bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular and functional analysis of the lipopolysaccharide biosynthesis locus wlb from Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica
Authors: Allen Andrew G. , Thomas Richard M. , Cadisch Joanna T. , Maskell Duncan J.
Published: March 2003

The Bordetella pertussis wlb locus (wlbpe, formerly bpl ) is required for the biosynthesis of a trisaccharide that, when attached to the B. pertussis lipopolysaccharide (LPS) core (band B), generates band A LPS. The equivalent loci in Bordetella bronchiseptica (wlbbr) and Bordetella parapertussis (wlbpa) were identified and cloned. The wlbbr and wlbpa loci differ from wlbpe in that they lack the insertion sequence that defines the right‐hand terminus of wlbpe. Deletion of 12 kb of DNA containing the whole wlb locus (Δwlb) by allelic exchange in each of the three bordetellae had no effect on band B biosynthesis, whereas band A biosynthesis was prevented in B. pertussis and B. bronchiseptica. In B. bronchiseptica and B. parapertussis, Δwlb mutants also lacked O‐antigen. Reintroduction of the wlbpe or wlbbr loci on a shuttle vector into the three Δwlb mutants restored the wild‐type LPS phenotype in the B. pertussis and B. bronchiseptica mutants. In the case of B. parapertussis, which normally does not synthesize an apparent band A structure, introduction of the wlbpe or wlbbr loci now enabled the generation of band A. This suggests that the attachment point for band A trisaccharide on the LPS core is present in B. parapertussis, and further suggests that the wild‐type wlbpa locus is not fully functional. Introduction of the wlbpa locus into the Δwlbpe, Δwlbbr and Δwlbpa mutants had interesting consequences. The B. bronchiseptica and B. parapertussis recipients were now able to biosynthesize O‐antigen, but no band A was generated. In the B. pertussis recipient, a truncated band A was expressed consistent with a mutation in the wlbH gene, but on Western blotting the expression of a small amount of full‐length band A was also seen. Evidence that the wlbHpa protein is not fully functional was provided by the failure of the wlbpa locus to fully complement a B. pertussis wlbH (ΔwlbHpe) mutant. This was supported by DNA sequence data showing that a single amino acid, conserved between homologous proteins from a range of bacteria, is altered in the B. parapertussis WlbH protein.
Characterization of a novel RNA regulator of Erwinia carotovora ssp. carotovora that controls production of extracellular enzymes and secondary metabolites
Authors: Liu Yang , Cui Yaya , Mukherjee Asita , Chatterjee Arun K.
Published: March 2003

The enterobacterium Erwinia carotovora ssp. carotovora strain 71 (hereafter Ecc71) produces extracellular enzymes such as pectate lyase isozymes (Pels), cellulase (Cel), polygalacturonase (Peh) and protease (Prt). These enzymes degrade plant cell wall components and are largely responsible for the elicitation of soft‐rot diseases in plants and plant products. Ecc71 also produces HarpinEcc, the elicitor of hypersensitive reaction (HR) and the quorum‐sensing signal, N‐(3‐oxohexanoyl)‐L‐homoserine lactone (OHL). OHL controls extracellular enzyme and HarpinEcc production. The levels of these enzymes, as well as the expression of hrpNEcc, the structural gene for HarpinEcc, and ohlI, the gene specifying OHL synthesis, are negatively regulated by RsmA. rsmB, formerly aepH, on the other hand, positively regulates extracellular enzyme production. 6His–RsmA recombinant protein purified from E. coli binds rsmB RNA as indicated by gel mobility shift assays. rsmB comprises 547 bp DNA, which is transcribed from a single start site immediately after a σ70‐like promoter. In Ecc71, two rsmB RNA species are detected: a full‐length 479 base rsmB RNA and a 259 base rsmB′ RNA. rsmB′ DNA hybridizes with the 259 base and the 479 base transcripts. A 3′ RNase protection assay revealed that the 259 base and the 479 base RNA species end at the same position immediately after the putative rho‐independent terminator. The expression of rsmB–lacZ transcriptional fusions established that the rsmB′ RNA is not produced because of the activation of an internal promoter. These data strongly suggest that the 259 base rsmB′ RNA is derived by processing of the primary rsmB RNA. In Ecc71, rsmB′ expression driven by the lac promoter causes overproduction of Pel, Peh, Cel and Prt, and accumulation of pel‐1, peh‐1, hrpNEcc and ohlI transcripts. By contrast, a plasmid with the rsmB′ DNA sequence deleted fails to cause overproduction of the extracellular enzymes in Ecc71. The rsmB′ effect also occurs in Escherichia coli as glycogen accumulation is stimulated in the presence of rsmB′. In vivo and in vitro translation as well as mutational analysis of rsmB′ have established that rsmB′ RNA does not yield a translational product. Therefore, we concluded that the rsmB′ RNA itself functions as the regulator. Indeed, the expression of rsmB′ DNA leads to neutralization of the negative effects of the RNA‐binding protein, RsmA, in Ecc71 and Serratia marcescens strain SM274. We propose a model that explains how RsmA and rsmB control the expression of genes for extracellular enzymes.
Role of the GlnK signal transduction protein in the regulation of nitrogen assimilation in Escherichia coli
Authors: Atkinson Mariette R. , Ninfa Alexander J.
Published: March 2003

