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PubMed1986

Alteration by mutation of the control by oxygen of the nar operon in Escherichia coli.

A nar-lac operon fusion was used to isolate a mutant in which the expression of the nar operon was no longer repressed by oxygen. The nard mutation, located upstream of the nar structural genes, was found to be cis dominant; it led to independence from the Fnr protein which, in the wild-type strain, exerts a strict positive control on the nar operon. Both other known controls, nitrate induction and autoregulation, were unaffected. It is proposed that molecular oxygen controls the expression of nar via Fnr and that the nard mutation affects the Fnr binding site of the narGHI control region.

Aerobiosis↗

PubMed1990

Five additional genes in the pulC-O operon of the gram-negative bacterium Klebsiella oxytoca UNF5023 which are required for pullulanase secretion.

DNA sequence analysis, Tnpho and Tntac-1, mutagenesis, deletion analysis, expression under bacteriophage T7 gene 10 promoter control, subcellular fractionation and complementation tests were used to study the function of DNA located in the centre of the pulC-O operon from Klebsiella oxytoca strain UNF5023. The characterized region of the operon includes five genes (pulG, pulH, pulI, pulJ and pulK) coding for apparently integral inner membrane proteins which are required for pullulanase secretion. The results presented here and previously show that the pulC-O operon contains at least 11 pullulanase secretion genes.

Amino Acid Sequence↗

PubMed1986

A new cell division operon in Escherichia coli.

At 76 min on the E. coli genetic map there is a cluster of genes affecting essential cellular functions, including the heat shock response and cell division. A combination of in-vivo and in-vitro genetic analysis of cell division mutants suggests that the cell division gene fts E is the second gene in a 3 gene operon. A cold-sensitive mutant, defective in the third gene, is also unable to divide at the restrictive temperature, and we designate this new cell division gene fts X. Another cell division gene, fts S, is very close to, but distinct from, the 3 genes of the operon. The fts E product is a 24.5 Kd polypeptide which shows strong homology with a small group of proteins involved in transport. Both the fts E product and the protein coded by the first gene (fts Y) in the operon have a sequence motif found in a wide range of heterogeneous proteins, including the Ras proteins of yeast. This common domain is indicative of a nucleotide-binding site.

Amino Acid Sequence↗

PubMed1989

Vitamin B12 transport in Escherichia coli K12 does not require the btuE gene of the btuCED operon.

Transport of vitamin B12 across the cytoplasmic membrane of Escherichia coli requires the products of btuC and btuD, two genes in the btuCED operon. The role of btuE, the central gene of this operon, was examined. Deletions within btuE were constructed by removal of internal restriction fragments and were crossed onto the chromosome by allelic replacement. In-frame deletions that removed 20% or 82% of the btuE coding region did not affect expression of the distal btuD gene. These nonpolar deletions had little effect on vitamin B12 binding (whole cells or periplasmic fraction) and transport. They did not affect the utilization of vitamin B12 or other cobalamins for methionine biosynthesis, even in strains with decreased outer membrane transport of vitamin B12. The btuE mutations did not impair adenosyl-cobalamin dependent catabolism of ethanolamine or repression of btuB expression. Thus, despite its genetic location in the transport operon, the btuE product plays no essential role in vitamin B12 transport.

Amino Acid Sequence↗

PubMed2002

Promoter analysis of the sodium-responsive V-ATPase (ntp) operon in Enterococcus hirae.

The Enterococcus hirae ntp operon encodes all subunits of the vacuolar-type ATPase (V-ATPase), which transports Na(+) or Li(+). This operon is expressed preferentially in response to Na(+), but not to Li(+). Deletion analysis of the ntp promoter region in plasmids indicated that the AT-tract between -198 and -132 is required for Na(+)-specific transcriptional regulation. In addition, lithium-tolerant (LTR) mutants were isolated in which functional V-ATPase levels were high even in Na(+)-depleted medium. Western blot and Northern blot experiments revealed an increase in basal Na(+)-independent transcription in one of the mutants (LTR1). The nucleotide sequences of the ntp promoter region of the LTR mutants showed mutational conversion of single base-pairs between positions -23 and +1. Na(+)-independent expression of a reporter gene linked to the ntp promoter in plasmids was elevated by base substitutions at -23 to +1, and promoter activity induced by these base substitutions was lost by deletion of the region between -198 and -132. These results suggest that the AT-tract between -198 and -132 is indispensable for transcription of the ntp operon.

