New mutations fts-36, lts-33, and ftsW clustered in the mra region of the Escherichia coli chromosome induce thermosensitive cell growth and division.

Three new mutants of Escherichia coli showing thermosensitive cell growth and division were isolated, and the mutations were mapped to the mra region at 2 min on the E. coli chromosome map distal to leuA. Two mutations were mapped closely upstream of ftsI (also called pbpB), in a region of 600 bases; the fts-36 mutant showed thermosensitive growth and formed filamentous cells at 42 degrees C, whereas the lts-33 mutant lysed at 42 degrees C without forming filamentous cells. The mutation in the third new thermosensitive, filament-forming mutant, named ftsW, was mapped between murF and murG. By isolation of these three mutants, about 90% of the 17-kilobase region from fts-36-lts-33 to envA could be filled with genes for cell division and growth, and the genes could be aligned.     

HrpA, a DEAH-box RNA helicase, is involved in mRNA processing of a fimbrial operon in Escherichia coli

Endonucleolytic cleavage of mRNA in the daa operon of Escherichia coli is responsible for co-ordinate regulation of genes involved in F1845 fimbrial biogenesis. Cleavage occurs by an unidentified endoribonuclease, is translation dependent and involves a unique recognition mechanism. Here, we present the results of a genetic strategy used to identify factors involved in daa mRNA processing. We used a reporter construct consisting of the daa mRNA processing region fused to the gene encoding green fluorescent protein (GFP). A mutant defective in daa mRNA processing and expressing high levels of GFP was isolated by flow cytometry. To determine the location of mutations, two different genetic approaches, Hfr crosses and P1 transductions, were used. The mutation responsible for the processing defect was subsequently mapped to the 32 min region of the E. coli chromosome. A putative DEAH-box RNA helicase-encoding gene at this position, hrpA, was able to restore the ability of the mutant to cleave daa mRNA. Site-directed mutagenesis of the hrpA regions predicted to encode nucleotide triphosphate binding and hydrolysis functions abolished the ability of the gene to restore the processing defect in the mutant. We propose that HrpA is a novel enzyme involved in mRNA processing in E. coli.     

Mutant isolation and molecular cloning of mre genes, which determine cell shape, sensitivity to mecillinam, and amount of penicillin-binding proteins in Escherichia coli.

A chromosomal region of Escherichia coli contiguous to the fabE gene at 71 min on the chromosomal map contains multiple genes that are responsible for determination of the rod shape and sensitivity to the amidinopenicillin mecillinam. The so-called mre region was cloned and analyzed by complementation of two closely related but distinct E. coli mutants characterized, respectively, by the mutations mre-129 and mre-678, that showed a rounded to irregular cell shape and altered sensitivities to mecillinam; the mre-129 mutant was supersensitive to mecillinam at 30 degrees C, but the mre-678 mutant was resistant. The mre-678 mutation also caused simultaneous overproduction of penicillin-binding proteins 1Bs and 3. A chromosomal region of the wild-type DNA containing the total mre region and the fabE gene was first cloned on a lambda phage; a 7-kilobase (kb) fragment containing the whole mre region, but not the fabE gene, was then recloned on a mini F plasmid, pLG339; and finally, a 2.8-kb fragment complementing only mre-129 was also cloned on this low-copy-number plasmid. The whole 7-kb fragment was required for complementing the mre-678 mutant phenotypes. Fragments containing fabE but not the mre-129 region could be cloned on a high-copy-number plasmid. Southern blot hybridization indicated that the mre-678 mutant had a large deletion of 5.25 kb in its DNA, covering at least part of the mre-129 gene.     

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.     

