bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatio‐temporal dynamics of theAllonemobius fasciatus–A. sociusmosaic hybrid zone: a 14‐year perspective
Authors: Britch S. C. , Cain M. L. , Howard D. J.
Published: March 2003

Long‐term studies of hybrid zones can provide valuable insight into a number of questions that have long attracted the attention of evolutionists. These questions range from the stability and fate of hybrid zones to the relative fitness of hybrids. In this paper we report the results of a 14‐year survey of theAllonemobius fasciatus–Allonemobius sociushybrid zone. Populations were collected intensively in 1986 and 1987 and then more sporadically through the end of the 1980s and throughout the 1990s. By documenting changes in the genetic composition of populations near and within the zone during this period of time we assessed: the strength of the reproductive isolation between the two species; the relative growth rates (which can be considered a surrogate of relative fitness) of genotype classes corresponding to hybrids and to pure species individuals; and, the power of single‐year and multi‐year measurements of relative growth rates to predict changes in the genetic composition of mixed populations through time. In brief, we found very large year‐to‐year variation in the relative growth rates of pure species and hybrid individuals. This variation may reflect the fact that both species are at the edge of their range and perhaps at the limits of their ability to deal with environmental perturbations. As a consequence of the variation, even multi‐year estimates of relative growth rates often provided imprecise predictions regarding the future genotypic composition of mixed populations. Despite our limited ability to predict the dynamics of individual populations, some trends are apparent.A. socius, the southern species, has clearly increased in frequency along a transect through the Appalachian Mountains, indicating that the zone is moving north in this region. In contrast, the zone appeared to be more stable along the East Coast transect. Within mixed populations, character‐index profiles are often bimodal and stable through time, indicating relatively strong reproductive isolation between the two species that is not being reinforced, nor is it breaking down.
Genetic differentiation in Eurasian populations of the postfire ascomycete Daldinia loculata
Authors: Johannesson H. , Vasiliauskas R. , Dahlberg A. , Penttilä R. , Stenlid J.
Published: March 2003

The genetic population structure of the postfire ascomycete Daldinia loculata was studied to test for differentiation on a continental scale. Ninety‐six samples of spore families, each comprising mycelia from six to 10 spores originating from single perithecia, were sampled from one Russian and six Fennoscandian forest sites. Allelic distribution was assayed for six nuclear gene loci by restriction enzyme analyses of polymerase chain reaction (PCR)‐amplified gene fragments. In addition, the full sequence of the gene fragment was analysed for a subset of haploid single‐ascospore isolates in a multiallelic approach. A third data set was generated by using arbitrary‐primed PCR with the core sequence of the phage M13 as primer. Although there was a reduction in heterozygosity in the total population from what would have been expected at random mating, the levels of genetic differentiation among the Eurasian subpopulations of D. loculata were low. All subpopulations were found to be in Hardy–Weinberg equilibrium and gametic equilibrium was observed between all investigated nuclear gene loci. The results obtained by the different markers were consistent; we confirmed low levels of genetic differentiation among the Eurasian subpopulations of D. loculata. The differentiation did not increase with distance; the Russian subpopulation, sampled more than 7000 km from the Fennoscandian subpopulations, was only moderately differentiated from the others (FST = 0.00–0.14). In contrast, one of the Swedish populations was the most highly differentiated from the others, with FST and GST values of 0.10–0.16. The results suggest that D. loculata consists of a long‐lived backround Eurasian population of latent mycelia in nonburned forests, established by sexual ascospores dispersed from scattered burned forest sites. Local differentiation is probably due to founder effects of populations in areas with low fire frequency. A tentative life cycle of D. loculata is presented.
Relatedness structure in Rhododendron metternichii var. hondoense revealed by microsatellite analysis
Authors: Kameyama Yoshiaki , Isagi Yuji , Nakagoshi Nobukazu
Published: March 2003

The relatedness structure of Rhododendron metternichii Sieb. et Zucc. var. hondoense Nakai was analysed in a 150 × 70‐m quadrat in Hiroshima Prefecture, western Japan. The population of R. metternichii occurred as three subpopulations at the study site (A1–A3). Pairwise relatedness based on microsatellite genotypes at eight loci and Mantel tests revealed a hierarchical structure of relatedness within and among subpopulations: (i) relatedness between individuals within 10 m of one another was significantly positive; (ii) relatedness between individuals in the same subpopulation was significantly positive, but negative between individuals in distant subpopulations; and (iii) relatedness was not significantly different from zero among neighbouring subpopulations. In detail, however, relatedness within each subpopulation was significantly positive in subpopulation A1, relatively weak but significantly positive in subpopulation A2, and not significantly different from zero in subpopulation A3. Relatedness within each subpopulation was inversely related to correlations between interindividual distance and relatedness. The aggregation of related individuals at short distances from one another may lead to decreasing relatedness within subpopulations as a whole. Moreover, negative correlations between interindividual distance and relatedness corresponded to high flowering densities at less than 10‐m distance, implying that high flowering densities reduce pollinator foraging distance and lead to stronger genetic structure within subpopulations. Small individuals (< 2.0 m in height) showed stronger genetic structure compared with that of large individuals (≥ 2.0 m in height). The different relatedness structure within and among subpopulations may be caused by various degrees of gene flow affected by distribution patterns of individuals and population density.
Genetic variation of symbiotic fungi cultivated by the macrotermitine termite Odontotermes formosanus (Isoptera: Termitidae) in the Ryukyu Archipelago
Authors: Katoh H. , Miura T. , Maekawa K. , Shinzato N. , Matsumoto T.
Published: March 2003

