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PubMed1988

UV-light-induced mutability in Salmonella strains containing the umuDC or the mucAB operon: evidence for a umuC function.

Multicopy plasmids carrying either the umuDC operon of Escherichia coli or its analog mucAB operon, were introduced into Ames Salmonella strains in order to analyze the influence of UmuDC and MucAB proteins on repair and mutability after UV irradiation. It was found that in uvr+ bacteria, plasmid pICV80:mucAB increased the frequency of UV-induced His+ revertants whereas pSE117:umuDC caused a smaller increase in UV mutagenesis. In delta uvrB bacteria, the protective role of pSE117 against UV killing was weak, and there was a great reduction in the mutant yield. In contrast, in these cells, pICV80 led to a large increase in both cell survival and mutation frequency. These results suggest that in Salmonella, as in E. coli, MucAB proteins mediate UV mutagenesis more efficiently than UmuDC proteins do. Plasmid pICV84:umuD+ C- significantly increased UV mutagenesis of TA2659: delta uvrB cells whereas in them, pICV77:mucA+ B- had no effect on mutability indicating the presence in Salmonella TA2659 of a gene functionally homologous to umuC.

Bacterial Proteins↗

PubMed1981

Differential translation efficiency explains discoordinate expression of the galactose operon.

We have used an mRNA-dependent E. coli S-30 translation system to compare the translation efficiencies of two polycistronic transcripts of the galactose operon, the CRP-cAMP-dependent mRNA (P1) and the CRP-cAMP-independent mRNA (P2). The RNAs were prepared in vitro, quantitated by hybridization or gel analysis and translated in a cell free system. The specific protein products were measured, and their quantities were compared with the amount of input mRNA. Our results show that the P2 mRNA synthesizes epimerase, the 5'-proximal gene product of the gal operon, four times more efficiently than the P1 mRNA. The 5'-distal gene products, transferase and kinase, are translated with the same efficiency from both transcripts. Thus the ratio of epimerase to kinase synthesis is four times higher for the P2 mRNA than for the P1 mRNA. This change in epimerase to kinase ratio is identical to that observed in vivo when the cellular cAMP level falls and gal transcription is believed to switch from P1 to P2. We suggest that it is the differential translation efficiency of the epimerase gene on the two different gal transcripts that accounts for this discoordinate expression. Moreover, since the P2 mRNA differs from the P1 mRNA only by the addition of five nucleotides at the 5' terminus and these nucleotides are outside the ribosome binding region we determine for epimerase, the selective difference in the translation efficiency of epimerase is probably mediated by RNA conformation.

Carbohydrate Epimerases↗

PubMed1981

Identification of ribosomal protein S7 as a repressor of translation within the str operon of E. coli.

A DNA-directed in vitro protein-synthesizing system was used to demonstrate that r protein S7 has the capacity to inhibit the translation of mRNA for the second and third gene products of the str operon (S7 and EF-G) but not for the first gene product (S12). Translation of mRNA of the last gene product in the operon (EF-Tu) is also probably not inhibited by S7. In addition, we localized the target site for S7 repressor action on the polycistronic str mRNA by examining the repressor activity of S7 in vitro using various template DNAs that contain the gene. The target site was found not to include a promoter-proximal portion of the mRNA for S12. To test for regulatory properties of S7 in vivo, we inserted the S7 gene into a plasmid vector containing the ara regulatory elements such that S7 synthesis was placed under ara control. A specific increase in S7 synthesis caused by stimulation in transcription originating from the arabinose promoter decreased the synthetic rate for EF-G but had no effect on S12 or EF-Tu synthesis.

Bacterial Proteins↗

PubMed1983

A control element within a structural gene: the gal operon of Escherichia coli.

The gal operon of Escherichia coli is transcribed from two overlapping promoters, PG1 and PG2. Cyclic AMP and its receptor protein (CRP) modulate the two promoters in opposite directions by binding to a single cat locus. Both the promoters are negatively regulated by a single repressor, the product of the galR gene. An operator site, defined by several mutations, has previously been located upstream from the cat locus. We have isolated and characterized a new set of cis-dominant constitutive mutations of the gal operon and determined their locations by DNA sequencing. From these studies, we propose the existence of a second functional gal operator element at an extraordinary site--within galE, the first structural gene. Both the operators, OE (exterior) and OI (interior), are involved in the repression of PG1 and PG2. This would be the first example of the presence of a functional operator element within a structural protein-coding region.

Base Sequence↗

PubMed1984

Dual promoter control of the Escherichia coli lactose operon.

