration was found to be invariant over a wide range of growth rates in the four E. coli K-12 strains and in S. typhimurium. In E. coli B/r the DnaA protein concentration was generally higher than in the K-12 strains, and it increased with decreasing growth rates. For all the strains, there appears to be a correlation between the DnaA protein concentration and the initiation mass. This supports the concept of the concentration of DnaA protein setting the initiation mass and, thus, that the DnaA protein is a key molecule in the regulation of initiation of chromosome replication in members of the family Enterobacteriaceae.     

Characterization of the Salmonella typhimurium phoE gene and development of Salmonella-specific DNA probes.

In Escherichia coli K-12, the phoE gene, encoding a phosphate-limitation-inducible outer membrane pore protein (PhoE), is closely linked to the genes proA and proB. When the corresponding fragment of the Salmonella typhimurium chromosome was transferred to E. coli K-12 using an RP4::miniMu plasmid, pULB113, no production of S. typhimurium PhoE could be detected. Nevertheless, DNA hybridization studies revealed that the corresponding plasmid did contain S. typhimurium phoE. Production of S. typhimurium PhoE in E. coli was detected only after subcloning the gene in a multicopy vector. Nucleotide (nt) sequence analysis showed extensive homology of S. typhimurium phoE to the E. coli gene and suggested possible explanations for the low expression of S. typhimurium phoE in E. coli. In addition, the sequence information was used to develop Salmonella-specific DNA probes. Two oligodeoxyribonucleotides were synthesized based on nt sequences encoding the fifth and eighth cell-surface-exposed regions of PhoE. When used in polymerase chain reactions, these probes turned out to be specific, i.e., no crossreactions occurred with the non-Salmonella strains, whereas 132 out of 133 tested Salmonella strains were recognized.     

Transfer of episome F'lac+ and chromosomal trp+ genes from Erwinia amylovora to Salmonella typhimurium.

The F'lac+ episome of Escherichia coli origin was transferred by conjugation with frequencies of 10(-7) to 10(-5) from Erwinia amylovora to 14 out of 15 Salmonella typhimurium trp female parents. The chromosomal trp+ genes were transferred with frequencies of 10(-7) to 10(-6) only to one trpB and 2 trpD female parents, which have a point mutation in the 2nd and fourth structural genes, respectively, of the tryptophan operon. The transferred male trp+ genes became integrated at the selected sites of the S. tryphimurium chromosome. The resulting Trp+ hybrids were phenotypically stable, lacked a cryptic trp allele selected against in the female parent, had high genetic homology values in the tryptophan region, and showed biochemical reactions and pathogenicity typical of S. typhimurium.     

Biosynthesis of bacterial glycogen: primary structure of Salmonella typhimurium ADPglucose synthetase as deduced from the nucleotide sequence of the glgC gene.

The nucleotide sequence of a 1.4-kilobase-pair fragment containing the Salmonella typhimurium LT2 glgC gene coding for ADPglucose synthetase was determined. The glgC structural gene contains 1,293 base pairs, having a coding capacity of 431 amino acids. The amino acid sequence deduced from the nucleotide sequence shows that the molecular weight of ADPglucose synthetase is 45,580. Previous results of the total amino acid composition analysis and amino acid sequencing (M. Lehmann and J. Preiss, J. Bacteriol. 143:120-127, 1980) of the first 27 amino acids from the N terminus agree with that deduced from nucleotide sequencing data. Comparison of the Escherichia coli K-12 and S. typhimurium LT2 ADPglucose synthetase shows that there is 80% homology in their nucleotide sequence and 90% homology in their deduced amino acid sequence. Moreover, the amino acid residues of the putative allosteric sites for the physiological activator fructose bisphosphate (amino acid residue 39) and inhibitor AMP (amino acid residue 114) are identical between the two enzymes. There is also extensive homology in the putative ADPglucose binding site. In both E. coli K-12 and S. typhimurium LT2, the first base of the translational start ATG of glgA overlaps with the third base TAA stop codon of the glgC gene.     

Ultraviolet Sensitivity Gene of Escherichia coli B

The ultraviolet sensitivity gene of Escherichia coli B was introduced into a K-12 recipient by transduction with phage P1. The uvs gene of E. coli B is cotransducible with the proC locus of K-12, is closely linked to tsx, is not linked to lacZ, and only rarely to purE. The transductants are mucoid, filamentous on irradiation, and show plating-medium response. The order of markers is lacZ proC tsx uvs purE.