bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel mechanism for control of antigenic variation in the haemagglutinin gene family of Mycoplasma synoviae
Authors: Noormohammadi A. H. , Markham P. F. , Kanci A. , Whithear K. G. , Browning G. F.
Published: March 2003

High‐frequency phase and antigenic variation of homologous lipoprotein haemagglutinins has been seen in both the major avian mycoplasma pathogens, Mycoplasma synoviae and Mycoplasma gallisepticum. The expression and, hence, antigenic variation of the pMGA gene family (encoding these lipoproteins in M. gallisepticum) is controlled by variation in the length of a trinucleotide repeat motif 5′ to the promoter of each gene. However, such a mechanism was not detected in preliminary observations on M. synoviae. Thus, the basis for control of variation in the vlhA gene family (which encodes the homologous haemagglutinin in M. synoviae) was investigated to enable comparison with its homologue in M. gallisepticum and with other lipoprotein gene families in mycoplasmas. The start point of transcription was identified 119 bp upstream of the initiation codon, but features associated with control of transcription in other mycoplasma lipoprotein genes were not seen. Comparison of three copies of vlhA revealed considerable sequence divergence at the 3′ end of the gene, but conservation of the 5′ end. Southern blot analysis of M. synoviae genomic DNA revealed that the promoter region and part of the conserved 5′ coding sequence occurred as a single copy, whereas the remainder of the coding sequence occurred as multiple copies. A 9.7 kb fragment of the genome was found to contain eight tandemly repeated regions partially homologous to vlhA, all lacking the putative promoter region and the single‐copy 5′ end of vlhA, but extending over one of four distinct overlapping regions of the 3′ coding sequence. Examination of sequential clones of M. synoviae established that unidirectional recombination occurs between the pseudogenes and the expressed vlhA, with duplication of pseudogene sequence and loss of the corresponding region previously seen in the expressed gene. Expression of the 5′ end of two variants of the vlhA gene showed that they differed in their reaction with monoclonal antibodies specific for this region. These data suggest that the control of vlhA antigenic variation in M. synoviae is achieved by multiple gene conversion events using a repertoire of coding sequences to generate a chimeric expressed gene, with the greatest potential for variation generated in the region encoding the haemagglutinin. Thus, completely distinct mechanisms have been adopted to control antigenic variation in homologous gene families.
Transfer of electrons across the cytoplasmic membrane by DsbD, a membrane protein involved in thiol–disulphide exchange and protein folding in the bacterial periplasm
Authors: Chung Jenny , Chen Thomas , Missiakas Dominique
Published: March 2003

Reduction of non‐native protein disulphides in the periplasm of Escherichia coli is catalysed by three enzymes, DsbC, DsbG and DsbE, each of which harbours a catalytic Cys–X–X–Cys dithiol motif. This dithiol motif requires continuous reduction for activity. Genetic evidence suggests that the source of periplasmic reducing power resides within the cytoplasm, provided by thioredoxin (trxA) and thioredoxin reductase (trxB). Cytoplasmic electrons donated by thioredoxin are thought to be transferred into the periplasm via the DsbD membrane protein. To understand the molecular nature of electron transfer, we have analysed the membrane topology of DsbD. DsbD is exported by an N‐terminal signal peptide. The N‐ and C‐terminal domains are positioned in the periplasmic space, connected by eight transmembrane segments. Electron transfer was shown to require five cysteine sulphydryl of DsbD. Trans complementation of mutant DsbD molecules revealed intermolecular electron transfer. We discuss a model whereby the membrane‐embedded disulphides of DsbD accept electrons from cytoplasmic thioredoxin and transfer them to the C‐terminal periplasmic dithiol motif of DsbD.
Repair of double‐strand breaks by incorporation of a molecule of homologous DNA
Authors: Lai Ying‐Ta , Masker Warren
Published: March 2003

An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to monitor repair of double‐strand breaks in the T7 genome. The efficiency of double‐strand break repair was markedly increased by DNA molecules (‘donor’ DNA) consisting of a 2.1 kb DNA fragment, generated by PCR, that had ends extending ≈ 1 kb on either side of the break site. Repair proceeded with greater than 10% efficiency even when T7 DNA replication was inhibited. When the donor DNA molecules were labelled with 32P, repaired genomes incorporated label only near the site of the double‐strand break. When repair was carried out with unlabelled donor DNA and [32P]‐dCTP provided as precursor for DNA synthesis the small amount of incorporated label was distributed randomly throughout the entire T7 genome. Repair was performed using donor DNA that had adjacent BamHI and PstI sites. When the BamHI site was methylated and the PstI site was left unmethylated, the repaired genomes were sensitive to PstI but not to BamHI endonuclease, showing that the methyl groups at the BamHI recognition site had not been replaced by new DNA synthesis during repair of the double‐strand break. These observations are most consistent with a model for double‐strand break repair in which the break is widened to a small gap, which is subsequently repaired by physical incorporation of a patch of donor DNA into the gap.
Biosynthesis of carbapenem antibiotic and prodigiosin pigment in Serratia is under quorum sensing control
Authors: Thomson N. R. , Crow M. A. , McGowan S. J. , Cox A. , Salmond G. P. C.
Published: March 2003

