Sequences of the envM gene and of two mutated alleles in Escherichia coli.

The nucleotide sequence of the Escherichia coli envM gene was determined. It codes for a protein of 262 amino acids. The sequences of the E. coli and Salmonella typhimurium EnvM proteins are 98% identical. Gene envM is preceded in E. coli by a 43-nucleotide-long structural element, termed 'box c', which occurs in several E. coli operons between structural genes. This sequence element is totally absent in S. typhimurium. Gene envM was mapped at coordinate position 1366.8 kb of the physical map of Kohara et al. (Cell, 1987, 50, 495-508). As in S. typhimurium, a Gly for Ser exchange at position 93 of the amino acid sequence leads to a diazaborine-resistant E. coli phenotype. A Ser for Phe exchange at position 241 of the EnvM protein results in a temperature-sensitive growth phenotype. Comparison of the EnvM amino acid sequence with sequences available in databases showed significant homology with the family of short-chain alcohol dehydrogenases.     

Genetic mapping of the chromosomal replication origin of Salmonella typhimurium.

Two hundred strains of Escherichia coli harboring Filv+ plasmids which carry a segment of the Salmonella typhimurium chromosome were isolated independently. Among them, two strains were found to harbor F' plasmids that are able to replicate in Hfr cells of E. coli; i.e., they carry a site designated poh (permissive on Hfr) of the S. typhimurium chromosome. The poh site is presumably identical with the replication origin (oriC) of the bacterial chromosome. These two plasmids carry the dnaA-uncA-rbs-ilv-cya-metE region of the chromosome of S. typhimurium. Other F' plasmids which only carried the ilv-cya-metE region were unable to be maintained in Hfr cells. The poh site (= oriC) of S. typhimurium thus is located in the uhp-ilv region of the chromosome. The two plasmids carrying the poh site of S. typhimurium can suppress the temperature-sensitive character of an E. coli mutant that carries the temperature-sensitive dnaA46 allele, when the plasmids exist in the mutant cells. This suggests that the dnaA chromosome in place of the dnaA gene product of E. coli itself. The ability of the plasmids carrying the poh site of S. typhimurium to replicate in Hfr cells of E. coli suggests that the replication system of E. coli can recognize the Salmonella replication origin.     

A single amino acid substitution in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase confers resistance to the herbicide glyphosate

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19), encoded by the aroA locus, is a target site of glyphosate inhibition in bacteria. A glyphosate-resistant aroA allele has been cloned in Escherichia coli from a mutagenized strain of Salmonella typhimurium. Subcloning of this mutant aroA allele shows the gene to reside on a 1.3-kilobase segment of S. typhimurium DNA. Nucleotide sequence analysis of this mutant gene indicates a protein-coding region 427 amino acids in length. Comparison of the mutant and wild type aroA gene sequences reveals a single base pair change resulting in a Pro to Ser amino acid substitution at the 101st codon of the protein. A hybrid gene fusion between mutant and wild type aroA gene sequences was constructed. 5-Enolpyruvylshikimate-3-phosphate synthase was prepared from E. coli cells harboring this construct. The glyphosate-resistant phenotype is shown to be associated with the single amino acid substitution described above.     

Biochemical and genetic analysis of Salmonella typhimurium and Escherichia coli mutants defective in specific incorporation of selenium into formate dehydrogenase and tRNAs

Mutation of a single gene, referred to as selA1 in Salmonella typhimurium and as selD in Escherichia coli, results in the inability of these organisms to insert selenium specifically into the selenopolypeptides of formate dehydrogenase and into the 2-selenouridine residues of tRNAs. The mutation does not involve transport of selenite into the cell or reduction of selenite to selenide since both mutant strains synthesize selenocysteine and selenomethionine from added selenite and incorporate these selenoamino acids non-specifically into numerous proteins of the bacterial cells. Complementation of the mutation in S. typhimurium with the selD gene from E. coli indicates functional identity of the selA1 and selD genes. Although the selA1 gene maps at approximately 21 min on the S. typhimurium chromosome and the selD gene at approximately 38 min on the E. coli chromosome, only a single gene in wild-type S. typhimurium hybridized to the E. coli selD gene probe. Transformation of the mutant Salmonella strain with a plasmid bearing the E. coli selD gene restored formate dehydrogenase activity, 75Se incorporation into formate dehydrogenase seleno-polypeptides and [75Se]seleno-tRNA synthesis. Transformation with an additional plasmid carrying an E. coli formate dehydrogenase selenopolypeptide-lacZ gene fusion showed that the selD gene allowed readthrough of the UGA codon and synthesis of beta-galactosidase in the Salmonella mutant.     