Adenosine Triphosphatases↗

PubMed2003

Gene structure and transcriptional regulation specific to the groESL operon from the psychrophilic bacterium Colwellia maris.

The groESL operon of a psychrophilic bacterium, Colwellia maris, was cloned and sequenced. The operon contains two ORFs of 291 bp and 1,650 bp separated by 210 bp. Northern blot analysis suggested that the groESL operon was transcribed as a bicistronic mRNA, and that the amount of mRNA markedly increased after the temperature was raised from 10 degrees C to 20 degrees C. Although the optimum temperatures for GroESL function are different in psychrophilic, mesophilic, and thermophilic bacteria, the deduced amino acid sequences of C. maris GroES and GroEL showed remarkably high similarity with those of GroES and GroEL from mesophilic and thermophilic bacteria. A putative promoter similar to the Escherichia coli sigma(32) consensus sequence was identified. One specific feature of C. maris groESL was that in the putative untranslated region the G+C content was about 24 mol%, which is much lower than that of mesophilic bacteria such as E. coli. The low G+C content may be important for maintaining transcription at low temperatures.

Alteromonadaceae↗

PubMed1997

Catabolite regulation of two Escherichia coli operons encoding nitrite reductases: role of the Cra protein.

The Escherichia coli nir and nrf operons, which encode alternative nitrite reductases expressed during anaerobic growth, are subject to catabolite regulation. Transcription from the nir promoter is maximal when bacteria are grown in rich media such as Lennox broth supplemented with glucose. Conversely, expression of the nrf operon is suppressed by rich media, but stimulated during growth in minimal medium with glycerol and fumarate. The role of the catabolite repressor-activator (Cra) protein in catabolite regulation of the nir and nrf promoters was investigated. Transcription from the nir promoter was repressed by Cra when cells were grown in minimal medium with glycerol and fumarate. Crude protein extracts from a strain overproducing Cra encoded on a multicopy plasmid retarded a nir promoter fragment in a mobility shift assay, confirming that the observed Cra-dependent repression was due to the direct interaction of Cra with the regulatory region of the nir operon. Furthermore, the inclusion of fructose 1-phosphate, an effector of Cra DNA-binding activity, in the assay decreased the ability of Cra to retard the nir promoter fragment. In contrast, transcription from the nrf promoter was not regulated by Cra under any of the growth conditions tested.

Bacterial Proteins↗

PubMed2001

Chloroplast transformation in Euglena gracilis: splicing of a group III twintron transcribed from a transgenic psbK operon.

The Escherichia coli aadA gene product, which confers resistance to spectinomycin and streptomycin, has been widely used as a dominant selectable marker for chloroplast transformation of Chlamydomonas and tobacco. An aadA transformation cassette was adapted for expression in Euglena gracilis chloroplasts by replacing the Chlamydomonas promoter and 3' untranslated region (UTR) with the E. gracilis psbA promoter and 3' UTR. Transgenic DNA was introduced into E. gracilis chloroplasts by biolistic transformation. Streptomycin- and spectinomycin-resistant colonies were obtained, which screened positively for the presence of the transforming vector by PCR amplification. Although integration of the transforming DNA into the chloroplast genome was not detected, transforming DNA was stably maintained in the chloroplast as an episomal element during continuous selection on antibiotics. The aadA cassette was also inserted into a transformation vector which contained the independently expressed psbK operon from either E. gracilis or a closely related species, E. stellata. The psbK operon contained at least two group III introns and a group III twintron, was highly expressed, and was only 1.5 kb in length. In transgenic E. gracilis chloroplasts, a truncated E. stellata psbK operon was transcribed, and the resultant pre-mRNA was accurately spliced. This system should allow the first direct analysis of group II and group III intron-splicing mechanisms. In addition, it could prove useful in the study of many other Euglena transcription and processing events.

Animals↗

PubMed1999

Organization and post-transcriptional processing of the psb B operon from chloroplasts of Populus deltoides.

Chloroplast genes are typically organized into polycistronic transcription units that give rise to complex sets of mono- and oligo-cistronic overlapping RNAs through a series of processing steps. The psbB operon contains genes for the PSII (psbB, psbT, psbH) and cytochrome b(6)f (petB and petD) complexes which are needed in different amounts during chloroplast biogenesis. The functional significance of gene organization in this polycistronic unit, containing information for two different complexes, is not known and is of interest. To determine the organization and expression of these complexes, studies have been carried out on crop plants by different groups, but not much information is known about trees. We present the nucleotide sequences of PSII genes and RNA profiles of the genes located in the psbB operon from Populus deltoides, a tree species. Although the gene organization of this operon in P. deltoides is similar to that in other species, a few variations have been observed in the processing scheme.

