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PubMed1985

Functional complementation between the two homologous genes, ops and tps, during differentiation of Myxococcus xanthus.

Protein S is a development-specific protein of Myxococcus xanthus encoded by the tps gene. It has been shown that there are two extensively homologous genes (ops and tps) tandemly repeated in the same direction with a 1.4 kb spacer fragment between them (Inouye et al. 1983). Seven deletion mutants were constructed by removing the ops gene, the tps gene, segments of the spacer sequence or combinations of these regions. The deleted regions were replaced with DNA fragments carrying the Tn5 gene for kanamycin resistance. The effects of deleting different regions on morphological changes and on patterns of protein synthesis during fruiting body formation were examined. The process of fruiting body formation was severely delayed when both the ops and the tps genes were deleted. However, this delay could be suppressed by either the ops gene or the tps gene, individually, although in the latter case, a slight delay was still observed. These results indicate that the ops gene is expressed during fruiting body formation and plays a role in the normal program of M. xanthus differentiation. Furthermore, the role of the ops gene can be complemented by the tps gene. The deletion of the ops and/or tps genes had no effect on glycerol-spore formation.

Bacterial Proteins↗
PubMed1985

Identification of a cis-acting regulatory region in the pncB locus of Salmonella typhimurium.

The pncB locus of the pyridine nucleotide cycle of NAD biosynthesis in Salmonella typhimurium was examined in terms of genetic structure and regulation. The gene appears to be regulated at the level of transcription in response to the end product of the pathway, NAD. Insertions into promotor proximal regions of the gene relinquish it from regulation, while insertions into more distal regions allow retention of regulation. Regulation cannot be restored in trans to strains containing promotor proximal insertions implicating the existence of a cis acting regulatory region.

Chromosome Deletion↗
PubMed1977

Transposition of TnA does not generate deletions.

We have examined the incidence of loss of the TnA unit, Tn801, from RP1 under conditions where transposition of Tn801 to another replicon. R388, was readily detected. We found that the frequency of transposition of Tn801 from RP1 to R388 exceeded, by at least a factor of one hundred, the frequency at which it was deleted from RP1. We conclude that, in general, transposition of Tn801 does not generate derivatives of the donor plasmid which specifically lack Tn801. The relevance of these findings to the mechanism of transposition is discussed.

Chromosome Aberrations↗
PubMed1978

Phage Mu-1 mediated transposition: a tool to study the organization of ribosomal protein genes in Escherichia coli.

Phage Mu-1 mediated transposition has been used to map genes coding for ribosomal proteins and elongation factor G inside transcriptional units. The data indicate that 1) the str A and fusA genes belong to the same operon, 2) the spcA and strA genes are expressed independently, 3) the spcA gene is located in a different transcriptional unit to that of the eryA and eryB genes.

Coliphages↗
PubMed1986

Pleiotropic mutations in appR reduce pH 2.5 acid phosphatase expression and restore succinate utilisation in CRP-deficient strains of Escherichia coli.

Several strains of Escherichia coli K12 were compared for activity of the periplasmic "pH 2.5 acid phosphatase", an enzyme whose expression is regulated negatively by cyclic AMP. Two distinct enzyme levels differing by about four-fold were observed. This strain-dependent difference does not involve modifications in the structure of the enzyme, but results from a difference in its expression. We show that strains with a high- or a low level of enzyme differ in the gene locus appR located in the 59 min region of the chromosome, a site remote from the structural gene appA; the appR+ versus appR enzyme ratio is 3-4 in wild-type strains, adenylate cyclase-deficient strains (cya) or cyclic AMP receptor protein-deficient strains (crp) grown in rich medium or in glucose minimal medium, but is close to 1 in cya strains in the presence of 0.1 mM cyclic AMP and in wild-type strains grown with succinate as carbon source; in a crp genetic background, appR strains, contrary to appR+ strains, are able to grow on minimal medium with succinate as the sole carbon source. The selection, from an appR+ crp strain, of clones growing on succinate-minimal medium, yielded mutations in the same region of the chromosome and showing the same phenotype as "naturally-occurring" appR strains. All appR strains analysed so far showed other similar deficiencies. The possibility that mutated appR gene products might function as weak substitutes for a functional cAMP-CRP complex is discussed.