Two structurally similar but functionally distinct PII‐like proteins, PII and GlnK, regulate nitrogen assimilation in Escherichia coli. Studies with cells indicated that both PII (the glnB product) and GlnK (the glnK product) acted through the kinase/phosphatase NRII [NtrB, the glnL (ntrB ) product] to reduce transcription initiation from Ntr promoters, apparently by regulating the phosphorylation state of the transcriptional activator NRI∼P (NtrC∼P, the phosphorylated form of the glnG (ntrC ) product). Both GlnK and PII also acted through adenylyltransferase (ATase, the glnE product) to regulate the adenylylation state of glutamine synthetase (GS). The activity of both GlnK and PII was regulated by the signal‐transducing uridylyltransferase/uridylyl‐removing enzyme (UTase/UR, glnD product). Our experiments indicate that either PII or GlnK could effectively regulate ATase, but that PII was required for the efficient regulation of NRII required to prevent expression of glnA, which encodes GS. Yet, GlnK also participated in regulation of NRII. Although cells that lack either PII or GlnK grew well, cells lacking both of these proteins were defective for growth on nitrogen‐rich minimal media. This defect was alleviated by the loss of NRII, and was apparently due to unregulated expression of the Ntr regulon. Also, mutations in glnK, designated glnK *, were obtained as suppressors of the Ntr− phenotype of a double mutant lacking PII and the UTase/UR. These suppressors appeared to reduce, but not eliminate, the ability of GlnK to prevent Ntr gene expression by acting through NRII. We hypothesize that one role of GlnK is to regulate the expression of the level of NRI∼P during conditions of severe nitrogen starvation, and by so doing to contribute to the regulation of certain Ntr genes.
Entry of OpaA+ gonococci into HEp‐2 cells requires concerted action of glycosaminoglycans, fibronectin and integrin receptors
Authors: Van Putten Jos P. M. , Duensing Thomas D. , Cole Robert L.
Published: March 2003

Heparan sulphate proteoglycans are increasingly implicated as eukaryotic cell surface receptors for bacterial pathogens. Here, we report that Neisseria gonorrhoeae adheres to proteoglycan receptors on HEp‐2 epithelial cells but that internalization of the bacterium by this cell type requires the serum glycoprotein fibronectin. Fibronectin was shown to bind specifically to gonococci producing the OpaA adhesin. Binding assays with fibronectin fragments located the bacterial binding site near the N‐terminal end of the molecule. However, none of the tested fibronectin fragments supported gonococcal entry into the eukaryotic cells; a 120 kDa fragment carrying the cell adhesion domain with the amino acid sequence RGD even inhibited the fibronectin‐mediated uptake of MS11‐OpaA. This inhibition could be mimicked by an RGD‐containing hexapeptide and by α5β1 integrin‐specific antibodies, suggesting that interaction of the central region of fibronectin with integrin receptors facilitated bacterial uptake. Fibronectin was unable to promote gonococcal entry into HEp‐2 cells that had been treated with the enzyme heparinase III, which degrades the glycosaminoglycan side‐chains of proteoglycan receptors. On the basis of these results, we propose a novel cellular uptake pathway for bacteria, which involves the binding of the pathogen to glycosaminoglycans that, in turn, act as co‐receptors facilitating fibronectin‐mediated bacterial uptake through integrin receptors. In this scenario, fibronectin would act as a molecular bridge linking the Opa–proteoglycan complex with host cell integrin receptors.
Characterization of polyamine synthesis pathway in Bacillus subtilis 168
Authors: Sekowska Agnieszka , Bertin Philippe , Danchin Antoine
Published: March 2003

The ubiquitous polyamines fulfil a variety of functions in all three kingdoms of life. However, little is known about the biosynthesis of these compounds in Gram‐positive bacteria. We show that, in Bacillus subtilisthere is a single pathway to polyamines, starting from arginine, with agmatine as an intermediate. We first identified the structural gene of arginine decarboxylase, speA (formerly cad ), and then described the speE speB operon, directing synthesis of spermidine synthase and agmatinase. This operon is transcribed into two messenger RNAs, a major one for the speE gene and a minor one for both speE and speB. The promoter of the operon was identified upstream from the speE gene by primer extension analysis. Transcription of this operon indicated that the level of agmatinase synthesis is very low, thus allowing a stringent control on the synthesis of putrescine and, therefore, of all polyamines. This is consistent with the level of polyamines measured in the cell.
Expression of ptsG, the gene for the major glucose PTS transporter in Escherichia coli, is repressed by Mlc and induced by growth on glucose
Authors: Plumbridge Jacqueline
Published: March 2003

The gene for the glucose‐specific transporter of the phosphotransferase system, ptsG, is expressed from two promoters separated by 141 bp. The expression of the major, shorter transcript is very strongly dependent upon cAMP/CAP. However, unlike other CAP‐activated genes, the expression of ptsG is higher in glucose media than in glycerol, implying that ptsG is controlled by a glucose‐inducible regulator. A mutation in the mlc gene greatly enhances ptsG expression in a glycerol‐grown culture but has no effect on ptsG expression during growth on glucose. The mlc gene encodes a transcriptional regulator that has been shown to affect the expression of manXYZ and malT. ptsG mRNA levels are lower in the mlc strain grown on glucose than in the same strain grown on glycerol. This is presumably because of the greater catabolite repression in the glucose culture than in glycerol. The final level of expression of ptsG in a mlc+ strain in glucose is a compromise between specific induction by glucose and generalized catabolite repression. The result is that ptsG expression is very similar in glucose‐grown cultures of wild‐type and mlc strains. The Mlc protein binds to two sites centred at −6 and −175 upstream of the major ptsG transcript. CAP binds at −40.5 compared with this site, typical of class II CAP‐regulated promoters, and the binding of CAP and Mlc is co‐operative.
Characterization of the Erwinia chrysanthemi expI–expR locus directing the synthesis of two N‐acyl‐homoserine lactone signal molecules
Authors: Nasser William , Bouillant Marie Louise , Salmond George , Reverchon Sylvie
Published: March 2003