Functional dissection of a mercuric ion transporter, MerC, from Acidithiobacillus ferrooxidans

Topological analysis with a phoA gene fusion suggested that Acidithiobacillus ferrooxidans MerC, a mercury transporter, has two periplasmic loops and four transmembrane domains. Cys-23 and Cys-26 of the protein were involved in Hg(2+)-recognition/uptake, but Cys-132 and Cys-137 were not. Escherichia coli cells producing the MerC were hypersensitive to CdCl(2). In this case, mutation of His72 rendered the host cells less CdCl(2) sensitive, whereas none of the Cys residues affected it. E. coli cells expressing the gene encoding a mercuric ion transporter (merC)-deletion mutant, in which the coding-sequence of the carboxy-terminal cytoplasmic region was removed, retained Hg(2+) hypersensitivity and showed about 55% HgCl(2) uptake ability compared to that of the one expressing the intact merC, indicating that the region is not essential for Hg(2+) uptake. Coexpression of A. ferrooxidans the gene encoding mercuric reductase (merA) and the merC deletion mutation conferred HgCl(2) tolerance to E. coli host cells. Under this condition, the merC deletion gene product was exclusively present as a monomer.     

Isolation and mapping of a mutation leading to damage in the cytoplasmic-specific component of the fructose transport system in Escherichia coli K-12 bacteria

A mutant delta ptsH of Escherichia coli was used to obtain a mutation which damages a function of cytoplasmic specific component of the fructose phosphotransferase system--FPr protein. This mutation was mapped using a number of genetical methods. In conjugational crosses the mutation was localized at 47 min of the E. coli chromosomal map. The gene responsible for this defect was designated fpr. In P1 mediated transduction and in conjugational three-factorial crosses the order of the markers in this region was established as: gyrA-ompC-fpr-ptsF-...-his. The frequency of cotransduction of the fpr gene was 4.5 and 35% with gyrA and ompC markers, respectively. In fructose containing minimal medium the doubling time of the ptsH+fpr mutant was lower than that of the delta ptsHfpr+ mutant. Also, the doubling time of the fpr mutant depends on concentration of fructose in growth medium but is independent of the amount of this sugar in the case of the delta ptsH mutant.     

Mechanism of D-cycloserine action: transport mutants for D-alanine, D-cycloserine, and glycine

The accumulation of d-alanine and the accumulation of glycine in Escherichia coli are related and appear to be separate from the transport of l-alanine. The analysis of four d-cycloserine-resistant mutants provides additional support for this conclusion. The first-step mutant from E. coli K-12 that is resistant to d-cycloserine was characterized by the loss of the high-affinity line segment of the d-alanine-glycine transport system in the Lineweaver-Burk plot. This mutation, which is linked to the met(1) locus, also resulted in the loss of the ability to transport d-cycloserine. The second-step mutation that is located 0.5 min from the first-step mutation resulted in the loss of the low-affinity line segment for the d-alanine-glycine transport system. The transport of l-alanine was decreased only 20 to 30% in each of these mutants. A multistep mutant from E. coli W that is 80-fold resistant to d-cycloserine lost >90% of the transport activity for d-alanine and glycine, whereas 75% of the transport activity for l-alanine was retained. E. coli W could utilize either d- or l-alanine as a carbon source, whereas the multistep mutant could only utilize l-alanine. Thus, a functioning transport system for d-alanine and glycine is required for both d-cycloserine action and growth on d-alanine.     

The mmsA locus of Streptococcus pneumoniae encodes a RecG-like protein involved in DNA repair and in three-strand recombination

