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PubMed1994

Crystal structure of AmiC: the controller of transcription antitermination in the amidase operon of Pseudomonas aeruginosa.

The crystal structure for the negative regulator (AmiC) of the amidase operon from Pseudomonas aeruginosa has been solved at a resolution of 2.1 A. AmiC is the amide sensor protein in the amidase operon and regulates the activity of the transcription antitermination factor AmiR, which in turn regulates amidase expression. The AmiC structure consists of two domains with an alternating beta-alpha-beta topology. The two domains are separated by a central cleft and the amide binding site is positioned in this cleft at the interface of the domains. The overall fold for AmiC is extremely similar to that for the leucine-isoleucine-valine binding protein (LivJ) of Escherichia coli despite only 17% sequence identity, however, the two domains of AmiC are substantially closed compared with LivJ. The closed structure of AmiC is stabilized significantly by the bound acetamide, suggesting a molecular mechanism for the process of amide induction. The amide binding site is extremely specific for acetamide and would not allow a closed conformation in the presence of the anti-inducer molecule butyramide.

Amidohydrolases↗

PubMed1995

Co-ordinated regulation of amino sugar biosynthesis and degradation: the NagC repressor acts as both an activator and a repressor for the transcription of the glmUS operon and requires two separated NagC binding sites.

The NagC repressor controls the expression of the divergently transcribed nagE-nagBACD operons involved in the uptake and degradation of the amino sugars, N-acetyl-D-glucosamine (GlcNAc) and glucosamine (GlcN). The glmUS operon, encoding proteins necessary for the synthesis of GlcN (glmS) and the formation of UDP-GlcNAc (glmU), is transcribed from two promoters located upstream of glmU. In the absence of amino sugars both promoters are active. However, in the presence of GlcNAc, the glmU proximal promoter, P1, is inactive while the upstream promoter, P2, is subject to weak induction. Two binding sites for the NagC repressor are located at -200 and -47 bp upstream of P1. Mutations which prevent NagC binding to either of these sites eliminate expression from the P1 promoter. This shows that binding of NagC is necessary for expression of the glmU P1 promoter and implies that NagC is playing the role of activator for this promoter. Moreover, the location of the distal NagC site suggests that this site is behaving like an upstream activating sequence (UAS).

Acetylglucosamine↗

PubMed2006

Molecular characterization of polyphosphate (PolyP) operon from Serratia marcescens.

The polyphosphate (polyP) operon was cloned from a genomic library of Serratia marcescens KCTC 2172 by Southern hybridization using E. coli ppk gene as a probe. The polyP operon was composed of a polyphosphate promoter, polyphosphate kinase (ppk) and exopolyphosphatase (ppx). A potential CRP binding site and pho box sequence were found in the region upstream of the putative promoter in the regulatory region. The ppk gene comprises 2,063 nucleotides and encodes 686 amino acids yielding a protein with a molecular mass of 70 kDa. The ppx gene contains 1611 nucleotides and encodes 536 amino acids with a molecular 58 kDa. An E. coli strain transformed with the ppk gene had a 16-fold increased in polyphosphate kinase activity, while introduction of the ppx gene produced a 25-fold increase in polyphosphatase activity. E. coli strains transformed with ppk and ppx genes also displayed increased accumulation of polyphosphate.

Bacterial Proteins↗

PubMed1992

Structural and functional analyses of the repressor, RbsR, of the ribose operon of Escherichia coli.

The DNA sequence encoding the rbs repressor protein, RbsR, has been determined. Amino acid sequence analyses of the product of an rbsR-lacZ fusion and of affinity-purified RbsR demonstrate that translation begins at an unusual codon, TTG, and that the initial amino acid is removed during maturation of the protein. DNA-binding assays indicate that RbsR binds to a region of perfect dyad symmetry spanning the rbs operon transcriptional start site and that the affinity for the rbs operator is reduced by addition of ribose, consistent with ribose being the inducer of the operon. RbsR is a member of a family of homologous repressor proteins having very similar DNA-binding sites and binding to similar operator sequences.