The plant pathogen Erwinia chrysanthemi produces three acyl‐homoserine lactones (acyl‐HSLs). One has been identified as N‐(3‐oxohexanoyl)‐homoserine lactone (OHHL), and the two others were supposed to be N‐(hexanoyl)‐homoserine lactone (HHL) and N‐(decanoyl)‐homoserine lactone (DHL). The genes for a quorum‐sensing signal generator (expI ) and a response regulator (expR ) were cloned. These genes are convergently transcribed and display high similarity to the expI–expR genes of Erwinia carotovora. ExpI is responsible for both OHHL and HHL production. Inactivation of expI had little effect on pectinase synthesis in E. chrysanthemi, as expression of only two of the pectate lyase genes, pelA and pelB, was decreased. E. chrysanthemi expR mutants still produced acyl‐HSL and pectinases. However, gel shift and DNAse I footprinting experiments showed that the purified E. chrysanthemi ExpR protein binds specifically to the promoter regions of the five major pel genes. Addition of OHHL modified the ExpR‐DNA bandshift profiles, indicating that ExpR interacts with OHHL and binds to DNA in different ways, depending on the OHHL concentration. Localization of the ExpR binding sites just upstream of promoter regions suggests that ExpR functions as an activator of pel expression in the presence of OHHL. The absence of a phenotype in expR mutants strongly suggests that at least an additional interchangeable ExpR homologue exists in E. chrysanthemi. Finally, transcription of expI ::uidA and expR ::uidA fusions is dependent on the population density, suggesting the existence of a quorum‐sensing hierarchy in E. chrysanthemi. These results suggest that the expI–expR locus is part of a complex autoregulatory system that controls quorum sensing in E. chrysanthemi.
Processing of the pectate lyase PelI by extracellular proteases of Erwinia chrysanthemi 3937
Authors: Shevchik Vladimir E. , Boccara Martine , Vedel Regine , Hugouvieux‐Cotte‐Pattat Nicole
Published: March 2003

Erwinia chrysanthemi causes soft rot on various plants. The maceration of plant tissues is mainly due to the action of endopectate lyases. The E. chrysanthemi strain 3937 produces eight endopectate lyases (PelA, PelB, PelC, PelD, PelE, PelI, PelL and PelZ) that are secreted by the Out pathway. The necrotic response elicited by the wild‐type E. chrysanthemi strain on tobacco leaves is due to an extracellular protein secreted by the Out machinery. Purification of the active factor revealed that it corresponds to a pectate lyase presenting immunological cross‐reaction with PelI. Analysis of pelI and out mutants indicated that the necrosis‐inducing pectate lyase results from a post‐translational modification of PelI occurring extracellularly both in culture media and in planta. This modification consists of the cleavage of 97 N‐terminal amino acids by the extracellular proteases of E. chrysanthemi. The enzymatic properties of the maturated form, PelI‐3, are not, or only weakly, modified. However, this maturation gives rise to a small size and basic form that is active as a defence elicitor in plants.
Inducer exclusion in Escherichia coli by non‐PTS substrates: the role of the PEP to pyruvate ratio in determining the phosphorylation state of enzyme IIAGlc
Authors: Hogema Boris M. , Arents Jos C. , Bader Rechien , Eijkemans Kevin , Yoshida Hiromi , Takahashi Hideyuki , Aiba Hiroji , Postma Pieter W.
Published: March 2003

The main mechanism causing catabolite repression in Escherichia coli is the dephosphorylation of enzyme IIAGlc, one of the enzymes of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS is involved in the uptake of a large number of carbohydrates that are phosphorylated during transport, phosphoenolpyruvate (PEP) being the phosphoryl donor. Dephosphorylation of enzyme IIAGlc causes inhibition of uptake of a number of non‐PTS carbon sources, a process called inducer exclusion. In this paper, we show that dephosphorylation of enzyme IIAGlc is not only caused by the transport of PTS carbohydrates, as has always been thought, and that an additional mechanism causing dephosphorylation exists. Direct monitoring of the phosphorylation state of enzyme IIAGlc also showed that many carbohydrates that are not transported by the PTS caused dephosphorylation during growth. In the case of glucose 6‐phosphate, it was shown that transport and the first metabolic step are not involved in the dephosphorylation of enzyme IIAGlc, but that later steps in the glycolysis are essential. Evidence is provided that the [PEP]–[pyruvate] ratio, the driving force for the phosphorylation of the PTS proteins, determines the phosphorylation state of enzyme IIAGlc. The implications of these new findings for our view on catabolite repression and inducer exclusion are discussed.
The S box regulon: a new global transcription termination control system for methionine and cysteine biosynthesis genes in Gram‐positive bacteria
Authors: Grundy Frank J. , Henkin Tina M.
Published: March 2003

