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B Rak

Publications and source records attributed to B Rak.

At least 19 recordsLinked to original sources
PubMed1999

Catabolite control of Escherichia coli regulatory protein BglG activity by antagonistically acting phosphorylations.

In bacteria various sugars are taken up and concomitantly phosphorylated by sugar-specific enzymes II (EII) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The phosphoryl groups are donated by the phosphocarrier protein HPr. BglG, the positively acting regulatory protein of the Escherichia coli bgl (beta-glucoside utilization) operon, is known to be negatively regulated by reversible phosphorylation catalyzed by the membrane spanning beta-glucoside-specific EIIBgl. Here we present evidence that in addition BglG must be phosphorylated by HPr at a distinct site to gain activity. Our data suggest that this second, shortcut route of phosphorylation is used to monitor the state of the various PTS sugar availabilities in order to hierarchically tune expression of the bgl operon in a physiologically meaningful way. Thus, the PTS may represent a highly integrated signal transduction network in carbon catabolite control.

Bacterial Proteins↗
PubMed1999

The gene bglH present in the bgl operon of Escherichia coli, responsible for uptake and fermentation of beta-glucosides encodes for a carbohydrate-specific outer membrane porin.

The cryptic gene bglH from the Escherichia coli chromosome was cloned into a tacOP-driven expression vector. The resulting plasmid was transferred into the porin-deficient E. coli strain KS26 and the protein was expressed by addition of IPTG. The BglH protein was localized in the outer membrane. It was purified to homogeneity using standard methods. Reconstitution experiments with lipid bilayer membranes defined BglH as a channel-forming component, i.e. it is an outer membrane porin. The single-channel conductance of BglH (560 pS in 1 M KCl) was only one-third of that of the general diffusion porins of E. coli outer membrane. The presence of carbohydrates in the aqueous phase led to a dose-dependent block of ion transport through the channel, similar to that found for LamB (maltoporin) of E. coli and Salmonella typhimurium, which means that BglH is a porin specific for the uptake of carbohydrates. The binding constants of a variety of different carbohydrates were calculated from titration experiments of the BglH-induced membrane conductance. The tightest binding was observed with the aromatic beta-D-glucosides arbutin and salicin, and with gentibiose and cellobiose. Binding of maltooligosaccharides to BglH was in contrast to their binding to LamB in that it was much weaker, indicating that the binding site of BglH for carbohydrates is different from that of LamB (maltoporin). The kinetics of cellopentaose binding to BglH was investigated using the carbohydrate-induced current noise and was compared with that of cellopentaose binding to LamB (maltoporin) and ScrY (sucroseporin).

Amino Acid Sequence↗
PubMed1996

Identification of a site in the phosphocarrier protein, HPr, which influences its interactions with sugar permeases of the bacterial phosphotransferase system: kinetic analyses employing site-specific mutants.

The permeases of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS), the sugar-specific enzymes II, are energized by sequential phosphoryl transfer from phosphoenolpyruvate to (i) enzyme I, (ii) the phosphocarrier protein HPr, (iii) the enzyme IIA domains of the permeases, and (iv) the enzyme IIBC domains of the permeases which transport and phosphorylate their sugar substrates. A number of site-specific mutants of HPr were examined by using kinetic approaches. Most of the mutations exerted minimal effects on the kinetic parameters characterizing reactions involving phosphoryl transfer from phospho-HPr to various sugars. However, when the well-conserved aspartyl 69 residue in HPr was changed to a glutamyl residue, the affinities for phospho-HPr of the enzymes II specific for mannitol, N-acetylglucosamine, and beta-glucosides decreased markedly without changing the maximal reaction rates. The same mutation reduced the spontaneous rate of phosphohistidyl HPr hydrolysis but did not appear to alter the rate of phosphoryl transfer from phospho-enzyme I to HPr. When the adjacent glutamyl residue 70 in HPr was changed to a lysyl residue, the Vmax values of the reactions catalyzed by the enzymes II were reduced, but the Km values remained unaltered. Changing this residue to alanine exerted little effect. Site-specific alterations in the C terminus of the beta-glucoside enzyme II which reduced the maximal reaction rate of phosphoryl transfer about 20-fold did not alter the relative kinetic parameters because of the aforementioned mutations in HPr. Published three-dimensional structural analyses of HPr and the complex of HPr with the glucose-specific enzyme IIA (IIAGlc) (homologous to the beta-glucoside and N-acetylglucosamine enzyme IIA domains) have revealed that residues 69 and 70 in HPr are distant from the active phosphorylation site and the IIAGlc binding interface in HPr. The results reported therefore suggest that residues D-69 and E-70 in HPr play important roles in controlling conformational aspects of HPr that influence (i) autophosphohydrolysis, (ii) the interaction of this protein with the sugar permeases of the bacterial phosphotransferase system, and (iii) catalysis of phosphoryl transfer to the IIA domains in these permeases.

