Suppression of recJ mutations of Escherichia coli by mutations in translation initiation factor IF3.

We have isolated genetic suppressors of mutations in the recJ gene of Escherichia coli in a locus we term srjA. These srjA mutations cause partial to complete alleviation of the recombination and UV repair defects conferred by recJ153 and recJ154 mutations in a recBC sbcA genetic background. The srjA gene was mapped to 37.5 min on the E. coli chromosome. This chromosomal region from the srjA5 strain was cloned into a plasmid vector and was shown to confer recJ suppression in a dominant fashion. Mutational analysis of this plasmid mapped srjA to the infC gene encoding translation initiation factor 3 (IF3). Sequence analysis revealed that all three srjA alleles cause amino acid substitutions of IF3. Suppression of recJ was shown to be allele specific: recJ153 and recJ154 mutations were suppressible, but recJ77 and the insertion allele recJ284::Tn10 were not. In addition, growth medium-conditional lethality was observed for strains carrying srjA mutations with the nonsuppressible recJ alleles. When introduced into recJ+ strains, srjA mutations conferred hyperrecombinational and hyper-UVr phenotypes. An interesting implication of these genetic properties of srjA suppression is that IF3 may regulate the expression of recJ and perhaps other recombination genes and hence may regulate the recombinational capacity of the cell.     

Identification of the phoM gene product and its regulation in Escherichia coli K-12.

Plasmids containing the chromosome region of Escherichia coli encoding phoM, whose product is a positive regulator of alkaline phosphatase expression, were isolated from the Clarke and Carbon plasmid bank. A 9.9-kilobase EcoRI fragment of plasmid pLC17-39 (subcloned into pBR322) was able to complement both phoM and thrB mutations. Restriction endonuclease analysis and in vitro mutagenesis of the hybird plasmids enabled the localization of the phoM gene locus to 3 kilobases of the cloned chromosomal fragment. The phoM gene product was identified, with maxicell techniques, as a protein with an approximate molecular weight of 55,000. A phoM-lacZ protein fusion was constructed by using a plasmid carrying the phoM gene and a derivative of phage lambda, lambda plac Mu2. Restriction endonuclease analysis of the plasmid carrying the fusion indicated that phoM is transcribed in a clockwise direction on the circular E. coli chromosome. Analysis of strains bearing the fusion on a multiple-copy plasmid or integrated at the lambda attachment site of the chromosome indicated that the synthesis of the phoM gene product was unaffected by phosphate limitation of growth. The expression of the phoM gene was studied in strains with mutations in genes encoding effectors of the pho regulon. A threefold increase in phoM expression was seen in a phoU strain in comparison with the wild-type strain.     

Cloning of the type VII trimethoprim-resistant dihydrofolate reductase gene and identification of a specific DNA probe

Abstract A 1.3 kb Hin III fragment encoding the type VII trimethoprim-resistant dihydrofolate reductase gene was cloned into pBR322. Unidirectional deletion of this cloned fragment with exonuclease III identified the start of the dihydrofolate reductase gene. An internal 300bp Eco RV fragment was identified which could be used as a specific non-radioactive DNA probe to distinguish bacteria carrying the type VII gene from those carrying genes encoding other known dihydrofolate reductase types.     

Suppression of Recj Exonuclease Mutants of Escherichia Coli by Alterations in DNA Helicases II (Uvrd) and IV (Held)

The recJ gene encodes a single-strand DNA-specific exonuclease involved in homologous recombination. We have isolated a pseudorevertant strain in which recJ mutant phenotypes were alleviated. Suppression of recJ was due to at least three mutations, two of which we have identified as alterations in DNA helicase genes. A recessive amber mutation, ``uvrD517(am),' at codon 503 of the gene encoding helicase II was sufficient to suppress recJ partially. The uvrD517(am) mutation does not eliminate uvrD function because it affects UV survival only weakly; moreover, a uvrD insertion mutation could not replace uvrD517(am) as a suppressor. However, suppression may result from differential loss of uvrD function: mutation rate in a uvrD517(am) derivative was greatly elevated, equal to that in a uvrD insertion mutant. The second cosuppressor mutation is an allele of the helD gene, encoding DNA helicase IV, and could be replaced by insertion mutations in helD. The identity of the third cosuppressor ``srjD' is not known. Strains carrying the three cosuppressor mutations exhibited hyperrecombinational phenotypes including elevated excision of repeated sequences. To explain recJ suppression, we propose that loss of antirecombinational helicase activity by the suppressor mutations stabilizes recombinational intermediates formed in the absence of recJ.     

