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PubMed1992

Locating essential Escherichia coli genes by using mini-Tn10 transposons: the pdxJ operon.

The mini-Tn10 transposon (delta 16 delta 17Tn10) confers tetracycline resistance. When inserted between a gene and its promoter, it blocks transcription and prevents expression of that gene. Tetracycline in the medium induces divergent transcription of the tetA and tetR genes within the transposon, and this transcription extends beyond the transposon in both directions into the bacterial genes. If the mini-Tn10 inserts between an essential bacterial gene and its promoter, the insertion mutation can cause conditional growth which is dependent on the presence of tetracycline. Two essential genes in adjacent operons of Escherichia coli have been detected by screening for tetracycline dependence among tetracycline-resistant insertion mutants. These essential genes are the era gene in the rnc operon and the dpj gene in the adjacent pdxJ operon. The pdxJ operon has not been described previously. It consists of two genes, pdxJ and dpj. Whereas the dpj gene is essential for E. coli growth in all media tested, pdxJ is not essential. The pdxJ gene encodes a protein required in the biosynthesis of pyridoxine (vitamin B6).

Amino Acid Sequence↗

PubMed1993

Regions of the Bacillus subtilis ilv-leu operon involved in regulation by leucine.

The ilv-leu operon of Bacillus subtilis is regulated in part by transcription attenuation. The cis-acting elements required for regulation by leucine lie within a 683-bp fragment of DNA from the region upstream of ilvB, the first gene of the operon. This fragment contains the ilv-leu promoter and 482 bp of the ilv-leu leader region. Spontaneous mutations that lead to increased expression of the operon were shown to lie in an imperfect inverted repeat encoding the terminator stem within the leader region. Mutations within the inverted repeat of the terminator destroyed most of the leucine-mediated repression. The remaining leucine-mediated repression probably resulted from a decrease in transcription initiation. A systematic analysis of other deletions within the ilv-leu leader region identified a 40-bp region required for the derepression that occurred during leucine limitation. This region lies within a potential RNA stem-and-loop structure that is probably required for leucine-dependent control. Deletion analysis also suggested that alternate secondary structures proximal to the terminator are involved in allowing transcription to proceed beyond the terminator. Additional experiments suggested that attenuation of the ilv-leu operon is not dependent on coupling translation to transcription of the leader region. Our data support a model proposed by Grundy and Henkin (F. J. Grundy and T. M. Henkin, Cell 74:475-482, 1993) in which uncharged tRNA acts as a positive regulatory factor to increase gene expression during amino acid limitation.

Bacillus subtilis↗

PubMed1994

Effect of cell growth rate on expression of the anaerobic respiratory pathway operons frdABCD, dmsABC, and narGHJI of Escherichia coli.

The fumarate reductase (frdABCD), dimethyl sulfoxide (DMSO)-trimethylamine-N-oxide (TMAO) reductase (dmsABC), and nitrate reductase (narGHJI) operons in Escherichia coli encode enzymes involved in anaerobic respiration to the electron acceptors fumarate, DMSO or TMAO, and nitrate, respectively. They are regulated in response to anaerobiosis and nitrate availability. To determine how each operon is regulated in response to changes in cell growth rate and in oxygen availability, expression of frdA-lacZ, dmsA-lacZ, and narG-lacZ fusion genes was examined during continuous culture. After a change in the cell growth rate, each anaerobic electron transport pathway operon fusion responded somewhat differently. Whereas frdA-lacZ expression increased by fivefold as the growth rate decreased from 0.60 to 0.12/hour during aerobic growth, little change was seen under anaerobic conditions. In contrast, growth rate-dependent expression of narG-lacZ expression occurred under anaerobic conditions but not under aerobic conditions. Finally, dmsA-lacZ expression did not vary greatly for any of the growth rates tested. When cells were shifted from aerobic to anaerobic growth conditions, expression of each fusion increased at a moderate rate and peaked or "overshot" before reaching a new equilibrium value. This "overshoot" phenomenon was independent of the fnr gene product, which functions as a transcriptional activator of each respiratory operon during anaerobic conditions. In contrast to the moderate rate of anaerobic induction seen for narG-lacZ expression, the addition of nitrate caused a rapid induction response. The cell appears to have many ways to adjust cell respiration in response to changes in cell growth conditions.