Acetylglucosamine↗
PubMed1996

LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family.

Gene licS of Bacillus subtilis encodes an excreted Beta-1,3-1,4-endoglucanase necessary for lichenan utilization. Upstream of licS we found a gene (termed licT) together with its promoter which encodes a transcriptional antiterminator of the BglG family. Genes licT and licS are separated by a palindromic sequence (lic-t) reminiscent of transcriptional terminators recognized by the antiterminator proteins of the BglG family. The LicT protein can prevent termination at terminator lic-t and also at terminator t2 of the Escherichia coli bgl operon and BglG prevents termination at lic-t. The role of LicT in licS regulation by preventing termination at its terminator lic-t appears to be limited since expression of licS is inducible only two- to threefold. This limited regulation is mainly due to a high basal level of licS expression which can in part be attributed to the presence of a second promoter preceding licS and located downstream of lic-t. However, disruption of gene licT leads not only to loss of inducibility of licS but also to loss of growth on lichenan or on its degradation products, indicating its stringent role in beta-glucan utilization.

Amino Acid Sequence↗
PubMed1992

IS5: a mobile enhancer of transcription in Escherichia coli.

The cryptic bgl operon of Escherichia coli is activated by the spontaneous insertion of mobile DNA elements. Screening of a collection of such mutations revealed insertion of the 1195-base-pair element IS5 into various positions both upstream and downstream of the bgl promoter P0. Activation of the operon was in all cases attributable to enhancement of P0 activity. Introduction of internal deletions into IS5 almost completely abolished P0 enhancement, demonstrating that enhancement is not simply the result of mutational inactivation of some inhibitory sequences. Intact copies of IS5 in trans restored the enhancing activity of the deletion derivatives. The trans-activator is encoded by IS5 gene ins5A, an essential transposition function. Activation of gene expression by means of interaction of a defective mobile element in cis with functions encoded by a nondefective element in trans has so far been described only for a maize controlling element.

Base Sequence↗
PubMed1991

High-level ribosomal frameshifting directs the synthesis of IS150 gene products.

IS150 contains two tandem, out-of-phase, overlapping genes, ins150A and ins150B, which are controlled by the same promoter. These genes encode proteins of 19 and 31 kD, respectively. A third protein of 49 kD is a transframe gene product consisting of domains encoded by both genes. Specific -1 ribosomal frameshifting is responsible for the synthesis of the large protein. Expression of ins150B also involves frameshifting. The IS150 frameshifting signals operate with a remarkably high efficiency, causing about one third of the ribosomes to switch frame. All of the signals required for this process are encoded in a 83-bp segment of the element. The heptanucleotide A AAA AAG and a potential stem-loop-forming sequence mark the frameshifting site. Similar sequence elements are found in -1 frameshifting regions of bacterial and retroviral genes. A mutation within the stem-loop sequence reduces the rate of frameshifting by about 80%. Artificial transposons carrying this mutation transpose at a normal frequency, but form cointegrates at a approximately 100-fold reduced rate.

