bluuebunny/crossref_metadata_2025_split huggingface dataset:
Engineering specificity of starter unit selection by the erythromycin‐producing polyketide synthase
Authors: Long Paul F. , Wilkinson Christopher J. , Bisang Christian P. , Cortés Jesús , Dunster Nicholas , Oliynyk Marko , McCormick Ellen , McArthur Hamish , Mendez Carmen , Salas José A. , Staunton James , Leadlay Peter F. 
Published: March 2003

SummaryChain initiation on many modular polyketide synthases is mediated by acyl transfer from the CoA ester of a dicarboxylic acid, followed by decarboxylation in situ
by KSQ, a ketosynthase‐like decarboxylase domain. Consistent with this, the acyltransferase
(AT) domains of all KSQ‐containing loading modules are shown here to contain a key
arginine residue at their active site. Site‐specific replacement of this arginine
residue in the oleandomycin (ole) loading AT domain effectively abolished
AT activity, consistent with its importance for catalysis. Substitution of the ole
PKS loading module, or of the tylosin PKS loading module, for the erythromycin (ery)
loading module gave polyketide products almost wholly either acetate derived or propionate
derived, respectively, instead of the mixture found normally. An authentic extension
module AT domain, rap AT2 from the rapamycin PKS, functioned appropriately
when engineered in the place of the ole loading AT domain, and gave rise to
substantial amounts of C13‐methylerythromycins, as predicted. The role of direct
acylation of the ketosynthase domain of ex‐tension module 1 in chain initiation was
confirmed by demonstrating that a mutant of the triketide synthase DEBS1‐TE, in which
the 4′‐phosphopante‐theine attachment site for starter acyl groups was specifically removed, produced triketide lactone pro‐ducts in detectable amounts.
Transcription of multiple var genes by individual, trophozoite‐stage Plasmodium falciparum cells expressing a chondroitin sulphate A binding phenotype
Authors: Duffy Michael F. , Brown Graham V. , Basuki Wanny , Krejany Efrosinia O. , Noviyanti Rintis , Cowman Alan F. , Reeder John C. 
Published: March 2003

SummaryIn this study, we detected multiple var gene transcripts within single,
mature trophozoite‐infected red blood cells (iRBCs) bound to chondroitin sulphate
A (CSA). Several of the var detected had previously been demonstrated to encode
Plasmodium falciparum erythrocyte membrane protein‐1 (PfEMP‐1) variants with
domains that mediated iRBC adhesion to receptors other than CSA. Parasites expressing
the CSA‐adherent phenotype transcribed far more of one var than of all others,
but this gene was different from the two other var previously purported to
encode adhesion to CSA. Previous work suggesting that only single var are transcribed by mature trophozoites needs re‐examination in the light of these data from single, infected cells.
Two DNA‐binding domains of Mga are required for virulence gene activation in the group A streptococcus
Authors: McIver Kevin S. , Myles Rhonda L. 
Published: March 2003

SummaryMga is a DNA‐binding protein that activates expression of several important virulence genes in the group A streptococcus (GAS), including those encoding M protein (emm),

C5a peptidase (scpA) and Mga (mga). To determine the functionality

of four potential helix–turn–helix DNA‐binding motifs (HTH1–HTH4)

identified within the amino‐terminus of Mga, alanine substitutions were introduced

within each domain in a MBP–Mga fusion allele and purified proteins were assayed

for binding to Mga‐specific promoter fragments (Pmga, PscpA and Pemm)

in vitro. Although HTH‐1 and HTH‐2 mutations showed wild type DNA‐binding

activity, an altered HTH‐3 domain resulted in reduced binding to the three promoters

and an HTH‐4 mutant was devoid of detectable binding activity. Plasmid‐encoded expression

of the HTH‐3 and HTH‐4 alleles from a constitutive promoter (Pspac) in the

mga‐deleted GAS strain JRS519 demonstrated that Mga‐regulated emm expression

correlated directly to the DNA‐binding activity observed for each mutant protein

in vitro. Single‐copy expression of HTH‐3 and HTH‐4 from their native Pmga

resulted in a dramatic reduction in autoregulated mga expression in both mutant

strains. Thus, Mga appears to contain two DNA‐binding domains (HTH‐3 and HTH‐4) that

are required for direct activation of the Mga virulence regulon in vivo.
Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer
Authors: Ast Vivian M. , Schoenhofen Ian C. , Langen Geoffrey R. , Stratilo Chad W. , Chamberlain M. Dean , Howard S. Peter 
Published: March 2003

Summary Aeromonas hydrophila secretes protein toxins via the type II pathway, involving

the products of at least two operons, exeAB (gspAB) and exeC‐N

(gspC‐N). In the studies reported here, aerolysin secretion was restored to

C5.84, an exeA::Tn5–751 mutant, by overexpression of exeD

alone in trans. Expression studies indicated that these results did not reflect

a role of ExeAB in the regulation of the exeC‐N operon. Instead, immunoblot

analysis showed that ExeD did not multimerize in C5.84, and fractionation of the

membranes showed that the monomeric ExeD remained in the inner membrane. Expression

of ExeAB, but not either protein alone, from a plasmid in C5.84 resulted in increases

in the amount of multimeric ExeD, which correlated with increases in aerolysin secretion.

