Rbs Operon Research Articles

RbsR Activates Capsule but Represses the rbsUDK Operon in Staphylococcus aureus.

Staphylococcus aureus capsule is an important virulence factor that is regulated by a large number of regulators. Capsule genes are expressed from a major promoter upstream of the cap operon. A 10-bp inverted repeat (IR) located 13 bp upstream of the -35 region of the promoter was previously shown to affect capsule gene transcription. However, little is known about transcriptional activation of the cap promoter. To search for potential proteins which directly interact with the cap promoter region (Pcap), we directly analyzed the proteins interacting with the Pcap DNA fragment from shifted gel bands identified by electrophoretic mobility shift assay. One of these regulators, RbsR, was further characterized and found to positively regulate cap gene expression by specifically binding to the cap promoter region. Footprinting analyses showed that RbsR protected a DNA region encompassing the 10-bp IR. Our results further showed that rbsR was directly controlled by SigB and that RbsR was a repressor of the rbsUDK operon, involved in ribose uptake and phosphorylation. The repression of rbsUDK by RbsR could be derepressed by D-ribose. However, D-ribose did not affect RbsR activation of capsule. Staphylococcus aureus is an important human pathogen which produces a large number of virulence factors. We have been using capsule as a model virulence factor to study virulence regulation. Although many capsule regulators have been identified, the mechanism of regulation of most of these regulators is unknown. We show here that RbsR activates capsule by direct promoter binding and that SigB is required for the expression of rbsR. These results define a new pathway wherein SigB activates capsule through RbsR. Our results further demonstrate that RbsR inhibits the rbs operon involved in ribose utilization, thereby providing an example of coregulation of metabolism and virulence in S. aureus. Thus, this study further advances our understanding of staphylococcal virulence regulation.

Involvement of the ribose operon repressor RbsR in regulation of purine nucleotide synthesis inEscherichia coli

Escherichia coli is able to utilize d-ribose as its sole carbon source. The genes for the transport and initial-step metabolism of d-ribose form a single rbsDACBK operon. RbsABC forms the ABC-type high-affinity d-ribose transporter, while RbsD and RbsK are involved in the conversion of d-ribose into d-ribose 5-phosphate. In the absence of inducer d-ribose, the ribose operon is repressed by a LacI-type transcription factor RbsR, which is encoded by a gene located downstream of this ribose operon. At present, the rbs operon is believed to be the only target of regulation by RbsR. After Genomic SELEX screening, however, we have identified that RbsR binds not only to the rbs promoter but also to the promoters of a set of genes involved in purine nucleotide metabolism. Northern blotting analysis indicated that RbsR represses the purHD operon for de novo synthesis of purine nucleotide but activates the add and udk genes involved in the salvage pathway of purine nucleotide synthesis. Taken together, we propose that RbsR is a global regulator for switch control between the de novo synthesis of purine nucleotides and its salvage pathway.

Ribose utilization by the human commensal Bifidobacterium breve UCC2003

SummaryGrowth of Bifidobacterium breve UCC2003 on ribose leads to the transcriptional induction of the rbsACBDK gene cluster. Generation and phenotypic analysis of an rbsA insertion mutant established that the rbs gene cluster is essential for ribose utilization, and that its transcription is likely regulated by a LacI‐type regulator encoded by rbsR, located immediately upstream of rbsA. Gel mobility shift assays using purified RbsRHis indicate that the promoter upstream of rbsABCDK is negatively controlled by RbsRHis binding to an 18 bp inverted repeat and that RbsRHis binding activity is modulated by d‐ribose. The rbsK gene of the rbs operon of B. breve UCC2003 was shown to specify a ribokinase (EC 2.7.1.15), which specifically directs its phosphorylating activity towards d‐ribose, converting this pentose sugar to ribose‐5‐phosphate.

Metabolic Changes Associated With Adaptive Diversification in Escherichia coli

During a 1000-generation evolution experiment, two types of morphologically and kinetically distinct bacteria repeatedly diverged from a common ancestor in a fully sympatric seasonal environment containing glucose and acetate. To investigate the metabolic modifications associated with this adaptive diversification, we compared transcription profiles of the two derived types and the common ancestor. Both derived types share a suite of common metabolic changes that may represent adaptation to the environment preceding the diversification event. These include improved translation efficiency, glucose uptake capacity via the mal/lamB genes, upregulation of various transporters during stationary phase, and likely the disruption of the rbs operon. The diversification event is associated with the overexpression of genes involved in the TCA cycle, glyoxylate shunt, acetate consumption, and anaerobic respiration in one type and in acetate excretion in the other. These results reveal that competition for both carbon and oxygen have likely played an important role in the adaptation of Escherichia coli during this adaptive diversification event, where one derived type mainly consumes glucose at a fast rate when oxygen is not limiting, and the other derived type consumes glucose and acetate at a slower rate, even when oxygen is limiting.

