bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Salmonella typhimurium virulence gene expression by cationic antimicrobial peptides
Authors: Bader Martin W. , Navarre William Wiley , Shiau Whitney , Nikaido Hiroshi , Frye Jonathan G. , McClelland Michael , Fang Ferric C. , Miller Samuel I.
Published: September 2003

Cationic antimicrobial peptides (CAMP) represent a conserved and highly effective component of innate immunity. During infection, the Gram‐negative pathogen Salmonella typhimurium induces different mechanisms of CAMP resistance that promote pathogenesis in animals. This study shows that exposure of S. typhimurium to sublethal concentrations of CAMP activates the PhoP/PhoQ and RpoS virulence regulons, while repressing the transcription of genes required for flagella synthesis and the invasion‐associated type III secretion system. We further demonstrate that growth of S. typhimurium in low doses of the α‐helical peptide C18G induces resistance to CAMP of different structural classes. Inducible resistance depends on the presence of PhoP, indicating that the PhoP/PhoQ system can sense sublethal concentrations of cationic antimicrobial peptides. Growth of S. typhimurium in the presence of CAMP also leads to RpoS‐dependent protection against hydrogen peroxide. Because bacterial resistance to oxidative stress and CAMP are induced during infection of animals, CAMP may be an important signal recognized by bacteria on colonization of animal tissues.
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

Trimeric 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

Detailed 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

Analysis 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

Most 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

Nutrient 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

The 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

In 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

The 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

PSA 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

Haem 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

Higher‐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

Multiple 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

Virulent 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

Replication 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.
Bacteriophage T4‐encoded Stp can be replaced as activator of anticodon nuclease by a normal host cell metabolite
Authors: Amitsur Michal , Benjamin Sima , Rosner Rachel , Chapman‐Shimshoni Daphne , Meidler Roberto , Blanga Shani , Kaufmann Gabriel
Published: September 2003

The bacterial tRNALys‐specific anticodon nuclease is known as a phage T4 exclusion system. In the uninfected host cell anticodon nuclease is kept latent due to the association of its core protein PrrC with the DNA restriction‐modification endonuclease EcoprrI. Stp, the T4‐encoded peptide inhibitor of EcoprrI activates the latent enzyme. Previous in vitro work indicated that the activation by Stp is sensitive to DNase and requires added nucleotides. Biochemical and mutational data reported here suggest that Stp activates the latent holoenzyme when its EcoprrI component is tethered to a cognate DNA substrate. Moreover, the activation is driven by GTP hydrolysis, possibly mediated by the NTPase domain of PrrC. The data also reveal that Stp can be replaced as the activator of latent anticodon nuclease by certain pyrimidine nucleotides, the most potent of which is dTTP. The activation by dTTP likewise requires an EcoprrI DNA substrate and GTP hydrolysis but involves a different form of the latent holoenzyme/DNA complex. Moreover, whereas Stp relays its activating effect through EcoprrI, dTTP targets PrrC. The activation of the latent enzyme by a normal cell constituent hints that anticodon nuclease plays additional roles, other than warding off phage T4 infection.
Shifting fungal reproductive mode by manipulation of mating type genes: obligatory heterothallism of Gibberella zeae
Authors: Lee Jungkwan , Lee Theresa , Lee Yin‐Won , Yun Sung‐Hwan , Turgeon B. Gillian
Published: September 2003

Fungi capable of sexual reproduction use heterothallic (self‐sterile) or homothallic (self‐fertile) mating strategies. In most ascomycetes, a single mating type locus, MAT, with two alternative forms (MAT1‐1 and MAT1‐2) called idiomorphs, controls mating ability. In heterothallic ascomycetes, these alternative idiomorphs reside in different nuclei. In contrast, most homothallic ascomycetes carry both MAT1‐1 and MAT1‐2 in a single nucleus, usually closely linked. An example of the latter is Gibberella zeae, a species that is capable of both selfing and outcrossing. G. zeae is a devastating cereal pathogen of ubiquitous geographical distribution, and also a producer of mycotoxins that threaten human and animal health. We asked whether G. zeae could be made strictly heterothallic by manipulation of MAT. Targeted gene replacement was used to differentially delete MAT1‐1 or MAT1‐2 from a wild‐type haploid MAT1‐1; MAT1‐2 strain, resulting in MAT1‐1; mat1‐2, mat1‐1; MAT1‐2 strains that were self‐sterile, yet able to cross to wild‐type testers and, more importantly, to each other. These results indicated that differential deletion of MAT idiomorphs eliminates selfing ability of G. zeae, but the ability to outcross is retained. They also indicated that both MAT idiomorphs are required for self‐fertility. To our knowledge, this is the first report of complete conversion of fungal reproductive strategy from homothallic to heterothallic by targeted manipulation of MAT. Practically, this approach opens the door to simple and efficient procedures for obtaining sexual recombinants of G. zeae that will be useful for genetic analyses of pathogenicity and other traits, such as the ability to produce mycotoxins.
Titration of the Escherichia coli DnaA protein to excess datA sites causes destabilization of replication forks, delayed replication initiation and delayed cell division
Authors: Morigen  , Løbner‐Olesen Anders , Skarstad Kirsten
Published: September 2003