Amino Acid Sequence↗
PubMed1990

Identification of catalytic residues in the beta-glucoside permease of Escherichia coli by site-specific mutagenesis and demonstration of interdomain cross-reactivity between the beta-glucoside and glucose systems.

beta-Glucoside Enzyme II (IIBgl) of the Escherichia coli phosphotransferase system transports and phosphorylates beta-glucosides, whereas the glucose Enzyme II-III pair (IIGlc-IIIGlc) transports and phosphorylates glucose as well as certain aliphatic alpha- and beta-glucosides. Comparisons of their respective amino acid sequences previously revealed that both systems are homologous and must be evolutionarily related. To gain more insight into the details of the transport mechanism, we made use of the observed homologies among phosphotransferase system permeases to design a suitable set of site-specific mutants within the gene encoding IIBgl. This set was used to study in vivo fermentation and to analyze in vitro P-enolpyruvate-dependent sugar phosphorylation as well as sugar phosphate-dependent sugar transphosphorylation. The following results were obtained. (i) IIBgl transports and phosphorylates glucose as well as aryl- and alkyl-beta-glucosides; (ii) histidyl 547 is essential for the phosphorylation of IIBgl by the histidine-containing phosphoryl carrier protein of the phosphotransferase system (HPr) (first phosphorylation site); (iii) both cysteyl 24 and histidyl 306 are essential for the transfer of the phosphoryl group to the sugar; (iv) replacement of Cys-24 by serine leads to uncoupling of sugar transport from phosphorylation; and (v) histidyl 183 is important for substrate specificity. Our studies also revealed heterologous phosphoryl transfer between the beta-glucoside and glucose permease components which probably occurs as follows: 1) HPr-P----IIBgl (His-547)----IIGlc----alkyl-alpha- or -beta-glucosides or glucose (but not aryl-beta-glucosides) and 2) HPr-P----IIIGlc----IIBgl (Cys-24 or His-306)----alkyl- or aryl-beta-glucosides or glucose (but not methyl-alpha-glucoside). In addition to the essential residues noted above, several residues in IIBgl were identified which when mutated reduced the in vitro catalytic efficiency of the enzyme more than 10-fold. Thus, aspartyl 551 and arginyl 625 appeared to function together with histidyl 547 in phosphoryl transfer involving the first phosphorylation site in the permease, whereas histidyl 183 appeared to function together with cysteyl 24 and histidyl 306 in phosphoryl transfer involving the second phosphorylation site in the permease.

Arbutin↗
PubMed1990

Beta-glucoside permease represses the bgl operon of Escherichia coli by phosphorylation of the antiterminator protein and also interacts with glucose-specific enzyme III, the key element in catabolite control.

The beta-glucoside (bgl) operon of Escherichia coli is subject to both positive control by transcriptional termination/antitermination and negative control by the beta-glucoside-specific transport protein, an integral membrane protein known as enzyme IIBgl. Previous results led us to speculate that enzyme IIBgl exerts its negative control by phosphorylating and thereby inactivating the antiterminator protein, BglG. Specifically, our model postulated that the transport protein enzyme IIBgl exhibits protein-phosphotransferase activity in the absence of beta-glucosides. We now present biochemical evidence that the phosphorylation of protein BglG does indeed occur in vivo and that it is accompanied by the loss of antitermination activity. BglG persists in the phosphorylated state in the absence of beta-glucosides but is rapidly dephosphorylated when beta-glucosides become available for transport. Our data also suggested specific interactions between the beta-glucoside transport protein and the glucose-specific enzyme III (enzyme IIIGlc), a component of glucose transport and a key element in regulation of catabolite repression. These observations indicate that enzyme IIIGlc may, in conjunction with enzyme IIBgl, modulate the transport of beta-glucosides and the phosphorylation of the antiterminator protein. In the absence of both sugars, when the catabolite-controlled promoter of the operon is derepressed, enzyme IIIGlc may mediate tight repression of antitermination.