Aerobiosis↗

PubMed1995

Roles of the three transcriptional attenuators of the Bacillus subtilis pyrimidine biosynthetic operon in the regulation of its expression.

Expression of the Bacillus subtilis pyr operon is regulated by exogenous pyrimidines and the protein product of the first gene of the operon, PyrR. It has been proposed that PyrR mediates transcriptional attenuation at three untranslated segments of the operon (R.J. Turner, Y. Lu, and R.L. Switzer, J. Bacteriol., 176:3708-3722, 1994). In this study, transcriptional fusions of the pyr promoter followed by the pyr attenuation sequences, either individually or in tandem to a lacZ reporter gene, were used to examine the physiological functions of all three attenuators through their ability to affect beta-galactosidase expression. These fusions were studied as chromosomal integrants in various B. subtilis strains to examine the entire range of control by pyrimidines, PyrR dependence, amd developmental control of pyr gene expression. The nutritional regulation of each attenuator separately was roughly equivalent to that of the other two and was totally dependent upon PyrR, and that of tandem attenuators was cumulative. The regulation of a fusion of the spac promoter followed by the pyrP:pyrB intercistronic region to lacZ produced results similar to those obtained with the corresponding fusion containing the pyr promoter, demonstrating that attenuator-dependent regulation is independent of the promoter. Extreme pyrimidine starvation gave rise to two- to threefold-higher levels of expression of a pyr-lacZ fusion that lacked attenuators, independent of PyrR, than were obtained with cells that were not starved. Increased expression of a similar spac-lacZ fusion during pyrimidine starvation was also observed, however, indicating that attenuator-independent regulation is not a specific property of the pyr operon. Conversion of the initiator AUG codon in a small open reading frame in the pyrP:pyrB intercistronic region to UAG reduced expression by about half but did not alter regulation by pyrimidines, which excludes the possibility of a coupled transcription-translation attenuation mechanism. Developmental regulation of pyr expression during early stationary phase was found to be dependent upon the attenuators and PyrR, and the participation of SpoOA was excluded.

Bacillus subtilis↗

PubMed1996

Transcriptional regulation of the proton-translocating ATPase (atpIBEFHAGDC) operon of Escherichia coli: control by cell growth rate.

The F0F1 proton-translocating ATPase complex of Escherichia coli, encoded by the atpIBEFHAGDC operon, catalyzes the synthesis of ATP from ADP and Pi during aerobic and anaerobic growth when respiratory substrates are present. It can also catalyze the reverse reaction to hydrolyze ATP during nonrespiratory conditions (i.e., during fermentation of simple sugars) in order to maintain a electrochemical proton gradient across the cytoplasmic membrane. To examine how the atp genes are expressed under different conditions of cell culture, atpI-lacZ operon fusions were constructed and analyzed in single copy on the bacterial chromosome or on low-copy-number plasmids. Expression varied over a relatively narrow range (about threefold) regardless of the complexity of the cell growth medium, the availability of different electron acceptors or carbon compounds, or the pH of the culture medium. In contrast to prior proposals, atp operon expression was shown to occur from a single promoter located immediately before atpI rather than from within it. The results of continuous-culture experiments suggest that the cell growth rate rather than the type of carbon compound used for growth is the major variable in controlling atp gene expression. Together, these studies establish that synthesis of the F0F1 ATPase is not greatly varied by modulating atp operon transcription.

Bacterial Outer Membrane Proteins↗

PubMed1997

Characterization of transcriptional regulation of the kdp operon of Salmonella typhimurium.

The transcriptional control of the kdpFABC (K+ transport) operon of Salmonella typhimurium was characterized with a lacZ fusion. The kdpFABC operon of this organism was induced by K+ limitation and high osmolality, and osmotic induction was antagonized by a high concentration of K+. In the trkA (sapG) kdp+ mutant background, high concentrations of K+ inhibited growth, along with repressing the kdp operon. This result, which has not been reported for Escherichia coli, is inconsistent with the model in which the signal for the induction of the kdp operon is turgor loss.

Biological Transport↗

PubMed1999

Identification and characterization of a DeoR-specific operator sequence essential for induction of dra-nupC-pdp operon expression in Bacillus subtilis.