In Escherichia coli, the level of the initiator protein DnaA is limiting for initiation of replication at oriC. A high‐affinity binding site for DnaA, datA, plays an important role. Here, the effect of extra datA sites was studied. A moderate increase in datA dosage (≈ fourfold) delayed initiation of replication and cell division, but increased the rate of replication fork movement about twofold. At a further increase in the datA gene dosage, the SOS response was induced, and incomplete rounds of chromosome replication were detected. Overexpression of DnaA protein suppressed the SOS response and restored normal replication timing and rate of fork movement. In the presence of extra datA sites, cells showed a dependency on PriA and RecA proteins, indicating instability of the replication fork. The results suggest that wild‐type replication fork progression normally includes controlled pausing, and that this is a prerequisite for normal replication fork function.
Negative feedback regulation ofdnaK,clpBandlonexpression by the DnaK chaperone machine inStreptomyces coelicolor, identified by transcriptome andin vivoDnaK‐depletion analysis
Authors: Bucca Giselda , Brassington Anna M. E. , Hotchkiss Graham , Mersinias Vassilios , Smith Colin P.
Published: September 2003

ThednaKoperon ofStreptomyces coelicolorencodes the DnaK chaperone machine and the negative autoregulator HspR, which confers repression of the operon by binding to several inverted repeat sequences in the promoter region,dnaKp. Previousin vitrostudies demonstrated that DnaK forms a specific complex with HspR bound to its operator sequences indnaKp, and a model was proposed in which DnaK functions as a corepressor of thednaKoperon (Bucca, G., Brassington, A., Schonfeld, H.J., and Smith, C.P. (2000)Mol Microbiol38: 1093–1103). Here we reportin vivoDnaK depletion experiments which demonstrate that DnaK is a negative regulator of thednaKoperon. Cellular depletion of the DnaK chaperone leads to high‐level transcription fromdnaKpat the normal growth temperature. DNA microarray‐based analysis of gene expression in wild‐type andhspR‐disruption mutant strains has identified a core cluster of genes regulated by HspR: thednaKandclpB‐SCO3660operons andlon. These three transcription units are considered to be the direct targets of HspR. Significantly, analysis of the entire genome sequence revealed that the promoter regions ofdnaK,clpBandlonare the only sequences that contain the HspR consensus binding sequence 5′‐TTGAGY‐N7‐ACTCAA. S1 nuclease mapping confirmed that transcription of bothclpBandlonis substantially enhanced at ambient temperature in strains depleted of DnaK, providing further evidence that these genes are members of the DnaK‐HspR regulon. From transcriptome analysis, 17 genes were shown to be upregulated more than twofold in anhspRdisruption mutant. This included the seven genes encoded by thednaK,clpBandlontranscription units. Significantly, the other 10 genes are not heat‐shock inducible in the wild type and their upregulation in thehspRmutant is considered to be an indirect consequence of enhanced synthesis of one or more components of the HspR regulon (the DnaK chaperone machine, ClpB and Lon protease).
Large‐scale essential gene identification in Candida albicans and applications to antifungal drug discovery
Authors: Roemer Terry , Jiang Bo , Davison John , Ketela Troy , Veillette Karynn , Breton Anouk , Tandia Fatou , Linteau Annie , Sillaots Susan , Marta Catarina , Martel Nick , Veronneau Steeve , Lemieux Sebastien , Kauffman Sarah , Becker Jeff , Storms Reginald , Boone Charles , Bussey Howard
Published: September 2003

