bluuebunny/crossref_metadata_2025_split huggingface dataset:
The G‐beta subunit MGB1 is involved in regulating multiple steps of infection‐related morphogenesis in Magnaporthe grisea
Authors: Nishimura Marie , Park Gyungsoon , Xu Jin‐Rong 
Published: September 2003

SummaryTrimeric G‐proteins transmit extracellular signals to various downstream effectors (e.g. MAP kinases) in eukaryotes. In the rice blast fungus Magnaporthe grisea, the Pmk1 MAP kinase is essential for appressorium formation and infectious growth. The pmk1 deletion mutant fails to form appressoria but still responds to exogenous cAMP for tip deformation. Since gene disruption mutants of three Gα subunits still form appressoria and are phenotypically different from pmk1 mutants, it is likely that the Pmk1 pathway is activated by Gβ in M. grisea. In this study, we isolated and characterized the MGB1 gene that encodes the G subunit in M. grisea. Mutants disrupted in MGB1 were reduced in conidiation. Conidia from mgb1 mutants were defective in appressorium formation and failed to penetrate or grow invasively on rice leaves. Exogenous cAMP induced appressorium formation in mgb1 mutants, but these appressoria were abnormal in shape and could not penetrate. The intracellular cAMP level was reduced in mgb1 mutants and the defects in conidiation and hyphal growth were partially suppressed with 1 mM cAMP. Transformants expressing multiple copies of MGB1 were able to form appressoria on hydrophilic surfaces. Our results suggest that MGB1 may be involved in the cAMP signalling for regulating conidiation, surface recognition and appressorium formation. The Pmk1 pathway may be the downstream target of MGB1 for regulating penetration and infectious hyphae growth in M. grisea.
Involvement of bacterial migration in the development of complex multicellular structures in Pseudomonas aeruginosa biofilms
Authors: Klausen Mikkel , Aaes‐Jørgensen Anders , Molin Søren , Tolker‐Nielsen Tim 
Published: September 2003

SummaryDetailed knowledge of the developmental process from single cells scattered on a surface to complex multicellular biofilm structures is essential in order to create strategies to control biofilm development. In order to study bacterial migration patterns during Pseudomonas aeruginosa biofilm development, we have performed an investigation with time‐lapse confocal laser scanning microscopy of biofilms formed by various combinations of colour‐coded P. aeruginosa wild type and motility mutants. We show that mushroom‐shaped multicellular structures in P. aeruginosa biofilms can form in a sequential process involving a non‐motile bacterial subpopulation and a migrating bacterial subpopulation. The non‐motile bacteria form the mushroom stalks by growth in certain foci of the biofilm. The migrating bacteria form the mushroom caps by climbing the stalks and aggregating on the tops in a process which is driven by type‐IV pili. These results lead to a new model for biofilm formation by P. aeruginosa.
Experimentally revised repertoire of putative contingency loci in Neisseria meningitidis strain MC58: evidence for a novel mechanism of phase variation
Authors: Martin P. , Van De Ven T. , Mouchel N. , Jeffries A. C. , Hood D. W. , Moxon E. R. 
Published: September 2003

SummaryAnalysis of the genome sequence of Neisseria meningitidis strain MC58 revealed 65 genes associated with simple sequence repeats. Experimental evidence of phase variation exists for only 14 of these 65 putatively phase variable genes. We investigated the phase variable potential of the remaining 51 genes. The repeat tract associated with 20 of these 51 genes was sequenced in 26 genetically distinct strains. This analysis provided circumstantial evidence for or against the phase variability of the candidate genes, based on the sequence and the length of the repeated motif. These predictions of phase variability were substantiated for three of these candidate genes using colony immunoblotting or β‐galactosidase as a reporter. This investigation identified a novel phase variable gene (NMB1994 or nadA) associated with a repeat tract (TAAA) not previously reported to be associated with phase variable genes in N. meningitidis. Analysis of the nadA transcript revealed that the repeat tract was located upstream of the putative −35 element of the nadA promoter. Semiquantitative RT‐PCR showed that variation in the number of repeats was associated with changes in the level of expression of nadA, findings consistent with a model whereby the variable number of (TAAA) repeats modulates the promoter strength.
Recombination, repair and replication in the pathogenic Neisseriae: the 3 R′s of molecular genetics of two human‐specific bacterial pathogens
Authors: Kline Kimberly A. , Sechman Eric V. , Skaar Eric P. , Seifert H. Steven 
Published: September 2003