The molecular mechanisms for regulation of the genes involved in the biosynthesis of methionine and cysteine are poorly characterized in Bacillus subtilis. Analyses of the recently completed B. subtilis genome revealed 11 copies of a highly conserved motif. In all cases, this motif was located in the leader region of putative transcriptional units, upstream of coding sequences that included genes involved in methionine or cysteine biosynthesis. Additional copies were identified in Clostridium acetobutylicum and Staphylococcus aureus, indicating conservation in other Gram‐positive genera. The motif includes an element resembling an intrinsic transcriptional terminator, suggesting that regulation might be controlled at the level of premature termination of transcription. The 5′ portion of all of the leaders could fold into a conserved complex structure. Analysis of the yitJ gene, which is homologous to Escherichia coli metH and metF, revealed that expression was induced by starvation for methionine and that induction was independent of the promoter and dependent on the leader region terminator. Mutation of conserved primary sequence and structural elements supported a model in which the 5′ portion of the leader forms an anti‐antiterminator structure, which sequesters sequences required for the formation of an antiterminator, which, in turn, sequesters sequences required for the formation of the terminator; the anti‐antiterminator is postulated to be stabilized by the binding of some unknown factor when methionine is available. This set of genes is proposed to form a new regulon controlled by a global termination control system, which we designate the S box system, as most of the genes are involved in sulphur metabolism and biosynthesis of methionine and cysteine.
Pho signal transduction network reveals direct transcriptional regulation of one two‐component system by another two‐component regulator: Bacillus subtilis PhoP directly regulates production of ResD
Authors: Birkey Stephanie M. , Liu Wei , Zhang Xiaohui , Duggan Mary Fran , Hulett F. Marion
Published: March 2003

The Bacillus subtilis ResD–ResE two‐component system is responsible for the regulation of a number of genes involved in cytochrome c biogenesis and haem A biosynthesis, and it is required for anaerobic respiration in this organism. We reported previously that the operon encoding these regulatory proteins, the resABCDE operon, is induced under several conditions, one of which is phosphate starvation. We report here that this transcription requires the PhoP–PhoR two‐component system, whereas other induction conditions do not. The PhoP∼P response regulator directly binds to and is essential for transcriptional activation of the resABCDE operon as well as being involved in repression of the internal resDE promoter during phosphate‐limited growth. The concentration of ResD in various phoP mutant strains corroborates the role of PhoP in the production of ResD. These interactions result in a regulatory network that ties together the cellular functions of respiration/energy production and phosphate starvation. Significantly, this represents the first evidence for direct involvement of one two‐component system in transcription of a second two‐component system.
SarA level is a determinant of agr activation in Staphylococcus aureus
Authors: Chien Yueh‐tyng , Manna Adhar C. , Cheung Ambrose L.
Published: March 2003

The control of virulence determinant expression in Staphylococcus aureus is a complex process involving global regulatory loci such as sar and agr. The sar locus consists of a 372 bp sarA open reading frame (ORF) preceded by a triple promoter region interspersed with two putative smaller ORFs (ORF3 and ORF4). The triple promoter system yields three overlapping sar transcripts (sarA, sarC and sarB of 0.56, 0.8 and 1.2 kb respectively). We have recently shown that the SarA protein binds to the agr promoter region to stimulate the transcription of RNAII and RNAIII, two major transcripts encoded within the agr locus. To assess the role of the region upstream of sarA in agr expression, we evaluated the contribution of ORF3 and ORF4 to SarA protein expression and to agr activation by introducing nonsense mutations into the respective ORFs. Northern analysis of sar mutant clones containing these mutations carried on a shuttle plasmid revealed that all three sar‐related transcripts are present. Using anti‐SarA monoclonal antibodies with defined epitopes in a competitive ELISA to determine the SarA protein level, we found that the introduction of a stop codon in ORF3 on a shuttle plasmid carrying the intact sar locus in a sar mutant led to a significant decrease in SarA protein level compared with the non‐mutated control. The effect of a nonsense mutation in ORF4 on SarA levels is much less. Likewise, an analogous sar mutant clone with a deletion in ORF3 also displayed a lower SarA level than its intact counterpart. The reduction in SarA expression correlated with a lower level of agr activation in the corresponding sar mutant clone. These data suggest that ORF3, and to a lesser degree ORF4, may affect agr expression by modulating SarA protein expression.
Cytosolic enzymes with a mitochondrial ancestry from the anaerobic chytrid Piromyces sp. E2
Authors: Akhmanova Anna , Voncken Frank G. J. , Harhangi Harry , Hosea Ken M. , Vogels Godfried D. , Hackstein Johannes H. P.
Published: March 2003

The anaerobic chytrid Piromyces sp. E2 lacks mitochondria, but contains hydrogen‐producing organelles, the hydrogenosomes. We are interested in how the adaptation to anaerobiosis influenced enzyme compartmentalization in this organism. Random sequencing of a cDNA library from Piromyces sp. E2 resulted in the isolation of cDNAs encoding malate dehydrogenase, aconitase and acetohydroxyacid reductoisomerase. Phylogenetic analysis of the deduced amino acid sequences revealed that they are closely related to their mitochondrial homologues from aerobic eukaryotes. However, the deduced sequences lack N‐terminal extensions, which function as mitochondrial leader sequences in the corresponding mitochondrial enzymes from aerobic eukaryotes. Subcellular fractionation and enzyme assays confirmed that the corresponding enzymes are located in the cytosol. As anaerobic chytrids evolved from aerobic, mitochondria‐bearing ancestors, we suggest that, in the course of the adaptation from an aerobic to an anaerobic lifestyle, mitochondrial enzymes were retargeted to the cytosol with the concomitant loss of their N‐terminal leader sequences.
The B regulatory subunit of protein phosphatase 2A is required for completion of macroconidiation and other developmental processes in Neurospora crassa
Authors: Yatzkan Einat , Yarden Oded
Published: March 2003