Serratia sp. ATCC 39006 produces the carbapenem antibiotic, carbapen‐2‐em‐3‐carboxylic acid and the red pigment, prodigiosin. We have previously reported the characterization of a gene, carR, controlling production of carbapenem in this strain. We now describe further characterization of the carR locus to locate the genes encoding carbapenem biosynthetic and resistance functions. A novel family of diverse proteins showing sequence similarity to the C‐terminal domain of CarF (required for carbapenem resistance) is described. We also report the isolation of the locus involved in the biosynthesis of the red pigment, prodigiosin. A cosmid containing ≈ 35 kb of the Serratia chromosome encodes synthesis of the pigment in the heterologous host, Erwinia carotovora, demonstrating, for the first time, that the complete prodigiosin biosynthetic gene cluster had been cloned and functionally expressed. We report the isolation of a third locus in Serratia, containing convergently transcribed genes, smaI and smaR, encoding LuxI and LuxR homologues respectively. SmaI directs the synthesis of N‐acyl homoserine lactones involved in the quorum sensing process. We demonstrate that biosynthesis of the two secondary metabolites, carbapenem antibiotic and prodigiosin pigment, is under pheromone‐mediated transcriptional regulation in this bacterium. Finally, we describe a new prodigiosin‐based bioassay for detection of some N‐acyl homoserine lactones.
Identification of alanine dehydrogenase and its role in mixed secretion of ammonium and alanine by pea bacteroids
Authors: Allaway D. , Lodwig E. M. , Crompton L. A. , Wood M. , Parsons R. , Wheeler T. R. , Poole P. S.
Published: March 2003

N2‐fixation by Rhizobium‐legume symbionts is of major ecological and agricultural importance, responsible for producing a substantial fraction of the biosphere's nitrogen. On the basis of 15N‐labelling studies, it had been generally accepted that ammonium is the sole secretion product of N2‐fixation by the bacteroid and that the plant is responsible for assimilating it into amino acids. However, this paradigm has been challenged in a recent 15N‐labelling study showing that soybean bacteroids only secrete alanine. Hitherto, nitrogen secretion has only been assessed from in vitro15N‐labelling studies of isolated bacteroids. We show that both ammonium and alanine are secreted by pea bacteroids. The in vitro partitioning between them will depend on whether the system is open or closed, as well as the ammonium concentration and bacteroid density. To overcome these limitations we identified and mutated the gene for alanine dehydrogenase (aldA) and demonstrate that AldA is the primary route for alanine synthesis in isolated bacteroids. Bacteroids of the aldA mutant fix nitrogen but only secrete ammonium at a significant rate, resulting in lower total nitrogen secretion. Peas inoculated with the aldA mutant are green and healthy, demonstrating that ammonium secretion by bacteroids can provide sufficient nitrogen for plant growth. However, plants inoculated with the mutant are reduced in biomass compared with those inoculated with the wild type. The labelling and plant growth studies suggest that alanine synthesis and secretion contributes to the efficiency of N2‐fixation and therefore biomass accumulation.
PriA and phage T4 gp59: factors that promote DNA replication on forked DNA substrates
Authors: Jones Jessica M. , Nakai Hiroshi
Published: March 2003

The initiation of DNA synthesis on forked DNA templates is a vital process in the replication and maintenance of cellular chromosomes. Two proteins that promote replisome assembly on DNA forks have so far been identified. In phage T4 development the gene 59 protein (gp59) assembles replisomes at D‐loops, the sites of homologous strand exchange. Bacterial PriA protein plays an analogous function, most probably restarting replication after replication fork arrest with the aid of homologous recombination proteins, and PriA is also required for phage Mu replication by transposition. Gp59 and PriA exhibit similar DNA fork binding activities, but PriA also has a 3′ to 5′ helicase activity that can promote duplex opening for replisome assembly. The helicase activity allows PriA’s repertoire of templates to be more diverse than that of gp59. It may give PriA the versatility to restart DNA replication without recombination on arrested replication forks that lack appropriate duplex openings.
S‐adenosylmethionine decarboxylase of Bacillus subtilis is closely related to archaebacterial counterparts
Authors: Sekowska Agnieszka , Coppée Jean‐Yves , Le Caer Jean‐Pierre , Martin‐Verstraete Isabelle , Danchin Antoine
Published: March 2003

Bacillus subtilis synthesizes polyamines by decarboxylating arginine to agmatine, which is subsequently hydrolysed to putrescine. Spermidine is synthesized from putrescine and decarboxylated S‐adenosylmethionine (dAdoMet). In Gram‐negative bacteria and in eukaryotes, AdoMet is decarboxylated by an unusual ‘pyruvoyl’ AdoMet decarboxylase (SpeD), the catalytic pyruvoyl moiety of which is generated by serinolysis of an internal serine with self‐cleavage of the protein at the upstream peptide bond. Neither the Gram‐positive bacterial nor the archaeal counterpart of the Escherichia coli SpeD enzyme were known. We have identified the corresponding B. subtilis speD gene (formely ytcF). Heterologous expression of the cognate Methanococcus jannaschii protein, MJ0315, demonstrated that it displays the same activity as B. subtilis SpeD, indicating that spermidine biosynthesis in Gram‐positive bacteria and in archaea follows a pathway very similar to that of Gram‐negatives and eukarya. In B. subtilis, transcription of speD is modulated by spermidine and methionine. Its expression is high under usual growth conditions. In contrast, the SpeD protein self‐cleaves slowly in vitro, a noticeable difference with its archaeal counterpart. Under certain growth conditions (minimal medium containing succinate and glutamate as a carbon source), speD is co‐transcribed with gapB, the gene encoding glyceraldehyde‐3‐phosphate dehydrogenase, an enzyme required for gluconeogenesis. This observation may couple polyamine metabolism to sulphur and carbon metabolism by a so far unknown mechanism.
Identification of SopE2 from Salmonella typhimurium, a conserved guanine nucleotide exchange factor for Cdc42 of the host cell
Authors: Stender Silke , Friebel Andrea , Linder Stefan , Rohde Manfred , Mirold Susanne , Hardt Wolf‐Dietrich
Published: March 2003