The control of transcription initiation at the lactose operon promoter was investigated in vitro. We found that an upstream promoter (termed lac P2) interfered with RNA polymerase binding at the principal promoter (termed lac P1). The start site for lac P2 was located at base pair position -22 relative to the P1 start site. The addition of cAMP receptor protein and cAMP was shown to repress lac P2 and to activate lac P1. Abortive initiation reactions for both promoters were used to investigate the coordinate repression-activation elicited by CRP-cAMP. The effects of lac promoter mutations (L8, Ps, and UV5) were consistent with an important RNA polymerase positioning role for CRP-cAMP in the activation of lac operon expression.

Cyclic AMP↗

PubMed1984

Conformational change in the DNA associated with an unusual promoter mutation in a tRNA operon of Salmonella.

We describe two novel types of mutations which decrease transcription of a tRNA operon in Salmonella typhimurium. One mutation consists of a 3 bp deletion at position -70 from the transcription start site. The second mutation is a single bp insertion between the -10 region and the transcription start site. The region around position -70 shows unusual physical properties. DNA fragments carrying the wild-type version of this region exhibit abnormally low electrophoretic mobility in polyacrylamide gels. This anomalous electrophoretic behavior is corrected if gels are run at high temperature or if the fragments contain the 3 bp deletion at -70. Our results indicate the existence of an unusual DNA conformation, probably involving DNA bending, in the region preceding the tRNA operon promoter. This structure is apparently disrupted by the 3 bp deletion. The transcriptional defect of the -70 mutant suggests a role for the unusual conformation in promoter function.

Base Sequence↗

PubMed1987

A bacterial gene involved in transcription antitermination: regulation at a rho-independent terminator in the bgl operon of E. coli.

We have investigated the mechanism of regulation of the bgl operon in Escherichia coli K-12. A regulatory region has been located downstream of the bgl promoter, revealing a 130 base leader containing a sequence from +64 to +112 characteristic of a rho-independent terminator. In vitro, over 90% of the transcripts initiated at the bgl promoter terminate within the leader, at the 3' end of the terminator. Transcriptional fusions containing the terminator require the bglC gene in trans for expression; fusions deleted for the sequence show bglC-independent expression. A mutation resulting in partially constitutive expression of the fusion maps within the terminator. We propose that the bglC gene product mediates positive regulation of the bgl operon by functioning as an antiterminator at the rho-independent terminator located within the leader.

Base Sequence↗

PubMed1990

Transcriptional antitermination in the bgl operon of E. coli is modulated by a specific RNA binding protein.

Regulation of bgl operon expression in E. coli occurs by a mechanism involving antitermination of transcription at two termination sites within the operon. The bglG gene product is absolutely required for this process. Here we provide evidence that BglG is an RNA binding protein that recognizes a specific sequence located just upstream of each of the terminators. The sequence was delimited using a series of specific oligonucleotide probes. Mutational analysis of this sequence indicates that the protein requires a specific RNA secondary structure for recognition. We propose that BglG prevents transcription termination by binding to nascent RNA and blocking formation of the terminator structure.

Bacterial Proteins↗

PubMed1986

Plasmid pU29, a vehicle for mutagenesis of the photosynthetic puf operon in Rhodopseudomonas capsulata.

Plasmid pU21, which carries the reaction center and light-harvesting genes (puf operon) of Rhodopseudomonas capsulata, has been redesigned by site-specific mutagenesis. Five restriction sites have been removed and three unique restriction sites have been introduced into this 11,589-bp pBR322 derivative. The modifications divide the puf structural genes into four regions separated by five unique and nonmutagenic restriction sites. These four fragments have been subcloned into the M13-mp series of vectors to facilitate oligonucleotide-mediated site-specific mutagenesis experiments on the photosynthetic apparatus structural genes. The inserts can then be returned from the M13 replicative form to the redesigned pU21 derivative. The modified plasmid, pU29, greatly facilitates in vitro mutagenesis experiments since previously described techniques and screening procedures are more efficient with M13 derivatives carrying smaller inserts. Additionally, tandem homologous sequences (the reaction center L and M subunits) within the puf operon are now separated on different phage vectors, eliminating problems encountered in the targeting of mutagenic oligonucleotides to only one of the two homologous sites.

Chromosome Mapping↗

PubMed1989

Host-specific effects of the korA-korB operon and oriT region on the maintenance of miniplasmid derivatives of broad host-range plasmid RK2.

