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PubMed1993

Transcriptional regulation of puc operon expression in Rhodobacter sphaeroides. Involvement of an integration host factor-binding sequence.

The putative overlapping consensus sequences (-129 to -105) for binding of fumarate nitrate reductase regulator- and integration host factor (IHF)-like proteins to puc operon upstream DNA of Rhodobacter sphaeroides was protected from DNase I digestion by purified Escherichia coli IHF. The binding of E. coli IHF to the purported IHF-binding site in the puc upstream DNA is highly sequence-specific. The recorded binding affinity was significantly lower than that of E. coli IHF to the lambda attP site. Employing site-directed changes in the DNA sequence within the -129 to -105 region, a loss in IHF binding, as monitored through gel retardation analysis, was correlated with alterations in puc operon expression monitored through the use of puc::lacZ transcriptional fusions. These results suggest that the IHF-binding site is involved in repression of puc operon transcription by oxygen as well as modulation of puc operon transcription levels by incident light intensity. Mutations specific to the upstream half of the putative fumarate nitrate reductase regulator-binding site of the puc upstream DNA did not show any physiological effects under the experimental conditions employed. Taken together, these studies reveal that the DNA sequence between -129 to -105 may involve facilitation of the interaction between upstream and downstream cis-acting regulatory sequences involved in puc operon expression.

Bacterial Proteins↗

PubMedDate not supplied

[Features of expression of the pertussis toxin operon under the control of its own and heterologous promotors in Bordetella bronchiseptica cells].

Expression of the cloned operon encoding the pertussis toxin synthesis under the control of operons own vir-dependent promoter or vir-independent promoter of Escherichia coli origin was studied. Proteins produced by the recombinant strains have been characterized. The pertussis toxin operon was shown to express under the control of both homologous and heterologous promoters in Bordetella bronchiseptica cells. Use of the lac-promoter increases the yield of produced toxin twofold. Copy number of operon in the cell does not influence the level of toxin production. The synthesized protein can be transported into the culture medium. The biological and physico-chemical properties of the protein are similar to the ones of the natural pertussis toxin. Bordetella bronchiseptica strain producing the toxin with decreased toxic activity has been obtained. Thus, a simple system for cellular expression of the toxin operon was constructed in Bordetella bronchiseptica. It permits one to construct new strains producing nontoxic derivatives of the pertussis toxin for construction of nonreactogenic vaccines.

Animals↗

PubMed1996

Frequency of pap and pil operons in Escherichia coli strains associated with urinary infections.

Strains of E. coli isolated from patients with urinary tract infection were examined for P and type 1 adhesin production by colony hybridization with pap and pil operons. The P pili probe detected 45 (46.4%) of the total of 97 strains studied and the type 1 pili probe detected 83 (85.6%). The pap operon was detected in 39 (53.4%) of 73 strains isolated from urine of patients with urinary disease and in 6 (25.0%) of 24 strains isolated from feces of healthy individuals employed as controls (P = 0.029), and the pil operon was detected in 67 (91.8%) of the urinary strains and in 16 (66.6%) of the fecal strains (P = 0.007). Our data did not show significant differences in frequency of P pili among isolates from pyelonephritis (78.5%), cystitis (45.8%) and asymptomatic bacteriuria (54.5%). Type 1 pili were not associated with the different types of infection; the frequency of these pili was 100% in pyelonephritis and in asymptomatic bacteriuria, and 87.5% in cystitis. The incidence of pap operon in strains isolated from pyelonephritis and from asymptomatic bacteriuria was higher in 11- to 40-year old women. These data show a high frequency of pap and pil operons among uropathogenic strains of E. coli, which seems to be an important factor in the development of urinary infection.

Adhesins, Bacterial↗

PubMed1998

Priority of pentose utilization at the level of transcription: arabinose, xylose, and ribose operons.

When E. coli cells were grown in minimal medium supplemented with D-ribose and D-xylose, a diauxic growth preferring D-xylose was observed. Transcription of the ribose (rbs) operon was repressed in the presence of D-xylose, phenotypically similar to catabolite repression by D-glucose, although D-ribose did not affect transcription of the xylose (xyl) operon. Complementation analysis with xylR revealed that the repression of the rbs operon by D-xylose is exerted at the transcriptional level through XylR, suggesting a novel mechanism for catabolite repression. Furthermore, it was shown that L-arabinose reduced transcriptions of both xyl and rbs operons, whereas the arabinose operon was not affected by D-xylose or D-ribose, suggesting a priority mechanism for pentose utilization.