Bacterial Proteins↗
PubMed1988

IS150: distribution, nucleotide sequence and phylogenetic relationships of a new E. coli insertion element.

Recently we identified the new insertion (IS) sequence IS150 in various strains of Escherichia coli K-12. We have screened other strains of E. coli and Salmonella typhimurium for the presence of homologous sequences. The strains of E. coli K-12 and W tested contain one or more copies of homology to IS150. We have also determined the complete nucleotide sequence of a copy of IS150 inserted into IS1. Comparison of nucleotide and deduced amino acid sequences of IS150, IS2, IS3, IS51, IS600 and IS629 reveals significant homologies suggesting that these elements are members of a family of phylogenetically related insertion sequences.

Amino Acid Sequence↗
PubMed1988

Sugar permeases of the bacterial phosphoenolpyruvate-dependent phosphotransferase system: sequence comparisons.

The amino acyl sequences of eight permeases (enzymes II and enzyme II-III pairs) of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) have been analyzed. All systems show similar sizes, and six of these systems exhibit the same molecular weight +/- 2%. Several exhibit sequence homology. Characteristic NH2-terminal and COOH-terminal sequences were found. The NH2-terminal leader sequences are believed to function in targeting of the permeases to the membrane, whereas the characteristic COOH-terminal sequences are postulated to mediate interaction with the energy-coupling protein phospho HPr. One of the systems, the one specific for mannose, exhibits distinctive characteristics. A pair of probable phosphorylation sites was detected in each of the five most similar systems, those specific for beta-glucosides, sucrose, glucose, N-acetylglucosamine, and mannitol. One of the two equivalent phosphorylation sites (proposed phosphorylation site 1) was located approximately 80 residues from the COOH terminus of each system. The other site (proposed phosphorylation site 2) was located approximately 440 residues from the COOH termini of the glucose and N-acetylglucosamine systems, approximately 320 residues from the COOH termini of the beta-glucoside and sucrose systems, and 381 residues from the COOH terminus of the mannitol system. Intragenic rearrangement during evolutionary history may account for the different positions of phosphorylation sites 2 in the different PTS permeases. More extensive intragenic rearrangements may have given rise to entirely different positions of phosphorylation in the glucitol, mannose, and lactose systems. A single, internal amphipathic alpha-helix with characteristic features was found in each of seven of the eight enzymes II. The lactose-specific enzyme III of Staphylococcus aureus was unique in possessing a COOH-terminal amphipathic alpha-helix rich in basic amino acyl residues. Possible functions for these amphipathic segments are discussed.

Amino Acid Sequence↗
PubMed1988

Regulation of the bgl operon of Escherichia coli by transcriptional antitermination.

The bgl operon of Escherichia coli encodes all functions necessary for the regulated uptake and utilization of aryl beta-glucosides. The operon is unusual, however, in that it is cryptic in wild-type strains, requiring activation by mutational events. The vast majority of these mutations are due to transposition of insertion elements into the promoter region of the operon. In this report we show that integration of IS5 into the vicinity of the bgl promoter (P0) enhances its activity by greater than 60-fold thereby activating the operon. In the activated state the operon is subject to induction by substrate. Recent studies have shown that induction of the bgl operon by substrate involves antitermination within the leader of the operon. We now show that substrate-dependent regulation involves specific termination/antitermination of transcription at two signal structures flanking the first gene of the operon, bglG. Antitermination is mediated by the product of gene bglG. In the absence of substrate this antitermination is prevented by the action of the product of gene bglF (the second gene of the operon), which encodes the beta-glucoside-specific transport protein (enzymeIIBgl of the phosphoenolpyruvate-dependent phosphotransferase system, PTS) resulting in repression of the operon. The bgl promoter (P0) is not subject to substrate-dependent regulation. The bgl operon has two additional promoters (P1 and P2) located within the terminators, which could also participate in regulation.