Pulse‐chase analysis also sug‐gested that the induction of ExeAB allowed multimerization

of previously accumulated monomer ExeD. In C5.84 cells overproducing ExeD, it multimerized

even in the absence of ExeAB and, although most remained in the inner membrane, an

amount similar to that in wild‐type outer membranes fractionated with the outer membrane

of the overproducing cells. These results indicate that the secretion defect of exeAB mutants is a result of an inability to assemble the ExeD secretin in the outer membrane. The localization and multimerization of overproduced ExeD in these mutants further suggests that the ExeAB complex plays either a direct or indirect role in the transport of ExeD into the outer membrane.
Role of rubrerythrin in the oxidative stress response of Porphyromonas gingivalis
Authors: Sztukowska Maryta , Bugno Marcin , Potempa Jan , Travis James , Kurtz Jr Donald M. 
Published: March 2003

SummaryRubrerythrins are non‐haem iron proteins that have been implicated in oxidative stress protection in anaerobic bacteria and archaea. However, up to now, this role has not been confirmed directly by inactivation of a rubrerythrin gene. Here we report generation of an rbr− mutant of Porphyromonas gingivalis, an obligately anaerobic gingival pathogenic bacterium. Characterization of the rbr− strain clearly showed that P. gingivalis produces a rubrerythrin‐like protein that is absent in the rbr− strain, and that the P. gingivalis rbr− strain is more dioxygen‐ and hydrogen peroxide‐sensitive than the wild type. The latter conclusion is based on two independent results, namely, deeper no‐growth zones upon diffusion of the oxidants through soft agar culture tubes and growth impairment of liquid cultures exposed to the oxidants. A same‐site rbr+ revertant showed increased hydrogen peroxide and dioxygen resistance relative to the rbr− strain. Transcription of the P. gingivalis rubrerythrin gene is induced above its constitutive anaerobic level in response to dioxygen or hydrogen peroxide exposures. Purified rubrerythrins from other organisms have been shown to catalyse reduction of hydrogen peroxide, while showing relatively sluggish reaction with dioxygen and little or no catalase or superoxide dismutase activities. Porphyromonas gingivalis contains a superoxide dismutase but lacks catalase and haem peroxidases. We therefore suggest that rubrerythrin provides oxidative stress protection via catalytic reduction of intracellular hydrogen peroxide.
Complete glycosylphosphatidylinositol anchors are required in Candida albicans for full morphogenesis, virulence and resistance to macrophages
Authors: Richard Mathias , Ibata‐Ombetta Stella , Dromer Françoise , Bordon‐Pallier Florence , Jouault Thierry , Gaillardin Claude 
Published: March 2003

SummaryGlycosylphosphatidylinositol (GPI)‐anchored proteins are involved in cell wall integrity and cell–cell interactions. We disrupted the Candida albicans
homologue of the Saccharomyces cerevisiae GPI7/LAS21 gene, which encodes a
GPI anchor‐modifying activity. In the mutant and on solid media, the yeast‐to‐hyphae
transition was blocked, whereas chlamydospore formation was enhanced. However, the
morphogenetic switch was normal in liquid medium. Abnormal budding patterns, cytokinesis
and cell shape were observed in both liquid and solid media. The cell wall structure
was also modified in the mutants, as shown by hypersensitivity to Calcofluor white.
In vitro and in vivo assays revealed that the mutant interacted with
its host in a modified way, resulting in reduced virulence in mice and reduced survival
in the gastrointestinal environment of mice. The mitogen‐activated protein (MAP)
kinase pathway of macrophages was downregulated by the wild‐type cells but not by
the ΔCagpi7 null strains. In agreement with this abnormal behaviour,
mutant cells were more sensitive to the lytic action of macrophages. Our results
indicate that a functional GPI anchor is required for full hyphal formation in C.
albicans, and that perturbation of the GPI biosynthesis results in hypersensitivity to host defences.
Identification of novel hrp‐regulated genes through functional genomic analysis of the Pseudomonas syringae pv. tomato DC3000 genome
Authors: Zwiesler‐Vollick Julie , Plovanich‐Jones Anne E. , Nomura Kinya , Bandyopadhyay Sruti , Joardar Vinita , Kunkel Barbara N. , He Sheng Yang 
Published: March 2003