We describe the characterization of a mutant strain of Streptococcus pneumoniae previously isolated on the basis of its sensitivity to Methyl Methane Sulphonate (MMS). The mutant strain also exhibited increased sensitivity to UV light and to X-rays, together with a reduced capacity for recombination and Hex-mediated generalized mismatch repair. We show that the original mutant contains two unlinked mutations in the mmsA and in the pms genes. The mmsA wild-type region was cloned and the nucleotide sequence of the mmsA gene was determined. mmsA encodes a polypeptide of 671 amino acids related to a large family of DNA–RNA helicases, with the highest similarity to Escheri-chia coli RecG, a protein involved in the branch migration of Holliday junctions. A plasmid carrying the intact mmsA coding region was shown to restore UV resistance to E. coli recG mutant strains. An mmsA -null mutant constructed by insertion of a chloramphenicol-resistance gene exhibited a 25-fold reduction in recombination during transformation. We suggest that MmsA recognizes and branch migrates three-strand transformation intermediates to extend donor–recipient heteroduplex regions. The mmsA -null mutant exhibited the other phenotypes of the original mutant, apart from mismatch-repair deficiency and, in addition, an alteration in colony-forming ability was noticed. In the pms mutant background, all phenotypes caused by the mmsA mutation were attenuated. Therefore, the pms mutation, although it affected mismatch repair and, to some extent, DNA repair and recombination, acted as a suppressor of the mmsA mutation.     

Mutation Preventing Capsular Polysaccharide Synthesis in Escherichia coli K-12 and Its Effect on Bacteriophage Resistance

A mutant strain of Escherichia coli K-12 was found in which spontaneous mutation to phage T7 resistance occurred at a very low frequency. T7 resistance in the parental strain from which this mutant was derived resulted from a mutation to excess capsular polysaccharide synthesis. The mutation preventing T7 resistance, non-9, inhibited capsule formation when transduced into capsulated strains. The non-9 mutation was cotransducible with his, the gene order in this region being non-9 his Su-1.     

Evidence for the essential function of 2,4-dienoyl-coenzyme A reductase in the beta-oxidation of unsaturated fatty acids in vivo. Isolation and characterization of an Escherichia coli mutant with a defective 2,4-dienoyl-coenzyme A reductase

For the purpose of assessing in vivo the importance of 2,4-dienoyl-CoA reductase (EC 1.3.1.34) in the beta-oxidation of unsaturated fatty acids, reductase mutants of Escherichia coli were isolated by selecting cells that were able to grow on oleate but not on petroselinic acid (6-cis-octadecenoic acid). One mutant (fadH) exhibited 12% of the 2,4-dienoyl-CoA reductase activity present in the parental strain with other beta-oxidation enzymes being essentially unaffected. Antireductase antibodies were used to show that the mutant contains a fadH gene product at a level similar to that observed in the parental strain. Thus, the mutation seems to have resulted in the synthesis of a fadH gene product with lower specific activity. The mutation was mapped in the 71-75-min region of the E. coli chromosome where no other gene for beta-oxidation enzymes has so far been located. Complementation of the mutation by F'141, which carries the 67-75.5-min region of the E. coli genome, resulted in an increase in the 2,4-dienoyl-CoA reductase activity to 80% of the level found in the parental strain. Measurements of respiration with petroselinic acid as the substrate showed rates to be linearly dependent on the 2,4-dienoyl-CoA reductase activity up to levels found in wild-type E. coli. 2,4-Dienoyl-CoA reductase, like other enzymes of beta-oxidation, was induced when E. coli was grown on a long chain fatty acid as the sole carbon source. It is concluded that 2,4-dienoyl-CoA reductase is required in vivo for the beta-oxidation of unsaturated fatty acids with double bonds extending from even-numbered carbon atoms.     

Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30

Deinococcus radiodurans strain rec30, which is a DNA damage repair-deficient mutant, has been estimated to be defective in the deinococcal recA gene. To identify the mutation site of strain rec30 and obtain information about the region flanking the gene, a 4.4-kb fragment carrying the wild-type recA gene was sequenced. It was revealed that the recA locus forms a polycistronic operon with the preceding cistrons (orf105a and orf105b). Predicted amino acid sequences of orf105a and orf105b showed substantial similarity to the competence-damage inducible protein (cinA gene product) from Streptococcus pneumoniae and the 2'-5' RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the genomic DNA of strain rec30, the mutation site in the strain was identified as a single G:C to A:T transition which causes an amino acid substitution at position 224 (Gly to Ser) of the deinococcal RecA protein. Furthermore, we succeeded in expressing both the wild-type and mutant recA genes of D. radiodurans in E. coli without any obvious toxicity or death. The gamma-ray resistance of an E. coli recA1 strain was fully restored by the expression of the wild-type recA gene of D. radiodurans that was cloned in an E. coli vector plasmid. This result is consistent with evidence that RecA proteins from many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from strain rec30 did not complement E. coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair.     