Fungus‐growing termites have a mutualistic relationship with their cultivated fungi. To improve understanding of genetic aspects of this relationship, we examined molecular markers in the fungus‐growing termite Odontotermes formosanus and its fungi Termitomyces spp. from the Ryukyu Archipelago. Based on the polymorphic band patterns obtained from arbitrarily primed polymerase chain reaction methods, we constructed cladograms for related colonies of the termites and fungi. The resulting trees indicated that the termites display little genetic variation among the colonies, while the symbiotic fungi consist of two major genetic types. In addition, molecular phylogenetic trees of the symbiotic fungi based on internal transcribed spacer and 18S rDNA suggested that these two types of fungi are different species. We also demonstrated that the fungi comprising the fruiting bodies and fungus combs are identical, and that fungus combs are probably a monoculture within a single termite colony. Our results indicate that horizontal transmission of symbiotic fungi among termite colonies occurred during the evolutionary history of this symbiosis.
Molecular genecology of temperature response in Lolium perenne: 1. preliminary analysis to reduce false positives
Authors: Hamilton N. R. Sackville , Skøt L. , Chorlton K. H. , Thomas I. D. , Mizen S.
Published: March 2003

Molecular genecology is the study of geographical clines in frequencies of molecular markers and their relationship to ecological clines in environmental conditions. This study outlines the principles underlying the selection of populations, focusing on avoiding ‘false positives’— noncausal correlations between allele frequency and the environment. The principles are illustrated by identifying a set of populations of Lolium perenne for the study of temperature responses. The selected set of populations encompasses a 20 °C range in mean January temperature. Their freezing tolerance shows a linear trend with winter temperature, LT50 decreasing by 0.25 °C for each 1 °C reduction in mean January temperature.
Molecular genecology of temperature response in Lolium perenne: 2. association of AFLP markers with ecogeography
Authors: Skøt L. , Sackville Hamilton N. R. , Mizen S. , Chorlton K. H. , Thomas I. D.
Published: March 2003

Improved winter hardiness is an important breeding objective in the forage grass Lolium perenne. This is a complex trait with several components, including the ability to survive and grow at low temperature, to acclimate to cold, tolerate wind, snow cover and ice encasement. Marker‐assisted selection has the potential to increase the efficiency of breeding for improved cold tolerance. Here we describe a genecological approach to identifying molecular markers that are associated with adaptation to low winter temperatures. AFLP was used to assess the genetic diversity in 29 wild populations of ryegrass (Lolium perenne) representing a pan‐European temperature cline in terms of their geographical origin. A further 18 populations from a temperature cline in Bulgaria were also analysed. In addition, two varieties and five populations representing parents of mapping families currently in use at IGER were included in the analysis. Principal coordinate (PCoA) and cluster analyses of the molecular marker data showed that the Bulgarian altitude cline populations could be distinguished clearly from the other populations. Two regression analyses were carried out; one to identify AFLP markers that correlated in frequency with low mean January temperature of the geographical origin of the population, and another to identify AFLP markers correlating in frequency with the cold tolerance phenotype of the populations, as determined by LT50 values in freezing tests. In the first analysis six AFLP markers showed significant type II trends with mean January temperature, and in the second analysis 28 bands had a significant univariate relationship with the LT50 value of the accessions. In steps 2 and 3 of the stepwise analysis a further 4 and 5 bands, respectively, improved the fit significantly. The results of the two types of regression analysis are discussed in relation to ecogeography and cold tolerance phenotype of the populations.
Chloroplast DNA variation, postglacial recolonization and hybridization in hazel, Corylus avellana
Authors: Palmé A. E. , G. G. Vendramin
Published: March 2003