Summary Pseudomonas syringae
pv.
tomato
(
Pst
) strain DC3000 infects the model plants
Arabidopsis thaliana
and tomato, causing disease symptoms characterized by necrotic lesions surrounded by chlorosis. One mechanism used by
Pst
DC3000 to infect host plants is the type III protein secretion system, which is thought to deliver multiple effector proteins to the plant cell. The exact number of type III effectors in
Pst
DC3000 or any other plant pathogenic bacterium is not known. All known type III effector genes of
P. syringae
are regulated by HrpS, an NtrC family protein, and the HrpL alternative sigma factor, which presumably binds to a conserved
cis
element (called the ‘hrp box’) in the promoters of type III secretion‐associated genes. In this study, we designed a search motif based on the promoter sequences conserved in 12 published
hrp
operons and putative effector genes in
Pst
DC3000. Seventy‐three predicted genes were retrieved from the January 2001 release of the
Pst
DC3000 genome sequence, which had 95% genome coverage. The expression of the 73 genes was analysed by microarray and Northern blotting, revealing 24 genes/operons (including eight novel genes), the expression of which was consistently higher in
hrp
‐inducing minimal medium than in nutrient‐rich Luria–Bertani broth. Expression of all eight genes was dependent on the
hrpS
gene. Most were also dependent on the
hrpL
gene, but at least one was dependent on the
hrpS
gene, but not on the
hrpL
gene. An AvrRpt2‐based type III translocation assay provides evidence that some of the
hrpS ‐regulated novel genes encode putative effector proteins.
Polymerase chain reaction‐based mutageneses identify key transporters belonging to multigene families involved in Na+ and pH homeostasis of Synechocystis sp. PCC 6803
Authors: Wang Hong‐Liang , Postier Bradley L. , Burnap Robert L. 
Published: March 2003

SummaryPrimary ion pumps and antiporters exist as multigene families in the Synechocystis

sp. PCC 6803 genome and show very strong homologies to those found in higher plants.

The gene knock‐outs of five putative Na+/H+ antiporters

(slr1727, sll0273, sll0689, slr1595 and slr0415)

and seven cation ATPases (sll1614, sll1920, slr0671–72,

slr0822, slr1507–08–09, slr1728– 29 and

slr1950) in the model cyanobacterium (http://www.kazusa.or.jp/cyano/cyano.html)

were performed in this study relying on homologous recombination with mutagenenic

fragments constructed using a fusion polymerase chain reaction (PCR) approach. The

impacts of these gene knock‐outs were evaluated in terms of Na+

and pH, and light‐induced acidification and alkalization that are asso‐ciated with

inorganic carbon uptake. Two of the five putative antiporter mutants exhibit a characteristic

interplay between the pH and Na+ dependence of growth, but only

one of the antiporters appears to be necessary for high NaCl tolerance. On the other

hand, the mutation of one of the two copper‐trafficking ATPases produces a cell line

that shows acute NaCl sensitivity. Additionally, disruptions of a putative Ca2+‐ATPase

and a gene cluster encoding a putative Na+‐ATPase subunit also

cause high NaCl sensitivity. The findings and possible mechanisms are discussed in

relation to the potential roles of these transporters in Synechocystis sp. PCC 6803.
Regulation of inducible peroxide stress responses
Authors: Mongkolsuk Skorn , Helmann John D. 
Published: March 2003

SummaryBacteria adapt to the presence of reactive oxygen species (ROS) by increasing the expression of detoxification enzymes and protein and DNA repair functions. These responses are co‐ordinated by transcription factors that regulate target genes in response to ROS. We compare three classes of peroxide‐sensing regulators: OxyR, PerR and OhrR. In all three cases, peroxides effect changes in the redox status of cysteine residues, but the molecular details are distinct. OxyR is converted into a transcriptional activator by the formation of a disulphide bond between two reactive cysteine residues. PerR is a metalloprotein that functions as a peroxide‐ sensitive repressor. Oxidation is modulated by metal ion composition and may also involve disulphide bond formation. OhrR represses an organic peroxide resistance protein and mediates derepression in response to organic peroxides. Peroxide sensing in this system requires a single conserved cysteine, which is oxidized to form a cysteine–sulphenic acid derivative.
Vibrio cholerae CytR is a repressor of biofilm development
Authors: Haugo Adam J. , Watnick Paula I. 
Published: March 2003