SummaryMost of the detailed mechanisms that have been established for the molecular biological processes that mediate recombination, repair and replication of DNA have come from studies of the Escherichia coli paradigm. The human specific pathogens, Neisseria gonorrhoeae and N. meningitidis, are Gram‐negative bacteria that have some molecular processes that are similar to E. coli and others that appear to be divergent. We propose that the pathogenic Neisseriae have evolved a specialized collection of molecular mechanisms to adapt to life limited to human hosts. In this MicroReview, we explore what is known about the basic processes of DNA repair, DNA recombination (genetic exchange and pilin variation) and DNA replication in these human specific pathogens.
Isolation and characterization from pathogenic fungi of genes encoding ammonium permeases and their roles in dimorphism
Authors: Smith David G. , Garcia‐Pedrajas Maria D. , Gold Scott E. , Perlin Michael H. 
Published: September 2003

SummaryNutrient sensing plays important roles in fungal development in general, and specifically in critical aspects of pathogenicity and virulence, for both animal and plant pathogens. Dimorphic pathogens such as the phytopathogenic smut fungi, Ustilago maydis and Microbotryum violaceum, must switch from a yeast‐like to a filamentous form in order to cause disease. Two genes encoding methylammonium permeases (MEPs) were identified from each of these latter fungi and all the encoded proteins were most similar to Mep2p, the high‐affinity permease from Saccharomyces cerevisiae that plays a direct role in pseudohyphal or filamentous growth for that organism. This is the first report of MEPs from pathogenic fungi. The two genes from U. maydis and one of the genes from M. violaceum were expressed in diploid S. cerevisiae mutants deleted for all three mep genes (mep1mep2mep3). Each of the heterologous genes could complement the severe growth defect of the S. cerevisiae mutant on low ammonium. Moreover, the U. maydis ump2 gene, initially detected as an upregulated gene in budding cells, was also able to complement the pseudohyphal defect characteristic of the mutant yeast. This gene is thus one of few heterologous MEP genes capable of efficiently restoring pseudohyphal growth in yeast. For U. maydis, disruption of ump2 eliminated the filamentous phenotype of haploid cells on low ammonium, while ump1 disruption only slightly reduced methylamine uptake. The most significant drop in methylamine uptake was seen for the ump2 and the ump1ump2 double mutants. Moreover, when grown in liquid medium, the ump1ump2 double mutant aggregated and sedimented. Also, the importance of a putative site for phosphorylation by protein kinase A was investigated in both Mep2p and Ump2p via site‐directed mutagenesis of the respective genes. A mutation predicted to prevent phosphorylation of either protein, still allowed each to provide growth on low ammonium, but eliminated their abilities to provide pseudohyphal growth for the S. cerevisiae triple mutant. These findings allow us to present a model of how ammonium transporters play a role in regulating dimorphic growth in fungi.
The Emb proteins of mycobacteria direct arabinosylation of lipoarabinomannan and arabinogalactan via an N‐terminal recognition region and a C‐terminal synthetic region
Authors: Zhang Nannan , Torrelles Jordi B. , McNeil Michael R. , Escuyer Vincent E. , Khoo Kay‐Hooi , Brennan Patrick J. , Chatterjee Delphi 
Published: September 2003