Crystal Structures of RbsD Leading to the Identification of Cytoplasmic Sugar-binding Proteins with a Novel Folding Architecture

RbsD is the only protein whose biochemical function is unknown among the six gene products of the rbs operon involved in the active transport of ribose. FucU, a paralogue of RbsD conserved from bacteria to human, is also the only protein whose function is unknown among the seven gene products of the l-fucose regulon. Here we report the crystal structures of Bacillus subtilis RbsD, which reveals a novel decameric toroidal assembly of the protein. Nuclear magnetic resonance and other studies on RbsD reveal that the intersubunit cleft of the protein binds specific forms of d-ribose, but it does not have an enzyme activity toward the sugar. Likewise, FucU binds l-fucose but lacks an enzyme activity toward this sugar. We conclude that RbsD and FucU are cytoplasmic sugar-binding proteins, a novel class of proteins whose functional role may lie in helping influx of the sugar substrates.

Autoinducer 2 activity in Escherichia coli culture supernatants can be actively reduced despite maintenance of an active synthase, LuxS.

Production of the signalling molecule (autoinducer-2) synthesized by LuxS has been proposed to be pivotal to a universal mechanism of inter-species bacterial cell-cell communication (quorum sensing); however recently the function of LuxS has been noted to be integral to central metabolism since it contributes to the activated methyl cycle. This paper shows that when Helicobacter pylori LuxS is overproduced in Escherichia coli, it forms cross-linkable multimers. These multimers persist at comparable levels after 24 h of growth if glucose is omitted from the growth medium; however, the levels of extracellular autoinducer-2 decline (Glucose Retention of AI-2 Levels: GRAIL). Glycerol, maltose, galactose, ribose and L-arabinose could substitute for glucose, but lactose, D-arabinose, acetate, citrate and pyruvate could not. Mutations in (i). metabolic pathways (glycolytic enzymes eno, pgk, pgm; galactose epimerase; the Pta-AckA pathway), (ii). sugar transport (pts components, rbs operon, mgl, trg), and (iii). regulators involved in conventional catabolic repression (crp, cya), cAMP-independent catabolite repression (creC, fruR, rpoS,) the stringent response (relA, spoT) and the global carbon storage regulator (csrA) did not prevent GRAIL. Although the basis of GRAIL remains uncertain, it is clear that the mechanism is distinct from conventional catabolite repression. Moreover, GRAIL is not due to inactivation of the enzymic activity of LuxS, since in E. coli, LuxS contained within stationary-phase cells grown in the absence of glucose maintains its activity in vitro.

Crystallization and preliminary X-ray crystallographic analysis of Escherichia coli RbsD, a component of the ribose-transport system with unknown biochemical function.

The Escherichia coli high-affinity ribose-transport system consists of six proteins encoded by the rbs operon (rbsD, rbsA, rbsC, rbsB, rbsK and rbsR). Of the six components, RbsD is the only one whose function is unknown. In order to gain insights into the function of RbsD by structural analysis, we overexpressed and crystallized the protein as a first step toward this goal. RbsD was overexpressed in E. coli and crystallized using the hanging-drop vapour-diffusion method at 296 K. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 285.9, b = 92.3, c = 93.3 A, beta = 105.0 degrees. The unit cell is likely to contain 64 molecules of RbsD, with a crystal volume per protein mass (V(M)) of 2.43 A(3) Da(-1) and a solvent content of about 49.3% by volume. An equilibrium centrifugation analysis demonstrated that RbsD (MW = 15 292 Da) exists as an octamer in solution, suggesting that the asymmetric unit contains two octameric assemblies of RbsD. A native data set to 2.7 A resolution was obtained from a flash-cooled crystal.

Adaptation of Lactobacillus sakei to meat: a new regulatory mechanism of ribose utilization?