Summary Vibrio cholerae is both a human pathogen and a natural inhabitant of aquatic environments. In the aquatic environment, microorganisms are found attached to surfaces in structures known as biofilms. We have identified a transcriptional repressor in V. cholerae that inhibits exopolysaccharide synthesis and biofilm development. Our studies show that this repressor is the V. cholerae homologue of Escherichia coli CytR, a protein that represses nucleoside uptake and catabolism when nucleosides are scarce. We propose that the role of CytR in V. cholerae biofilm development is to co‐ordinate bacterial biofilm accumulation with the presence of nucleosides. Thus, nucleosides may be a signal to planktonic cells to join the biofilm.
Control of the glycolytic gapA operon by the catabolite control protein A in Bacillus subtilis: a novel mechanism of CcpA‐mediated regulation
Authors: Ludwig Holger , Rebhan Nicole , Blencke Hans‐Matti , Merzbacher Matthias , Stülke Jörg 
Published: March 2003

SummaryGlycolysis is one of the main pathways of carbon catabolism in Bacillus subtilis. Expression of the gapA gene encoding glyceraldehyde‐3‐phosphate dehydrogenase, the key enzyme of glycolysis from an energetic point of view, is induced by glucose and other sugars. Two regulators are involved in induction of the gapA operon, the product of the first gene of the operon, the CggR repressor, and catabolite control protein A (CcpA). CcpA is required for induction of the gapA operon by glucose. Genetic evidence has demonstrated that CcpA does not control the expression of the gapA operon by binding directly to a target in the promoter region. Here, we demonstrate by physiological analysis of the inducer spectrum that CcpA is required only for induction by sugars transported by the phosphotransferase system (PTS). A functional CcpA is needed for efficient transport of these sugars. This interference of CcpA with PTS sugar transport results from an altered phosphorylation pattern of HPr, a phosphotransferase of the PTS. In a ccpA mutant strain, HPr is nearly completely phosphorylated on a regulatory site, Ser‐46, and is trapped in this state, resulting in its inactivity in PTS phosphotransfer. A mutation in HPr affecting the regulatory phosphorylation site suppresses both the defect in PTS sugar transport and the induction of the gapA operon. We conclude that a low‐molecular effector derived from glucose that acts as an inducer for the repressor CggR is limiting in the ccpA mutant.
GabR, a member of a novel protein family, regulates the utilization of γ‐aminobutyrate in Bacillus subtilis
Authors: Belitsky Boris R. , Sonenshein Abraham L. 
Published: March 2003

SummaryThe Bacillus subtilis ycnG (gabT) and ycnH (gabD) genes were shown to encode γ‐aminobutyrate (GABA) aminotransferase and succinic semi‐aldehyde dehydrogenase, respectively, and to form a GABA‐inducible operon. Null mutations in gabT, gabD or the divergently transcribed ycnF (gabR) gene blocked the utilization of GABA as sole nitrogen source. GabR proved to be a transcriptional activator of the gabTD operon and a negative autoregulator. The target of GabR action was localized to an 87 bp region that includes both gabR and gabT promoters. GabR is a member of a novel but widespread family of chimeric bacterial proteins that have apparent DNA‐binding and aminotransferase domains. Mutations in conserved residues of the putative aminotransferase domain abolished GabR function as a transcriptional activator, but did not affect its activity as a negative autoregulator.
Action at a distance in CI repressor regulation of the bacteriophage 186 genetic switch
Authors: Dodd Ian B. , Egan J. Barry 
Published: March 2003

SummaryThe non‐lambdoid coliphage 186 provides an alternative model to the lytic‐lysogenic switch of phage λ. Like λ, the key switch regulator, the CI repressor, associates to octamers. Unlike λ, the lytic promoter (pR) and the lysogenic promoter (pL) are face‐to‐face, 62 bp apart and are flanked by distal CI binding sites (FL and FR) located ≈ 300 bp away. Using reporter and footprinting studies, we show that the outcome, but not the mechanism, of regulation by 186 CI is very similar to λ. 186 CI stimulates pL transcription indirectly by repressing convergent interfering transcription from pR. However, in the absence of the flanking FL and FR sites, CI bound at pR interacts co‐operatively with a weak CI binding site at pL and represses both promoters. FL and FR play a critical role; they assist repression of pR and simultaneously alleviate repression of pL, thus allowing high pL activity. We propose that the 186 switch is regulated by a novel mechanism in which a CI octamer bound at pR forms alternative DNA loops to pL or to a flanking site, depending on CI concentration.
Iron transport and regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas
Authors: Visca Paolo , Leoni Livia , Wilson Megan J. , Lamont Iain L. 
Published: March 2003