Arabinose↗

PubMed1992

Cloning and sequencing of the petBD operon from the cyanobacterium Synechococcus sp. PCC 7002.

The genes encoding the photosynthetic cytochrome b6 (petB) and subunit 4 (petD) have been cloned and sequenced from the unicellular, photoheterotrophic, transformable cyanobacterium Synechococcus sp. PCC 7002, formerly designated Agmenellum quadruplicatum. The gene arrangement was found to be similar to that reported in the cyanobacterium Nostoc PCC 7906. The DNA and derived protein sequences were compared to chloroplast and the other cyanobacterial sequences. By pulsed-field electrophoresis, the petBD operon and the petCA operon, encoding the Rieske iron-sulfur protein and cytochrome f, were found to be located on separate, unlinked, Not I-digested DNA fragments. The petBD operon was found on the third largest Not I fragment (NC-325) while the petCA operon was found on the second largest Not I fragment (NB-370). These results suggest the two operons are not in proximity. The 1.35 kb transcript was shown to be light-regulated. Transcripts from cells grown under constant illumination showed a decrease in petB transcript levels to undetectable levels within 2 h after the cells were placed in the dark. Upon reillumination, transcript levels rose to three-fold over that seen initially under constant illumination.

Amino Acid Sequence↗

PubMed1995

Insertional Mutations in the Hydrogenase vhc and frc Operons Encoding Selenium-Free Hydrogenases in Methanococcus voltae.

Methanococcus voltae, which contains four different gene groups that encode [NiFe]-hydrogenases, was transformed with integration vectors to achieve polar inactivation of two of the four hydrogenase operons that encode the selenium-free enzymes Vhc and Frc. Transformants which were selected by their acquired puromycin resistance showed site-specific insertions in either the vhc or frc operon by single crossover events. Southern hybridization revealed tandem integrations of whole vectors in the vhc operon, whereas only one vector copy was found in the frc operon. Northern (RNA) hybridizations showed a pac transcript of defined size, indicating strong termination in front of the hydrogenase genes downstream. In spite of the apparent abolition of expression of selenium-free hydrogenases through these polar insertions, they were not lethal to cells upon growth in selenium-deprived minimal medium, which we had previously shown to strongly induce transcription of the respective operons in M. voltae. Instead, like wild-type control cultures, transformants responded to selenium deprivation only with a reduction in growth rate. We conclude that loss of the potential to express a selenium-free hydrogenase can nevertheless be balanced by very small amounts of selenium hydrogenases under laboratory conditions in which the hydrogen supply is not likely to be a limiting growth factor.

Journal Article↗

PubMed1971

Evidence that tryptophanyl transfer ribonucleic acid is not the corepressor of the tryptophan operon of Escherichia coli.

When a growing culture of a tryptophan auxotroph of Escherichia coli is transferred to a tryptophan-free medium, the bacteria exhaust their supply of Trp-transfer ribonucleic acid (tRNA). Under these conditions transcription of the trp operon is derepressed. When l-tryptophan, dl-4-methyltrytophan, dl-6-methyltryptophan, or dl-7-azatryptophan is added to a tryptophan-starved culture, charging to tRNA(Trp) can be detected with each of these compounds except 6-methyltryptophan. Under these conditions, transcription initiations on the trp operon are repressed completely by tryptophan, 4-methyltryptophan, and 6-methyltryptophan, but only slightly by 7-azatryptophan. If a culture of a bacterium containing an altered tryptophanyl-tRNA synthetase (trpS(-)) is starved for tryptophan in the same manner, tRNA(Trp) is uncharged, but transcription of the operon is derepressed to only about one-third the level observed in trpS(+) cultures. When tryptophan, 4-methyltryptophan, 6-methyltryptophan, or 7-azatryptophan is added to a tryptophan-starved trpS(-) culture, tRNA(Trp) is charged with tryptophan but apparently not with the analogues. However, tryptophan, 4-methyltryptophan, and 6-methyltryptophan completely repress transcription initiations, whereas 7-azatryptophan derepresses transcription initiations to approximately the level of partial repression observed in trpS(+) cultures in the presence of 7-azatryptophan. When tryptophan is added to a tryptophan-starved trpS10110 culture, there is a 2-min delay before tRNA(Trp) appears to be charged. However, under these conditions, transcription initiations on the trp operon are repressed immediately by the addition of tryptophan. We interpret these results as indicating that Trp-tRNA is not the corepressor of the trp operon.