The deoR gene located just upstream the dra-nupC-pdp operon of Bacillus subtilis encodes the DeoR repressor protein that negatively regulates the expression of the operon at the level of transcription. The control region upstream of the operon was mapped by the use of transcriptional lacZ fusions. It was shown that all of the cis-acting elements, which were necessary for full DeoR regulation of the operon, were included in a 141-bp sequence just upstream of dra. The increased copy number of this control region resulted in titration of the DeoR molecules of the cell. By using mutagenic PCR and site-directed mutagenesis techniques, a palindromic sequence located from position -60 to position -43 relative to the transcription start point was identified as a part of the operator site for the binding of DeoR. Furthermore, it was shown that a direct repeat of five nucleotides, which was identical to the 3' half of the palindrome and was located between the -10 and -35 regions of the dra promoter, might function as a half binding site involved in cooperative binding of DeoR to the regulatory region. Binding of DeoR protein to the operator DNA was confirmed by a gel electrophoresis mobility shift assay. Moreover, deoxyribose-5-phosphate was shown to be a likely candidate for the true inducer of the dra-nupC-pdp expression.

Aldehyde-Lyases↗

PubMed2002

Vibrio parahaemolyticus scrABC, a novel operon affecting swarming and capsular polysaccharide regulation.

Swarming is an adaptation of many bacteria to growth on surfaces. A search for genes controlling swarmer cell differentiation of Vibrio parahaemolyticus identified a novel three-gene operon that potentially encodes a pyridoxal-phosphate-dependent enzyme, an extracellular solute-binding protein, and a membrane-bound GGDEF- and EAL-motif sensory protein. The functions of these motifs, which are named after conserved amino acid sequences, are unknown, although the domains are found singly and in combination in a variety of bacterial signaling proteins. Studies with translational fusions supported the predicted localization of the gene products. When the operon was overexpressed, swarmer cell gene transcription was induced in liquid culture. Mutants with defects in any of the three genes exhibited decreased swarming and lateral flagellar (laf) gene expression. Complementation studies confirmed an operon organization and suggested that all three genes participated in laf regulation. The lesions that decreased swarming increased capsular polysaccharide (CPS) production, and overexpression of the operon inhibited transcription of the CPS gene cpsA. Thus, the scrABC locus appears to inversely regulate two gene systems that are pertinent to colonization of surface swarming and CPS.

ATP-Binding Cassette Transporters↗

PubMed2000

The inhibition of Escherichia coli lac operon gene expression by antigene oligonucleotides-mathematical modeling.

Gene transcription is regulated by transcription factors that can bind to specific regions on DNA. Antigene oligonucleotides (oligos) can bind to specific regions on DNA and form a triplex with the double-stranded DNA. The triplex can competitively inhibit the binding of transcription factors and, as a result, transcription can be inhibited. A genetically structured model has been developed to quantitatively describe the inhibition of the Escherichia coli lac operon gene expression by triplex-forming oligos. The model predicts that the effect of triplex-forming oligos on the lac operon gene expression depends on their target sites. Oligonucleotides targeted to the operator are much more effective than those targeted to other regulatory sites on the lac operon. In some cases, the effect of oligo binding is similar to that of a mutation in the lac operon. The model provides insight as to the specific binding site to be targeted to achieve the most effective inhibition of gene expression. The model is also capable of predicting the oligo concentration needed to inhibit gene expression, which is in general agreement with results reported by other investigators.

Computer Simulation↗

PubMed1984

Transcription of the TOL plasmid toluate catabolic pathway operon of Pseudomonas putida is determined by a pair of co-ordinately and positively regulated overlapping promoters.

Expression of the meta-cleavage pathway operon of TOL plasmid pWW0 of Pseudomonas putida is positively regulated by the xylS gene product. We have sequenced the promoter region of this operon and localized the transcription initiation sites. Two overlapping promoters, designated Pm1 and Pm2, are responsible for the positively regulated expression of the meta-pathway operon. Mutants of P. putida were isolated that expressed the meta-cleavage pathway operon constitutively. Several plasmid-located mutations that led to constitutivity were characterized by sequencing and the transcription initiation sites on mutant plasmids localized. This resulted in the identification of newly created promoters whose functioning did not require the xylS product. Comparison of the promoter sequences obtained suggests a tentative consensus sequence for promoters of P. putida which is significantly different from that of E. coli.