SummaryThe arabinans of the mycobacterial cell wall are key structural and immunological polymers in the context of arabinogalactan (AG) and lipoarabinomannan (LAM) respectively. The three homologous membrane proteins EmbA, EmbB and EmbC are known to be involved in the synthesis of arabinan but their biochemical functions are not understood. Herein we show, that synthesis of LAM, but not AG, ceases after inactivation of embC in Mycobacterium smegmatis by insertional mutagenesis. LAM synthesis is restored upon complementation with the embC wild‐type gene. Previously we have shown that the synthesis of the arabinan of AG is affected by embA or embB disruption. Thus the Emb proteins are capable of differential recognition of the galactan or mannan acceptors prior to appropriate arabinosylation. In addition, a combination of genetic and biochemical approaches have allowed us to assign some specific functions to the regions of emb gene products. Complementation of the embC¯ mutant with a hybrid gene encoding the N‐terminus of EmbC and the C‐terminus of EmbB resulted in LAM with a lower molecular weight than the wild‐type LAM. Structural studies involving enzyme digestion, chromatography and mass spectrometry analyses revealed that the arabinan of the ‘LAM’ formed in the hybrid was of AG kind rather than LAM type of arabinan.
Multiple GATA sites: protein binding and physiological relevance for the regulation of the proline transporter gene of Aspergillus nidulans
Authors: Gómez Dennis , García Irene , Scazzocchio Claudio , Cubero Beatriz 
Published: September 2003

SummaryIn Aspergillus nidulans, proline can serve both as a carbon and a nitrogen source. The transcription of the prnB gene, encoding the proline transporter, is efficiently repressed only by the simultaneous presence of ammonium and glucose. Thus, repression of this gene demands the activation of the CreA repressor and the inactivation of the positive‐acting GATA factor AreA. Repression of all other prn structural genes results largely from inducer exclusion. In an areA null mutation background, prnB is repressible by the sole presence of glucose. We have determined by EMSA and missing‐base interference experiments that there are 15 AreA‐binding sites in the prnD‐prnB intergenic region. Only sites 13/14, in the proximity of the prnB TATA box, are clearly involved in transcriptional activation and regulation. Mutation of these sites mimics qualitatively the regulatory effect of an areA null mutation. The deletion of the TATA box has a measurable effect on the maximal level of prnB transcription but does not alter the regulation pattern of this gene.
Eclipse period during replication of plasmid R1: contributions from structural events and from the copy‐number control system
Authors: Olsson Jan A. , Berg Otto G. , Dasgupta Santanu , Nordström Kurt 
Published: September 2003

SummaryThe eclipse period (the time period during which a newly replicated plasmid copy is not available for a new replication) of plasmid R1 in Escherichia coli was determined with the classic Meselson–Stahl density‐shift experiment. A mini‐plasmid with the wild‐type R1 replicon and a mutant with a thermo‐inducible runaway‐replication phenotype were used in this work. The eclipses of the chromosome and of the wild‐type plasmid were 0.6 and 0.2 generation times, respectively, at temperatures ranging from 30°C to 42°C. The mutant plasmid had a similar eclipse at temperatures up to 38°C. At 42°C, the plasmid copy number increased rapidly because of the absence of replication control and replication reached a rate of 350–400 plasmid replications per cell and cell generation. During uncontrolled replication, the eclipse was about 3 min compared with 10 min at controlled replication (the wild‐type plasmid at 42°C). Hence, the copy‐number control system contributed significantly to the eclipse. The eclipse in the absence of copy‐number control (3 min) presumably is caused by structural requirements: the covalently closed circular plasmid DNA has to regain the right degree of superhelicity needed for initiation of replication and it takes time to assemble the initiation factors.
Genome and proteome of Listeria monocytogenes phage PSA: an unusual case for programmed + 1 translational frameshifting in structural protein synthesis
Authors: Zimmer Markus , Sattelberger Elke , Inman Ross B. , Calendar Richard , Loessner Martin J. 
Published: September 2003