Bacterial Proteins↗

PubMed1971

D-Fucose as a gratuitous inducer of the L-arabinose operon in strains of Escherichia coli B-r mutant in gene araC.

d-Fucose, a nonmetabolizable analogue of l-arabinose, prevents growth of Escherichia coli B/r on a mineral salts medium plus l-arabinose by inhibiting induction of the l-arabinose operon. Mutations giving rise to d-fucose resistance map in gene araC and result in constitutive expression of the l-arabinose operon. Most of these mutations also permit d-fucose to serve as a gratuitous inducer. It is concluded that d-fucose-resistant mutants produce an araC gene product with an altered inducer specificity. Addition of l-arabinose to cells induced with the gratuitous inducer, d-fucose, resulted in severe transient repression of operon expression followed by permanent catabolite repression. Transient repression but no permanent catabolite repression was obtained when cells unable to metabolize l-arabinose were employed. It is concluded that transport of l-arabinose alone is sufficient to achieve transient repression of its own operon, but that metabolism of l-arabinose must occur to achieve permanent catabolite repression of the l-arabinose operon. This general effect has been termed "self-catabolite repression."

Arabinose↗

PubMed1992

Regulation of the gua operon of Escherichia coli by the DnaA protein.

The guaBA operon determines production of the two enzymes required to convert hypoxanthine to guanine at the nucleotide level during guanine nucleotide biosynthesis. Two DnaA boxes, binding sites for the DNA replication-initiating DnaA protein, are present in the gua operon, one at the gua promoter (guaP) and the other within the guaB coding sequence. Regulation of the guaBA operon by DnaA protein was studied using strains carrying chromosomal gua-lacZ fusions. In these strains beta-galactosidase acts as a reporter enzyme for transcription initiated at guaP. When the intracellular levels of DnaA were increased (by induction of a multicopy plasmid carrying the dnaA gene fused to the tac promoter) transcription from the gua promoter was repressed. Reducing the intracellular level of DnaA, either by sequestration with an oriC plasmid or by placing a temperature-sensitive dnaA mutant at the restrictive temperature, resulted in increased transcription from guaP. Thus the transcriptional activity of the gua operon is coupled, through the DnaA protein, to the DNA replication cycle. Repression of guaP by DnaA was dependent on the presence of both boxes in the gua-lacZ fusion; constructs containing only the box at guaP were unaffected by DnaA.

Amino Acid Sequence↗

PubMed1988

Analysis of transcription and processing signals in the 5' regions of the two Mycoplasma capricolum rRNA operons.

The transcription and RNA processing signals of the rRNA operons (rrnA and rrnB) of Mycoplasma capricolum were analyzed by mapping the 5' ends of in vivo and in vitro synthesized RNAs. The results of both in vitro and in vivo analyses point to the rrnA operon being transcribed from two promoters (P1 and P2) into large precursor RNAs. Transcripts initiating at P1 contain two tRNAs, and probably 16 S, 23 S, and 5 S rRNAs, whereas the transcripts starting from P2 consist only of the three rRNAs. The precursor RNAs are processed via distinct intermediates into mature tRNAs and rRNAs. In vivo experiments indicated that the rrnB operon is transcribed only from one promoter, although a second promoter could be identified using cell free extracts. The rrnB operon does not contain tRNA genes, but the precursor is still processed in the same way as the rrnA precursor that is synthesized from P2.

Base Sequence↗

PubMed2005

Gene replacement in Haloarcula marismortui: construction of a strain with two of its three chromosomal rRNA operons deleted.

Site-directed mutagenesis were done in Haloarcula marismortui using the strategy that Khorana and coworkers devised for deleting the bacteriorhodopsin gene from Halobacterium halobium [Krebs et al. Proc Natl Acad Sci USA 90:1987-1991 (1993)]. Strains have been prepared from H. marsimortui, which normally has three rRNA operons, that are missing either its rrnB operon or both its rrnB and rrnC operons. In rich media, both strains grow at about the same rate as wild type. The G2099 in the 23S rRNA gene of the single operon strain was changed to A, and a three amino acid deletion was introduced into the gene for ribosomal protein L22 of the wild-type organism. The structural consequences of these and other such mutations can be determined with unusual accuracy because crystals of the large ribosomal subunit of H. marismortui diffract to atomic resolution.