To unravel the postglacial migration history of hazel, Corylus avellana, the genetic variation at two types of chloroplast DNA markers, polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) and microsatellites, was assessed in 26 natural hazel populations distributed across the range of C. avellana. In addition a sequence of 2468 base pairs, which contains the matK gene, was analysed in seven individuals. Very little variation was detected overall [hT:PCR‐RFLP= 0.091, hT:microsatellite= 0.423, π (nucleotide diversity) = 0.00093] but the microsatellite markers, which have the highest levels of variation, show a clear geographical structure that divides Europe into two areas: (i) Italy and the Balkans, on one hand and (ii) the rest of Europe, on the other hand. These data exclude Italy and the Balkans as possible origins of the postglacial recolonization but cannot unambiguously show which other area is the origin, since the genetic data does not indicate the direction of spread. If we take the pollen record into account, the most likely scenario would be an expansion from southwestern France into most of Europe except Italy and the Balkans, and then a local expansion in the latter area. The two main haplotypes identified with both PCR‐RFLP and sequencing, A and B, were found not only in C. avellana but also in other European Corylus species and cultivars. Haplotype A, which is dominating all investigated natural populations of C. avellana, is also found in the European tree hazel (C. colurna) and haplotype B, which is rare in C. avellana, has been identified in the filbert (C. maxima) and C. avellana cultivars. This pattern seems to indicate a history of past hybridization among the European Corylus species and cultivars.
Geographic patterns of population genetic structure inMytilocypris(Ostracoda: Cyprididae): interpreting breeding systems, gene flow and history in species with differing distributions
Authors: Finston Terrie
Published: March 2003

Samples from 83 populations of salt lake Ostracods belonging to the genusMytilocypriswere collected from 74 saline lakes and ponds in the semi‐arid regions of Australia. These populations were examined for variation at six polymorphic enzyme loci to diagnose breeding systems and to measure population structure, to investigate relative levels of gene flow in species with differing distributions and hence different presumed dispersal capabilities. Despite the occurrence of some populations in disjunct, peripheral, and recent ephemeral habitats, all populations of each species were found to reproduce sexually. Gene flow does occur on a local basis and appears to be facilitated by occupation in the same drainage basin for some species. There was considerable gene pool fragmentation among peripheral populations of four of the five species. Only one species,M. mytiloides, was relatively homogeneous across its range. It may be that gene flow is non‐existent into peripheral populations because of poor dispersal abilities, or it may not be frequent enough to overcome local selective pressures. Regardless of these possibilities, the observed gene pool fragmentation has implications for allopatric speciation.
Genetic differentiation in the winter pine processionary moth (Thaumetopoea pityocampa — wilkinsoni complex), inferred by AFLP and mitochondrial DNA markers
Authors: Salvato Paola , Battisti Andrea , Concato Silvia , Masutti Luigi , Patarnello Tomaso , Zane Lorenzo
Published: March 2003

The winter pine processionary moth has become an important pine pest in the last century, as a consequence of the spread of pine cultivation in the Mediterranean region. The pattern of genetic differentiation of this group, that includes two sibling species (Thaumetopoea pityocampa and Th. wilkinsoni), has been studied in nine populations using amplified fragment length polymorphism (AFLP) and single strand conformation polymorphism‐sequence analysis (SSCP) of the mitochondrial cytochrome oxidase 1 (COI) and cytochrome oxydase 2 (COII). Results indicate the existence of strong genetic differentiation between the two species that became separated before the Quaternary ice ages. Moreover data indicate that Th. pityocampa has a strong geographical structure, particularly evident at the nuclear level, where all pairwise φST resulted to be highly significant and individuals from the same population resulted to be strongly clustered when an individual tree was reconstructed. The estimates of the absolute number of migrants between populations (Nm), obtained from mitochondrial and nuclear DNA markers, suggest that gene flow is low and that a gender‐related dispersal could occur in this species. The males appear to disperse more than females, contributing to the genetic diversity of populations on a relatively wide range, reducing the risks of inbreeding and the genetic loss associated with bottlenecks occurring in isolated populations.
A novel DNA element that controls bacterial heat shock gene expression
Authors: Narberhaus Franz , Käser Roman , Nocker Andreas , Hennecke Hauke
Published: March 2003

The hspArpoH1 and hspBCdegP heat shock operons of Bradyrhizobium japonicum are preceded by a novel, conserved DNA element of approximately 100 bp, which is responsible for the temperature‐regulated transcription of their σ70‐type promoters. We designated this motif ROSE for repression of heat shock gene expression and found additional ROSE elements upstream of two newly identified heat shock operons. A critical core region in the hspA‐associated ROSE1 was defined by introducing insertions or deletions. While four mutants retained the ability to repress transcription of the hspArpoH1 operon, five deletion mutants produced elevated hspA mRNA levels under low‐temperature growth conditions. Derepression was confirmed by increased RpoH1 levels in non‐heat‐shocked cells from one of these mutants and by strains that contained a translational hspA–lacZ fusion associated with mutated ROSE1 elements. The hspArpoH1 operon was efficiently transcribed in vitro, and a deletion of ROSE1 did not impair this activity. Gel retardation experiments demonstrated that a protein in non‐heat‐shocked cells specifically binds to the intact ROSE1 element but not to a mutated element lacking the core region. Taken together, these results indicate that a central region of ROSE serves as a binding site for a repressor protein under standard growth conditions in order to prevent the undesired transcription of heat shock genes.
NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli
Authors: Blasco Francis , Dos Santos Jean‐Philippe , Magalon Axel , Frixon Chantal , Guigliarelli Bruno , Santini Claire‐Lise , Giordano Gérard
Published: March 2003