Sequence and complementation analysis of recF genes from Escherichia coli, Salmonella typhimurium, Pseudomonas putida and Bacillus subtilis: evidence for an essential phosphate binding loop.

We have compared the recF genes from Escherichia coli K-12, Salmonella typhimurium, Pseudomonas putida, and Bacillus subtilis at the DNA and amino acid sequence levels. To do this we determined the complete nucleotide sequence of the recF gene from Salmonella typhimurium and we completed the nucleotide sequence of recF gene from Pseudomonas putida begun by Fujita et al. (1). We found that the RecF proteins encoded by these two genes contain respectively 92% and 38% amino acid identity with the E. coli RecF protein. Additionally, we have found that the S. typhimurium and P. putida recF genes will complement an E. coli recF mutant, but the recF gene from Bacillus subtilis [showing about 20% identity with E. coli (2)] will not. Amino acid sequence alignment of the four proteins identified four highly conserved regions. Two of these regions are part of a putative phosphate binding loop. In one region (position 36), we changed the lysine codon (which is essential for ATPase, GTPase and kinase activity in other proteins having this phosphate binding loop) to an arginine codon. We then tested this mutation (recF4101) on a multicopy plasmid for its ability to complement a recF chromosomal mutation and on the E. coli chromosome for its effect on sensitivity to UV irradiation. The strain with recF4101 on its chromosome is as sensitive as a null recF mutant strain. The strain with the plasmid-borne mutant allele is however more UV resistant than the null mutant strain. We conclude that lysine-36 and possibly a phosphate binding loop is essential for full recF activity. Lastly we made two chimeric recF genes by exchanging the amino terminal 48 amino acids of the S. typhimurium and E. coli recF genes. Both chimeras could complement E. coli chromosomal recF mutations.     

Construction and characterization of mutations in hupB, the gene encoding HU-beta (HU-1) in Escherichia coli K-12.

Plasmid pJMC21 contains Escherichia coli chromosomal DNA encoding Lon protease, HU-beta (HU-1), and an unidentified 67,000-dalton protein. A kanamycin resistance cassette was used in the construction of insertion and deletion mutations in hupB, the gene encoding HU-beta on plasmid pJMC21. The reconstructed plasmids were linearized and used to introduce hupB chromosomal mutations into JC7623 (recBC sbcBC). These mutations, as expected, mapped in the 9.8-min region of the E. coli chromosome by P1 transduction (16% linkage to proC+). Southern blot hybridization of chromosomal fragments verified that hupB+ was replaced by the mutant allele, with no indication of gene duplication. All the mutant strains had growth rates identical to that of wild-type E. coli, were resistant to UV irradiation and nitrofurantoin, and supported the in vivo transposition-replication of bacteriophage Mu, Mu lysogenization, Tn10 transposition from lambda 1098, and lambda replication-lysogenization. The only observable phenotypic variation was a reduced Mu plaque size on the hupB mutant strains; however, the yield of bacteriophage Mu in liquid lysates prepared from the mutant strains was indistinguishable from the yield for the wild type.     

Phenol: a complex chemoeffector in bacterial chemotaxis.

Earlier observations that phenol is a repellent for Salmonella typhimurium but an attractant for Escherichia coli were confirmed. This behavioral difference was found to correlate with a difference in the effect phenol had on receptor methylation levels; it caused net demethylation in S. typhimurium but net methylation in E. coli. On the basis of mutant behavior and measurement of phenol-stimulated methylation, the attractant response of E. coli was shown to be mediated principally by the Tar receptor. In S. typhimurium, two receptors were found to be sensitive to phenol, namely, an unidentified receptor, which mediated the repellent response and showed phenol-stimulated demethylation; and the Tar receptor, which (as with E. coli) mediated the attractant response and showed phenol-stimulated methylation. In wild-type S. typhimurium, the former receptor dominated the Tar receptor, with respect to both behavior and methylation changes. However, when the amount of Tar receptor was artificially increased by the use of Tar-encoding plasmids, S. typhimurium cells exhibited an attractant response to phenol. No protein analogous to the phenol-specific repellent receptor was evident in E. coli, explaining the different behavioral responses of the two species toward phenol.     