Candida albicans is the primary fungal pathogen of humans. Despite the need for novel drugs to combat fungal infections [Sobel, J.D. (2000) Clin Infectious Dis 30: 652], antifungal drug discovery is currently limited by both the availability of suitable drug targets and assays to screen corresponding targets. A functional genomics approach based on the diploid C. albicans genome sequence, termed GRACETM (gene replacement and conditional expression), was used to assess gene essentiality through a combination of gene replacement and conditional gene expression. In a systematic application of this approach, we identify 567 essential genes in C. albicans. Interestingly, evaluating the conditional phenotype of all identifiable C. albicans homologues of the Saccharomyces cerevisiae essential gene set [Giaever, G., Chu, A.M., Ni, L., Connelly, C., Riles, L., Veronneau, S., et al. (2002) Nature 418: 387–391] by GRACE revealed only 61% to be essential in C. albicans, emphasizing the importance of performing such studies directly within the pathogen. Construction of this conditional mutant strain collection facilitates large‐scale examination of terminal phenotypes of essential genes. This information enables preferred drug targets to be selected from the C. albicans essential gene set by phenotypic information derived both in vitro, such as cidal versus static terminal phenotypes, as well as in vivo through virulence studies using conditional strains in an animal model of infection. In addition, the combination of phenotypic and bioinformatic analyses further improves drug target selection from the C. albicans essential gene set, and their respective conditional mutant strains may be directly used as sensitive whole‐cell assays for drug screening.
CcpA regulation of aerobic and respiration growth in Lactococcus lactis
Authors: Gaudu Philippe , Lamberet Gilles , Poncet Sandrine , Gruss Alexandra
Published: September 2003

The catabolic control protein CcpA is the highly conserved regulator of carbon metabolism in Gram‐positive bacteria. We recently showed that Lactococcus lactis, a fermenting bacterium in the family of Streptococcaceae, is capable of respiration late in growth when haem is added to aerated cultures. As the start of respiration coincides with glucose depletion from the medium, we hypothesized that CcpA is involved in this metabolic switch and investigated its role in lactococcal growth under aeration and respiration conditions. Compared with modest changes observed in fermentation growth, inactivation of ccpA shifts metabolism to mixed acid fermentation under aeration conditions. This shift is due to a modification of the redox balance via derepression of NADH oxidase, which eliminates oxygen and decreases the NADH pool. CcpA also plays a decisive role in respiration metabolism. Haem addition to lag phase ccpA cells results in growth arrest and cell mortality. Toxicity is due to oxidative stress provoked by precocious haem uptake. We identify the repressor of the haem transport system and show that it is a target of CcpA activation. We propose that CcpA‐mediated repression of haem uptake is a means of preventing oxidative damage at the start of exponential growth. CcpA thus appears to govern a regulatory network that coordinates oxygen, iron and carbon metabolism.
The Vibrio fischeri quorum‐sensing systems ain and lux sequentially induce luminescence gene expression and are important for persistence in the squid host
Authors: Lupp Claudia , Urbanowski Mark , Greenberg E. Peter , Ruby Edward G.
Published: September 2003

Bacterial quorum sensing using acyl‐homoserine lactones (acyl‐HSLs) as cell‐density dependent signalling molecules is important for the transcriptional regulation of many genes essential in the establishment and the maintenance of bacteria–host associations. Vibrio fischeri, the symbiotic partner of the Hawaiian bobtail squid Euprymna scolopes, possesses two distinct acyl‐HSL synthase proteins, LuxI and AinS. Whereas the cell density‐dependent regulation of luminescence by the LuxI‐produced signal is a well‐described phenomenon, and its role in light organ symbiosis has been defined, little is known about the ain system. We have investigated the impact of the V. fischeri acyl‐HSL synthase AinS on both luminescence and symbiotic colonization. Through phenotypic studies of V. fischeri mutants we have found that the AinS‐signal is the predominant inducer of luminescence expression in culture, whereas the impact of the LuxI‐signal is apparent only at the high cell densities occurring in symbiosis. Furthermore, our studies revealed that ainS regulates activities essential for successful colonization of E. scolopes, i.e. the V. fischeri ainS mutant failed to persist in the squid light organ. Mutational inactivation of the transcriptional regulator protein LuxO in the ainS mutant partially or completely reversed all the observed phenotypes, demonstrating that the AinS‐signal regulates expression of downstream genes through the inactivation of LuxO. Taken together, our results suggest that the two quorum‐sensing systems in V. fischeri, ain and lux, sequentially induce the expression of luminescence genes and possibly other colonization factors.
Predicting yield loss in maize fields and developing decision support for post‐emergence herbicide applications
Authors: Lemieux C , Vallée L , Vanasse A
Published: September 2003