Acid Phosphatase↗
PubMed1989

Analysis of structural and biological parameters affecting plasmid deletion formation in Bacillus subtilis.

Deletions generated following stimulation by the deletion hot spot of plasmid pHV15-1 were studied in Bacillus subtilis. Nucleotide sequencing showed that deletions extend between short direct repeat sequences. Such direct repeat sequences may have homology to the sequence of the hot spot. Deletion formation is recE-independent, but requires an active exonuclease V (AddAB) enzyme. Other structural parameters like plasmid size and structure influence deletion formation.

Bacillus subtilis↗
PubMed1989

asgB, a gene required early for developmental signalling, aggregation, and sporulation of Myxococcus xanthus.

The asgB genetic locus of Myxococcus xanthus specifies a function which is required early in the developmental pathway leading to aggregation and sporulation in fruiting bodies. The developmental defect of asgB mutants can be compensated by extracellular complementation using either intact wild-type cells or cell-free supernatants conditioned by developing wild-type cells. A Tn5 insertion was isolated closely linked to asgB480 and facilitated the cloning of both the wild-type (asgB+) and the mutant (asgB480) alleles in Escherichia coli plasmid. Tandem duplications of the asgB locus were constructed in M. xanthus; the completely wild-type phenotype of asgB+/asgB480 partial diploids implies that the asgB480 allele is recessive. This finding, along with extracellular complementation by wild-type cells, is consistent with the hypothesis that the asgB+ locus is required to produce a substance with an intercellular signalling function. At least part of the asgB gene was found to lie within a 1.2 kb SmaI DNA fragment. This 1.2 kb fragment, as well as smaller fragments derived from it, were used as DNA probes in RNA/DNA hybrid analyses of transcription in the asgB region. Two small mRNA species were detected, one about 650 bp long, and the other about 500 bp; the two species of mRNAs apparently overlap. Both mRNAs are present in low, but approximately equal amounts, in vegetatively growing cells. This is consistent with the observation that asg mutants display a mutant vegetative phenotype (a change in colony color and spreading behavior) as well as defective development.

Blotting, Northern↗
PubMed1984

New regulatory genes involved in the control of transcription initiation at the thr and ilv promoters of Escherichia coli K-12.

The gene ileR+, considered to encode a transacting protein involved in the regulation of the thr and ilv operons of Escherichia coli, has been cloned and localized to a 1.2 Kb BglII-SalI fragment of DNA. In strains harboring attenuation-defective fusions of lacZ to the promoter regions of the thr and ilv operons, ileR mutations lead to beta-galactosidase levels higher than those of the deattenuated parental strains. Reduced utilization of the thr and ilv promoters was observed in ileR cells harboring either ilvR+ plasmids or plasmids leading to the hyperproduction of Trp repressor. These results support the idea that ileR+ encodes a repressor protein that negatively affects the expression of the thr and ilv operons. Two additional trans-acting positive regulatory elements that act at the thr and ilv promoters have been identified by an analysis of deletion mutants. It thus appears that there exist positive as well as negative controlling elements that can act independently of attenuation to modulate the ilv and thr operons.

Cloning, Molecular↗
PubMed1984

Regulation of the trimethylamine N-oxide (TMAO) reductase in Escherichia coli: analysis of tor::Mud1 operon fusion.

Mud1 insertion mutants of Escherichia coli were obtained in which the lac structural genes were fused to the promoter of torA, a gene encoding the trimethylamine N-oxide (TMAO) reductase. Expression of the fusion is induced by TMAO and repressed by oxygen. However, in contrast to the nar operon which codes for the nitrate reductase structural genes, the tor::Mud1 fusion was found to be independent of the positive control exerted by the nirR gene product and not repressed by the molybdenum cofactor. The torA gene which is strongly linked to pyrF (28.3U) is different from any tor gene already described in E. coli or in Salmonella typhimurium.

Crosses, Genetic↗
PubMed1976

Overproduction of phage lambda repressor under control of the lac promotor of Escherichia coli.