Amino Acid Sequence↗

PubMed1998

Purification and characterization of the heteromeric transcriptional activator MvaT of the Pseudomonas mevalonii mvaAB operon.

The mvaAB operon of Pseudomonas mevalonii encodes HMG-CoA reductase (EC 1.1.1.88) and HMG-CoA lyase (EC 4.1.3.4), enzymes that catalyze the initial reactions of mevalonate catabolism in this organism. Expression of this operon is regulated by the constitutively expressed transcriptional activator protein MvaT that binds in vitro to an upstream regulatory element. Mevalonate is essential for activation of transcription in vivo, and in vitro data demonstrated that MvaT binds to the mvaAB cis-regulatory element in the absence of mevalonate with a Kd,app of 2 nM. Purification of MvaT enriched for two polypeptides of approximate molecular mass 15 kDa and 16 kDa, designated P15 and P16. MvaT, assayed by its DNA-binding activity, comigrated with P15 and P16 during DNA-affinity chromatography, size-exclusion chromatography, and sucrose density gradient centrifugation. P15 and P16 also comigrated during denaturing isoelectric focusing of purified MvaT. Treatment of MvaT with dimethylsuberimidate formed a 31-kDa polypeptide complex that contained N-terminal sequences from P15 and P16. The apparent association of P15 and P16 in solution and their copurification with MvaT activity strongly suggests that MvaT is comprised of these two subunits. Size-exclusion chromatography gave an estimated molecular mass for MvaT of 33 kDa. A partial DNA sequence of the P16 gene was obtained using PCR employing degenerate primers directed against the N-termini of P15 and P16. P16 appears to be comprised of at least 128 aminoacyl residues having a predicted molecular mass of 14.3 kDa.

Amino Acid Sequence↗

PubMed1993

Fast and sensitive laser-based enzymatic detection of the lactose operon in microorganisms.

This work investigates the ability of laser-based, time-resolved fluorometry to detect the lactose operon genetic marker in microorganisms and to study protein-DNA interactions. In the first study, rapid detection of the Escherichia coli lacZY operon inserted in two strains of Pseudomonas proposed as fungal biological control organisms was achieved. Optimization of incubation time, immobilization apparatus size, and reagent volumes, along with the laser-based instrumentation, yielded an assay capable of detecting 10(4) immobilized lac+ Pseudomonas fluorescens cells within a 30-min incubation time. In the second study, the synthesis of E. coli beta-galactosidase was monitored in "real-time" with observable enzymatic activity beginning 4 to 5 min after induction with isopropylthiogalactoside.

DNA Transposable Elements↗

PubMed1996

Atomic force microscopy proposes a novel model for stem-loop structure that binds a heat shock protein in the Staphylococcus aureus HSP70 operon.

The Staphylococcus aureus HSP70 operon produces a polycistronic RNA in response to heat shock, and ORF37 is the first protein to be translated. The promoter of this operon contains a palindromic nucleotide sequence that may form a stem-loop structure. Structural analysis of the promoter regions by atomic force microscopy (AFM) revealed a quadruplet that consists of a pair of stem-loops. A novel "SL2S' (Stem-Loop-Loop-Stem) model was proposed for this structure. AFM also revealed the binding of ORF37 to the quadruplet, establishing a molecular mechanism for this heat shock gene expression; ORF37 acts as a regulator by binding to the SL2S structure in the promoter.

Base Sequence↗

PubMed1997

The pkI gene encoding pyruvate kinase I links to the luxZ gene which enhances bioluminescence of the lux operon from Photobacterium leiognathi.