Two genetic determinants are sufficient for small derivatives of broad host-range plasmid RK2 to replicate in different Gram-negative bacteria: trfA, which encodes a replication initiator, and oriV, the origin of replication. In this study, nonessential RK2 determinants in the region encoding oriT, the origin of conjugative transfer, and the korA-korB operon, whose products regulate trfA expression, were tested for their effects on the stability of mini-RK2 plasmids in eight different hosts. We found that determinants of both regions can substantially alter plasmid stability, but the effects are not uniform in all hosts. The results also indicate that the effects of the korA-korB operon extend beyond that of the regulation of trfA transcription. This study further illustrates the different requirements for stable plasmid maintenance in diverse bacteria and the ability of wild-type RK2 to adapt to a variety of intracellular environments. The data also provide further evidence for the involvement of different regions of RK2 for stable maintenance in various hosts.

Conjugation, Genetic↗

PubMed1990

A phasmid shuttle vector for the cloning of complex operons in Salmonella.

Phasmid (phage plasmid hybrid) P4 vir1 can be propagated in Escherichia coli as a helper-dependent lytic phage, as a plasmid, or as a prophage. On the basis of an understanding of these modes of propagation, derivatives of P4 have been constructed for use as cloning vectors. In this report we demonstrate that phasmid P4 (i) will propagate as a helper-dependent lytic phage and as a plasmid in Salmonella spp. and (ii) can be used as a high efficiency phage shuttle vector for the reversible transfer of cloned genes between Salmonella spp. and E. coli. For both E. coli and Salmonella spp., P4 phage-mediated gene transfer proved to be only 10-fold lower than plaquing efficiency. For the case of Salmonella spp., this frequency is ca. 10(4)-fold more efficient than is typically found for the transformation of DNA molecules. The usefulness of this cloning vector system for analyses of pathogenic virulence factors is demonstrated by the cloning and expression of both the P pilus adhesin operon and the hemolysin operon of uropathogenic E. coli.

Cloning, Molecular↗

PubMed1994

Cyclic AMP-dependent expression of the Escherichia coli serC-aroA operon.

The Escherichia coli serC-aroA operon encodes biosynthetic enzymes for unrelated amino acid biosynthetic pathways leading to the synthesis of serine and the aromatic amino acids. A serC-aroA-lac translational fusion was constructed in the vector pMC1403. Synthesis of beta-galactosidase from the serC-aroA-lac fusion was found to be enhanced in the presence of lactose as the sole carbon source. This enhancement was not observed in strains containing a cya or crp mutant. However, the exogenous addition of cAMP greatly increased the beta-galactosidase synthesis in the cya mutant strain. The serC-aroA mRNA content, analyzed by a dot blot assay, also appeared to increase in the serC+ aroA+ cells after the exogenous addition of cAMP. These findings unambiguously indicate that the expression of the serC-aroA operon is positively controlled by cAMP.

Base Sequence↗

PubMed1988

Evidence for a translation-mediated attenuation of a spinach chloroplast rDNA operon.

The presence of potential hairpin structures H1, H2, H3 in the leader region of a spinach rDNA operon led us to postulate that this operon is regulated by premature termination. The mechanism would be controlled by the presence or absence of ribosomes translating a leader peptide. In vitro synchronized transcription by E. coli RNA polymerase shows that pauses do occur in the leader region. By their sizes, the transient transcripts could correspond to pauses on H1 and H2 as predicted by the model in the absence of ribosomes. The complete leader sequence (pKOPH) and the leader sequence with the hairpin structures deleted (pKOP) have been used to the GalK gene in the pK01 plasmid. The resulting plasmids have been used to transform a GalK- E. coli strain. Measurements of GalK expression show that the promoter region of spinach chloroplast rDNA is neither subjected to the growth rate nor to the stringent control. However, under growth conditions leading to an excess of free ribosomes, the expression of GalK gene appears systematically to be reduced in pKOPH when compared with that of pKOP. These results are consistent with a role of the leader region in a translation-mediated attenuation of the chloroplast rDNA expression.

Chloroplasts↗

PubMedDate not supplied

Utilization of acetate in Escherichia coli: structural organization and differential expression of the ace operon.

Growth of Escherichia coli on acetate as the sole source of carbon and energy requires operation of the glyoxylate bypass in connection with the expression of the polycistronic ace operon. The structural organization of this operon is presented, including the 3 structural genes coding respectively for malate synthase (aceB), isocitrate lyase (aceA) and isocitrate dehydrogenase kinase/phosphatase (aceK), and the surrounding genes iclR and metA. In addition, the differential expression of genes aceB, aceA, and aceK has been tested both in vivo in a minicell system and in vitro in a plasmid-directed transcription-translation coupled system. Moreover, the codon usage and adaptation to transfer RNA frequencies during translation of the corresponding messenger RNAs have been measured.