rgb‐1, encoding the tentative B regulatory subunit of the type 2A Ser/Thr phosphatase in Neurospora crassa, was isolated from cDNA and genomic libraries. Based on analysis of cDNA and genomic clones, rgb‐1 is 3387 nucleotides in length, contains seven putative introns and encodes a 461‐amino‐acid polypeptide. Intron I, which is 5′ to the presumed translation initiation codon, contains a uORF encoding 34 amino acids. Intron VI undergoes alternative splicing. Inactivation of rgb‐1 by the repeat‐induced point (RIP) mutation procedure produced progeny that grow slowly, have abnormal hyphal morphology, are female sterile and produce abundant amounts of arthroconidia. The rgb‐1RIP strain does not produce major constriction chains or mature macroconidia. Minor constriction chains are formed, yet the growth process reverts to hyphal elongation. Microscopic and genetic analyses indicate that rgb‐1 is a regulator of the budding subroutine of the macroconidiation process and that arthroconidiation, which shares common early and late events with macroconidiation, is induced as a default mechanism for asexual reproduction in this fungus.
Cardiolipin synthase expression is essential for growth at elevated temperature and is regulated by factors affecting mitochondrial development
Authors: Jiang Feng , Gu Zhiming , Granger John M. , Greenberg Miriam L.
Published: March 2003

Cardiolipin (CL) is a unique dimeric phospholipid localized primarily in the mitochondrial membrane. In eukaryotes, the enzyme CL synthase catalyses the synthesis of CL from two lipid substrates, CDP‐diacylglycerol and phosphatidylglycerol. In earlier studies, we reported the purification of CL synthase from Saccharomyces cerevisiae and the cloning of the gene CRD1 (previously called CLS1) that encodes the enzyme. Because CL is an important component of the mitochondrial membrane, knowledge of its regulation will provide insight into the biogenesis of this organelle. To understand how CL synthesis is regulated, we analysed CRD1 expression by Northern blot analysis of RNA extracted from cells under a variety of growth conditions. CRD1 expression is regulated by mitochondrial development factors. CRD1 levels were 7‐ to 10‐fold greater in stationary than in logarithmic growth phase, and threefold greater in wild‐type than in ρ0 mutants. Expression was somewhat elevated during growth in glycerol/ethanol versus glucose media. In contrast, CRD1 expression was not regulated by the phospholipid precursors inositol and choline, and was not altered in the regulatory mutants ino2, ino4 and opi1. Mutations in cytochrome oxidase assembly, which led to reduced Crd1p enzyme activity, did not affect CRD1 expression. The crd1 null mutant makes a truncated CRD1 message. Although the null mutant can grow on both fermentable and non‐fermentable carbon sources at lower temperatures, it cannot form colonies at 37°C. In conclusion, CRD1 expression is controlled by factors affecting mitochondrial development, but not by the phospholipid precursors inositol and choline. Expression of CRD1 is essential for growth at elevated temperatures, suggesting that either CL or Crd1p is required for an essential cellular function.
Autogenous regulation of transcription termination factor Rho and the requirement for Nus factors in Bacillus subtilis
Authors: Ingham Colin J. , Dennis Joanne , Furneaux Peter A.
Published: March 2003

The expression and activity of transcription termination factor Rho and the requirement for transcription elongation factors NusA and NusG was investigated in Bacillus subtilis. Rho was present at < 5% of the level found in Escherichia coli, but Rho factors from these two bacteria had similar properties as RNA‐activated ATPases and in vitro termination of transcription on the λtR1 terminator. The B. subtilis rho gene was autoregulated at the level of transcription; autoregulation required sequences within the rho mRNA leader region and gene. To date, the B. subtilis rho is the only gene from a Gram‐positive bacterium found to be regulated by Rho. Rho was not involved in bulk mRNA decay in B. subtilis. The E. coli elongation factors NusA and NusG target Rho, and the importance of these proteins in B. subtilis was examined by gene disruption. The B. subtilis NusG was inessential for both the viability and the autoregulation of Rho, whereas NusA was essential, and the requirement for NusA was independent of Rho. This contrasts with E. coli in which NusG is essential but NusA becomes dispensable if Rho terminates transcription less efficiently.
Lipid modification of prelipoproteins is dispensable for growth but essential for efficient protein secretion in Bacillus subtilis: characterization of the lgt gene
Authors: Leskelä Soile , Wahlström Eva , Kontinen Vesa P. , Sarvas Matti
Published: March 2003

We have identified and characterized the lgt gene of Bacillus subtilis. The prelipoprotein diacylglycerol transferase enzyme (Lgt) catalyses the first reaction in lipomodification of bacterial lipoproteins. Inactivation of lgt in B. subtilis by a nonsense mutation (prs‐11 mutation) or by disruption was shown here to abolish lipomodification of prelipoproteins completely, as well as the cleavage of signal peptide. However, unlike in Gram‐negative bacteria, the lgt mutants of B. subtilis were fully viable. In agreement with this observation, studies of two lipoproteins, PrsA and BlaP, indicated that non‐lipomodified precursors of these proteins were functional and translocated across the cytoplasmic membrane. However, there was release of both precursors from cells, resulting in a reduced level of the cell‐bound form. We have shown that the reduced level of the PrsA lipoprotein, a foldase involved in protein secretion, caused impaired protein secretion, a prominent phenotype of lgt mutants. There was no indication that non‐lipomodified PrsA displayed reduced activity.
The dimerization and topological specificity functions of MinE reside in a structurally autonomous C‐terminal domain
Authors: King Glenn F. , Rowland Susan L. , Pan Borlan , Mackay Joel P. , Mullen Gregory P. , Rothfield Lawrence I.
Published: March 2003