Lactobacillus sakei is a lactic acid bacterium commonly found on meat and meat products. Meat is a rich substrate but contains small amounts of sugars, mainly glucose and ribose. The phosphotransferase system (PTS) is a multienzymatic complex responsible for sugar uptake. It is also involved in the regulation of metabolism through various mechanisms (catabolite activation and repression, inducer expulsion and exclusion). The ptsHI operon of L. sakei, encoding the general enzymes of the PTS, was studied and mutants were constructed. On ribose, these mutants grow twice as fast as the wild-type strain. This phenotype was never described in other bacteria and suggests that the PTS regulates ribose utilization. When compared to what is known from the regulations involving the PTS in other bacteria, this mechanism might be new. In Bacillus subtilis and Escherichia coli, in which ribose catabolism was investigated, ribose is transported by an ABC transporter, encoded by rbsABCD genes and then phosphorylated by the rbsK encoded ribose kinase. Ribose-5P is then metabolized through the pentose-P pathway involving xylulose-5P phosphoketolase and acetate kinase. Whereas phosphoketolase and acetate kinase activities remained unchanged in L. sakei ptsI mutants, ribose kinase activity and uptake were increased by a factor of 2.5 and 1.5, respectively. The target of the PTS regulation would thus be transport and/or phosphorylation of ribose. The gene cluster encoding a ribose transporter, ribose kinase and a regulator was cloned and sequenced. In L. sakei no gene encoding RbsA, RbsB or RbsC could be found. However, rbsD was present as well as a new gene rbsU, encoding a protein homologous to a glucose transporter responsible for facilitated diffusion of glucose. The rbsUDK operon is induced by ribose via the regulator RbsR encoded by rbsR located downstream of rbsUDK. In ptsI mutants, this operon was not overexpressed on ribose. This shows that the regulation of ribose utilization is not a transcriptional regulation. Upstream from the rbs operon, a gene encoding acetate kinase (ackA) was found. In other bacteria in which these genes were identified, ackA and the rbs operon are not linked. Moreover, in B. subtilis, ackA is regulated by catabolite activation whereas the rbs operon is repressed by catabolite repression, two mechanisms involving the PTS. In L. sakei, ackA and the rbs operon are adjacent on the

The proteins encoded by the rbs operon of escherichia coli: II. Use of chimeric protein constructs to isolate and characterize RbsC

Chimeric genes encoding full-length copies of rbsA and rbsC connected by segments coding for short bridge peptides were constructed and expressed in Escherichia coli. Surprisingly, the chimeric genes complemented the strain in which rbsA and rbsC were deleted. The chimeric proteins were overproduced, and the products were purified by affinity chromatography. In order to obtain highly purified protein, a poly-His leader peptide was incorporated so that Ni-chelate affinity chromatography could be employed. The leader peptide and the bridge peptide were designed with factor Xa-cleavable sites to permit recovery of the individual RbsA and RbsC protein. A rbsC gene encoding a poly-His leader was also constructed and expressed. Both the chimeric RbsA-C species and the poly-HisRbsC were produced at levels that permitted isolation of the equivalent of milligram quantities of RbsC per liter of culture. This is a substantial increase in amounts from any previous RbsC production vectors. All proteins from the rbs operon have now been overproduced and substantially purified.

Identification of the genes in multicopy plasmids affecting ompC and ompF expression in Escherichia coli

Osmoregulation of the porin genes, ompF and ompC of Escherichia coli, occurs at the level of transcription through the action of EnvZ and OmpR proteins as well as at the level of translation through micF antisense RNA. In this study, we used a genetic screening approach to identify new genes which interfere with the expression of ompC or ompF. Using an E. coli genomic library in pUC19, we identified three clones whose products altered expression of ompC and ompF in response to medium osmolarity. One clone carrying the secB gene was found to block ompC and inhibit ompF expression. One clone carrying gcvA, a transcriptional regulator for the gvcA operon, was found to block ompF expression at high osmolarity and elevate ompC expression at low osmolarity. One clone carrying rbsR, a repressor for the rbs operon, was found to block ompF expression at both low and high osmolarities and elevate ompC expression at low osmolarity. These results suggest that ompF and ompC expression is associated with other physiological regulating systems in addition to osmoregulation.

Identification of the genes in multicopy plasmids affecting ompC and ompF expression in Escherichia coli.

Osmoregulation of the porin genes, ompF and ompC of Escherichia coli, occurs at the level of transcription through the action of EnvZ and OmpR proteins as well as at the level of translation through micF antisense RNA. In this study, we used a genetic screening approach to identify new genes which interfere with the expression of ompC or ompF. Using an E. coli genomic library in pUC19, we identified three clones whose products altered expression of ompC and ompF in response to medium osmolarity. One clone carrying the secB gene was found to block ompC and inhibit ompF expression. One clone carrying gcvA, a transcriptional regulator for the gvcA operon, was found to block ompF expression at high osmolarity and elevate ompC expression at low osmolarity. One clone carrying rbsR, a repressor for the rbs operon, was found to block ompF expression at both low and high osmolarities and elevate ompC expression at low osmolarity. These results suggest that ompF and ompC expression is associated with other physiological regulating systems in addition to osmoregulation.