SummaryPSA is a temperate phage isolated from Listeria monocytogenes strain Scott A. We report its complete nucleotide sequence, which consists of a linear 37 618 bp DNA featuring invariable, 3′‐protruding single stranded (cohesive) ends of 10 nucleotides. The physical characteristics were confirmed by partial denaturation mapping and electron microscopy of DNA molecules. Fifty‐seven open reading frames were identified on the PSA genome, which are apparently organized into three major transcriptional units, in a life cycle‐specific order. Functional assignments could be made to 33 gene products, including structural proteins, lysis components, DNA packaging proteins, lysogeny control functions and replication proteins. Bioinformatics demonstrated relatedness of PSA to phages infecting lactic acid bacteria and other low G + C Gram‐positives, but revealed only few similarities to Listeria phage A118. Virion proteins were analysed by amino acid sequencing and mass spectrometry, which enabled identification of major capsid and tail proteins, a tape measure and a putative portal. These analyses also revealed an unusual form of translational frameshifting, which occurs during decoding of the mRNAs specifying the two major structural proteins. Frameshifting yields different length forms of Cps (gp5) and Tsh (gp10), featuring identical N‐termini but different C‐termini. Matrix‐assisted laser‐desorption ionization mass spectrometry (MALDI‐MS) and electrospray ionization mass spectrometry (ESI‐MS) of tryptic peptide fragments was used to identify the modified C‐termini of the longer protein species, by demonstration of specific sequences resulting from + 1 programmed translational frameshifting. A slippery sequence with overlapping proline codons near the 3′ ends of both genes apparently redirects the ribosomes and initiates the recoding event. Two different cis‐acting factors, a shifty stop and a pseudoknot, presumably stimulate frameshifting efficiency. PSA represents the first case of + 1 frameshifting among dsDNA phages, and appears to be the first example of a virus utilizing a 3′ pseudoknot to stimulate such an event.
Ligand delivery by haem carrier proteins: the binding of Serratia marcescens haemophore to its outer membrane receptor is mediated by two distinct peptide regions
Authors: Létoffé Sylvie , Debarbieux Laurent , Izadi Nadia , Delepelaire Philippe , Wandersman Cécile 
Published: September 2003

SummaryHaem is involved in essential processes. It is toxic and thus is not found free in living organisms but almost entirely sequestered by haem carrier proteins. We investigated the mechanisms of haem transfer between the proteins of a bacterial haem acquisition system involving haemophores. Haemophores are secreted by several Gram‐negative bacteria and are able to extract haem (assimilated as an iron source) from haemoproteins and deliver it to specific outer membrane receptors. The Serratia marcescens haemophore (HasA) is folded into a globular form and tyrosine and histidine are involved in haem ligation. Interaction with the receptor is of high affinity (5 nM) and does not involve haem. Identification and study of mutants with altered binding properties led to the description of two regions of the haemophore that bind to the receptor. They consist of residues involved in two β strands located on the same side of HasA. Each region is sufficient for high affinity binding. The synthetic peptide corresponding to one β strand competes with the corresponding haemophore region for binding to the receptor, suggesting that the two binding regions are independent binding sites. We propose a model for haem release and transfer to the receptor.
The molecular basis of co‐operative DNA binding between lambda integrase and excisionase
Authors: Swalla Brian M. , Cho Eun Hee , Gumport Richard I. , Gardner Jeffrey F. 
Published: September 2003

SummaryHigher‐order nucleoprotein complexes often stabilize catalytic proteins in appropriate conformations for optimal activity and contribute to regulation during reactions requiring association of proteins and DNA. Formation of such complexes, known as intasomes, is required for site‐specific recombination catalysed by bacteriophage Lambda Integrase protein (Int). Int‐catalysed recombination is regulated by a second bacteriophage‐encoded protein, Excisionase (Xis), which both stimulates excision and inhibits integration. To exert its effect, Xis binds co‐operatively with Int, thereby inducing and stabilizing a DNA bend that alters the intasome structures formed during recombination. A rare int mutant, int 2268 ts, was reported (Enquist, L.W. and Weisberg, R.A. (1984) Mol Gen Genet 195: 62–69) to be more defective for excision than integration. Here, we have determined that this mutant Int protein contains an E47K substitution, and that the resultant excision‐specific defect is due, at least in part, to destabilized interactions between Int and Xis. Analysis of several engineered substitutions at Int position 47 showed that a negatively charged residue is required for co‐operative DNA binding between Int and Xis, and suggest that the Int‐E47 residue may contact Xis directly. Substitutions at Int position 47 also affect co‐operative binding among Int proteins at arm‐type DNA sites, and thereby reduce the efficiency of both integration and excision. Collectively, these results suggest that a single surface of the Int amino‐terminal domain mediates two alternate types of co‐operative binding interactions.
Quorum sensing controls biofilm formation in Vibrio cholerae
Authors: Hammer Brian K. , Bassler Bonnie L. 
Published: September 2003