The formation of active membrane‐bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase. In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex. A significant activation of this complex was observed in vitro after adding purified NarJ‐6His polypeptide to the cell supernatant of a narJ strain. Once the apo‐enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro. NarJ protein specifically recognizes the catalytic NarG subunit. Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex. We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent‐open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme. Upon insertion of the molybdenum cofactor into the apo‐nitrate reductase, NarJ is then dissociated from the activated enzyme.
Hyperthermophiles and the problem of DNA instability
Authors: Grogan Dennis W.
Published: March 2003

Rates of chemical decomposition of DNA at the optimal growth temperatures of hyperthermophiles seem incongruent with the requirements of accurate genome replication. The peculiar physiology, ecology and phylogeny of hyperthermophiles combine to suggest that these prokaryotes have solved a molecular problem (spontaneous loss of native DNA structure) of a magnitude that well‐studied microorganisms do not face. The failure of DNA base composition to correlate with optimal growth temperature among hyperthermophiles provides indirect evidence that other mechanisms maintain their chromosomal DNA in the duplex form. Studies in vitro indicate that DNA primary structure is more difficult to maintain at extremely high temperature than is secondary structure, yet hyperthermophiles exhibit only modest levels of spontaneous mutation. Radiation sensitivity studies also indicate that hyperthermophiles repair their DNA efficiently in vivo, and underlying mechanisms are beginning to be examined. Several enzymes of DNA metabolism from hyperthermophilic archaea exhibit unusual biochemical features that may ultimately prove relevant to DNA repair. However, genomic sequencing results suggest that many DNA repair genes of hyperthermophilic archaea may not be recognized because they are not sufficiently related to those of well‐studied organisms.
A 45 bp inverted repeat is required for cell cycle regulation of the Escherichia coli nrd operon
Authors: Jacobson Blake A. , Fuchs James A.
Published: March 2003

Expression of β‐galactosidase from a nrd–lacZ fusion was used to determine the role in nrd regulation of an inverted sequence upstream of the promoter. Removal or replacement of a 45 bp inverted repeat with an altered sequence including a 48 bp perfect inverted repeat resulted in a mutant phenotype that was low in nrd expression in an exponentially growing culture and that did not increase during DNA synthesis inhibition. Changing the 22 bp in the upstream half of the inverted repeat resulted in the same phenotype, whereas changing the 22 bp in the downstream half of the inverted repeat decreased nrd expression to a lesser extent in an exponentially growing culture and had only a smaller effect on nrd expression during DNA synthesis inhibition. As other mutants with the phenotype of the upstream inverted repeat mutant were found to lack cell cycle regulation, expression of nrd–lac mRNA produced from a plasmid with this mutation in the nrd–lacZ fusion gene was compared with nrd mRNA produced from the chromosomal nrd gene in a synchronized culture. The results indicated that the upstream half of the nrd inverted repeat contains a cis‐acting element essential for nrd cell cycle regulation.
The cell wall porin of Nocardia farcinica: biochemical identification of the channel‐forming protein and biophysical characterization of the channel properties
Authors: Rieß Franziska G. , Lichtinger Thomas , Cseh Richard , Yassin Atteyet F. , Schaal Klaus P. , Benz Roland
Published: March 2003

A channel‐forming protein was identified in cell wall extracts of the Gram‐positive, strictly aerobic bacterium Nocardia farcinica. The cell wall porin was purified to homogeneity and had an apparent molecular mass of about 87 kDa on tricine‐containing SDS–PAGE. When the 87 kDa protein was boiled for a longer time in sodium dodecylsulphate (SDS) it dissociated into two subunits with molecular masses of about 19 and 23 kDa. The 87 kDa form of the protein was able to increase the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine (PC) phosphatidylserine (PS) mixtures by the formation of ion‐permeable channels. The channels had on average a single‐channel conductance of 3.0 nS in 1 M KCl, 10 mM Tris‐HCl, pH 8, and were found to be cation selective. Asymmetric addition of the cell wall porin to lipid bilayer membranes resulted in an asymmetric voltage dependence. The single‐channel conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated point charge effects on the channel properties. The analysis of the single‐channel conductance data in different salt solutions using the Renkin correction factor, and the effect of negative charges on channel conductance suggested that the diameter of the cell wall porin is about 1.4–1.6 nm. Channel‐forming properties of the cell wall porin of N. farcinica were compared with those of mycobacteria and corynebacteria. The cell wall porins of these members of the order Actinomycetales share common features because they form large and water‐filled channels that contain negative point charges.
MglA and MglB are required for the intramacrophage growth of Francisella novicida
Authors: Baron Gerald S. , Nano Francis E.
Published: March 2003