DNA Transposable Elements↗
PubMed1988

Second-element turn-on of gene expression in an IS1 insertion mutant.

To learn more about the ways in which genes silenced by insertion mutations can be reactivated, we have undertaken a systematic investigation of Gal+ revertants of the polar mutant galOP-306::IS1 in Escherichia coli K12. The selective conditions used excluded reversion to wild type by precise excision of IS1. In this system (which resided on a multi-copy plasmid) reversion to the Gal+ phenotype occurred with a frequency of about 10(-7) per cell and per generation. Analysis of the revertants revealed that - with the single exception of the previously published chromosomal mutant sis1 - alterations in the structure of IS1 lead to reactivation of gal operon expression. These events fall into four classes: (I) insertion of IS2 at position 327 in IS1, insertion of IS2 at position 687 in IS1, (III) insertion of a hitherto undetected mobile element, IS150, at position 387, (IV) a 16-bp deletion encompassing IS1 coordinates 553-568. Of some 200 independent reversion events studied, all but one were of types I-III i.e. they involved the intervention of a second mobile element.

Base Sequence↗
PubMed1988

Bacterial proteins with N-terminal leader sequences resembling mitochondrial targeting sequences of eukaryotes.

Amphipathic, alpha-helical, leader sequences, analogous to those that direct nuclear-encoded eukaryotic proteins into mitochondria, have been found in one and only one class of bacterial integral membrane proteins. These bacterial proteins are the sugar permeases of the phosphoenolpyruvate-dependent phosphotransferase system. The amphipathic leader sequence in each of these proteins is terminated by a helix breaker, either a prolyl residue or 2 adjacent glycyl residues. Preliminary evidence suggests that these leader sequences function to target the proteins to the envelope fraction of the prokaryotic cell during their biosynthesis.

Amino Acid Sequence↗
PubMed1987

Genetic organization of insertion element IS2 based on a revised nucleotide sequence.

We identified a transposable element resident in the chromosome of Escherichia coli K-12 strain HB101. This is an approx. 4400-bp-long transposon flanked by two copies of insertion sequence (IS) 1 element in direct orientation. One of the IS1 elements was found to be integrated into an IS2 element between IS2 bp 139 and bp 140 with the large moiety of IS2 within the transposon. The sequence of this part of IS2 differs from the published sequence of galOP-308::IS2 at a number of positions. Restriction analysis of the published allele, however, indicated that both alleles may in fact be identical. Since six of the eight differences found alter open reading frames, the revised sequence presents a new outlook for the potential genetic organization of IS2.

Base Composition↗
PubMed1987

Beta-glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes.

Wild-type Escherichia coli cells are unable to grow on beta-glucosides. Spontaneous mutants arise, however, which are able to utilize certain aromatic beta-glucosides such as salicin or arbutin as carbon sources, revealing the presence of a cryptic operon called bgl. Mutations activating the operon map within (or close to) the promoter region of the operon and are due to the transposition of an IS1 or IS5 insertion element into this region. This operon was reported to consist of three genes coding for a phospho-beta-glucosidase, a specific transport protein (enzyme IIBgl), and a positively regulating protein. We have defined the extent and location of three structural genes, bglC, bglS, and bglB, and have determined their DNA sequence. The amino acid sequences deduced from the open reading frames together with deletion and subcloning analyses suggest that the first gene, bglC, codes for the regulatory protein, the second, bglS, codes for the transport protein, and the third, bglB, for phospho-beta-glucosidase. A fourth gene may exist which codes for a product of unknown function. We discuss structural features of the DNA sequence which may bear on the regulation of the operon. Homologies to sequences preceding the gene for an excreted levansucrase of Bacillus subtilis, which are known to be involved in the regulation of this gene, and to sequences preceding the gene for an excreted beta-endoglucanase of B. subtilis, for which data pertaining to regulation are not yet available, suggest a close evolutionary relationship among the regulatory components of all three systems.

Amino Acid Sequence↗