DNA primase of plasmid ColIb is involved in conjugal DnA synthesis in donor and recipient bacteria.

The sog gene of the IncI alpha group plasmid ColIb is known to encode a DNA primase that can substitute for defective host primase in dnaG mutants of Escherichia coli during discontinuous DNA replication. The biological significance of this enzyme was investigated by using sog mutants, constructed from a derivative of ColIb by in vivo recombination of previously defined mutations in a cloned sog gene. The resultant Sog- plasmids failed to specify detectable primase activity and were unable to suppress a dnaG lesion. These mutants were maintained stably in E. coli, implying that the enzyme is not involved in vegetative replication of ColIb. However, the Sog- plasmids were partially transfer deficient in E. coli and Salmonella typhimurium matings, consistent with the hypothesis that the normal physiological role of this enzyme is in conjugation. This was confirmed by measurements of conjugal DNA synthesis. Studies of recipient cells have indicated that plasmid primase is required to initiate efficient synthesis of DNA complementary to the transferred strand, with the protein being supplied by the donor parent and probably transmitted between the mating cells. Primase specified by the dnaG gene of the recipient can substitute partially for the mutant enzyme, thus providing an explanation for the partial transfer proficiency of the mutant plasmids. Conjugal DNA synthesis in dnaB donor cells was deficient in the absence of plasmid primase, implying that the enzyme also initiates synthesis of DNA to replace the transferred material.     

Mutants carrying conditionally lethal mutations in outer membrane genes omsA and firA (ssc) are phenotypically similar, and omsA is allelic to firA.

We have previously identified the gene (the ssc gene) defective in the thermosensitive and antibiotic-supersusceptible outer membrane permeability mutant SS-C of Salmonella typhimurium and shown that this gene is analogous to the Escherichia coli gene firA (L. Hirvas, P. Koski, and M. Vaara, EMBO J. 10:1017-1023, 1991). Others have tentatively implicated firA in a different function, mRNA synthesis. Here we report that the defect in the thermosensitive outer membrane omsA mutant of E. coli (T. Tsuruoka, M. Ito, S. Tomioka, A. Hirata, and M. Matsuhashi, J. Bacteriol. 170:5229-5235, 1988) is due to a mutation in firA; this mutation changed codon 271 from serine to asparagine. The omsA-induced phenotype was completely reverted by plasmids containing wild-type firA or ssc. Plasmids carrying the omsA allele, or an identical mutant allele prepared by localized mutagenesis, under the control of lac elicited partial complementation. Transcomplementation studies with plasmids carrying various mutant alleles of the S. typhimurium gene indicated that the ability of these plasmids to complement the omsA mutation was similar to their ability to complement the ssc mutation. The antibiotic-supersusceptible phenotype of the omsA mutant closely resembled that of the ssc mutant, i.e., the omsA mutant was supersusceptible to hydrophobic antibiotics and large-peptide antibiotics against which the intact outer membrane is an effective permeability barrier. As previously demonstrated with the omsA mutant, the outer membrane of the ssc mutant became selectively ruptured after incubation for 1 h at the growth-nonpermitting temperature; 82% of the periplasmic beta-lactamase and less than 3% of the cytoplasmic marker enzyme were released into the medium. All of these findings are consistent with our concept that firA is an essential gene involved in generation of the outer membrane.     

Complementation of argG and hisA mutations of Escherichia coli by DNA cloned from the archaebacterium Methanococcus voltae

DNA derived from the methanogenic archaebacterium Methanococcus voltae was digested with PstI restriction endonuclease and cloned into the PstI site of pBR322. The recombinant plasmids generated were used to transform a multiply auxotrophic strain of Escherichia coli with selection for tetracycline resistance. Plasmids complementing the argG(pAW1) or hisA(pAW2) mutations were isolated and characterized. Nick-translated pAW1 and pAW2 hybridized to the predicted M. voltae PstI fragments but not to digested E. coli DNA. A novel 55,000-dalton protein was synthesized in UV-irradiated cells by pAW1, whereas pAW2 synthesized a novel 26,000-dalton protein. Derivatives of pAW1 carrying insertion elements no longer complemented the argG mutation and failed to produce the 55,000-dalton protein. When an AccI fragment was deleted from pAW2, complementation of hisA did not occur and no 26,000-dalton protein was synthesized. The effect of orientation of the cloned DNA within the vector on complementation and polypeptide synthesis was examined.     