Amino Acid Sequence↗

PubMed2002

Transcriptional analysis of the pst operon of Escherichia coli.

The pst operon of Escherichia coli, which encodes the phosphate-specific transport system, is composed of five genes, pstS, pstC, pstA, pstB and phoU, whose transcription is induced by phosphate starvation. A phosphate-regulated promoter located upstream of the most proximal gene ( pstS) controls the transcription of the entire operon. Though the full-length pst mRNA could be detected by an improved RT-PCR protocol, Northern analysis using several pst-specific probes failed to reveal this transcript. Instead, smaller but distinct pst mRNA species were evident. Primer-extension experiments localized the 5' ends of pst mRNAs within the operon. The data suggest that the full-length mRNA is rapidly processed post-transcriptionally.

ATP-Binding Cassette Transporters↗

PubMed1999

Isolation and characterization of a purC(orf)QLF operon from Lactococcus [correction of Lactobacillus] lactis MG1614.

We have isolated genes encoding enzymes of the de novo purine nucleotide biosynthesis pathway from Lactococcus lactis MG1614 by colony hybridization using DIG-labeled DNA probes. The organization of the genes needed for the de novo biosynthesis of purine nucleotides in L. lactis differs from that found in other organisms. In L. lactis there is a gene cluster, which contains five out of the 11 genes needed for the de novo biosynthesis of IMP, namely purC, orf, purQ, purL and purF. These genes were shown to be transcribed as a single transcription unit by Northern hybridization analysis. The 5' end of the transcript of the purC(orf)QLF operon was determined by primer extension analysis using fluorescently end-labeled probes. The purC(orf)QLF operon of L. lactis is transcribed in Escherichia coli, and the gene product of the purF gene, glutamine phosphoribosylpyrophosphate amidotransferase (glutamine PRPP ATase, EC 2.4.2.14), can functionally complement the E. coli purF mutant strain TX158. We also show that the promoter of the purC(orf)QLF operon is regulated in response to exogenously added purines.

Amino Acid Sequence↗

PubMed2000

Expression of the hemolysin operon in Escherichia coli is modulated by a nucleoid-protein complex that includes the proteins Hha and H-NS.

The Escherichia coli protein Hha is a temperature- and osmolarity-dependent modulator of the expression of the hemolysin operon. The Hha protein was purified and its DNA-binding properties analyzed. Hha binds in a non-specific manner throughout the upstream regulatory region of the hemolysin operon in the recombinant hemolytic plasmid pANN202-312. A search for interacting proteins revealed that Hha interacts with H-NS. DNA-binding studies showed that, in vitro, Hha and H-NS together form a complex with DNA that differs from those formed with either protein alone. These data, together with the effects of hha and hns mutations on the expression of the hemolysin genes, suggest that in vivo H-NS and Hha form a nucleoid-protein complex that accounts for the thermo-osmotic regulation of the hemolysin operon in E. coli.

Bacterial Proteins↗

PubMed2001

The cyo operon of Pseudomonas putida is involved in carbon catabolite repression of phenol degradation.

A bicistronic reporter consisting of the promoterless genes aacC1 (conferring gentamycin resistance) and lacZ fused to the catabolic promoter of the phenol degradation genes was used to identify and analyse mutants of Pseudomonas putida with altered carbon catabolite repression (CR) of phenol degradation. Out of approximately 2500 mini-Tn5 mutants analysed so far, 12 mutants that were resistant to gentamycin during growth on succinate were identified. In eight of these mutants mini-Tn5 was inserted into one of the genes of the cyo operon. The cyo operon encodes the cytochrome o ubiquinol oxidase, the terminal oxidase of the cyanide-sensitive branch of the respiratory chain. In these mutants the activity of the PphlA promoter was significantly increased during growth on succinate and reached 15-20% of that found during growth with the non-repressing carbon source pyruvate. During growth on glucose the reduction of CR was less obvious, during growth on lactate CR was unchanged. The possible significance of the cyo operon for the generation of signal(s) for carbon catabolite repression is discussed.