Identification of a mutation causing increased expression of the tas gene in Escherichia coli FX-11

Studies of N-ethyl-N-nitrosourea (ENU)-induced mutagenesis with a tyrosine auxotroph of Escherichia coli revealed a new type of revertant. This mutant strain was interesting because: (i) it was not a true revertant of the nonsense (ochre) defect nor a tRNA suppressor mutation; and (ii) it was induced by ENU to greater extent in a UmuC-defective host. Genetic mapping located the probable mutation to a region of the E. coli chromosome containing a newly described gene called tas. To investigate this mutation, the upstream region of the tas gene from both wild-type and mutant cells was cloned into a promoterless lacZ expression vector and recombined onto a lambda bacteriophage. Recombinant bacteriophage were inserted into the bacterial chromosome and beta-galactosidase (betaGal) assays were performed. These assays revealed an almost three-fold greater expression of betaGal from the mutant DNA than from the wild-type DNA. Sequence analysis of the region directly upstream of the tas gene revealed a G:C to A:T transition at base number 2263 (numbering based on GenBank Accession #AE000367), located within a potential promoter site. Further sequencing indicated no other mutations within the 1454bp region analyzed; however, there were several nucleotide differences seen in our B/r strain of E. coli, when compared with the published E. coli K-12 sequence. A total of 10 base differences were discovered; one in mutH, six within a potential open reading frame (ORF-o237) and three in non-coding regions. Yet, none of the changes altered the predicted amino acid sequences. These results provide evidence of a mechanism for increased expression of the novel gene tas and support the neutral drift hypothesis for the evolution of DNA sequences.     

beta-Alanine auxotrophy associated with dfp, a locus affecting DNA synthesis in Escherichia coli.

Strains containing the conditional-lethal dfp-707 mutation, which have a defect in DNA synthesis at 42 degrees C, were found to require either pantothenate or its precursor, beta-alanine, for growth at 30 degrees C. The auxotrophy and conditional lethality were corevertible. Through localized mutagenesis of the dfp-pyrE region of Escherichia coli, another mutation, dfp-1, was obtained. It conferred the auxotrophy but not the conditional lethality of dfp-707. Complementation analysis, performed with a set of plasmid-borne deletion and insertion mutations, revealed a correspondence between the complementation of each mutant phenotype and the production of the dfp gene product, previously identified as a 45-kilodalton flavoprotein. The dfp mutants had a normal level of aspartate-1-decarboxylase, which is the only enzyme known to produce beta-alanine in E. coli and which is specified by the distant panD gene. A prototrophic pseudorevertant of a dfp-1 strain was found to have retained the dfp mutation, to be genetically unstable, and to have an elevated level of aspartate-1-decarboxylase, suggesting that it had acquired a duplication of panD. It is not known what steps in pantothenate or DNA metabolism are affected by the mutant dfp product or how its flavin moiety may be involved.     