This work was initiated to integrate an image analysis system and a prediction equation to support decisions for post‐emergence herbicide applications under field conditions. Data were collected from 1999 to 2001 in 32 commercial fields to obtain weed cover data at the three to four leaf stage of maize (Zea mays L.), and crop yield at maturity. Relative crop yield was predicted using a non‐linear sigmoidal equation with relative weed cover as the predictor variable (P < 0.0001; R2 = 0.39). The decision procedure consists of using the equation within the limits of a yield loss threshold that represents the loss one is willing to tolerate. The tolerance threshold (TT) allows determination of a weed threshold (WT). The procedure considers the variability around the prediction equation by setting the WT at the intersection between the lower 95% confidence interval of the prediction line and the TT. It also considers the variability around the weed cover estimate. For a given field, the decision is made by comparing the average weed cover corrected for sampling error, to the WT. We tested the performance of the decision procedure and found it could lead to a saving of 25% of herbicide use. We also computed a probability table showing the chances of getting relative yield above or below the TT. We suggest using the probability table in combination with the decision procedure to manage risks. The proposed approach does not offer a set ‘yes’ or ‘no’ answer but rather provides a framework to support decisions by producers who ultimately must manage the risks.
Mechanisms of resistance to simazine in Sonchus oleraceus
Authors: Fraga M I , Tasende M G
Published: September 2003

Sonchus oleraceus biotypes resistant to simazine were identified by chlorophyll fluorescence analysis of intact leaves. The mechanisms of resistance were determined on the basis of cpDNA and glutathione S‐transferase (GST) activity analyses. The results of the cpDNA analysis showed that most of the resistant biotypes had a Ser 264 Gly mutation in the psbA gene, which is responsible for an amino acid substitution in the D‐1 protein sequence. The cpDNA fragments were amplified by polymerase chain reaction and digested with restriction enzyme MaeI. Two restriction bands of 338 and 75 bp were recorded in biotypes with the target mutation, while three bands (218, 120 and 75 bp) were present in biotypes without this mutation. A second mechanism of resistance in this species was through the detoxification of simazine by conjugation with glutathione. In resistant biotypes without the above‐mentioned mutation, the average level of GST (simazine) activity in leaves was 4.5‐fold greater than in the resistant biotypes with the target mutation and 8.3‐fold greater than in the susceptible biotypes. Resistance as a result of the target mutation was more common than that achieved through detoxification by glutathione conjugation of simazine.
Natural tolerance of Cuscuta campestris to herbicides inhibiting amino acid biosynthesis
Authors: Nadler‐Hassar T , Rubin B
Published: September 2003

The response of Cuscuta campestris Yuncker, a non‐specific above‐ground holoparasite, to amino acid biosynthesis inhibitor (AABI) herbicides, was compared with other resistant and sensitive plants in dose–response assays carried out in Petri dishes. Cuscuta campestris was found to be much more resistant to all AABI herbicides tested. The I50 value of C. campestris growth inhibition by glyphosate was eightfold higher than that of transgenic, glyphosate‐resistant cotton (RR‐cotton). The I50 value for C. campestris shoot growth inhibition by sulfometuron was above 500 μM, whereas that of sorghum roots was only 0.004 μM. Cuscuta campestris exposed to glyphosate gradually accumulated shikimate, confirming herbicide penetration into the parasite and interaction with an active form of the target enzyme of the herbicide, 5‐enolpyruvylshikimate‐3‐phosphate synthase. More than half of the C. campestris plants associated with transgenic, glyphosate‐resistant sugarbeet (RR‐sugarbeet) treated with glyphosate or with transgenic, sulfometuron‐resistant tomato (SuR‐tomato) treated with sulfometuron recovered and resumed regular growth 20–30 days after treatment. New healthy stems developed, followed by normal flowering and seed setting. The results of the current study demonstrate the unique capacity of C. campestris to tolerate high rates of AABI. The mechanism of this phenomenon is yet to be elucidated.
Changes in the abundance of weeds in spring cereal fields in Finland
Authors: Hyvönen T , Ketoja E , Salonen J
Published: September 2003

The data of two weed surveys of spring cereals (conducted in 1982–84 and 1997–99) in Finland were applied to study changes in the total abundance (the number of individuals and dry weight) of the weed community as well as in the abundance and frequency of occurrence of single species. The estimated overall mean for the differences in total dry weight was 31.1 kg ha−1 and the differences in the total number of individuals was 46.6 individuals m−2 with higher numbers in the 1990s. Most of the overall variability in dry weight (63%) and in number of individuals (57%) was attributable to variability between fields within the same farm. None of the environmental variables could explain the variability in the difference in dry weight, or in the number of individuals between decades. Descriptive analysis of the single species data revealed a higher abundance and frequency of occurrence of most species in the 1990s than in the 1980s. No statistically significant differences were detected in the number of individuals of species tolerant of sulfonylureas (pooled data on Galium spurium and Viola arvensis) between decades. The results are discussed in relation to changes in the use of herbicides.
Combining physical, cultural and biological methods: prospects for integrated non‐chemical weed management strategies
Authors: Hatcher P E , Melander B
Published: September 2003