Partial 3'-end nucleotide sequence of the pkI gene (GenBank accession No. AF019143) from Photobacterium leiognathi ATCC 25521 has been determined, and the encoded pyruvate kinase I is deduced. Pyruvate kinase I is the key enzyme of glycolysis, which converts phosphoenol pyruvate to pyruvate. Alignment and comparison of pyruvate kinase Is from P. leiognathi, E. coli and Salmonella typhimurium show that they are homologous. Nucleotide sequence reveals that the pkI gene is linked to the luxZ gene that enhances bioluminescence of the lux operon from P. leiognathi. The gene order of the pkI and luxZ genes is-pk1-ter-->-R&R"-luxZ-ter"-->, whereas ter is transcriptional terminator for the pkI and related genes, and R&R" is the regulatory region and ter" is transcriptional terminator for the luxZ gene. It clearly elicits that the pkI gene and luxZ gene are divided to two operons. Functional analysis confirms that the potential hairpin loop omega T is the transcriptional terminator for the pkI and related genes. It infers that the pkI and related genes are simply linked to the luxZ gene in P. leiognathi genome.

Amino Acid Sequence↗

PubMed1993

Some base substitutions in the leader of an Escherichia coli ribosomal RNA operon affect the structure and function of ribosomes. Evidence for a transient scaffold function of the rRNA leader.

Recently, we reported on the creation of a systematic series of C to T transition mutations, located between 19 and 45 nucleotides upstream of the mature 16 S RNA 5' end of a complete, plasmid encoded ribosomal RNA operon. We showed that some of these base transitions have pronounced effects on the growth phenotype of mutant cells, and on the stability of their 16 S RNA as well as on the association capability of their ribosomal subunits. From these observations we concluded that the mutated leader region has post-transcriptional functions in the biogenesis of ribosomes. To further substantiate our conclusions we have now analyzed the growth phenotypes of some leader mutants in more detail, and show here that they are temperature dependent. Furthermore, we have isolated ribosomal RNA, 70 S ribosomes and ribosomal subunits from wild-type and mutant strains and subjected them to a detailed structural and functional analysis. We show that processing and maturation of the 16 S RNA is not altered as a consequence of the base transitions in the leader. In contrast, comparison of the protein composition of wild-type and mutant 30 S particles by two-dimensional gel electrophoresis revealed specific differences. Wild-type 30 S subunits, which are not tightly bound to 50 S, are lacking many ribosomal proteins, while the same fraction of ribosomes from mutant cells has an approximately complete r-protein set, and instead contains some additional non-ribosomal proteins. The translational activity of mutant and wild-type total ribosome preparations was analyzed in vitro. Ribosomes from slowly growing mutants show a significantly reduced in vitro translational activity, which is caused by the 30 S subunits. In contrast, the defects in association reside mainly in the 50 S subunits. Our results demonstrate that some base substitutions in the leader of an Escherichia coli rRNA operon affect the structure and function of ribosomes, although the mutated region is not part of the particles finally formed. This finding implies that at least part of the leader region assists the structure formation of functional 30 S subunits, before it is cut away and discarded. We argue that the rrn leader thus fulfills the functional criteria of a transient molecular scaffold.

Base Sequence↗

PubMed1995

Role of NusA in L4-mediated attenuation control of the S10 r-protein operon of Escherichia coli.

The transcription of the 11 gene S10 operon of Escherichia coli is autogenously regulated by one of the operon's products, ribosomal protein L4. This protein stimulates termination of transcription in vivo at a specific site within the S10 leader. The in vivo effect can be reproduced in a purified transcription system but requires an additional factor, NusA. Our earlier in vitro studies showed that NusA is required for RNA polymerase pausing at the termination site; such paused complexes are further stabilized by L4, which presumably accounts for L4's stimulation of termination in vivo. Here we show that NusA is not absolutely required for RNA polymerase to recognize the attenuation site: at low (5 microM) UTP concentration, RNA polymerase pauses at the site, although the paused transcription complex formed in the absence of NusA can be further stabilized by subsequent addition of the protein. Furthermore, RNA polymerase pausing at the attenuation site is not sufficient for the L4 effect, since L4 cannot stabilize a transcription complex paused at the attenuation site in the absence of NusA. We have been able to isolate paused complexes formed without NusA and/or L4; such complexes are active upon re-addition of NTPs, and respond as expected to the addition of L4 or NusA. Our experiments are consistent with the notion that L4 is a stable component of a paused transcription complex.