Salmonella typhimurium translocates effector proteins into host cells via the SPI1 type III secretion system to induce responses such as membrane ruffling and internalization by non‐phagocytic cells. Activation of the host cellular RhoGTPase Cdc42 is thought to be a key event during internalization. The translocated Salmonella protein SopE is an activator for Cdc42. Because SopE is absent from most S. typhimurium strains it remains unclear whether all S. typhimurium strains rely on activation of Cdc42 to invade host cells. We have identified SopE2, a translocated effector protein common to all S. typhimurium strains. SopE2 is a guanine nucleotide exchange factor for Cdc42 and shows 69% sequence similarity to SopE. Analysis of S. typhimurium mutants demonstrated that SopE2 plays a role in recruitment of the actin‐nucleating Arp2/3 complex to the membrane ruffles and in efficient host cell invasion. Transfection experiments showed that SopE2 is sufficient to activate host cellular Cdc42, to recruit the actin‐nucleating Arp2/3 complex and to induce actin cytoskeletal rearrangements and internalization. In conclusion, as a result of SopE2 all S. typhimurium strains tested have the capacity to activate Cdc42 signalling inside host cells which is important to ensure efficient entry.
A chromosomally encoded type III secretion pathway in Yersinia enterocolitica is important in virulence
Authors: Haller Jon C. , Carlson Sharon , Pederson Kristin J. , Pierson Dorothy E.
Published: March 2003

Numerous Gram‐negative bacteria use a type III, or contact dependent, secretion system to deliver proteins into the cytosol of host cells. All of these systems identified to date have been shown to have a role in pathogenesis. We have identified 13 genes on the Yersinia enterocolitica chromosome that encode a type III secretion apparatus plus two associated putative regulatory genes. In order to determine the function of this chromosomally‐encoded secretion apparatus, we created an in frame deletion of a gene that has homology to the hypothesized inner membrane pore, ysaV. The ysaV mutant strain failed to secrete eight proteins, called Ysps, normally secreted by the parental strain when grown at 28°C in Luria–Bertani (LB) broth supplemented with 0.4 M NaCl. Disruption of the ysaV gene had no effect on motility or phospholipase activity, suggesting this chromosomally encoded type III secretion pathway is distinct from the flagella secretion pathway of Y. enterocolitica. Deletion of the ysaV gene in a virulence plasmid positive strain had no effect on in vitro secretion of Yops by the plasmid‐encoded type III secretion apparatus. Secretion of the Ysps was unaffected by the presence or absence of the virulence plasmid, suggesting the chromosomally encoded and plasmid‐encoded type III secretion pathways act independently. Y. enterocolitica thus has three type III secretion pathways that appear to act independently. The ysaV mutant strain was somewhat attenuated in virulence compared with the wild type in the mouse oral model of infection (an approximately 0.9 log difference in LD50). The ysaV mutant strain was nearly as virulent as the wild type when inoculated intraperitoneally in the mouse model. A ysaV probe hybridized to sequences in other Yersinia spp. and homologues were found in the incomplete Y. pestis genome sequence, indicating a possible role for this system throughout the genus.
Transcriptional induction of the conserved alternative sigma factor RpoS in Escherichia coli is dependent on BarA, a probable two‐component regulator
Authors: Mukhopadhyay Suman , Audia Jonathon P. , Roy Rabindra N. , Schellhorn Herb E.
Published: March 2003

The stationary phase expression of many conserved, adaptive bacterial proteins is dependent on RpoS, a second vegetative sigma factor. The regulation of RpoS itself, however, is complex and not fully understood, particularly at the level of transcription. In this report, we show that the observed hydrogen peroxide sensitivity of a mutant defective in expression of barA, a bacterial virulence factor, can be explained by a reduction in catalase activity, an RpoS‐controlled function. Levels of katE mRNA, encoding the major catalase of Escherichia coli, were much lower in the barA mutant, suggesting that BarA is required for the expression of this RpoS‐regulated gene. Expression of another RpoS‐regulated gene, osmY, was also found to be severely reduced in the barA mutant. Employing Western analyses with anti‐RpoS antisera and Northern analyses using probes specific for rpoS, we found that BarA is required for the exponential phase induction of RpoS itself. Operon lacZ fusion expression studies and Northern analyses indicate that BarA itself is maximally expressed in early exponential phase cultures immediately preceding the transcriptional induction of RpoS. Results of primer extension studies indicate that exponential phase expression from the rpoSp1 promoter is reduced by more than 85% in a barA mutant but could be efficiently complemented by a plasmid‐borne copy of barA in trans. These results suggest that regulatory signals that are operant in exponentially growing cultures play an important role in effecting stationary phase gene expression.
The role of lateral gene transfer in the evolution of isoprenoid biosynthesis pathways
Authors: Boucher Yan , Doolittle W. Ford
Published: March 2003