Physical map of the Salmonella typhimurium histidine transport operon: correlation with the genetic map.

A detailed restriction map of a 12.4-kilobase EcoRI fragment of Salmonella typhimurium deoxyribonucleic acid (DNA) containing the entire histidine transport operon and the argT gene is presented. Subclones of specific regions of the transport operon of S. typhimurium were constructed in plasmid vectors. An accurate correlation between the restriction map and the location of genetically defined deletions was obtained by hybridizing restriction digests of chromosomal DNA from strains carrying each deletion with cloned transport operon DNA as a probe. These data were used to position the histidine transport genes on the cloned 12.4-kilobase fragment of DNA.     

Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes.

A gene cluster encoding biphenyl- and chlorobiphenyl-degrading enzymes was cloned from a soil pseudomonad into Pseudomonas aeruginosa PAO1161. Chromosomal DNA from polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes KF707 was digested with restriction endonuclease XhoI and cloned into the unique XhoI site of broad-host-range plasmid pKF330. Of 8,000 transformants tested, only 1, containing the chimeric plasmid pMFB1, rendered the host cell able to convert biphenyls and chlorobiphenyls to ring meta cleavage compounds via dihydrodiols and dihydroxy compounds. The chimeric plasmid contained a 7.9-kilobase XhoI insert. Subcloning experiments revealed that the genes bphA (encoding biphenyl dioxygenase), bphB (encoding dihydrodiol dehydrogenase), and bphC (encoding 2,3-dihydroxybiphenyl dioxygenase) were coded for by the 7.9-kilobase fragment. The gene order was bphA-bphB-bphC. The hydrolase activity, which converted the intermediate meta cleavage compounds to the final product, chlorobenzoic acids, and was encoded by a putative bphD gene, was missing from the cloned 7.9-kilobase fragment.     

Identification and nucleotide sequence determination of the gene responsible for Ogawa serotype specificity of V. cholerae 01.

The gene encoding a protein of 27 kDa, which is specifically expressed in Vibrio cholerae of serotype Ogawa, was identified and its nucleotide sequence determined. The plasmid carrying this gene was found to convert serotype specificity from Inaba to Ogawa when introduced into the Escherichia coli DH5(pVCI112) cell which harbors a cloned 20-kilobase genomic DNA fragment of V. cholerae NIH35A3 and expresses the 01 antigen of Inaba serotype.     

Plasmid genes required for microcin B17 production.

The production of the antibiotic substance microcin B17 (Mcc) is determined by a 3.5-kilobase DNA fragment from plasmid pMccB17. Several Mcc- mutations on plasmid pMccB17 were obtained by both transposon insertion and nitrosoguanidine mutagenesis. Plasmids carrying these mutations were tested for their ability to complement Mcc- insertion or deletion mutations on pMM102 (pMM102 is a pBR322 derivative carrying the region encoding microcin B17). Results from these experiments indicate that at least four plasmid genes are required for microcin production.     

Cloning and expression of a Bacillus subtilis division initiation gene for which a homolog has not been identified in another organism.

The Bacillus subtilis 168 division initiation genes defined by the temperature-sensitive mutations ts-1 and ts-12 were cloned into a 10.5-kilobase EcoRI fragment of DNA in the lambda EMBL4 vector. The two genes were separated by approximately 3 kilobases. The gene in which the ts-1 mutation resides was shown to be the same as the B. subtilis homolog of the Escherichia coli ftsZ gene. The other gene was named divIB. It showed no homology to any previously identified gene and coded for a protein of 30.1 kilodaltons which was probably membrane bound.     

Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli

We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.     

Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7

The genes encoding the enzymes responsible for conversion of naphthalene to 2-hydroxymuconic acid (nahA through nahI) are contained on a 25-kilobase EcoRI fragment of an 85-kilobase NAH plasmid of Pseudomonas putida. These genes were cloned into the plasmid vectors pBR322 and RSF1010 to obtain the recombinant plasmids pKGX505 and pKGX511, respectively. To facilitate cloning and analysis, an NAH7 plasmid containing a Tn5 transposon in the salicylate hydroxylase gene (nahG) was used to derive the EcoRI fragment. The genes for naphthalene degradation were expressed at a low level in Escherichia coli strains containing the fragment on the recombinant plasmids pKGX505 or pKGX511. This was shown by the ability of whole cells to convert naphthalene to salicylic acid and by in vitro enzyme assays. The expression of at least two of these genes in E. coli appeared to be regulated by the presence of the inducer salicylic acid. In addition, high-level expression and induction appear to be mediated by an NAH plasmid promoter and a regulatory gene located on the fragment. A restriction endonuclease cleavage map of the cloned fragment was generated, and the map positions of several nah genes were determined by analysis of various subcloned DNA fragments.     

Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli.

We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.     

Cloning of genes encoding extracellular metalloproteases from Erwinia chrysanthemi EC16.

A 14-kilobase BamHI-EcoRI DNA fragment cloned from Erwinia chrysanthemi EC16 contained a gene encoding a metalloprotease inhibitor as well as three tandem prt genes encoding metalloproteases. The prt genes were separated from the inhibitor gene by a ca. 4-kilobase region that was necessary for extracellular secretion of the proteases. When individually subcloned downstream from vector promoters, the three prt genes each led to substantial extracellular secretion of the proteases by Escherichia coli cells, provided that the 4-kilobase required region was supplied in cis or trans. One of the protease structural genes, prtC, was sequenced and had high homology to a metalloprotease gene previously described from Serratia species as well as to the prtB gene of E. chrysanthemi B374. Marker exchange mutants of E. chrysanthemi EC16 defective in production of one or all of the extracellular proteases were not impaired in virulence on plant tissue.     

Cloning and sequence of the gene encoding the major structural component of mannose-resistant fimbriae of Serratia marcescens.

Serratia marcescens US46, a human urinary tract isolate, exhibits mannose-resistant hemagglutination and agglutinates yeast cells, thereby indicating that it has two types of adhesins. We constructed a cosmid library for the DNA of this organism and isolated DNA clones carrying genes for mannose-sensitive (MS) and mannose-resistant (MR) fimbriae. On introduction of the cloned genes into Escherichia coli K-12, MS and MR fimbriae were formed. These fimbriae were functionally and morphologically indistinguishable from those of S. marcescens. Subcloning of these gene clusters revealed that the genes encoding MS fimbriae reside on a 9-kilobase (kb) DNA fragment, while those encoding MR fimbriae are present on a 12-kb fragment. Transposon insertion and maxicell analyses revealed that formation of MR fimbriae is controlled by several genes which reside on the 9-kb fragment. The nucleotide sequence of smfA, the gene encoding the major structural component of MR fimbriae, revealed that this gene encodes a 174-amino-acid polypeptide with a typical procaryotic signal peptide. The primary structure of the smfA product showed significant homology with the primary structure of the E. coli fimbrial subunit.     

Characterization of two Azospirillum brasilense Sp7 plasmid genes homologous to Rhizobium meliloti nodPQ

Bacteria belonging to the Azospirillum genus are nitrogen fixers that colonize the roots of grasses, but do not cause the formation of differentiated structures. Sequences from total DNA of several Azospirillum strains are homologous to restriction fragments containing Rhizobium meliloti nodulation genes. A 10-kilobase (kb) EcoRI fragment from A. brasilense Sp7, sharing homology with a 6.8-kb EcoRI fragment carrying nodGEFH and part of nodP of R. meliloti 41, was cloned in pUC18 to yield pAB502. The nucleotide sequence of a 3.5-kb EcoRI-SmaI fragment of the pAB502 insert revealed 60% homology with R. meliloti nodP and nodQ genes. The nodP gene product shares no homology to any known protein sequence. The Azospirillum nodQ gene product shares homology with a family of initiation and elongation factors as does the R. meliloti nodQ gene product. Since the nodQ gene overlaps the nodP gene, the two genes might be cotranscribed. Azospirillum contains large plasmids, and the nodPQ genes were found on the 90-MDa plasmid (p90). A translational nodP-lacZ fusion was constructed in the broad host range plasmid pGD926. No beta-galactosidase activity was detected in Escherichia coli, but the fusion was functional in Azospirillum and constitutively expressed. Deletions and mutations of nodPQ did not modify growth, nitrogen fixation, or interaction with wheat seedlings.