Francisella novicida is a facultative intracellular pathogen capable of growing in macrophages. A spontaneous mutant of F. novicida defective for growth in macrophages was isolated on LB media containing the chromogenic phosphatase substrate 5‐bromo‐4‐chloro‐3‐indolyl phosphate (X‐p) and designated GB2. Using an in cis complementation strategy, four strains were isolated that are restored for growth in macrophages. A locus isolated from one of these strains complements GB2 for both the intracellular growth defect and the colony morphology on LB (X‐p) media. The locus consists of an apparent operon of two genes, designated mglAB, for macrophage growth locus. Both mglA and mglB transposon insertion mutants are defective for intracellular growth and have a phenotype similar to GB2 on LB (X‐p) media. Sequencing of mglA cloned from GB2 identified a missense mutation, providing evidence that both mglA and mglB are required for the intramacrophage growth of F. novicida. mglB expression in GB2 was confirmed using antiserum against recombinant MglB. Cell fractionation studies revealed several differences in the protein profiles of mgl mutants compared with wild‐type F. novicida. The deduced amino acid sequences of mglA and mglB show similarity to the SspA and SspB proteins of Escherichia coli and Haemophilus spp. In E. coli, SspA and/or SspB influence the levels of multiple proteins under conditions of nutritional stress, and SspA can associate with the RNA polymerase holoenzyme. Taken together, these observations suggest that in Francisella MglA and MglB may affect the expression of genes whose products contribute to survival and growth within macrophages.
Coupled structure change of SecA and SecG revealed by the synthetic lethality of the secAcsR11 and ΔsecG::kan double mutant
Authors: Suzuki Hirofumi , Nishiyama Ken‐ichi , Tokuda Hajime
Published: March 2003

An Escherichia coli strain carrying either the secAcsR11 or ΔsecG::kan mutation is unable to grow at low temperature owing to cold‐sensitive protein translocation but grows normally at 37°C. However, introduction of the two mutations into the same cells caused a severe defect in protein translocation and the cells were unable to grow at any temperature examined, indicating that secG is essential for the secAcsR11 mutant. The mutant SecA (csSecA) was found to possess a single amino acid substitution in the precursor‐binding region and was defective in the interaction with the precursor protein. Furthermore, the membrane insertion of SecA and the membrane topology inversion of SecG, both of which took place upon the initiation of protein translocation, were significantly retarded even at 37°C, when csSecA was used instead of the wild‐type SecA. The insertion of the wild‐type SecA was also significantly defective when SecG‐depleted membrane vesicles were used in place of SecG‐containing ones. No insertion of csSecA occurred into SecG‐depleted membrane vesicles. Examination of in vitro protein translocation at 37°C revealed that SecG is essential for csSecA‐dependent protein translocation. We conclude that SecG and SecA undergo a coupled structure change, that is critical for efficient protein translocation.
Inhibition of a naturally occurring rolling‐circle replicon in derivatives of the theta‐replicating plasmid pIP501
Authors: Pujol Catherine , Chédin Frédéric , Ehrlich S. Dusko , Jannière Laurent
Published: March 2003

The mechanisms ensuring regulation of DNA replication in genomes containing multiple replicons are poorly understood. In this report, we addressed this question by analysing in Bacillus subtilis the replication of a derivative of the promiscuous plasmid pIP501 that carries a rolling‐circle and a theta replicon. Genetic analyses revealed that the rolling‐circle replicon is strongly inhibited in the derivative and that inhibition requires three elements involved in theta replication: the replication origin, the initiator RepR protein and strong transcription of the repR gene. Inhibition is, however, independent of DNA synthesis at the theta origin. We conclude that rolling‐circle inhibition is caused by an inhibitory signal encoded by the theta replicon and propose that the signal is composed, at least, of the RepR protein bound to its cognate origin.
Characterization of the essential cell division gene ftsL (yllD ) of Bacillus subtilis and its role in the assembly of the division apparatus
Authors: Daniel R. A. , Harry E. J. , Katis V. L. , Wake R. G. , Errington J.
Published: March 2003

We have identified the Bacillus subtilis homologue of the essential cell division gene, ftsL, of Escherichia coli. Repression of ftsL in a strain engineered to carry a conditional promoter results in cell filamentation, with a near immediate arrest of cell division. The filaments show no sign of invagination, indicating that division is blocked at an early stage. FtsL is also shown to be required for septation during sporulation, and depletion of FtsL blocks the activation but not the synthesis of the prespore‐specific sigma factor, σF. Immunofluorescence microscopy shows that depletion of FtsL has little or no effect on FtsZ ring formation, but the assembly of other division proteins, DivIB and DivIC, at the site of division is prevented. Repression of FtsL also results in a rapid loss of DivIC protein, indicating that DivIC stability is dependent on the presence of FtsL, in turn suggesting that FtsL is intrinsically unstable. The instability of one or more components of the division apparatus may be important for the cyclic assembly/disassembly of the division apparatus.
Evidence that a globular conformation is not compatible with FhaC‐mediated secretion of the Bordetella pertussis filamentous haemagglutinin
Authors: Guédin Sandrine , Willery Eve , Locht Camille , Jacob‐Dubuisson Françoise
Published: March 2003