Lateral gene transfer (LGT) is a major force in microbial genome evolution. Here, we present an overview of lateral transfers affecting genes involved in isopentenyl diphosphate (IPP) synthesis. Two alternative metabolic pathways can synthesize this universal precursor of isoprenoids, the 1‐deoxy‐d‐xylulose 5‐phosphate (DOXP) pathway and the mevalonate (MVA) pathway. We have surveyed recent genomic data and the biochemical literature to determine the distribution of the genes composing these pathways within the bacterial domain. The scattered distribution observed is incompatible with a simple scheme of vertical transmission. LGT (among and between bacteria, archaea and eukaryotes) more parsimoniously explains many features of this pattern. This alternative scenario is supported by phylogenetic analyses, which unambiguously confirm several cases of lateral transfer. Available biochemical data allow the formulation of hypotheses about selective pressures favouring transfer. The phylogenetic diversity of the organisms involved and the range of possible causes and effects of these transfer events make the IPP biosynthetic pathways an ideal system for studying the evolutionary role of LGT.
Acid activation of Helicobacter pylori vacuolating cytotoxin (VacA) results in toxin internalization by eukaryotic cells
Authors: McClain Mark S. , Schraw Wayne , Ricci Vittorio , Boquet Patrice , Cover Timothy L.
Published: March 2003

Helicobacter pylori VacA is a secreted toxin that induces multiple structural and functional alterations in eukaryotic cells. Exposure of VacA to either acidic or alkaline pH (‘activation’) results in structural changes in the protein and a marked enhancement of its cell‐vacuolating activity. However, the mechanism by which activation leads to increased cytotoxicity is not well understood. In this study, we analysed the binding and internalization of [125I]‐VacA by HeLa cells. We detected no difference in the binding of untreated and activated [125I]‐VacA to cells. Binding of acid‐activated [125I]‐VacA to cells at 4°C was not saturable, and was only partially inhibited by excess unlabelled toxin. These results suggest that VacA binds either non‐specifically or to an abundant, low‐affinity receptor on HeLa cells. To study internalization of VacA, we used a protease protection assay. Analysis by SDS–PAGE and autoradiography indicated that the intact 87 kDa toxin was internalized in a time‐dependent process at 37°C but not at 4°C. Furthermore, internalization of the intact toxin was detected only if VacA was acid or alkaline activated before being added to cells. The internalization of activated [125I]‐VacA was not substantially inhibited by the presence of excess unlabelled toxin, but was blocked if cells were depleted of cellular ATP by the addition of sodium azide and 2‐deoxy‐d‐glucose. These results indicate that acid or alkaline pH‐induced structural changes in VacA are required for VacA entry into cells, and that internalization of the intact 87 kDa toxin is required for VacA cytotoxicity.
Activation and silencing of leu‐500 promoter by transcription‐induced DNA supercoiling in the Salmonella chromosome
Authors: Hanafi Driss El , Bossi Lionello
Published: March 2003

The notion that transcription can generate supercoils in the DNA template largely stems from work with small circular plasmids. In the present work, we tested this model in the bacterial chromosome using a supercoiling‐sensitive promoter as a functional sensor of superhelicity changes. The leu‐500 promoter of Salmonella typhimurium is a mutant and inactive variant of the leucine operon promoter that regains activity if negative DNA supercoiling rises above normal levels, typically as a result of mutations affecting DNA topoisomerase I (topA mutants). Activation of the leu‐500 promoter was analysed in topA mutant cells harbouring transcriptionally inducible tet or cat gene cassettes inserted in the region upstream from the leu operon. Some insertions inhibited leu‐500 promoter activation in the absence of inducer. This effect is dramatic in the interval between 1.7 kb and 0.6 kb from the leu operon, suggesting that the insertions physically interfere with the mechanism responsible for activation. Superimposed on these effects, transcription of the inserted gene stimulated or inhibited leu‐500 promoter activity depending on whether this gene was oriented divergently from the leu operon or in the same direction respectively. Interestingly, transcription‐mediated inhibition of leu‐500 promoter was observed with inserts as far as 5 kb from the leu operon, and it could be relieved by the introduction of a strong gyrase site between the inserted element and the leu‐500 promoter. These results are consistent with the idea that transcriptionally generated positive and negative supercoils can diffuse along chromosomal DNA and, depending on their topological sign, elicit opposite responses from the leu‐500 promoter.
Inhibition of glutamine synthetase II expression by the product of the gstI gene
Authors: Spinosa M. , Riccio A. , Mandrich L. , Manco G. , Lamberti A. , Iaccarino M. , Merrick M. , Patriarca E. J.
Published: March 2003

We report the identification of a previously unrecognized gene that is involved in the regulation of the Rhizobium leguminosarum glnII (glutamine synthetase II) gene. This gene, which is situated immediately upstream of glnII, was identified by means of a deletion/complementation analysis performed in the heterologous background of Klebsiella pneumoniae. It has been designated gstI (glutamine synthetase translational Inhibitor) because, when a complete version of gstI is present, it is possible to detect glnII‐specific mRNA, but neither GSII activity nor GSII protein. The gstI gene encodes a small (63 amino acids) protein, which acts in cis or in trans with respect to glnII and is transcribed divergently with respect to glnII from a promoter that was found to be strongly repressed by the nitrogen transcriptional regulator NtrC. A mutated version of GstI lacking the last 14 amino acids completely lost its capacity to repress glnII expression. Our results indicate that gstI mediates the translation inhibition of glnII mRNA and, based on in silico analyses, a mechanism for GstI action is proposed.
The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is required for antiphagocytic function and virulence
Authors: Black Deborah S. , Bliska James B.
Published: March 2003