Evidence for the ability of L10 ribosomal proteins of Salmonella typhimurium and Klebsiella pneumoniae to regulate rplJL gene expression in Escherichia coli

Genes rplJ, coding for ribosomal protein L10 of Salmonella typhimurium and Klebsiella pneumoniae, have been cloned on pUC plasmid. The resultant multicopy recombinant plasmids were detrimental for the growth of normal JM101 E. coli host cells and harmless for the mutant JF3029 host. This negative effect is the evidence for the ability of heterologous L10 proteins to regulate expression of rplJL genes in E. coli. Nucleotide sequence was determined completely for S. typhimurium rplJL' DNA portion and partially for rplJL' genes of K. pneumoniae. According to the nucleotide sequence data obtained three amino acid substitutions differ L10 proteins of S. typhimurium and E. coli and the long range, providing for the coupled translations of L10 and L7/L12 cistrons in E. coli mRNA is also valid for S. typhimurium and K. pneumoniae.     

Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities.

Salmonella typhimurium has a SOS regulon which resembles that of Escherichia coli. recA mutants of S. typhimurium have already been isolated, but no mutations in lexA have been described yet. In this work, two different lexA mutants of S. typhimurium LT2 have been constructed on a sulA background to prevent cell death and further characterized. The lexA552 and lexA11 alleles contain an insertion of the kanamycin resistance fragment into the carboxy- and amino-terminal regions of the lexA gene, respectively. SOS induction assays indicated that both lexA mutants exhibited a LexA(Def) phenotype, although SOS genes were apparently more derepressed in the lexA11 mutant than in the lexA552 mutant. Like lexA(Def) of E. coli, both lexA mutations only moderately increased the UV survival of S. typhimurium, and the lexA552 strain was as mutable as the lexA+ strain by UV in the presence of plasmids encoding MucAB or E. coli UmuDC (UmuDCEc). In contrast, a lexA11 strain carrying any of these plasmids was nonmutable by UV. This unexpected behavior was abolished when the lexA11 mutation was complemented in trans by the lexA gene of S. typhimurium. The results of UV mutagenesis correlated well with those of survival to UV irradiation, indicating that MucAB and UmuDCEc proteins participate in the error-prone repair of UV damage in lexA552 but not in lexA11. These intriguing differences between the mutagenic responses of lexA552 and lexA11 mutants to UV irradiation are discussed, taking into account the different degrees to which the SOS response is derepressed in these mutants.     

Cloning of the him genes encoding the integration host factor from Salmonella typhirmurium in E. coli

Abstract By hybridization of him A and him D gene probes from E. coli to chromosomal DNA of Salmonella typhimurium cross-hybridization was obtained in both cases. A gene bank of Salmonella DNA was isolated using the mini-Mu cloning system. This gene bank was transformed into either a prototrophic E. coli him A or him D mutant. Transformants complementing either the him A or him D defect were isolated on minimal medium plates supplemented with 40 μg/ml leucin at 42°C. The Salmonella him genes on these plasmids were further verified by their ability to plate phage Mu and to yield turbid plaques with phage lambda and by the ability of the recombinant plasmids to hybridize to E. coli him gene probes.     

Genetic and molecular analyses of Escherichia coli N-acetylneuraminate lyase gene.

Two plasmids containing the N-acetylneuraminate lyase (NALase) gene (nanA) of Escherichia coli, pNL1 and pNL4, were constructed. Immunoprecipitation analysis indicated that the 35,000-dalton protein encoded in pNL4 was NALase. The synthesis of NALase in E. coli carrying these plasmids was constitutive.     