The 220 kDa Bordetella pertussis filamentous haemagglutinin (FHA) is the major extracellular protein of this organism. It is exported using a signal peptide‐dependent pathway, and its secretion depends on one specific outer membrane accessory protein, FhaC. In this work, we have investigated the influence of conformation on the FhaC‐mediated secretion of FHA using an 80 kDa N‐terminal FHA derivative, Fha44. In contrast to many signal peptide‐dependent secretory proteins, no soluble periplasmic intermediate of Fha44 could be isolated. In addition, cell‐associated Fha44 synthesized in the absence of FhaC did not remain competent for extracellular secretion upon delayed expression of FhaC, indicating that the translocation steps across the cytoplasmic and the outer membrane might be coupled. A chimeric protein, in which the globular B subunit of the cholera toxin, CtxB, was fused at the C‐terminus of Fha44, was not secreted in B. pertussis or in Escherichia coli expressing FhaC. The hybrid protein was only secreted when both disulphide bond‐forming cysteines of CtxB were replaced by serines or when it was produced in DsbA−E. coli. The Fha44 portion of the secretion‐incompetent hybrid protein was partly exposed on the cell surface. These results argue that the Fha44–CtxB hybrid protein transited through the periplasmic space, where disulphide bond formation is specifically catalysed, and that secretion across the outer membrane was initiated. The folded CtxB portion prevented extracellular release of the hybrid, in contrast to the more flexible CtxB domain devoid of cysteines. We propose a secretion model whereby Fha44 transits through the periplasmic space on its way to the cell surface and initiates its translocation through the outer membrane before being released from the cytoplasmic membrane. Coupling of Fha44 translocation across both membranes would delay the acquisition of its folded structure until the protein emerges from the outer membrane. Such a model would be consistent with the extensive intracellular proteolysis of FHA derivatives in B. pertussis.
Identification of an amino‐terminal substrate‐binding domain in the Yersinia tyrosine phosphatase that is required for efficient recognition of focal adhesion targets
Authors: Black Deborah S. , Montagna Lee G. , Zitsmann Sabine , Bliska James B.
Published: March 2003

YopH is a protein tyrosine phosphatase (PTP) that is delivered into host mammalian cells via a type III secretion pathway in pathogenic Yersinia species. Although YopH is a highly active PTP, it preferentially targets a subset of tyrosine‐phosphorylated proteins in host cells, including p130Cas. Previous in vitro studies have indicated that the carboxy‐terminal PTP domain contributes specificity to the interaction of YopH with substrates. However, it is not known if the PTP domain is sufficient for substrate recognition by YopH. Here, we have identified paxillin as an additional substrate of YopH in HeLa cells. In addition, we have identified a domain in the amino‐terminal region of YopH that binds to both p130Cas and paxillin and is required for the efficient recognition of substrates by the wild‐type enzyme. This ‘substrate‐binding’ domain exhibits a ligand specificity that is similar to that of the Crk Src homology 2 (SH2) domain, and it binds substrates directly in a phosphotyrosine‐dependent manner. The substrate‐binding domain of YopH may represent a novel type of protein–protein interaction module, as it lacks significant sequence similarity with any known SH2 or phosphotyrosine‐binding (PTB) domain.
Integration of the quorum‐sensing system in the regulatory networks controlling virulence factor synthesis in Erwinia chrysanthemi
Authors: Reverchon Sylvie , Bouillant Marie Louise , Salmond George , Nasser William
Published: March 2003

The expI–expR locus drives a quorum‐sensing system in the phytopathogenic bacterium, Erwinia chrysanthemi. Purified ExpR, an N‐acyl homoserine lactone‐responsive regulatory protein, binds to the promoter/operator region of the expI and expR genes. DNase I footprinting experiments showed that ExpR protects the regions between −66 and −40 from the P1 transcription initiation site of expI and between −54 and −18 from the expR transcription initiation site P1. The protected region overlaps the two expR promoters, P1 and P2, suggesting that ExpR exerts a negative control on its own gene expression. This assertion is reinforced by the fact that the addition of OHHL dissociates the ExpR–expR DNA complex. In contrast, the location of the ExpR binding site on the expI gene suggests an activator function, as reported for the pel genes. Moreover, ExpR is able to induce DNA bending. In vivo and in vitro studies revealed that CRP functions as an activator of expR expression, but as a repressor of expI transcription. A second level of control of expR and expI occurs through the PecS repressor, a regulator of pectinase synthesis. PecS represses expI expression, while ExpR activates pecS transcription, suggesting the existence of a mutual control between pecS and the expI–expR system in E. chrysanthemi. Regulation of pectinase synthesis in soft rot Erwinia appears to be a complex network of multiple cross‐acting regulatory elements. A model that integrates these regulatory elements is proposed.
In vivo analysis of the interactions of the LysR‐like regulator SpvR with the operator sequences of the spvA and spvR virulence genes of Salmonella typhimurium
Authors: Sheehan Brian J. , Dorman Charles J.
Published: March 2003