Base Sequence↗

PubMed1996

Expression and regulation of the rnc and pdxJ operons of Escherichia coli.

Escherichia coli rnc-era-recO operon (rnc operon) expression is negatively autoregulated at the level of message stability by ribonuclease III (RNase III), which is encoded by the rnc gene. RNase III, a double-stranded RNA-specific endoribonuclease involved in rRNA and mRNA processing and degradation, cleaves a stemloop structure in the 5' untranslated leader, initiating rapid decay of the rnc operon mRNA. Here, we examine rnc operon expression and regulation in greater detail. Northern, primer extension, and lacZ fusion analyses show that a single promoter (rncP) specifies two principal mRNAs: the 1.9 kb rnc-era transcript and the less-abundant 3.7 kb RNA encoding rnc-era-recO and the downstream pdxJ and acpS genes. A 1.3 kb pdxJ-acpS RNA is transcribed from a promoter (pdxP) located within recO. About 70% of pdxJ transcription depends on transcription from rncP. Both promoters were characterized genetically. RNase III reduces 1.9 kb and 3.7 kb transcript levels and stability, and corresponding effects are seen with genetic fusions. These detailed studies enabled us to show that the first 378 nucleotides of the rnc transcript comprise a portable RNA stability element (rncO) that contains all of the cis-acting elements required for RNase III-initiated decay of the rnc mRNA as well as the heterologous lacZ transcript. Moreover, mutations in rncO that block RNase III cleavage also block control, showing that RNase III initiates mRNA decay by cleaving at a single site.

Bacterial Proteins↗

PubMed1996

Expression of the groESL operon is cell-cycle controlled in Caulobacter crescentus.

The Caulobacter crescentus groESL operon was cloned, sequenced and found to be homologous to previously described groES and groEL genes and proteins. The size of the groESL-specific transcript (2.3 kb) suggested that groES and groEL of C. crescentus are organized in a bicistronic operon. Heat-shock induction of groESL mRNA is not transient, high levels of the transcript can be observed after 2 h at 40 degrees C. Primer extension experiments showed that transcription initiated at two sites. Only the start site closer to the groES coding region was highly induced during heat shock. The promoter corresponding to the heat-shock-inducible transcript has -10 and -35 regions very similar to Escherichia coli sigma 32 promoters. At normal temperatures, transcription of the groESL operon is cell-cycle controlled and both transcripts increase co-ordinately in pre-divisional cells. Transcription fusions with a lacZ reporter gene and deletions within the promoter region of the groESL operon have shown that no sequences upstream of the heat-shock promoter are necessary for temporal control. An 11 bp inverted repeat, located between the heat-shock promoter and the translation start site of groES and very similar to inverted repeats found in front of several heat-shock genes of other bacteria, may play a role in cell-cycle control of C. crescentus groESL expression.

Base Sequence↗

PubMed1998

tRNATrp as a key element of antitermination in the Lactococcus lactis trp operon.

The expression of the trp operon of Lactococcus lactis is regulated in response to tryptophan availability by a mechanism of transcription antitermination. We present evidence in support of a previously described model involving tRNATrp as a key element in the sensing of tryptophan levels and the realization of the regulatory response to tryptophan limitation. In agreement with this model, two sites of presumed direct interaction between the trp leader transcript and tRNATrp are found to be of crucial importance for efficient antitermination. These correspond to the specifier codon, which presumably interacts with the anticodon in the tRNA, and a sequence complementary to, and presumably interacting with, the acceptor stem of the tRNA. Through these interactions, uncharged tRNATrp is believed to stabilize an antiterminator conformation of the trp leader transcript, thus allowing transcription and expression of the structural genes of the operon. For the first time, we present direct evidence that it is the ratio of uncharged to charged tRNA that is important for the regulation of antitermination, rather than the absolute amount of uncharged tRNA. In addition, our results indicate that the codon-anticodon interaction, although contributing largely to the efficiency of the regulatory response, is not strictly indispensable, which suggests the existence of additional interactions between mRNA and tRNA. Finally, we describe a possible additional level of regulation, superimposed and dependent on tRNA-mediated anti-termination control, that is based on the processing of the trp leader transcript. Together with the regulation mechanisms described earlier for the Escherichia coli and Bacillus subtilis trp operons, this constitutes the third different mechanism of transcript elongation control found to be involved in the regulation of an operon of which the structural genes are highly conserved.