Cloning, sequencing, and species relatedness of the Escherichia coli cca gene encoding the enzyme tRNA nucleotidyltransferase

The Escherichia coli cca gene which encodes the enzyme tRNA nucleotidyltransferase has been cloned by taking advantage of its proximity to the previously cloned dnaG locus. A series of recombinant bacteriophages, spanning the chromosomal region between the dnaG and cca genes at 66 min on the E. coli linkage map, were isolated from a lambda Charon 28 partial Sau3A E. coli DNA library using recombinant plasmids containing regions between dnaG and cca as probes. Two of the recombinant phage isolates, lambda c1 and lambda c4, contained the cca gene. A BamHI fragment from lambda c1 was subcloned into pBR328, and cells containing this recombinant plasmid, pRH9, expressed tRNA nucleotidyltransferase activity at about 10-fold higher level than the wild type control. The cca gene was further localized to a 1.4-kilobase stretch of DNA by Bal31 deletion analysis. The nucleotide sequence of the cca gene was determined by the dideoxy method, and revealed an open reading frame extending for a total of 412 codons from an initiator GTG codon that would encode a protein of about 47,000 daltons. Southern analysis using genomic blots demonstrated that the cca gene is present as a single copy on the E. coli chromosome and that there is no homology on the DNA level between the E. coli cca gene, and the corresponding gene in the Bacillus subtilis, Saccharomyces cerevisiae, Petunia hybrida, or Homo sapiens genomes. Homology was found only with DNA from the closely related species, Salmonella typhimurium. These studies have also allowed exact placement of the cca gene on the E. coli genetic map, and have shown that it is transcribed in a clockwise direction.     

sn-Glycerol-3-phosphate transport in Salmonella typhimurium

Salmonella typhimurium contains a transport system for sn-glycerol-3-phosphate that is inducible by growth on glycerol and sn-glycerol-3-phosphate. In fully induced cells, the system exhibited an apparent Km of 50 microM and a Vmax of 2.2 nmol/min . 10(8) cells. The corresponding system in Escherichia coli exhibits, under comparable conditions, a Km of 14 microM and a Vmax of 2.2 nmol/min . 10(8) cells. Transport-defective mutants were isolated by selecting for resistance against the antibiotic fosfomycin. They mapped in glpT at 47 min in the S. typhimurium linkage map, 37% cotransducible with gyrA. In addition to the glpT-dependent system, S. typhimurium LT2 contains, like E. coli, a second, ugp-dependent transport system for sn-glycerol-3-phosphate that was derepressed by phosphate starvation. A S. typhimurium DNA bank containing EcoRI restriction fragments in phage lambda gt7 was used to clone the glpT gene in E. coli. Lysogens that were fully active in the transport of sn-glycerol-3-phosphate with a Km of 33 microM and a Vmax of 2.0 nmol/min . 10(8) cells were isolated in a delta glpT mutant of E. coli. The EcoRI fragment harboring glpT was 3.5 kilobases long and carried only part of glpQ, a gene distal to glpT but on the same operon. The fragment was subcloned in multicopy plasmid pACYC184. Strains carrying this hybrid plasmid produced large amounts of cytoplasmic membrane protein with an apparent molecular weight of 33,000, which was identified as the sn-glycerol-3-phosphate permease. Its properties were similar to the corresponding E. coli permease. The presence of the multicopy glpT hybrid plasmid had a strong influence on the synthesis or assembly of other cell envelope proteins of E. coli. For instance, the periplasmic ribose-binding protein was nearly absent. On the other hand, the quantity of an unidentified E. coli outer membrane protein usually present only in small amounts increased.     

Cloning, expression, and mapping of the Aeromonas hydrophila aerolysin gene determinant in Escherichia coli K-12.

DNA sequences corresponding to the aerolysin gene (aer) of Aeromonas hydrophila AH2 DNA were identified by screening a cosmid gene library for hemolytic and cytotoxic activities. A plasmid containing a 5.8-kilobase EcoRI fragment of A. hydrophila DNA was required for full expression of the hemolytic and cytotoxic phenotype in Escherichia coli K-12. Deletion analysis and transposon mutagenesis allowed us to localize the gene product to 1.4 kilobases of Aeromonas DNA and define flanking DNA regions affecting aerolysin production. The reduced hemolytic activity with plasmids lacking these flanking regions is associated with a temporal delay in the appearance of hemolytic activity and is not a result of a loss of transport functions. The aerolysin gene product was detected as a 54,000-dalton protein in E. coli maxicells harboring aer plasmids and by immunoblotting E. coli whole cells carrying aer plasmids. We suggest that the gene coding aerolysin be designated aerA and that regions downstream and upstream of aerA which modulate its expression and activity be designated aerB and aerC, respectively.