A variety of pathogenic bacteria use type III secretion pathways to translocate virulence proteins into host eukaryotic cells. YopE is an important virulence factor that is translocated into mammalian cells via a plasmid‐encoded type III system in Yersinia spp. YopE action in mammalian cells promotes the disruption of actin filaments, cell rounding and blockage of phagocytosis. It was reported recently that two proteins with sequence similarity to YopE, SptP of Salmonella typhimurium and ExoS of Pseudomonas aeruginosa, function as GTPase‐activating proteins (GAPs) for Rho GTPases. YopE contains an ‘arginine finger’ motif that is present in SptP, ExoS and other Rho GAPs and is essential for catalysis by this class of proteins. We show here that a GST–YopE fusion protein stimulated in vitro GTP hydrolysis by the Rho family members Cdc42, RhoA and Rac1, but not by Ras. Conversion of the essential arginine in the arginine finger motif to alanine (R144A) eliminated the in vitro GAP activity of GST–YopE. Infection assays carried out with a Yersinia pseudotuberculosis strain producing YopER144A demonstrated that GAP function was essential for the disruption of actin filaments, cell rounding and inhibition of phagocytosis by YopE in HeLa cells. Furthermore, the GAP function of YopE was important for Y. pseudotuberculosis pathogenesis in a mouse infection assay. Transfection of HeLa cells with a vector that produces a constitutively active form of RhoA (RhoA‐V14) prevented the disruption of actin filaments and cell rounding by YopE. Production of an activated form of Rac1 (Rac1‐V12), but not RhoA‐V14, in HeLa cells interfered with YopE antiphagocytic activity. These results demonstrate that YopE functions as a RhoGAP to downregulate multiple Rho GTPases, leading to the disruption of actin filaments and inhibition of bacterial uptake into host cells.
A single amino acid substitution in region 1.2 of the principal σ factor of Streptomyces coelicolor A3(2) results in pleiotropic loss of antibiotic production
Authors: Aigle Bertrand , Wietzorrek Andreas , Takano Eriko , Bibb Mervyn J.
Published: March 2003

Antibiotic production in streptomycetes generally occurs in a growth phase‐dependent and developmentally co‐ordinated manner, and is subject to pathway‐specific and pleiotropic control. Streptomyces coelicolor A3(2) produces at least four chemically distinct antibiotics, including actinorhodin (Act) and undecylprodigiosin (Red). afsB mutants of S. coelicolor are deficient in the production of both compounds and in the synthesis of a diffusible γ‐butyrolactone, SCB1, that can elicit precocious Act and Red production. Clones encoding the principal and essential σ factor (σHrdB) of S. coelicolor restored Act and Red production in the afsB mutant BH5. A highly conserved glycine (G) at position 243 of σHrdB was shown to be replaced by aspartate (D) in BH5. Replacement of G243 by D in the afsB+ strain M145 reproduced the afsB phenotype. The antibiotic deficiency correlated with reduced transcription of actII‐ORF4 and redD, pathway‐specific regulatory genes for Act and Red production respectively. Exogenous addition of SCB1 to the G‐243D mutants failed to restore Act and Red synthesis, indicating that loss of antibiotic production was not a result of the deficiency in SCB1 synthesis. The G‐243D substitution, which lies in the highly conserved 1.2 region of undefined function, had no effect on growth rate or morphological differentiation, and appears specifically to affect antibiotic production.
The N‐terminal prepeptide is required for the production of spore cortex‐lytic enzyme from its inactive precursor during germination of Clostridium perfringens S40 spores
Authors: Okamura S. , Urakami K. , Kimata M. , Aoshima T. , Shimamoto S. , Moriyama R. , Makino S.
Published: March 2003

A spore cortex‐lytic enzyme of Clostridium perfringens S40 is synthesized during sporulation as a precursor consisting of four domains. After cleavage of an N‐terminal preregion and a C‐terminal proregion, inactive proenzyme (termed C35) is converted to active enzyme by processing of an N‐terminal prosequence with germination‐specific protease (GSP) during germination. The present results demonstrated that the cleaved N‐terminal prepeptide remained associated with C35. After the isolated complex was denatured and dissociated in 6 M urea solution, removal of urea regenerated a prepeptide–C35 complex which produces active enzyme when incubated with GSP. However, isolated C35 alone could not be activated by GSP. The prepeptide–C35 complex was more heat stable than active enzyme. Thus, non‐covalent attachment of the prepeptide to C35 is required to assist correct folding of C35 and to stabilize its conformation, suggesting that the prepeptide functions as an intramolecular chaperone. Recombinant proteins, which have prepeptide covalently bonded to C35, were processed by GSP as well as the in vivo prepeptide–C35 complex, and the full length of the N‐terminal presequence was needed to fulfil its role. Although the C‐terminal prosequence is present as an independent domain which is not involved in the activation process of the enzyme, it appears that the N‐terminal prosequence contributes to the regulation of enzyme activity as an inhibitor of the enzyme.
Analysis of interactions between Activating Region 1 of Escherichia coli FNR protein and the C‐terminal domain of the RNA polymerase α subunit: use of alanine scanning and suppression genetics
Authors: Lee David J. , Wing Helen J. , Savery Nigel J. , Busby Stephen J. W.
Published: March 2003