Correct placement of the division septum in Escherichia coli requires the co‐ordinated action of three proteins, MinC, MinD and MinE. MinC and MinD interact to form a non‐specific division inhibitor that blocks septation at all potential division sites. MinE is able to antagonize MinCD in a topologically sensitive manner, as it restricts MinCD activity to the unwanted division sites at the cell poles. Here, we show that the topological specificity function of MinE residues in a structurally autonomous, trypsin‐resistant domain comprising residues 31–88. Nuclear magnetic resonance (NMR) and circular dichroic spectroscopy indicate that this domain includes both α and β secondary structure, while analytical ultracentrifugation reveals that it also contains a region responsible for MinE homodimerization. While trypsin digestion indicates that the anti‐MinCD domain of MinE (residues 1–22) does not form a tightly folded structural domain, NMR analysis of a peptide corresponding to MinE1–22 indicates that this region forms a nascent helix in which the peptide rapidly interconverts between disordered (random coil) and α‐helical conformations. This suggests that the N‐terminal region of MinE may be poised to adopt an α‐helical conformation when it interacts with the target of its anti‐MinCD activity, presumably MinD.
Lipid domains of mycobacteria studied with fluorescent molecular probes
Authors: Christensen Henriette , Garton Natalie J. , Horobin Richard W. , Minnikin David E. , Barer Michael R.
Published: March 2003

The complex mycobacterial cell envelope is recognized as a critical factor in our failure to control tuberculosis, leprosy and other non‐tuberculous pathogens. Although its composition has been extensively determined, many details regarding the organization of the envelope remain uncertain. This is particularly so for the non‐covalently bound lipids, whose natural distribution may be disrupted by conventional biochemical or cytological techniques. In order to study the native organization of lipid domains in the mycobacterial envelope, we have applied a range of fluorescent lipophilic probes to live mycobacteria, including Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium gadium and Mycobacterium aurum, and analysed the resultant signals by fluorescence microscopy and digital image processing. Five key features were observed: (i) the presence of both envelope and intracellular lipid domains; (ii) differential localization of probes into these domains influenced predominantly by their hydrophobicity, as modelled by their calculated octanol:water partition coefficients and by their amphiphilicities; (iii) uneven distribution of lipophilic material in the envelope; (iv) selective labelling of septal regions of the envelope; and (v) modification of labelling patterns by additional treatments such as fluorescence quenching antibodies, detergents and solvents. Using this last approach, a coherent cell envelope lipid domain was demonstrated outside the cytoplasmic membrane and, for the first time, the proposed covalently linked mycolyl‐arabinogalactan‐peptidoglycan macromolecular complex was imaged directly. The use of fluorescent probes and high‐resolution fluorescence microscopy has enabled us to obtain a coherent view of distinct lipid domains in mycobacteria. Further application of this approach will facilitate understanding of the role of lipids in the physiology of these organisms.
Alteration of the synthesis of the Clp ATP‐dependent protease affects morphological and physiological differentiation in Streptomyces
Authors: De Crécy‐Lagard Valérie , Servant‐Moisson Pascale , Viala Julie , Grandvalet Cosette , Mazodier Philippe
Published: March 2003

The genes of Streptomyces coelicolor A3(2) encoding catalytic subunits (ClpP) and regulatory subunits (ClpX and ClpC) of the ATP‐dependent protease family Clp were cloned, mapped and characterized. S. coelicolor contains at least two clpP genes, clpP1 and clpP2, located in tandem upstream from the clpX gene, and at least two unlinked clpC genes. Disruption of the clpP1 gene in S. lividans and S. coelicolor blocks differentiation at the substrate mycelium step. Overexpression of clpP1 and clpP2 accelerates aerial mycelium formation in S. lividans, S. albus and S. coelicolor. Overproduction of ClpX accelerates actinorhodin production in S. coelicolor and activates its production in S. lividans.
Physical mapping of an origin of bidirectional replication at the centre of the Borrelia burgdorferi linear chromosome
Authors: Picardeau Mathieu , Lobry Jean R. , Hinnebusch B. Joseph
Published: March 2003

The Borrelia burgdorferi chromosome is linear, with telomeres characterized by terminal inverted repeats and covalently closed single‐stranded hairpin loops. The replication mechanism of these unusual molecules is unknown. Previous analyses of bacterial chromosomes for which the complete sequence has been determined, including that of B. burgdorferi, revealed an abrupt switch in polarity of CG skew at known or putative origins of replication. We used nascent DNA strand analysis to physically map the B. burgdorferi origin to within a 2 kb region at the centre of the linear chromosome, and to show that replication proceeds bidirectionally from this origin. The results are consistent with replication models in which termination occurs at the telomeres after bidirectional, symmetrical elongation from the central origin. Sequences typical of origins of other bacterial chromosomes were not found at the origin of this spirochete. The most likely location of the replication origin of the linear chromosome is the 240 bp sequence between dnaA and dnaN where the switch in CG skew occurs.
Genetic organization of sulphur‐controlled aryl desulphonation in Pseudomonas putida S‐313
Authors: Vermeij Paul , Wietek Claudia , Kahnert Antje , Wüest Thomas , Kertesz Michael A.
Published: March 2003