Base Sequence↗

PubMed1993

Depletion of functional ribosomal RNA operons in Escherichia coli causes increased expression of the remaining intact copies.

The synthesis of ribosomal RNA is a complex and highly regulated process. To study this process, we have used deletion-insertions to disrupt sequentially from one to four of the seven rRNA (rrn) operons on the Escherichia coli genome. Inactivation of four rrn operons caused a 2.3-fold increase in the expression of a chloramphenicol acetyl transferase reporter gene fused to the tandem promoters of rrnA and a similar increase in the expression of the trp tRNA gene at the end of rrnC. This reflected enhanced expression of the remaining operons to compensate for having only three intact copies. The elevated expression was caused by an increase in both transcription initiation and RNA polymerase elongation rates specifically on rrn operons and occurred in the absence of changes in the intracellular concentration of ppGpp, suggesting that ppGpp is not involved in the regulation of this phenomenon. We discuss these results in relation to the ribosome feedback inhibition model described by Nomura and coworkers.

Base Sequence↗

PubMed1994

Unique dicistronic operon (ptsI-crr) in Mycoplasma capricolum encoding enzyme I and the glucose-specific enzyme IIA of the phosphoenolpyruvate:sugar phosphotransferase system: cloning, sequencing, promoter analysis, and protein characterization.

The region of the genome of Mycoplasma capricolum encompassing the genes for Enzymes I and IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was cloned and sequenced. Examination of the sequence revealed a unique arrangement of the pts operon. In all other bacterial species characterized thus far, the gene encoding Enzyme I (ptsI) in the pts operon is located immediately downstream of the gene (ptsH) encoding HPr, a general energy coupling protein of the PTS. In M. capricolum, ptsH and ptsI reside on 2 distinct operons at separate loci on the chromosome (Zhu PP, Reizer J, Reizer A, Peterkofsky A, 1993, J Biol Chem 268:26531-26540). In the present work, it is shown that the Mycoplasma Enzyme I gene is preceded by an open reading frame homologous to the product of the Escherichia coli kdtB gene and is followed by the gene (crr) encoding Enzyme IIAglc. Northern blot analysis indicated that ptsI and crr constitute a dicistronic operon that includes an independent promoter for the crr gene. Primer extension studies established the transcription start sites for the ptsI and crr genes. The products of the ptsI and crr genes are homologous to previously sequenced Enzymes I and IIAglc proteins but are more similar to the counterpart proteins from gram-positive than to those from gram-negative organisms. The deduced amino acid sequence of the Mycoplasma Enzyme I shows that it differs from other Enzymes I by having fewer acidic amino acids and more basic, amidated, and aromatic amino acids. The deduced amino acid sequence of the Mycoplasma Enzyme IIAglc indicates that it is the shortest (154 residues) of the proteins in this class and it is the only Enzyme IIAglc with a tryptophan and a cysteine residue. In vitro sugar phosphorylation studies with extracts from E. coli and Bacillus subtilis and purified proteins indicated that the Mycoplasma HPr is not a phosphoacceptor from the E. coli Enzyme I, whereas the Mycoplasma Enzyme IIAglc accepts and transfers phosphate from both E. coli and B. subtilis PTS components.

Amino Acid Sequence↗

PubMed1996

Nucleotide sequence and functional analysis of the luxE gene encoding acyl-protein synthetase of the lux operon from Photobacterium leiognathi.