Purification and characterization of a mutant tRNA nucleotidyltransferase

tRNA nucleotidyltransferase has been extensively purified from a mutant strain of Escherichia coli which displays greatly decreased AMP incorporation, but normal CMP incorporation. The defect in AMP incorporation is retained throughout the purification of the mutant protein. The mutant protein behaves identically to the wild-type protein with regard to elution position on various chromatographic columns, and both have similar molecular weights of about 50000. The defect in the mutant protein is accentuated by the use of yeast tRNA rather than E. coli tRNA-C--C as substrate, by decreased pH, by increased ionic strength and by decreased divalent cation concentration. Substitution of MN2+ for Mg2+ greatly increases the relative activity of the mutant enzyme. In all these cases, CMP incorporation by the mutant enzyme remains the same as the wild-type enzyme. The Km values of the mutant enzyme for its tRNA and triphosphate substrates are unchanged, and the mutant protein is as stable as the wild type with respect to temperature inactivation. These results strongly suggest that the mutation is in the structural gene for tRNA nucleotidyltransferase, and that the mutation probably does not affect the overall structure of the mutant protein, but only a localized region near the AMP-incorporating site.     

Cloning, characterization and expression in Escherichia coli of a leucine biosynthetic gene from Streptomyces rochei

Leucine and histidine biosynthetic genes from Streptomyces rochei HP1 that complemented auxotrophic mutations in S. lividans TK54 were cloned in pIJ61. DNA from one leucine recombinant plasmid was subcloned into pBR322. From the latter, a recombinant plasmid was obtained that complemented the leuA mutation in Escherichia coli CV512 but not other leucine markers in E. coli. Analysis of this and several subclones, including mutant plasmids constructed in vitro, established that the cloned S. rochei gene was expressed in E. coli from the tetracycline promoter of pBR322 to produce a polypeptide of 67 kDa; the corresponding coding region was shown to be within a 1.7 kbp DNA fragment. Blot hybridization revealed corresponding homologous genes in several other streptomycetes.     

Identification of the uvrA6 mutation of Escherichia coli.

The uvrA6 mutation has been cloned on a multicopy plasmid by using a chloramphenicol resistance marker introduced next to the uvrA gene in the Escherichia coli chromosome. The mutation was shown to reside in the N-terminal part of the uvrA gene. Sequencing part of this region of the mutant gene revealed a frameshift mutation at positions 207 to 209, which leads to a stop codon at position 262. A marker rescue experiment showed that this frameshift is the only mutation responsible for the UV-sensitive phenotype of the UvrA6 mutant. The method presented is suitable for the cloning of every chromosomal uvrA mutation and can be useful for the study of the functional domains of the UvrA protein.     

Restoration by ribosomal protein S1 of the defective translation in a temperature-sensitive mutant of Escherichia coli K-12: characterization and genetic studies.

A temperature-sensitive mutant of Escherichia coli was isolated that had a temperature-sensitive defect in ribosomal-wash protein(s) required for translation in vitro of E. coli endogenous messenger ribonucleic acid. It was found that 30S ribosomal protein S1 rescued the defect in the ribosomal-wash protein(s) of the mutant and that the complete restoration to the wild-type level was attained when 1 mol of protein S1 was added to 1 mol of 70S ribosome. The mutation, tss, causing such a defect was mapped at 21 min and was closely linked to the pyrD locus, the region of which was entirely different from that of the other genes coding for the many ribosomal proteins of E. coli. These results indicate that the gene specified by this mutation is involved in the function of the 30S ribosomal protein S1.     

A single base mutation at position 2661 in E. coli 23S ribosomal RNA affects the binding of ternary complex to the ribosome.

A single base substitution mutation from guanine to cytosine was constructed at position 2661 of Escherichia coli 23S rRNA and cloned into the rrnB operon of the multi-copy plasmid pKK3535. The mutant plasmid was transformed into E.coli to determine the effect of the mutation on cell growth as well as the structural and functional properties of the mutant ribosomes in vivo and in vitro. The results show that the mutant ribosomes have a slower elongation rate and an altered affinity for EF-Tu-tRNA-GTP ternary complex. This supports previous findings which indicated that position 2661 is part of a region of 23S rRNA that forms a recognition site for binding of the ternary complex in the ribosomal A site. Combinations of the 2661 mutation with various mutations in ribosomal protein S12 also demonstrate that elements of both ribosomal subunits work in concert to form this binding site.