SummaryMultiple quorum‐sensing circuits function in parallel to control virulence and biofilm formation in Vibrio cholerae. In contrast to other bacterial pathogens that induce virulence factor production and/or biofilm formation at high cell density in the presence of quorum‐sensing autoinducers, V. cholerae represses these behaviours at high cell density. Consistent with this, we show here that V. cholerae strains ‘locked’ in the regulatory state mimicking low cell density are enhanced for biofilm production whereas mutants ‘locked’ in the regulatory state mimicking high cell density are incapable of producing biofilms. The quorum‐sensing cascade we have identified in V. cholerae regulates the transcription of genes involved in exopolysaccharide production (EPS), and variants that produce EPS and form biofilms arise at high frequency from non‐EPS, non‐biofilm producing strains. Our data show that spontaneous mutation of the transcriptional regulator hapR is responsible for this effect. Several toxigenic strains of V. cholerae possess a naturally occurring frameshift mutation in hapR. Thus, the distinct environments occupied by this aquatic pathogen presumably include niches where cell‐cell communication is crucial, as well as ones where loss of quorum sensing via hapR mutation confers a selective advantage. Bacterial biofilms could represent a complex habitat where such differentiation occurs.
Deletion of vapA encoding Virulence Associated Protein A attenuates the intracellular actinomycete Rhodococcus equi
Authors: Jain Shruti , Bloom Barry R. , Hondalus Mary K. 
Published: September 2003

SummaryVirulent strains of the facultative intracellular bacterium Rhodococcus equi isolated from young horses (foals) with R. equi pneumonia, carry an 80–90 kb virulence plasmid and express a highly immunogenic 15–17 kDa protein of unknown function called VapA (Virulence Associated Protein A). Recent sequencing of the virulence plasmid identified a putative pathogenicity island encoding a novel family of seven Vap proteins including VapA. These proteins exhibit a significant sequence similarity to each other but have no homologues in other organisms. In this study, we describe the construction of an R. equi mutant lacking a 7.9 kb DNA region spanning five vap genes (vapA, ‐C, ‐D, ‐E and ‐F ). This vap locus mutant was attenuated for virulence in mice as it was unable to replicate in vivo and was rapidly cleared in comparison to the virulent wild‐type strain. Complementation analysis of the vap locus mutant showed that expression of vapA alone could restore full virulence, whereas expression of vapC, ‐D and ‐E could not. We subsequently constructed an R. equi strain lacking only the vapA gene and found that it was attenuated for growth in vivo to the same degree as the vap locus mutant. Unlike wild‐type R. equi which replicates intracellularly, both of the mutant strains exhibited a growth defect in macrophages although their attachment to the macrophages was unaffected. These studies provide the first proof of a role for vapA in the virulence of R. equi, and demonstrate that its presence is essential for intracellular growth in macrophages.
A biochemical analysis of the interaction of DNA gyrase with the bacteriophage Mu, pSC101 and pBR322 strong gyrase sites: the role of DNA sequence in modulating gyrase supercoiling and biological activity
Authors: Oram Mark , Howells Alison J. , Maxwell Anthony , Pato Martin L. 
Published: September 2003

SummaryReplication of bacteriophage Mu DNA, a process requiring efficient synapsis of the prophage ends, takes place within the confines of the Escherichia coli nucleoid. Critical to ensuring rapid synapsis is the function of the SGS, a strong gyrase site, located at the centre of the Mu genome. Replacement of the SGS by the strong gyrase sites from pSC101 or pBR322 fails to support efficient prophage replication. To probe the unique SGS properties we undertook a biochemical analysis of the interaction of DNA gyrase with the Mu SGS, pSC101 and pBR322 sites. In binding and cleavage assays the order of efficacy was pSC101 > Mu SGS >> pBR322. However, in supercoiling assays the Mu SGS (cloned into pUC19) exhibited a strong enhancement of gyrase‐catalysed supercoiling over pUC19 alone; the pSC101 site showed none and the pBR322 site gave a moderate improvement. Most striking was the Mu SGS‐dependent increase in processivity of the gyrase reaction. This highly processive supercoiling coupled with efficient binding may account for the unique biological properties of the SGS. The results emphasize the importance of the DNA substrate as an active component in modulating the gyrase supercoiling reaction, and in determining the biological roles of specialized gyrase sites.