Nucleotide sequence of the luxE gene GenBank Accession No. U66407 from Photobacterium leiognathi PL741 has been determined, and the amino acid sequence of acyl-protein synthetase encoded by the luxE gene is deduced. Nucleotide sequence reveals that the luxE gene encodes acyl-protein synthetase, which is a component of the fatty acid reductase complex that is responsible for converting fatty acid to aldehyde as substrate in the luciferase-catalyzed bioluminescence reaction. The acyl-protein synthetase encoded by the luxE gene has a calculated M, 43,128 and comprises 373 amino acid residues. Alignment and comparison of acyl-protein synthetases from P. leiognathi, P. phosphoreum, Vibrio fischeri, V. harveyi and Xenorhabdus luminescens shows that they are homologous; there is 75.5% homologous (44.2% identity and 31.3% similarity) among these species. Functional analysis illustrates that the specific segment sequence lying before or in the luxE gene might from potential loops omega o omega e1, omega e2 as mRNA stability loop and/or for sub-regulation by alternative modulation in the lux operon. The gene order of the luxE gene in the lux and the lum operons is<--ter-lumQ-lumP-R&R-luxC-luxD-luxA-luxB -luxN-luxE-->(R&R: regulatory region; ter; transcriptional terminator), whereas the R&R is the regulatory region for the lum and the lux operons, and ter is the transcriptional terminator for the lum operon.

Acyltransferases↗

PubMed1999

Effects of promoter mutations on the in vivo regulation of the cop operon of Enterococcus hirae by copper(I) and copper(II).

The cop operon of Enterococcus hirae encodes a repressor, CopY, a copper chaperone, CopZ, and two copper ATPases, CopA and CopB. Regulation of the cop operon is bi-phasic, with copper addition as well as copper chelation leading to induction. Using a plasmid-borne system with a reporter gene, induction of wild-type and mutant cop promoters by high and low copper conditions was investigated. Only mutations that impaired the interaction of CopY with both DNA binding sites had a marked effect on regulation, leading to hyperinduction by copper(I) or copper(II). Chelation of copper(II), but not copper(I), also induced the operon, but induction by copper chelation was not significantly affected by the mutations. E. hirae mutants with reduced extracellular copper reductase activity exhibited the same induction kinetics as wild-type cells. These results show that copper addition and copper chelation induce the cop operon by different routes.

Adenosine Triphosphatases↗

PubMed1997

Role of the spacer boxA of Escherichia coli ribosomal RNA operons in efficient 23 S rRNA synthesis in vivo.

A boxA sequence, known to be important for transcriptional antitermination, is found in both the leader region and in the spacer between the 16 S and 23 S genes of Escherichia coli ribosomal RNA operons. We have shown that a functional leader boxA is important for efficient completion of 16 S rRNA transcription. In this study, point mutations were introduced into the 16S-23S spacer boxA of a plasmid-encoded E. coli rrnB operon in order to study the contribution of this conserved sequence element to ribosomal RNA synthesis in vivo. The rrnB mutant constructs contained an additional point mutation in each of the 16 S and 23 S genes, which were used to distinguish rRNA derived from plasmid and chromosomal rrn operons by primer extension analysis. Mutations in the spacer boxA reduced the proportion of plasmid-derived 23 S rRNA without affecting synthesis of plasmid-derived 16 S rRNA or spacer boxA RNA, indicating that premature termination of transcription occurred during 23 S rRNA synthesis. Reductions in plasmid-derived 23 S rRNA were very similar for total cellular RNA, 50 S subunits and 70 S ribosomes, suggesting that plasmid-derived rRNAs from mutant operons were functional in ribosome biogenesis. In the presence of a wild-type leader boxA, single nucleotide exchanges in the spacer boxA reduced the proportion of plasmid-derived 23 S rRNA from 70% to about 55% under conditions of exponential growth in rich medium. This proportion further decreased to 20 to 25% with an additional point mutation in the leader boxA. We conclude that modification of RNA polymerase into a termination-resistant form has to be renewed at the spacer boxA in order to ensure the faithful completion of full-length 23 S rRNA.

Escherichia coli↗

PubMed1998

NasR, a novel RNA-binding protein, mediates nitrate-responsive transcription antitermination of the Klebsiella oxytoca M5al nasF operon leader in vitro.