Bacterial Proteins↗

PubMed2006

Relation between induction of the mar operon and cyclohexane tolerance and reduction in fluoroquinolone susceptibility in Salmonella spp.

The relationship between cyclohexane tolerance and induction of the mar operon and a decrease in susceptibility to ciprofloxacin and moxifloxacin in isolates of Salmonella spp. from food and clinical isolates of Salmonella spp. was studied. We studied the influence of the mar operon using an inductor (acetylsalicylic acid) and we also studied the cyclohexane resistance. Induction was seen to produce an increase in the minimum inhibitory concentration (MIC) of these quinolones, which suggested that there was overexpression of the AcrAB type active efflux systems due to induction of the mar operon. Cyclohexane susceptibility was not shown to be a very sensitive method for studying this process, as only 3% (5/176) of the clinical isolates studied were cyclohexane-resistant; most of these belonged to the Hadar serotype. This study confirmed the participation of active efflux systems in the decrease in fluoroquinolone susceptibility in Salmonella spp. Furthermore, the study has indicated that these mechanisms (i.e., active efflux systems) are present in strains that are susceptible to the fluoroquinolone compounds, so their stimulation may be one of the mechanisms involved in the reduction in fluoroquinolone susceptibility. This suggests that the exposure of Salmonella spp. to antibiotics should be limited in order to prevent these active efflux systems from being activated. Consequently, the use of fluoroquinolones, both in the treatment of humans and in veterinary practice, should be controlled and rationalized in an attempt to curb the increase in the number of strains that are resistant to these compounds.

Anti-Bacterial Agents↗

PubMed1977

Genetic regulation of the constitutive D-ribose operon in Escherichia coli B/r.

Merodiploid complementation analysis of the constitutive synthesis of the D-ribokinase and the D-ribose permease in Escherichia coli B/r has shown that the constitutive D-ribose operon is genetically controlled by a transdominant regulatory gene closely linked to the D-ribokinase and D-ribose permease structural genes. The regulatory mechanism for this operon shows no requirement for operator-repressor interaction, rather a truly positive control mechanism and thus suggests an extension of the operon model in its application to constitutive enzyme regulation in bacteria.

Chromosome Mapping↗

PubMed1987

Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters. Interactions with RNA polymerase and the cyclic AMP receptor protein.

We report in vitro studies of the interactions between purified E. coli RNA polymerase and DNA from the regulatory region of the E. coli galactose operon which carries a point mutation that simultaneously stops transcription initiation at the two normal start points, S1 and S2. In the presence of this point mutation, transcription initiates at a third start point 14/15 bp downstream of S1, showing that inactivation of the two normally active promoters, P1 and P2, unmasks a third weaker promoter, P3. Transcription initiation in the gal operon is normally regulated by the cyclic AMP receptor protein, CRP, that binds to the gal regulatory region and switches transcription from P2 to P1. With the point mutation, CRP binding switches transcription from P3 to P1, although the formation of transcriptionally competent complexes at P1 is very slow. The results are discussed with respect to the mechanism of transcription activation by the CRP factor and the similarities between the regulatory regions of the galactose and lactose operons.

DNA-Directed RNA Polymerases↗

PubMed1989

Characterization of the two rRNA gene operons present in Thiobacillus ferrooxidans.

The organization of rRNA genes from the autotrophic, acidophilic bacterium Thiobacillus ferrooxidans has been examined. Two rRNA operons were found in this microorganism by means of genomic hybridization studies. Recombinant plasmids, pTR-3 and pTR-1 that carry a portion of 16/23 S rDNA from one operon and the 5'-flanking region of the second operon, respectively, were identified and characterized.

Cloning, Molecular↗

PubMed1991

Nitrite activates the transcription of the Pseudomonas aeruginosa nitrite reductase and cytochrome c-551 operon under anaerobic conditions.

The transcription of the Pseudomonas aeruginosa denAB operon, which consists of the nitrite reductase and cytochrome c-551 genes, is induced under anaerobic conditions. However, under anaerobic non-denitrifying conditions (anaerobic growth on arginine), the promoter activity of the operon was approximately one-fifth of that under anaerobic denitrifying conditions (anaerobic growth in the presence of nitrite or nitrate). This result clearly demonstrates that the presence of nitrite or nitrate activates the transcription of P. aeruginosa denAB operon under anaerobic conditions.

Anaerobiosis↗