Artificial Gene Fusion↗

PubMed2001

Mutational analysis of regulatory cis-acting elements for the transcriptional activation of the dmsCBA operon in Rhodobacter sphaeroides f. sp. denitrificans.

Four direct repeats of a 10-nt sequence, called dms boxes, are located upstream of the dmsCBA operon encoding dimethyl sulfoxide (DMSO) reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. Two dms boxes 1 and 2 have been shown to be binding sites of DmsR protein, a response regulator of a two-component system involved in the anaerobic induction by DMSO of DMSO reductase synthesis. In this study, functions of four dms boxes in the transcriptional regulation of the dmsCBA operon were investigated. The transcription start site of the dmsCBA genes was identified at the distance of 23 nt downstream of the closest dms box 4. Expression of the dmsC-lacZ gene fusion which included the dmsCBA promoter region containing the dms boxes was examined and its anaerobic induction by DMSO and DmsR-dependency were demonstrated in the phototroph. The examination with nucleotide substitutions in the four respective dms boxes showed that the set of four dms boxes is required for the dmsCBA operon activation. Moreover, the importance of the nucleotide sequence of TTCAC in dms box 4 and of A at the center in dms box 1 was significantly shown. These facts suggest that the pentad nucleotides TTCAC and TTAAC in the dms boxes serve as cis-acting elements in the transcriptional activation of the dmsCBA operon.

Anaerobiosis↗

PubMed1990

DNA looping in cellular repression of transcription of the galactose operon.

Communication between distant DNA sites is a central feature of many DNA transactions. Negative regulation of the galactose (gal) operon of Escherichia coli requires repressor binding to two operator sites located on opposite sides of the promoter. The proposed mechanism for regulation involves binding of the repressor to both operator sites, followed by a protein-protein association that loops the intervening promoter DNA (double occupancy plus association). To assess these requirements in vivo, we have previously converted gal operator sites to lac and shown that both operator sites must be occupied by the homologous repressor protein (Lac or Gal) for negative regulation of the gal operon. We have now addressed more directly the need for protein-protein association by the use of the converted operator sites and a mutant Lac repressor defective in association of the DNA-binding dimers. We have compared the biological and biochemical activity of two Lac repressors: the wild-type (tetramer) I+ form, in which the DNA-binding dimer units are tightly associated; and the mutant Iadi repressor, in which the dimer units do not associate effectively. The I+ repressor is an efficient negative regulator of the gal operon in vivo, but the Iadi mutant is an ineffective repressor. Purified I+ repressor efficiently forms DNA loops between operator sites that we have visualized by electron microscopy; the Iadi repressor fails to form DNA loops, although the protein binds effectively to both operator sites. From the clear correlation between looping in vitro and repression in vivo, we conclude that regulation of the gal operon depends on the association of repressor proteins bound to the two operator sites.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Bacterial↗

PubMed1988

Instability of rRNA operons in Bacillus subtilis.

Many laboratory strains of Bacillus subtilis contain 9 rather than 10 rRNA operons due to deletions occurring within the rrnJ-rrnW or rrnI-rrnH-rrnG gene cluster. These operons are members of two sets of closely spaced clusters located in the cysA-aroI region. Analysis of rescued DNA from integrants with insertions into rrnG and rrnH indicated that these tandemly arranged operons allowed frequent deletions of an rrn operon equivalent. These events may arise spontaneously by intrachromosomal recombination or by simultaneous double crossovers with a multimeric integrative plasmid.

Bacillus subtilis↗

PubMed1992

A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.

This report describes a set of Escherichia coli and Salmonella typhimurium strains that permits the reversible transfer of lac fusions between a plasmid and either bacterial chromosome. The system relies on homologous recombination in an E. coli recD host for transfer from plasmid to chromosome. This E. coli strain carries the S. typhimurium put operon inserted into trp, and the resulting fusions are of the form trp::put::[Kanr-X-lac], where X is the promoter or gene fragment under study. The put homology flanks the lac fusion segment, so that fusions can be transduced into S. typhimurium, replacing the resident put operon. Subsequent transduction into an S. typhimurium strain with a large chromosomal deletion covering put allows selection for recombinants that inherit the fusion on a plasmid. A transposable version of the put operon was constructed and used to direct lac fusions to novel locations, including the F plasmid and the ara locus. Transductional crosses between strains with fusions bearing different segments of the hemA-prfA operon were used to determine the contribution of the hemA promoter region to expression of the prfA gene and other genes downstream of hemA in S. typhimurium.

Chromosome Deletion↗