In Klebsiella oxytoca (pneumoniae), enzymes required for nitrate assimilation are encoded by the nasFEDCBA operon. Previous genetic studies led to the conclusion that nitrate and nitrite induction of nasF operon expression is determined by a transcriptional antitermination mechanism. In the presence of nitrate or nitrite, the nasR gene product is hypothesized to inhibit transcription termination at the factor-independent terminator site located in the nasF operon leader region. To test this model in vitro, we first purified NasR as both a maltose binding protein fusion form (MBP-NasR) and a His6-tagged form (His6-NasR). Templates for in vitro transcription contained the nasF operon leader region, with a substitution of the sigma70-dependent tac promoter for the native sigmaN-dependent promoter. We found that in vitro transcription of the leader template terminated at the terminator site, and that MBP-NasR and His6-NasR proteins both caused transcription readthrough of this site in response to nitrate or nitrite. Half-maximal antitermination required nitrate or nitrite at moderate (1 to 10 microM) concentrations, and several other anions tested, including chlorate, were without effect. Previous in vivo analysis of leader deletions identified regions required for both negative regulation (the terminator) and for positive regulation. Results from in vitro transcription of these deletion templates correlated fully with the in vivo analysis. Finally, electrophoresis mobility shift analysis revealed that His6-NasR bound specifically to nasF leader RNA. This binding was independent of nitrate in vitro. These results strongly support the conclusions drawn from previous in vivo analysis, and establish that NasR mediates ligand-responsive transcription antitermination through interaction with nasF leader RNA.

5' Untranslated Regions↗

PubMed1999

The structure and evolution of the ribosomal proteins encoded in the spc operon of the archaeon (Crenarchaeota) Sulfolobus acidocaldarius.

The genes for nine ribosomal proteins, L24, L5, S14, S8, L6, L18, S5, L30, and L15, have been isolated and sequenced from the spc operon in the archaeon (Crenarchaeota) Sulfolobus acidocaldarius, and the putative amino acid sequence of the proteins coded by these genes has been determined. In addition, three other genes in the spc operon, coding for ribosomal proteins S4E, L32E, and L19E (equivalent to rat ribosomal proteins S4, L32, and L19), were sequenced and the structure of the putative proteins was determined. The order of the ribosomal protein genes in the spc operon of the Crenarchaeota kingdom of Archaea is identical to that present in the Euryarchaeota kingdom of Archaea and also identical to that found in bacteria, except for the genes for r-proteins S4E, L32E, and L19E, which are absent in bacteria. Although AUG is the initiation codon in most of the spc genes, GUG (val) and UUG (leu) are also used as initiation codons in S. acidocaldarius. Over 70% of the codons in the Sulfolobus spc operon have A or U in the third position, reflecting the low GC content of Sulfolobus DNA. Phylogenetic analysis indicated that the archaeal r-proteins are a sister group of their eucaryotic counterparts but did not resolve the question of whether the Archaea is monophyletic, as suggested by the L6P, L15P, and L18P trees, or the question of whether the Crenarchaeota is separate from the Euryarchaeota and closer to the Eucarya, as suggested by the S8P, S5P, and L24P trees. In the case of the three Sulfolobus r-proteins that do not have a counterpart in the bacterial ribosome (S4E, L32E, and L19E), the archaeal r-proteins showed substantial identity to their eucaryotic equivalents, but in all cases the archaeal proteins formed a separate group from the eucaryotic proteins.

Amino Acid Sequence↗

PubMed1993

The bacteriophage Mu middle operon: essential and nonessential functions.

Transcription during the bacteriophage Mu lytic cycle occurs in three phases: early, middle, and late. Previous DNA sequence analysis of the middle operon revealed five potential open reading frames (ORFs) with lengths of 39, 42, 72, 120, and 140 amino acids. The distal 140-amino-acid ORF encodes C, the activator of late transcription. Expression of the middle operon under the control of a T7 promoter and T7 RNA polymerase resulted in production of two polypeptides of 15 (ORF 120) and 16.5 kDa (C). Introduction of a linker containing a translation terminator into ORF120 resulted in the production of a truncated form of the ORF120 polypeptide. When the ORF120 linker mutation and several middle operon deletion mutations were assayed for their effect on Mu growth in Escherichia coli K12, the deletions caused 6- to 22-min delays in lysis, and two resulted in a smaller plaque morphology, but all gave normal plating efficiencies and burst sizes. The plating efficiencies for all the mutants were also similar to that of wild-type Mu on alternate hosts E. coli C, Citrobacter freundii, Shigella sonnei, and Shigella flexneri. These results indicate that, with the exception of C, the middle operon ORFs are not essential for phage development.

Amino Acid Sequence↗