Bacterial Proteins↗

PubMed1994

The organization of intercistronic regions of the aerobactin operon of pColV-K30 may account for the differential expression of the iucABCD iutA genes.

The complete nucleotide sequence of the 8.3 kilobase operon of the enterobacterial virulence plasmid pColV-K30, which encodes a high-affinity iron transport system mediated by the hydroxamate siderophore aerobactin, has been determined. The region includes five open reading frames which correspond to the genes iucA, iucB, iucC and iucD, encoding the enzymes of the biosynthetic pathway for aerobactin, and iutA for the outer membrane receptor of ferri-aerobactin complexes. The sequences of the iucABCD genes are tightly coupled without any intervening non-coding sequence. The predicted secondary mRNA structures at the gene junctions within the iucABCD cluster, along with their codon usage, may account for the differential expression of each of the protein products, as observed in vivo with minicells. The genes iucA and iucC, which determine the two subunits of the aerobactin synthetase complex, showed a considerable homology within three stretches of their amino acid sequence. A potential operator sequence (iron box) for the binding of the iron(II)-responsive Fur repressor protein was found within the iucA coding region, suggesting that the operon is subjected to an additional level of transcriptional repression by iron (II).

Amino Acid Sequence↗

PubMed1994

Transcription of the xyl operon is controlled in Bacillus subtilis by tandem overlapping operators spaced by four base-pairs.

The expression of xylose utilization in Bacillus subtilis is regulated at the level of transcription by xylose dependent Xyl repressor-xyl operator interaction. We have structurally and functionally characterized the binding sites of Xyl repressor in the xyl regulatory region. Methylation and hydroxyl radical protection and ethylation interference of binding suggests tandem overlapping xyl operators spaced by four base-pairs. A mutational inactivation of each and both operators was performed. DNA retardation experiments with these mutants confirmed the existance of two binding sites. They can be simultaneously occupied, despite their overlapping, intertwined organization. In vivo repressor titration and regulation of indicator gene expression by the xylO mutants confirmed that both binding sites contribute to regulation of the xyl operon. The protection and interference patterns of both sites are identical and indicate binding of a repressor oligomer to one side of B-form DNA of each operator. A tandem overlapping arrangement of two operators is also found in the xyl regulatory sequences of Bacillus megaterium, Staphylococcus xylosus and Lactobacillus pentosus. The xyl operon of Bacillus licheniformis contains a similar element in which the second operator is more diverged. This high degree of conservation among bacteria of different genera supports the conclusion that a tandem overlapping arrangement of xyl operators contributes to efficient regulation.

Amino Acid Sequence↗

PubMed1995

carP, involved in pyrimidine regulation of the Escherichia coli carbamoylphosphate synthetase operon encodes a sequence-specific DNA-binding protein identical to XerB and PepA, also required for resolution of ColEI multimers.

The carP gene involved in pyrimidine-specific regulation of the upstream P1 promoter of the Escherichia coli carAB operon has been cloned in vivo on a mini-Mu replicon, sequenced and shown to be identical to the xerB (pepA) gene encoding aminopeptidase A, a protein also involved in the Xer-mediated site-specific recombination at ColEI cer. The trans-dominant allele carP6 was cloned as well and shown to bear a single G-->A transition that converts the TGG codon (Trp473) into a TAG amber stop codon. The truncated mutant protein, missing the 31 C-terminal amino acid residues, was shown to be partially active; in the multicopy state the carP6 allele can restore pyrimidine repressibility of the carAB promoter P1. The trans-dominant character of the single copy carP6 allele was found to be suppressed in the presence of multiple copies of the wild-type gene. The carP (pepA) control region was sequenced and transcription shown to be initiated at three promoters, the most upstream one of which was shown to be subject to negative autoregulation. The aminopeptidase activity of CarP (PepA) was found to be dispensable for its role in pyrimidine-mediated repression of carAB transcription. CarP (PepA) was shown to be a sequence-specific DNA-binding protein that does not require, at least not in vitro, any pyrimidine cofactor to bind to the DNA. Mobility-shift and DNase I footprinting experiments have revealed a specific binding of purified CarP (PepA) to two sites in each one of the control regions of the E. coli and Salmonella typhimurium carAB operons and to a single site in the carP (pepA) control region. We propose that integration host factor and CarP/PepA-induced structural modifications in the carAB control region cause conformational changes required to assemble a pyrimidine-specific nucleo-protein regulatory complex.