Pseudomonas putida S‐313 is able to desulphonate a broad range of aromatic sulphonates to provide sulphur for growth by monooxygenolytic cleavage to yield the corresponding phenol. After miniTn5 transposon mutagenesis of this strain, 11 mutants were isolated that were no longer able to utilize benzenesulphonate as a sulphur source. Three of these mutants were defective in the utilization of all aromatic sulphonates tested, but they grew normally with other sulphur sources. These strains contained independent insertions in the novel 4.2 kb asfRABC gene cluster, encoding a putative reductase (AsfA), a ferredoxin (AsfB), a putative periplasmic binding protein (AsfC), which was localized to the periplasm using alkaline phosphatase fusions, and a divergently oriented fourth gene, asfR, that encoded a LysR‐type regulator protein. A further mutant was interrupted in the ssu locus, which includes the gene for a putative desulphonative monooxygenase. Transformation of Pseudomonas aeruginosa with the asfRAB genes was sufficient to allow arylsulphonate utilization by this species, which does not normally use these compounds, suggesting that the AsfAB proteins may constitute an arylsulphonate‐specific electron transport system that interacts with a less specific oxygenase. Expression of the asfABC genes in P. putida was induced by benzenesulphonate or toluenesulphonate, and it was repressed in the presence of sulphate in the growth medium. AsfR was a negative regulator of asfABC expression, and toluenesulphonate induced expression of these genes indirectly by reducing the expression of the asfR gene.
A Saccharomyces cerevisiae G‐protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose
Authors: Kraakman Leon , Lemaire Katleen , Ma Pingsheng , Teunissen Aloys W.R.H. , Donaton Monica C.V. , Van Dijck Patrick , Winderickx Joris , De Winde Johannes H. , Thevelein Johan M.
Published: March 2003

In the yeast Saccharomyces cerevisiae the accumulation of cAMP is controlled by an elaborate pathway. Only two triggers of the Ras adenylate cyclase pathway are known. Intracellular acidification induces a Ras‐mediated long‐lasting cAMP increase. Addition of glucose to cells grown on a non‐fermentable carbon source or to stationary‐phase cells triggers a transient burst in the intracellular cAMP level. This glucose‐induced cAMP signal is dependent on the G alpha‐protein Gpa2. We show that the G‐protein coupled receptor (GPCR) Gpr1 interacts with Gpa2 and is required for stimulation of cAMP synthesis by glucose. Gpr1 displays sequence homology to GPCRs of higher organisms. The absence of Gpr1 is rescued by the constitutively activated Gpa2Val‐132 allele. In addition, we isolated a mutant allele of GPR1, named fil2, in a screen for mutants deficient in glucose‐induced loss of heat resistance, which is consistent with its lack of glucose‐induced cAMP activation. Apparently, Gpr1 together with Gpa2 constitute a glucose‐sensing system for activation of the cAMP pathway. Deletion of Gpr1 and/or Gpa2 affected cAPK‐controlled features (levels of trehalose, glycogen, heat resistance, expression of STRE‐controlled genes and ribosomal protein genes) specifically during the transition to growth on glucose. Hence, an alternative glucose‐sensing system must signal glucose availability for the Sch9‐dependent pathway during growth on glucose. This appears to be the first example of a GPCR system activated by a nutrient in eukaryotic cells. Hence, a subfamily of GPCRs might be involved in nutrient sensing.
Inducible prophages contribute to Salmonella virulence in mice
Authors: Figueroa‐Bossi Nara , Bossi Lionello
Published: March 2003

We show that Salmonella typhimurium harbours two fully functional prophages, Gifsy‐1 and Gifsy‐2, that can be induced by standard treatments or, more effectively, by exposing bacteria to hydrogen peroxide. Curing bacteria for the Gifsy‐2 prophage significantly reduces Salmonella's ability to establish a systemic infection in mice. Cured strains recover their virulence properties upon relysogenization. Phage Gifsy‐2 carries the sodC gene for a periplasmic [Cu,Zn]‐superoxide dismutase previously implicated in the bacterial defences against killing by macrophages. The contribution of the Gifsy‐1 prophage to virulence — undetectable in the presence of Gifsy‐2 as prophage — becomes significant in cells that lack Gifsy‐2 but carry the sodC gene integrated in the chromosome. This confirms the involvement of Gifsy‐2‐encoded SodC protein in Salmonella pathogenicity and suggests that the Gifsy‐1 prophage carries one or more additional virulence genes that have a functional equivalent on the Gifsy‐2 genome.
The heat shock response in yeast: differential regulations and contributions of the Msn2p/Msn4p and Hsf1p regulons
Authors: Boy‐Marcotte Emmanuelle , Lagniel Gilles , Perrot Michel , Bussereau Françoise , Boudsocq Anne , Jacquet Michel , Labarre Jean
Published: March 2003

The heat shock transcription factor Hsf1p and the stress‐responsive transcription factors Msn2p and Msn4p are activated by heat shock in the yeast Saccharomyces cerevisiae. Their respective contributions to heat shock protein induction have been analysed by comparison of mutants and wild‐type strains using [35S]‐methionine labelling and two‐dimensional gel electrophoresis. Among 52 proteins induced by a shift from 25°C to 38°C, half of them were found to be dependent upon Msn2p and/or Msn4p (including mostly antioxidants and enzymes involved in carbon metabolism), while the other half (including mostly chaperones and associated proteins) were dependent upon Hsf1p. The two sets of proteins overlapped only slightly. Three proteins were induced independently of these transcription factors, suggesting the involvement of other transcription factor(s). The Ras/cAMP/PKA signalling pathway cAMP had a negative effect on the induction of the Msn2p/Msn4p regulon, but did not affect the Hsf1p regulon. Thus, the two types of transcription factor are regulated differently and control two sets of functionally distinct proteins, suggesting two different physiological roles in the heat shock cellular response.
Entamoeba histolytica : a novel cysteine protease and an adhesin form the 112 kDa surface protein
Authors: García‐Rivera G. , Rodríguez M. A. , Ocádiz R. , Martínez‐López M. C. , Arroyo R. , González‐Robles A. , Orozco E.
Published: March 2003