The interaction of the Salmonella typhimurium virulence gene regulator, SpvR, with its operator sites upstream of the spvA and spvR genes was analysed in vivo by dimethyl sulphate (DMS) footprinting and site‐directed mutagenesis. DMS methylation protection assays showed that, in vivo, SpvR forms direct protein–DNA contacts with nucleotides clustered in two regions (+1 to −27 and −51 to −71) of the spvA regulatory region. These regions were subjected to site‐directed mutagenesis and the effects on SpvR binding and gene activation assessed. Mutations that prevented occupancy of the promoter distal site (−51 to −71) in vivo also prevented occupancy of the promoter proximal site (+1 to −27), whereas mutations in the proximal site affected binding only at the proximal site and not the distal site. SpvR binding at the promoter proximal site was an essential prerequisite for transcription activation. These findings demonstrated a hierarchy of SpvR binding in which the promoter distal site is dominant to the proximal. The spvR gene was found to possess an operator site that resembled closely the distal SpvR binding site of the spvA operator. Nonetheless, SpvR interaction with the spvR operator was difficult to detect in vivo. When the nucleotide sequence of the spvR operator was altered at two nucleotides so that it corresponded more precisely to that of the distal site of the spvA operator, strong SpvR–DNA interactions were detected, with nucleotides in the region −31 to −67 being protected from DMS methylation in vivo. However, despite the improved interaction with the transcriptional activator, the altered regulatory region was poorer at promoting spvR gene transcription than the wild type. We describe a two‐step model for activation of the spvA promoter and discuss the possibility that a specific cofactor in addition to sigma factor RpoS is required for SpvR action at this promoter in vivo.
Macrophage‐dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival
Authors: Cirillo Daniela Maria , Valdivia Raphael H. , Monack Denise M. , Falkow Stanley
Published: March 2003

Salmonella pathogenicity island 2 (SPI‐2) encodes a putative type III secretion system necessary for systemic infection in animals. We have investigated the transcriptional organization and regulation of SPI‐2 by creating gfp fusions throughout the entire gene cluster. These gfp fusions demonstrated that SPI‐2 genes encoding structural, regulatory and previously uncharacterized putative secreted proteins are preferentially expressed in the intracellular environment of the host macrophage. Furthermore, the transcription of these genes within host cells was dependent on the two‐component regulatory system SsrA/SsrB and an acidic phagosomal environment. Most SPI‐2 mutants failed to replicate to the same level as wild‐type strains in murine macrophages and human epithelial cells. In orally infected mice, SPI‐2 mutants colonized the Peyer's patches but did not progress to the mesenteric lymph nodes. We conclude that SPI‐2 genes are specifically expressed upon entry into mammalian cells and are required for intracellular growth in host cells in vivo and in vitro.
SOS and Mayday: multiple inducible mutagenic pathways in Escherichia coli
Authors: Humayun M. Zafri
Published: March 2003

Environmental and physiological stress conditions can transiently alter the fidelity of DNA replication. The DNA damage‐mediated SOS response in Escherichia coli is the best‐known example of such an ‘inducible mutagenesis’ or ‘transient mutator’ pathway. Emerging evidence suggests the existence of a number of other stress‐inducible pathways that also affect the fidelity of replication. Among the more provocative recent findings are UVM, an SOS‐independent damage‐inducible mutagenic pathway, and a new recA‐dependent but umuD/C‐independent pathway that appears to be provoked by translational stress. These findings alter our view of inducible mutagenesis, and anticipate the existence of previously unrecognized links between protein synthesis and DNA replication.
The cytoplasmic kinase domain of PhoR is sufficient for the low phosphate‐inducible expression of Pho regulon genes in Bacillus subtilis
Authors: Shi Lei , Hulett F. Marion
Published: March 2003

PhoP–PhoR, one of three two‐component systems known to be required to regulate the pho regulon in Bacillus subtilis, directly regulates the alkaline phosphatase genes that are used as pho reporters. Biochemical studies showed that B. subtilis PhoR, purified from Escherichia coli, was autophosphorylated in vitro in the presence of ATP. Phosphorylated PhoR showed stability under basic conditions but not acidic conditions, indicating that the phosphorylation probably occurs on a conserved histidine residue. Phospho–PhoR phosphorylated its cognate response regulator, PhoP in vitro. B. subtilis phoR was placed in the Bacillus chromosome under the control of the Pspac promoter, which is IPTG inducible. The wild‐type phoR, under either native promoter or Pspac promoter with IPTG induction, resulted in a similar level of alkaline phosphatase production. Under high phosphate conditions, strains containing wild‐type phoR, or phoR mutant gene products that lacked either the periplasmic domain, or both N‐terminal transmembrane PhoR sequences or various extended N‐terminal sequences, showed no significant APase production. Under phosphate starvation conditions, in the presence of IPTG, all strains containing mutated phoR genes showed alkaline phosphatase induction patterns similar to that of the wild‐type strain, although the fully induced level was lower in the mutants. The decrease in total alkaline phosphatase production in these mutant strains can be compensated completely or partially by increasing the copy number of the mutant phoR gene. These in vivo results suggest that the C‐terminal kinase domain of PhoR is sufficient for the induction of alkaline phosphatase expression under phosphate‐limited conditions, and that the regulation for repression of APase under phosphate‐replete conditions remains intact.
A co‐activator of nitrogen‐regulated transcription in Saccharomyces cerevisiae
Authors: Soussi‐Boudekou Saïd , André Bruno
Published: March 2003