Activating Region 1 of Escherichia coli FNR protein is proposed to interact directly with the C‐terminal domain of the RNA polymerase α subunit (αCTD) during transcription activation at FNR‐regulated promoters. Using an αCTD alanine scan mutant library, we have identified the residues of αCTD that are important for FNR‐dependent transcription activation. Residues Asp‐305, Gly‐315, Arg‐317, Leu‐318 and Asp‐319 are proposed to be the key residues in the contact site on αCTD for Activating Region 1 of FNR. In previous work, it had been shown that Activating Region 1 of FNR is a large surface‐exposed patch and that the two crucial amino acid residues are Thr‐118 and Ser‐187. In this work, we have constructed Arg‐118 FNR and Arg‐187 FNR and shown that both FNR derivatives are defective in transcription activation. However, the activity of FNR carrying Arg‐118 can be partially restored by substitutions of Lys‐304 in αCTD. Similarly, the activity of FNR carrying Arg‐187 can be partially restored by substitutions of Arg‐317 or Leu‐318 in αCTD. The specificity of the restoration suggests that, during transcription activation by FNR, the side‐chain of residue 118 in Activating Region 1 of FNR is located close to Lys‐304 and Asp‐305 in αCTD. Similarly, the side‐chain of residue 187 in Activating Region 1 of FNR is located close to Arg‐317 and Leu‐318 in αCTD. These results can be used to model the interface between Activating Region 1 of FNR and its contact target in αCTD, and permit comparison of this interface with the interface between Activating Region 1 of the related transcription activator, CRP and αCTD.
Excision from tRNA genes of a large chromosomal region, carrying avrPphB, associated with race change in the bean pathogen, Pseudomonas syringae pv. phaseolicola
Authors: Jackson Robert W. , Mansfield John W. , Arnold Dawn L. , Sesma Ane , Paynter Christopher D. , Murillo Jesús , Taylor John D. , Vivian Alan
Published: March 2003

Pseudomonas syringae pv. phaseolicola (Pph) race 4 strain 1302A carries avirulence gene avrPphB. Strain RJ3, a sectoral variant from a 1302A culture, exhibited an extended host range in cultivars of bean and soybean resulting from the absence of avrPphB from the RJ3 chromosome. Complementation of RJ3 with avrPphB restored the race 4 phenotype. Both strains showed similar in planta growth in susceptible bean cultivars. Analysis of RJ3 indicated loss of > 40 kb of DNA surrounding avrPphB. Collinearity of the two genomes was determined for the left and right junctions of the deleted avrPphB region; the left junction is ≈ 19 kb and the right junction > 20 kb from avrPphB in 1302A. Sequencing revealed that the region containing avrPphB was inserted into a tRNALYS gene, which was re‐formed at the right junction in strain 1302A. A putative lysine tRNA pseudogene (ΨtRNALYS) was found at the left junction of the insertion. All tRNA genes were in identical orientation in the chromosome. Genes near the left junction exhibited predicted protein homologies with gene products associated with a virulence locus of the periodontal pathogen Actinobacillus actinomycetemcomitans. Specific oligonucleotide primers that differentiate 1302A from RJ3 were designed and used to demonstrate that avrPphB was located in different regions of the chromosome in other strains of Pph. Deletion of a large region of the chromosome containing an avirulence gene represents a new route to race change in Pph.
Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by Ler
Authors: Sperandio Vanessa , Mellies Jay L. , Delahay Robin M. , Frankel Gad , Crawford J. Adam , Nguyen William , Kaper James B.
Published: March 2003

Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing lesions (AE) on epithelial cells. The genes involved in the formation of the AE lesions are contained within a pathogenicity island named the locus of enterocyte effacement (LEE). The LEE comprises 41 open reading frames organized in five major operons: LEE1, LEE2, LEE3, LEE4 and tir. The first gene of the LEE1 operon encodes a transcription activator of the other LEE operons that is called the LEE‐encoded regulator (Ler). The LEE2 and LEE3 operons are divergently transcribed with overlapping −10 promoter regions, and gene fusion studies have shown that they are both activated by Ler. Deletion analysis, using lacZ reporter fusions, of the LEE2 and LEE3 promoters demonstrated that deletions extending closer to the LEE2 transcription start site than −247 bp lead to loss of activation by Ler, whereas only 70 bp upstream of the LEE3 transcription start site is required for Ler‐mediated activation. We have purified Ler as a His‐tagged protein and used it to perform DNA‐binding assays with LEE2 and LEE3. We observed that Ler bound to a DNA fragment containing the −300 to +1 region of LEE2; however, it failed to bind to a DNA fragment containing the −300 to +1 region of LEE3, suggesting that Ler activates both operons by only binding to the regulatory region upstream of LEE2. The Ler‐activatable LEE3::lacZ fusions extended to what would be −246 bp of the LEE2 operon. A lacZ fusion from the −300 to +1 region of LEE3 failed to be activated by Ler, consistent with our hypothesis that Ler activates the expression of LEE2 and LEE3 by binding to a region located downstream of the LEE3 transcription start site. DNase I footprinting revealed that Ler protected a region of 121 bp upstream of LEE2. Purified Ler mutated in the coiled‐coil domain was unable to activate transcription and to bind to the LEE2 regulatory region. These data indicate that Ler may bind as a multimer to LEE2 and activate both divergent operons by a novel mechanism potentially involving changes in the DNA structure.
New prospects in studying the bacterial signal recognition particle pathway
Authors: Herskovits Anat A. , Bochkareva Elena S. , Bibi Eitan
Published: March 2003