Here, we present evidence that a cysteine protease (EhCP112) and a protein with an adherence domain (EhADH112) form the Entamoeba histolytica 112 kDa adhesin. Immunoelectron microscopy and immunofluorescence assays using monoclonal antibodies (mAbAdh) revealed that, during phagocytosis, the adhesin is translocated from the plasma membrane to phagocytic vacuoles. mAbAdh inhibited 54% adherence, 41% phagocytosis, and 35% and 62% destruction of MDCK cell monolayers by live trophozoites and their extracts respectively. We cloned a 3587 bp DNA fragment (Eh112 ) with two open reading frames (ORFs) separated by a 188 bp non‐coding region. The ORF at the 5′ end (Ehcp112 ) encodes a protein with a cysteine protease active site, a transmembranal segment and an RGD motif. The second ORF (Ehadh112 ) encodes a protein recognized by mAbAdh with three putative transmembranal segments and four glycosylation sites. Northern blot, primer extension and Southern blot experiments revealed that Ehcp112 and Ehadh112 are two adjacent genes in DNA. Ehcp112 and Ehadh112 genes were expressed in bacteria. The recombinant peptides presented protease activity and inhibited adherence and phagocytosis, respectively, and both were recognized by mAbAdh. The EhCP112 and EhADH112 peptides could be joined by covalent or strong electrostatic forces, which are not broken during phagocytosis.
An RGS protein regulates the pheromone response in the fission yeast Schizosaccharomyces pombe
Authors: Watson Peter , Davis Kevin , Didmon Mark , Broad Peter , Davey John
Published: March 2003

The rate and extent of a cell's response to an extracellular stimulus is influenced by regulators that act on the intracellular signalling machinery. Although not directly involved in propagating the intracellular signal, regulators control the activity of the proteins that transmit the signals. To understand this aspect of cell signalling, we have studied the pheromone response pathway in the fission yeast Schizosaccharomyces pombe, a relatively simple signalling system in a genetically tractable organism. We demonstrate this approach by investigating the role of Rgs1, a member of the Regulator of G protein Signalling (RGS) family of proteins. The rgs1 gene was identified through the Sz. pombe genome sequencing project (accession number Q09777) and recognized as having similarity to RGS proteins [Tesmer et al. (1997) Cell 89: 251–261], but this is the first report concerning the activity of the protein. Strains lacking rgs1 (Δrgs1) are hypersensitive to pheromone stimulation and unable to conjugate with a mating partner. Inhibition of mating occurs at a relative late stage in the process as Δrgs1 strains exhibit pheromone‐dependent transcription and form shmoos. Expression of SST2 (an RGS protein that regulates pheromone signalling in the budding yeast Saccharomyces cerevisiae) overcomes the hypersensitivity of the Δrgs1 strains but fails to rescue their mating defect.
Escherichia coli cells bearing mutA, a mutant glyV tRNA gene, express a recA‐dependent error‐prone DNA replication activity
Authors: Al Mamun Abu Amar M. , Rahman M. Sayeedur , Humayun M. Zafri
Published: March 2003

A base substitution mutation (mutA) in the Escherichia coli glyV tRNA gene potentiates asp → gly mistranslation and confers a strong mutator phenotype that is SOS independent, but requires recA, recB and recC genes. Here, we demonstrate that mutA cells express an error‐prone DNA polymerase by using an in vitro experimental system based on the conversion of phage M13 single‐stranded viral DNA bearing a model mutagenic lesion to the double‐stranded replicative form. Amplification of the newly synthesized strand followed by multiplex DNA sequence analysis revealed that mutation fixation at 3,N4‐ethenocytosine (ɛC) was ≈3% when the DNA was replicated by normal cell extracts, ≈48% when replicated by mutA cell extracts and ≈3% when replicated by mutA recA double mutant cell extracts, in complete agreement with previous in vivo results. Mutagenesis at undamaged DNA sites was significantly elevated by mutA cell‐free extracts in the M13 lacZ(α) forward mutagenesis system. Neither polA (DNA polymerase I) nor polB (DNA polymerase II) genes are required for the mutA phenotype, suggesting that the phenotype is mediated through a modification of DNA polymerase III or the activation of a previously unidentified DNA polymerase. These findings define the major features of a novel mutagenic pathway and imply the existence of previously unrecognized links between translation, recombination and replication.
Formation and transposition of the covalently closed IS30 circle: the relation between tandem dimers and monomeric circles
Authors: Kiss János , Olasz Ferenc
Published: March 2003

In the present study, we demonstrate that a circular IS30 element acts as an intermediate for simple insertion. Covalently closed IS and Tn circles constructed in vitro are suitable for integration into the host genome. Minicircle integration displays all the characteristics of transpositional fusion mediated by the (IS30 )2 dimer regarding target selection and target duplication. Evidence is provided for in vivo circularization of the element located either on plasmids or on the genome. It is shown that circle formation can occur through alternative pathways. One of them is excision of IS30 from a hot spot via joining the IRs. This reaction resembles the site‐specific dimerization that leads to (IS30 )2 establishment. The other process is the dissolution of (IS30 )2 dimer, when the element is excised from an IR–IR joint. These pathways differ basically in the fate of the donor replicon: only dimer dissolution gives rise to resealed donor backbone. Analysis of minicircles and the rearranged donor replicons led us to propose a molecular model that can account for differences between the circle‐generating processes. Our focus was to the dissolution of IR–IR joints located on the host genome, because these events promoted extensive genomic rearrangements and accompanied minicircle formation. The results present the possibility of host genome reorganization by IS30‐like transposition.