The gene coding for bacteriophage Lambda repressor (cI gene) has been fused to the lac operon of Escherichia coli. In some of the fusions Lambda repressor synthesis can be controlled by the lac operator and promoter. Upon induction of the lac operon the amount of Lambda repressor is increased by a factor of 7 over that found in a single lysogen. In combination with the polarity suppressor suA the induction factor rises to 20. Transducing phages of one fusion were constructed. After thermal induction of this phage the final level of Lambda repressor was enhanced by a factor of 150.

Chromosome Deletion↗
PubMed1978

Origin of replication, oriC, or the Escherichia coli chromosome on specialized transducing phages lambda asn.

Specialized transducing phages lambda asn harboring chromosomal DNA and genetic markers on either side of the asn gene were isolated. Phages carrying chromosomal DNA counterclockwise of the asn gene can upon infection establish themselves as self-replicating plasmids in asn, recA hosts lysogenic for lambda. It is concluded that this bypassing of normal lambda immunity is due to the presence of the chromosomal replication origin, oriC, in this class of phages. Genetic analysis and the determination of restriction endonuclease cleavage patterns of the different lambda asn lead to the allocation of oriC within 1.5 megadaltons of the asn gene towards the uncA, uncB genes at 82 min on the genetic map of E. coli. The clockwise order of genes on the chromosomes is found to be: bglB, (pst, glmS), (uncA, uncB), oriC, asn, trkD, rbs, rrnC, ilv.

Chromosome Mapping↗
PubMed1979

Cloning the trpR gene.

In Escherichia coli, the structural gene for purine nucleoside phosphorylase, deoD, is subject to insertional inactivation by prophage lambda. From one such secondary site lambda lysogen, strain SP265, one may isolate deletions that remove all or part of the trpR gene and other genes in the deo-thr sector of the E. coli chromosome. Specialized transducing phages harboring serB+ and trpR+ were liberated following induction of SP265. All such phages were N-defective, bio-type pseudolysogens whose DNA persisted in the form of plasmids. A collection of transducing phages, differing in their complement of bacterial DNA, was used to locate cleavage sites for BamHI, SalI, and PvuI within the deoD-trpR region of the E. coli genome. The trpR gene lies within a specific 950 base pair BamHI-PvuI segment. A 1250 base pair BamHI fragment carrying a functional trpR gene was cloned into the amplifiable plasmid pBR322. A single SalI site in this fragment was shown to lie within the TrpR gene. In two situations where increased gene dosage might generate elevated amounts of Trp repressor (N-defective trpR+ pseudolysogens and strains harboring pBR322 trpR+ plasmids) neither tryptophan auxotrophy, enhanced sensitivity to DL-5-methyl-tryptophan, nor super repression of the tryptophan biosynthetic enzymes was observed.

Bacteriophage lambda↗
PubMed1986

Physical and genetic structure of the glpD-malT interval of the Escherichia coli K-12 chromosome. Identification of two new structural genes of the glp-regulon.

A transducing lambda phage carrying glpD''lacZ, glpR, and malT was isolated from a strain harboring a glpD''lacZ fusion. Comparison of restriction endonuclease cleavage patterns of DNA isolated from this phage with that of the previously cloned malT region (Raibaud and Schwartz 1980) facilitated the construction of recombinant plasmids carrying different portions of the glpD-malT region. Results of minicell analysis and complementation studies showed that this region of the chromosome encodes at least five polypeptides. These included the previously identified glpD, glpR, and malT gene products. In addition, two new structural genes of the glp regulon (glpE and glpG) located between the glpD and glpR genes were identified. Hybrid plasmids carrying glpD''lacZ and glpR''lacZ fusions were constructed. Restriction endonuclease cleavage analysis of these two plasmids demonstrated that glpD and glpR are divergently transcribed.

Bacteriophage lambda↗
PubMed1982

An amber mutation in the gene rpsA for ribosomal protein S1 in Escherichia coli.

An amber mutation has been induced in the gene rpsA (which codes for ribosomal protein S1) of Escherichia coli K-12 strain in the presence of an amber suppressor (supD) and mutations sueA, sueB and sueC that additively enhance the efficiency of suppression. That the amber mutation has occurred in the gene rpsA was confirmed by complementation with a plasmid which carried the wild-type allele of rpsA. The mutation is lethal in the absence of an amber suppressor, indicating that ribosomal protein S1 is indispensable to E. coli.

Bacteriophage lambda↗