Amino Acid Sequence↗

PubMed1996

Deletion analysis of the che operon in the archaeon Halobacterium salinarium.

Halobacterium salinarium is a chemo- and phototactic archaeon whose signal transduction pathway includes the classical two-component system made up of CheA and CheY. Deletion analysis of the che operon in H. salinarium has been undertaken. Following the removal of the entire operon, the importance of each of the four individual members, cheY, cheB, cheA, and the novel member cheJ, was evaluated by their replacement in combinations of three. The mutant strains were investigated for their motility, their chemo- and phototactic signalling, and the rotational bias of their flagella. Loss of cheA, cheY or cheB led to the complete loss of chemo- and phototaxis, whereas the absence of cheJ caused a reduction in chemo- and phototactic ability. Reverse swimming and counterclockwise rotation of the flagella required the presence of cheA and CheY. The wild-type 50:50 distribution of forward and reverse swimming was observed in the strain lacking cheB, whereas this distribution was perturbed to 88:12 in the strain lacking cheJ. These results are compared with the corresponding deletion strains in Escherichia coli and provide new insights into the eu- and archeabacterial flagellar switch.

Bacterial Proteins↗

PubMed1996

Isolation, cloning, sequence analysis and localization of the operon encoding dimethyl sulfoxide/trimethylamine N-oxide reductase from Rhodobacter capsulatus.

The operon encoding the periplasmic enzymes dimethyl sulfoxide reductase (DMSOR) and trimethylamine N-oxide reductase (TMAOR) from the purple, non-sulfur, photosynthetic bacterium Rhodobacter capsulatus was isolated, cloned and sequenced, and its chromosomal location determined. It was shown by analytical and crystallographic data that DMSOR and TMAOR are identical enzymes. Degenerate primers were derived from short peptide sequences generated by automated Edman degradation and a 700 bp fragment was amplified by nested PCR, subsequently cloned and radioactively labeled to screen a prepared lambda DASH library. Positive lambda clones were subcloned into pBluescript and subsequently transformed into Escherichia coli to sequence the DMSOR/ TMAOR operon. The promoter consisted of an A + T-rich region followed by a -35 region, a putative ribosome binding site, and a leader sequence of 13 amino acid residues. The transcription terminator was a G + C-rich dyad sequence capable of forming a hairpin structure, which may act rho-independently. An optimized protein purification of the wild-type enzyme is also described, giving high yields (5 mg protein per liter of culture) and a specific activity of 30 units/mg. The molecular mass was determined by electrospray mass spectrometry to be 85,034 Da; from the deduced amino acid sequence the molecular mass of the apoenzyme was 85,033 Da.

Amino Acid Sequence↗

PubMed1997

In vitro binding of the response regulator CitB and of its carboxy-terminal domain to A + T-rich DNA target sequences in the control region of the divergent citC and citS operons of Klebsiella pneumoniae.