In vivo and in vitro studies have suggested that the bacterial version of the mammalian signal recognition particle (SRP) system plays an essential and selective role in protein biogenesis. The bacterial SRP system consists of at least two proteins and an RNA molecule (termed Ffh, FtsY and 4.5S RNA, respectively, in Escherichia coli). Recent evidence suggests that other putative bacterial‐specific SRP components may also exist. In vitro experiments confirmed the expected basic features of the bacterial SRP system by demonstrating interactions among the SRP components themselves, between them and ribosomes, ribosome‐linked hydrophobic nascent polypeptides or inner membranes. The availability of a conserved (and essential) bacterial SRP version has facilitated the implementation of powerful genetic and biochemical approaches for studying the cascade of events during the SRP‐mediated targeting process in vivo and in vitro as well as the three‐dimensional structures and the properties of each SRP component and complex.
Group A streptococcal growth phase‐associated virulence factor regulation by a novel operon (Fas) with homologies to two‐component‐type regulators requires a small RNA molecule
Authors: Kreikemeyer Bernd , Boyle Michael D. P. , Buttaro Bettina A. (Leonard) , Heinemann Markus , Podbielski Andreas
Published: March 2003

A novel growth phase‐associated two‐component‐type regulator, Fas (fibronectin/fibrinogen binding/haemolytic activity/streptokinase regulator), of Streptococcus pyogenes was identified in the M1 genome sequence, based on homologies to the histidine protein kinase (HPK) and response regulator (RR) part of the Staphylococcus aureus Agr and Streptococcus pneumoniae Com quorum‐sensing systems. The fas operon, present in all 12 tested M serotypes, was transcribed as polycystronic message (fasBCA) and contained genes encoding two potential HPKs (FasB and FasC) and one RR (FasA). Downstream of fasBCA, we identified a small 300 nucleotide monocistronic transcript, designated fasX, that did not appear to encode true peptide sequences. Measurements of luciferase promoter fusions revealed a growth phase‐associated transcription of fasBCA and fasX, with peak activities during the late exponential phase. Insertional mutagenesis disrupting fasBCA and fasA led to a phenotype similar to agr‐null mutations in S. aureus, with prolonged expression of extracellular matrix protein‐binding adhesins and reduced expression of secreted virulence factors such as streptokinase and streptolysin S. In addition, fasX transcription was dependent on the RR FasA; however, deletion mutagenesis of fasX resulted in a similar phenotype to that of the fasBCA or fasA mutants. Complementation of the fasX deletion mutant, with the fasX gene expressed in trans from a plasmid, restored the wild‐type fasBCA regulation pattern. This strongly suggested that fasX, a putative non‐translated RNA, is the main effector molecule of the fas regulon. However, using spent culture supernatants from wild‐type and fas mutant strains, we were not able to show an influence on the logarithmic growth phase expression of fas and dependent genes. Thus, despite structural and functional similarities between fas and agr, to date the fas operon appears not to be involved in group A streptococcal (GAS) quorum‐sensing regulation.
Comparative phage genomics and the evolution of Siphoviridae: insights from dairy phages
Authors: Brüssow Harald , Desiere Frank
Published: March 2003

Comparative phage genomics can retrace part of the evolutionary history of phage modules encoding phage‐specific functions such as capsid building or establishment of the lysogenic state. The diagnosis of relatedness is not based exclusively on sequence similarity, but includes topological considerations of genome organization. The gene maps from the λ‐, ψM2‐, L5‐, Sfi21‐, Sfi11‐, φC31‐, sk1‐ and TM4‐like phages showed a remarkable synteny of their structural genes defining a λ supergroup within Siphoviridae (Caudovirales with long non‐contractile tails). A hierarchy of relatedness within the λ supergroup suggested elements of vertical evolution in the capsid module of Siphoviridae. Links to P22‐like Podoviridae and P2‐like Myoviridae were also detected. Numerous cases of horizontal gene transfer were observed, but recent transfers were limited to interbreeding phage populations. We suggest that tailed phages are the result of both vertical and horizontal evolution and are thus a good model system for web‐like phylogenies.
Emerging strategies in microbial haem capture
Authors: Genco Caroline Attardo , Dixon Dabney White
Published: March 2003

Gram‐negative pathogenic bacteria have evolved novel strategies to obtain iron from host haem‐sequestering proteins. These include the production of specific outer membrane receptors that bind directly to host haem‐sequestering proteins, secreted haem‐binding proteins (haemophores) that bind haem/haemoglobin/haemopexin and deliver the complex to a bacterial cell surface receptor and bacterial proteases that degrade haem‐sequestering proteins. Once removed from haem‐sequestering proteins, haem may be transported via the bacterial outer membrane receptor into the cell. Recent studies have begun to define the steps by which haem is removed from bacterial haem proteins and transported into the cell. This review describes recent work on the discovery and characterization of these systems. Reference is also made to the transport of haem in serum (via haemoglobin, haemoglobin/haptoglobin, haemopexin, albumin and lipoproteins) and to mechanisms of iron removal from the haem itself (probably via a haem oxygenase pathway in which the protoporphyrin ring is degraded). Haem protein–receptor interactions are discussed in terms of the criteria that govern protein–protein interactions in general, and connections between haem transport and the emerging field of metal transport via metallochaperones are outlined.
Characterization and analysis of an NAD(P)H dehydrogenase transcriptional regulator critical for the survival of cyanobacteria facing inorganic carbon starvation and osmotic stress
Authors: Figge R. M. , Cassier‐Chauvat C. , Chauvat F. , Cerff R.
Published: March 2003