Physical, cultural and biological methods for weed control have developed largely independently and are often concerned with weed control in different systems: physical and cultural control in annual crops and biocontrol in extensive grasslands. We discuss the strengths and limitations of four physical and cultural methods for weed control: mechanical, thermal, cutting, and intercropping, and the advantages and disadvantages of combining biological control with them. These physical and cultural control methods may increase soil nitrogen levels and alter microclimate at soil level; this may be of benefit to biocontrol agents, although physical disturbance to the soil and plant damage may be detrimental. Some weeds escape control by these methods; we suggest that these weeds may be controlled by biocontrol agents. It will be easiest to combine biological control with fire and cutting in grasslands; within arable systems it would be most promising to combine biological control (especially using seed predators and foliar pathogens) with cover‐cropping, and mechanical weeding combined with foliar bacterial and possibly foliar fungal pathogens. We stress the need to consider the timing of application of combined control methods in order to cause least damage to the biocontrol agent, along with maximum damage to the weed and to consider the wider implications of these different weed control methods.
The control of Achillea millefolium in the Snowy Mountains of Australia
Authors: Sanecki G M , Sanecki K L , Wright G T , Johnston F M
Published: September 2003

The response of Achillea millefolium L. to herbicides was measured to determine the effectiveness of the current recommendations and to test alternative herbicides. Five plots at each of the three replicate sites were selected and randomly treated with one of the four herbicides: dicamba/2,4‐D, glyphosate, metsulfuron‐methyl and triclopyr/picloram. After 12 months, the recommended treatment, dicamba/2,4‐D, did not cause a significant reduction in aerial biomass compared with untreated controls and the number of flowering stems was significantly increased in treated plots. Metsulfuron‐methyl and triclopyr/picloram caused a significant reduction in A. millefolium aerial biomass but did not affect the number of flowering stems. Glyphosate produced a significant reduction in aerial biomass and was less effective. These results suggest that the current recommendation for A. millefolium might be improved. Site‐specific effects such as aspect may also influence the effectiveness of herbicides on this species.
Deposition of spray liquid on the soil below cereal crops after applications during the growing season
Authors: Jensen P K , Spliid N H
Published: September 2003

The aim of this study was to investigate how big a proportion of the spray liquid applied in a typical application was deposited on the soil surface below the crop. From an agronomical point of view this part of the spray is an undesirable loss of foliar acting pesticides during application and it also constitutes a primary source of leaching and surface run‐off. The investigation was carried out during 1998–2001 in winter wheat and in spring barley. Applications were carried out at approximate intervals of 10 days during the whole growing season in the two cereal crops. The results showed that a high proportion of the spray liquid reaches the soil below the crop during applications at early growth stages corresponding to the growth stages where weed control on farms is carried out. At later growth stages soil deposition declined and reached values below 5% of the applied dose in winter wheat after inflorescence emergence (50–59 BBCH). The spring barley crops remained more open and minimum soil deposit values were approximately 15% of the applied dose at growth stages (50–59 BBCH). During ripening soil deposition values increased again as crop cover decreased.
Herbicides used in combination can reduce the probability of herbicide resistance in finite weed populations
Authors: Diggle A J , Neve P B , Smith F P
Published: September 2003

A simulation study was conducted to examine the effect of pattern of herbicide use on development of resistance to two herbicides with different modes of action in finite weed populations. The effects of the size of the treatment area (analogous to initial weed population), germination fraction and degree of self‐pollination in the weed were investigated. The results indicate that the probability of developing resistance to one or both herbicides decreases as the size of the area/initial population decreases. For treatment areas of 100 ha or less with an initial weed seedbank of 100 seeds m−2 and initial frequencies of the resistance genes of 10−6, development of resistance to both herbicides (double‐resistance) is uncommon within 50 years for all types of weeds if both herbicides are used in all years (used in combination). If herbicides are used in alternate years (rotated) double‐resistance almost always occurs in 100 ha areas but is uncommon in areas of 1 ha or less. The results suggest that adoption of practices that limit movement of weeds in conjunction with using herbicides in combination rather than in rotation can substantially delay development of herbicide resistance.