Ribokinase from Escherichia coli K12. Nucleotide sequence and overexpression of the rbsK gene and purification of ribokinase.

The nucleotide sequence of a 1455-base pair TaqI-HinfI fragment of the rbs operon of Escherichia coli K12 has been determined. It includes the 3' terminus of rbsB (the gene for ribose-binding protein) and the entire rbsK gene, encoding ribokinase. Potential consensus promoter sequences and a stable stem-loop structure are present in the rbsB-rbsK intercistronic region. The regulatory significance of these sequence features is discussed with respect to the rbs operon. rbsK has been cloned downstream from the Serratia marcescens trp promoter on a multicopy plasmid. Cells harboring this plasmid, when grown on minimal ribose plus ampicillin, express ribokinase at the level of 2% of the soluble protein, and induction with indoleacrylic acid raises ribokinase levels another 8-fold. Ribokinase has been purified to homogeneity (216 mumol/min/mg) from a strain harboring this plasmid. Protein sequence analyses of peptides generated by cyanogen bromide cleavage and o-iodosobenzoic acid cleavage confirmed the translation initiation site and the reading frame of the DNA sequence. Amino acid compositions of native ribokinase and the C-terminal dodecapeptide agree with the predicted amino acid compositions, confirming the accuracy of the DNA sequence and the translation termination site.

The nucleotide sequences of the rbsD, rbsA, and rbsC genes of Escherichia coli K12.

The nucleotide sequences of rbsD, rbsA, and rbsC have been determined. These genes encode components of the high affinity ribose transport system in Escherichia coli, and together with the sequences of rbsB (Groarke, J.M., Mahoney, W.C., Hope, J.N., Furlong, C.E., Robb, F.T., Zalkin, H., and Hermodson, M.A. (1983) J. Biol. Chem. 258, 12952-12956) and rbsK (Hope, J.N., Bell, A.W., Hermodson, M.A., and Groarke, J.M. (1986) J. Biol. Chem. 261, 7663-7668), they complete the nucleotide sequence of the first five genes of the rbs operon. Nuclease S1 mapping places the transcriptional start site for the operon 29 base pairs upstream from the most likely translational start site for rbsD. The open reading frames of rbsD, rbsA, and rbsC encode proteins of 139, 501, and 321 amino acid residues, respectively. The character of the proteins varies widely, from very hydrophilic for the rbsA product to exceedingly hydrophobic for the rbsC product. The intercistronic spaces between the three genes are very short, with the stop codons of the upstream genes overlapping the ribosome-binding sites of the downstream genes. This may imply translational control of expression of these genes, the products of which presumably form a membrane-bound transport complex.

D-ribose metabolism in Escherichia coli K-12: genetics, regulation, and transport.

We have isolated mutants defective in high-affinity D-ribose transport. The mutations map in rbsT or rbsB , the structural gene for ribose binding protein. rbsT consists of at least one gene coding for a protein required for high-affinity transport. The high-affinity transport-defective mutants were able to utilize D-ribose, indicating that at least a second, low-affinity transport system for D-ribose is present in Escherichia coli K-12. rbsT and rbsB are located at min 84 on the E. coli genetic map and, together with rbsK , the gene coding for ribokinase , constitute an rbs operon. The order of genes is rbsP /O rbsT rbsB rbsK . The rbs operon is subject to negative control by the product of the rbsR gene. rbsR is located distal to the rbs operon and appears to form a separate transcriptional unit.

Evolution of the D-ribose operon on Escherichia coli B/r.

The D-ribose operon (rbs) of Escherichia coli K-12 maps at 83 min and is inducible. The rbs operon of E. coli B/r maps at 2 min and is constitutive. Evidence is presented showing that a second inducible copy of the rbs operons is present in E. coli B/r mapping at 83 min. The data indicated that the duplication of the rbs operon represented a transposition of the 83-min region to 2 min. The identification of a second copy of the rbs operon in B/r and the determination of its inducibility were based on the reactivation, through mutagenesis, of inducible rbs expression, mapping by P1 transduction of the mutation site to 83 min, and merodiploid complementation analysis of the D-ribokinase expression in E. coli B/r. We also show that the rbs transposition to 2-min continued to generate transposable elements coding for the 1- to 2-min region of the chromosome and transposing onto extrachromosomal DNA target molecules such as pBR322.