The three Synechocystis PCC6803 genes homologous to proteobacterial Calvin cycle regulators (cbbR) have been analysed. sll0998 appeared to be crucial to cell viability, whereas both sll0030 and sll1594 were found to be dispensable for cell growth. In spite of their sequence homology, Sll0030 and Sll1594 did not appear to regulate the transcription of Calvin cycle key genes. Further analysis of Sll1594 showed that this protein plays an important role in the adaptation to inorganic carbon starvation and osmotic stress. Sll1594 mediates the response to these stress conditions by regulating the transcription of a new regulon including the monocistronic genes sll1594 and slr1727 (encoding a presumptive Na+/H+ antiporter), as well as the ndh3 operon encoding the NAD(P)H‐dehydrogenase subunits F3 and D3 and a protein of unknown function. The sll1594 gene and the ndh3 operon are negatively controlled by Sll1594, which also regulates the expression of the slr1727 gene. Sequence alignment of the diverse Sll1594 DNA binding sites led us to propose the TCAATG‐(N10)‐ATCAAT sequence as the consensus motif. To our knowledge, this is the first report on the characterization and analysis of a transcriptional regulator for ndh genes in a photoautotrophic organism.
An increase in expression of a Mycobacterium tuberculosis mycolyl transferase gene (fbpB) occurs early after infection of human monocytes
Authors: Wilkinson Robert J. , DesJardin Lucy E. , Islam Najmul , Gibson Brandon M. , Kanost R. Andrew , Wilkinson Katalin A. , Poelman David , Eisenach Kathleen D. , Toossi Zahra
Published: March 2003

Changes in the mRNA levels of two Mycobacterium tuberculosis genes (fbpB known as antigen 85B, and hspX known as Acr) were studied in infected human monocytes. Antigen 85B is an enzyme involved in cell wall biosynthesis and is also a major target of the immune response. Acr is a stress protein believed to be involved in the bacillary response to adverse conditions and in non‐replicating persistence. During the first 24 h of intracellular infection, the intramonocyte 85B mRNA level increased 54‐fold (P = 0.00001) and 14.6 times in comparison with the 16S ribosomal rRNA. In contrast, the Acr mRNA fell 14.3 times. Although monocyte cytokine production was very variable, the 24 h secretion of tumour necrosis factor (TNF)‐α correlated with the 85B−16S RNA ratio at 24 h (r = 0.77, Pcorr < 0.01). Furthermore, the addition of exogenous TNF‐α to cultures was associated with a twofold increase in the 85B−16S ratio and, conversely, neutralization of endogenous TNF‐α reduced the ratio. As antigen 85B also induces TNF‐α, the positive feedback implied by our findings suggests a previously unsuspected role for this protein in the immunopathogenesis of tuberculosis.
The X philes: structure‐specific endonucleases that resolve Holliday junctions
Authors: Sharples Gary J.
Published: March 2003

Genetic recombination is a critical cellular process that promotes evolutionary diversity, facilitates DNA repair and underpins genome duplication. It entails the reciprocal exchange of single strands between homologous DNA duplexes to form a four‐way branched intermediate commonly referred to as the Holliday junction. DNA molecules interlinked in this way have to be separated in order to allow normal chromosome transmission at cell division. This resolution reaction is mediated by structure‐specific endonucleases that catalyse dual‐strand incision across the point of strand cross‐over. Holliday junctions can also arise at stalled replication forks by reversing the direction of fork progression and annealing of nascent strands. Resolution of junctions in this instance generates a DNA break and thus serves to initiate rather than terminate recombination. Junction resolvases are generally small, homodimeric endonucleases with a high specificity for branched DNA. They use a metal‐binding pocket to co‐ordinate an activated water molecule for phosphodiester bond hydrolysis. In addition, most junction endonucleases modulate the structure of the junction upon binding, and some display a preference for cleavage at specific nucleotide target sequences. Holliday junction resolvases with distinct properties have been characterized from bacteriophages (T4 endo VII, T7 endo I, RusA and Rap), Bacteria (RuvC), Archaea (Hjc and Hje), yeast (CCE1) and poxviruses (A22R). Recent studies have brought about a reappraisal of the origins of junction‐specific endonucleases with the discovery that RuvC, CCE1 and A22R share a common catalytic core.
Identification of the teichoic acid phosphorylcholine esterase in Streptococcus pneumoniae
Authors: Vollmer Waldemar , Tomasz Alexander
Published: March 2003

Streptococcus pneumoniae is a major human pathogen and many interactions of this bacterium with its host appear to be mediated, directly or indirectly, by components of the bacterial cell wall, specifically by the phosphorylcholine residues which serve as anchors for surface‐located choline‐binding proteins and are also recognized by components of the host response, such as the human C‐reactive protein, a class of myeloma proteins and PAF receptors. In the present study, we describe the identification of the pneumococcal pce gene encoding for a teichoic acid phosphorylcholine esterase (Pce), an enzymatic activity capable of removing phosphorylcholine residues from the cell wall teichoic acid and lipoteichoic acid. Pce carries an N‐terminal signal sequence, contains a C‐terminal choline‐binding domain with 10 homologous repeating units similar to those found in other pneumococcal surface proteins, and the catalytic (phosphorylcholine esterase) activity is localized on the N‐terminal part of the protein. The mature protein was overexpressed in Escherichia coli and purified in a one‐step procedure by choline‐affinity chromatography and the enzymatic activity was followed using the chromophoric p‐nitrophenyl‐phosphorylcholine as a model substrate. The product of the enzymatic digestion of 3H‐choline‐labelled cell walls was shown to be phosphorylcholine. Inactivation of the pce gene in S. pneumoniae strains by insertion‐duplication mutagenesis caused a unique change in colony morphology and a striking increase in virulence in the intraperitoneal mouse model. Pce may be a regulatory element involved with the interaction of S. pneumoniae with its human host.