SummaryA variety of bacterial species secrete and take up chelating compounds that enable acquisition of iron (siderophores). It has become clear that a common feature in regulation of different iron acquisition systems is the involvement of alternative sigma factor proteins of the extracytoplasmic function (ECF) family. Two of these proteins, PvdS from Pseudomonas aeruginosa and FecI from Escherichia coli K‐12, have been studied extensively. PvdS directs transcription of genes required for the biosynthesis of a siderophore, pyoverdine, and FecI causes ex‐pression of genes for uptake of ferric citrate. FecI forms part of a signalling system that responds to the presence of ferric citrate. Here, we review recent advances in understanding of PvdS and of the Fec signalling system. PvdS and FecI are part of a distinct subfamily of ECF sigma factors involved in iron acquisition and hence named the iron‐starvation sigmas. Analysis of microbial genome sequences shows that Fec‐like signalling systems are present in a wide range of species and many such systems may be present in a single species. The availability of tools for large‐scale genome analysis is likely to lead to rapid advances in our understanding of this expanding family of proteins.
Controlling pore assembly of staphylococcal γ‐haemolysin by low temperature and by disulphide bond formation in double‐cysteine LukF mutants
Authors: Nguyen Vananh T. , Higuchi Hideo , Kamio Yoshiyuki 
Published: March 2003

SummaryStaphylococcal LukF and Hlg2 are water‐soluble monomers of γ‐haemolysin that assemble into oligomeric pores on the erythrocyte membranes. Here, we have created double‐cysteine LukF mutants, in which single disulphide bonds connect either the prestem domain and the cap domain (V12C‐T136C, Cap–Stem), or two β‐strands within the prestem domain (T117C‐T136C, Stem–Stem) to control pore assembly of γ‐haemolysin at intermediate stages. The disulphide‐trapped mutants were inactive in erythrocyte lysis, but gained full haemolytic activity if the disulphide bonds were reduced. The disulphide bonds blocked neither the membrane binding ability nor the intermediate prepore oligomerization, but efficiently inhibited the transition from prepores to pores. The prepores of Cap–Stem were dissociated into monomers in 1% SDS. In contrast, the prepores of Stem–Stem were stable in SDS and had ring‐shaped structures similar to those of wild‐type LukF, as observed by transmission electron microscopy. The transition of both mutants from prepores to pores could even be achieved by reducing disulphide bonds at low temperature (2°C), whereas prepore oligomerization was effectively inhibited by low temperature. Finally, real‐time transition of Stem–Stem from prepores to pores on ghost cells, visualized using a Ca2+‐sensitive fluorescent indicator (Rhod2), was shown by the sequential appearance of fluorescence spots, indicating pore‐opening events. Taken together, these data indicate that the prepores are legitimate intermediates during γ‐haemolysin pore assembly, and that conformational changes around residues 117 and 136 of the prestem domain are essential for pore formation, but not for membrane binding or prepore oligomerization. We propose a mechanism for γ‐haemolysin pore assembly based on the demonstrated intermediates.
A new mutation delivery system for genome‐scale approaches in Bacillus subtilis
Authors: Fabret Céline , Dusko Ehrlich S. , Noirot Philippe 
Published: March 2003

SummaryIn Bacillus subtilis, although many genetic tools have been developed, gene replacement remains labour‐intensive and not compatible with large‐scale appro‐aches. We have developed a new one‐step gene replacement procedure that allows rapid alteration of any gene sequence or multiple gene sequences in B. subtilis without altering the chromosome in any other way. This novel approach relies on the use of upp, which encodes uracil phosphoribosyl‐transferase, as a counter‐selectable marker. We fused the upp gene to an antibiotic‐resistance gene to create an ‘upp‐cassette’. A polymerase chain reaction (PCR)‐generated fragment, consisting of the target gene with the desired mutation joined to the upp‐cassette, was integrated into the chromosome by homologous recombination, using positive selection for antibiotic resistance. Then, the eviction of the upp‐cassette from the chromosome by recombination between short repeated chromosomal sequences, included in the design of the transforming DNA molecule, was achieved by counter‐selection of upp. This procedure was successfully used to deliver a point mutation, to generate in‐frame deletions with reduced polar effects, and to combine deletions in three paralogous genes encoding two‐component sensor kinases. Also, two chromosome regions carrying previously unrecognized essential functions were identified, and large deletions in two dispensable regions were combined. This work outlines a strategy for identifying essential functions that could be used at genome scale.