Acetates↗

PubMedDate not supplied

Positive and negative regulatory elements in the dnaA-dnaN-recF operon of Escherichia coli.

The recF gene of E coli lies within a cluster of genes which play essential roles in DNA replication; the gene order is dnaA dnaN recF gyrB. Each of these genes has its own promoters which, with the exception of dnaA promoters, reside entirely within the translated region of the respective preceding gene. In this report, we analyze the effect of the dnaA and dnaN promoters on recF expression by translational fusions between recF and the lacZ reporter gene. Our results indicate that recF is a distal gene of the dnaA operon, and support the previous proposal that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters. They also suggest that dnaA, dnaN and recF are predominantly expressed from the same mRNA although transcriptional and/or post-transcriptional mechanisms should be specifically involved in lowering expression of the recF gene. Recently, we have localized 3 tandem transcription termination sites in the second half of the dnaN gene, downstream from the recF promoters. Neither of them shows the typical features of simple terminators and apparently they do not work in a minimal system of in vitro transcription. In this report, we present evidence that only one of them is dependent on the Rho protein. Although the operon structure allows coordinate expression of dnaA, dnaN and recF, the presence of internal promoters (the dnaN and recF promoters), which appear to be inducible by DNA damage, and intracistronic terminators, whose activity is inversely proportional to the efficiency of translation, permits expression of individual genes to be independently regulated in response to altered growth conditions.

Bacterial Proteins↗

PubMedDate not supplied

Cloning and sequencing of the Bacillus megaterium spoIIA operon.

The spoIIA operon of Bacillus megaterium has been cloned and the nucleotide sequence determined. The spoIIA sequence contains three open reading frames coding for putative proteins of 116 aa, 147 aa, and 253 aa; the first and the third genes are preceded by a ribosomal binding site. The genes are in the same order as those of B subtilis and B licheniformis. The deduced amino acid sequences of these three open reading frames show 78-92% homology with SpoIIAA, SpoIIAB and SpoIIAC of B subtilis and B licheniformis. Northern hybridization revealed that B megaterium also has two spoIIA transcripts, 1.77 kb and 2.92 kb, attaining maximum expression at t1 and t3, respectively. In addition, homology to a possible penicillin binding protein gene upstream and the first part of a spoVA operon downstream has been identified on the 3.34-kb fragment. The spoIIA and the downstream spoVA promoter regions are highly conserved among these three species. Sequence analysis of the spoVA promoter revealed a region upstream to the -35 that is highly conserved across Bacillus species.

Amino Acid Sequence↗

PubMed1991

Analysis of Bordetella pertussis cya operon regulation by use of cya-lac fusions.

In Bordetella pertussis virulence-associated genes, including adenylate cyclase toxin (Cya), are coordinately regulated in response to environmental signals by proteins coded by the bvg-locus. We have constructed cya-lac fusions in Escherichia coli and have shown that the cya operon is not expressed in E. coli, neither is it activated by bvg, when introduced in trans. The cya-lac fusion is fully active when returned to B. pertussis by homologous recombination and responds to bvg-dependent activation and environmental regulation. These results indicate that in B. pertussis the activation of the cya operon by bvg is indirect.

Adenylate Cyclase Toxin↗

PubMed1991

Analysis of the first two genes of the CS1 fimbrial operon in human enterotoxigenic Escherichia coli of serotype 0139:H28.

An oligonucleotide, derived from the N-terminal amino acid sequence of the CS1 fimbrial subunit protein was used to identify the subunit gene on recombinant plasmid pDEP23 containing the structural genes of the CS1 fimbrial operon. The nucleotide sequence of the subunit gene (csoA), encoding a protein of 171 amino acids, was determined. Flanking it upstream, a gene (csoB) encoding a protein of 238 amino acids was found. The CsoB and CsoA proteins are homologous to the CfaA and CfaB proteins in the CFA/I fimbrial operon. For all the CS1 producing strains investigated the structural genes are located on plasmids. Like CFA/I fimbriae, CS1 fimbriae are only expressed in the presence of a positive regulator, CfaD for CFA/I and Rns for CS1, respectively. The promoter region upstream of the csoB gene was cloned in front of the promoterless alkaline phosphatase (phoA) gene of the promoter-probe vector pCB267. PhoA activity was enhanced approximately two-fold by the introduction of compatible plasmids containing either rns or cfaD.

Amino Acid Sequence↗

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