The genes specifically required for citrate fermentation in Klebsiella pneumoniae form a cluster on the chromosome consisting of two divergently transcribed groups, citCDEFG and citS-oadGAB-citAB. Northern blot analyses described here and elsewhere indicate that each group forms an operon. The transcriptional start sites of citC and citS, which were mapped in this work by primer extension, are separated by a stretch of 193 bp with an extraordinary high A + T content of 67%. Expression of the citrate fermentation genes was recently shown to be positively controlled by a two-component signal transduction system encoded by the promoter-distal genes of the citS operon, citA (sensor kinase) and citB (response regulator). As a first step towards the functional characterization of CitB, we analysed its DNA-binding properties. To this end, the entire CitB, its N-terminal receiver domain (CitBN), and its C-terminal output domain (CitBC), all modified by a (His)6-tag, were purified. CitB(His) and CitBN(His) could be phosphorylated either with acetylphosphate or with ATP plus MalE-CitAC. The latter protein contains the kinase domain of CitA fused to the C terminus of the maltose-binding protein. Upon phosphorylation, CitB(His) became more resistant towards limited proteolysis by trypsin, reflecting substantial changes in tertiary structure. In gel retardation assays, CitB(His) bound specifically to the citC-citS intergenic region. The retardation pattern changed significantly upon phosphorylation and the apparent binding affinity increased 10 to 100-fold. Depending on the protein concentration, four different phospho-CitB(His)-DNA complexes could be resolved, suggesting the presence of multiple binding sites between citC and citS. DNase I footprints revealed two protected regions extending maximally from -55 to -89 relative to the citS transcription start and from -50 to -96 relative to the citC transcription start. Gel retardation and DNase I footprint assays with CitBC(His) showed that the C-terminal domain is sufficient for specific DNA binding. Since its properties were similar to that of unphosphorylated CitB(His), an essential role of the N-terminal receiver domain in high-affinity DNA binding was indicated. The positions of the binding sites for CitB and of putative recognition sequences for the cAMP receptor protein suggested a model for the interaction of these activators with RNA polymerase.

Bacterial Proteins↗

PubMed1999

Regulatory features of the trp operon and the crystal structure of the trp RNA-binding attenuation protein from Bacillus stearothermophilus.

Characterization of both the cis and trans -acting regulatory elements indicates that the Bacillus stearothermophilustrp operon is regulated by an attenuation mechanism similar to that which controls the trp operon in Bacillus subtilis. Secondary structure predictions indicate that the leader region of the trp mRNA is capable of folding into terminator and anti- terminator RNA structures. B. stearothermophilus also encodes an RNA-binding protein with 77% sequence identity with the RNA-binding protein (TRAP) that regulates attenuation in B. subtilis. The X-ray structure of this protein has been determined in complex with L-tryptophan at 2.5 A resolution. Like the B. subtilis protein, B. stearothermophilus TRAP has 11 subunits arranged in a ring-like structure. The central cavities in these two structures have different sizes and opposite charge distributions, and packing within the B. stearothermophilus TRAP crystal form does not generate the head-to-head dimers seen in the B. subtilis protein, suggesting that neither of these properties is functionally important. However, the mode of L-tryptophan binding and the proposed RNA binding surfaces are similar, indicating that both proteins are activated by l -tryptophan and bind RNA in essentially the same way. As expected, the TRAP:RNA complex from B. stearothermophilus is significantly more thermostable than that from B. subtilis, with optimal binding occurring at 70 degrees C.

Amino Acid Sequence↗

PubMed1995

Nucleotide sequence within Tn3926 confirms this as a Tn21-like transposable element and provides evidence for the origin of the mer operon carried by plasmid pKLH2.

The DNA sequence of the resolvase gene, resolution sites, and the region between the transposition functions and the end of the mercury resistance operon of the bacterial transposon, Tn3926, is presented. The sequence of Tn3926 upstream of the resolution sites is homologous to that bordering the 11.2-kb insert of Tn21, supporting the idea that this insert transposed into a progenitor of Tn3926. This region of Tn3926 also shows 97.0% identity to the mercury-resistant determinant of the plasmid, pKLH2, suggesting that this plasmid once harbored a close relative of Tn3926. It is proposed that the mercury resistance operon of Tn3926 will have a structure very similar to that found on pKLH2.

Base Sequence↗