In Saccharomyces cerevisiae, the transcription factors Gln3p and Nil1p of the GATA family play a determinant role in expression of genes that are subject to nitrogen catabolite repression. Here we report the isolation of a new yeast mutant, gan1‐1, exhibiting dramatically decreased NAD‐linked glutamate dehydrogenase (NAD‐GDH) and glutamine synthetase (GS) activities. The GAN1 gene was cloned and found to encode a 488‐amino‐acid polypeptide bearing no typical DNA binding domain. Gan1p is required for full expression of GLN1, GDH2 and also other nitrogen utilization genes, including GAP1, PUT4, MEP2 and GDH1. The extent to which Gan1p is required, however, varies according to the gene and to the nitrogen source available. We show that Gan1p is in fact involved in Gln3p‐ and Nil1p‐dependent transcription. In the case of Gln3p‐dependent transcription, the degree to which Gan1p is required appears to be gene specific. The contribution of Gan1p to gene expression is also influenced by the nitrogen status of the cell. We found that GAN1 is identical to ADA1, which encodes a component of the ADA/GCN5 co‐activator complex. Ada1/Gan1p thus represents the first reported case of an accessory protein (a co‐activator) linking the GATA‐binding proteins Gln3p and Nil1p, mediating nitrogen‐regulated transcription, to the basal transcription machinery.
Expression and properties of an aerolysin–Clostridium septicum alpha toxin hybrid protein
Authors: Diep Dzung B. , Nelson Kim L. , Lawrence Tracy S. , Sellman Bret R. , Tweten Rodney K. , Buckley J. Thomas
Published: March 2003

Aerolysin is a bilobal channel‐forming toxin secreted by Aeromonas hydrophila. The alpha toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosylphosphatidylinositol‐anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.
The WD protein Cpc2p is required for repression of Gcn4 protein activity in yeast in the absence of amino‐acid starvation
Authors: Hoffmann Bernd , Mösch Hans‐Ulrich , Sattlegger Evelyn , Barthelmess Ilse Babette , Hinnebusch Alan , Braus Gerhard H.
Published: March 2003

The CPC2 gene of the budding yeast Saccharomyces cerevisiae encodes a Gβ‐like WD protein which is involved in regulating the activity of the general control activator Gcn4p. The CPC2 gene encodes a premRNA which is spliced and constitutively expressed in the presence or absence of amino acids. Loss of CPC2 gene function suppresses a deletion of the GCN2 gene encoding the general control sensor kinase, but not a deletion in the GCN4 gene. The resulting phenotype has resistance against amino‐acid analogues. The Neurospora crassa cpc‐2 and the rat RACK1 genes are homologues of CPC2 that complement the yeast cpc2 deletion. The cpc2Δ mutation leads to increased transcription of Gcn4p‐dependent genes under non‐starvation conditions without increasing GCN4 expression or the DNA binding activity of Gcn4p. Cpc2p‐mediated transcriptional repression requires the Gcn4p transcriptional activator and a Gcn4p recognition element in the target promoter. Frameshift mutations resulting in a shortened Gβ‐like protein cause a different phenotype that has sensitivity against amino‐acid analogues similar to a gcn2 deletion. Cpc2p seems to be part of an additional control of Gcn4p activity, independent of its translational regulation.
Expression of different group A streptococcal M proteins in an isogenic background demonstrates diversity in adherence to and invasion of eukaryotic cells
Authors: Berkower Carol , Ravins Miriam , Moses Allon E. , Hanski Emanuel
Published: March 2003

The M protein of group A streptococcus (GAS) is considered to be a major virulence factor because it renders GAS resistant to phagocytosis and allows bacterial growth in human blood. There are more than 80 known serotypes of M proteins, and protective opsonic antibodies produced during disease in humans are serotype specific. M proteins also mediate bacterial adherence to epithelial cells of skin and pharynx. GAS strains vary in the genomic organization of the mga regulon, which contains the genes encoding M and M‐like proteins and other virulence factors. This diversity of organization makes it difficult to assess virulence of M proteins of different serotypes, unless they can be expressed in an isogenic background. Here, we express M proteins of different serotypes in the M protein‐ and protein F1‐deficient GAS strain, SAM2, which also lacks M‐like proteins. Genes encoding M proteins of different serotypes (emmXs) have been integrated into the SAM2 chromosome in frame with the emm6.1 promoter and its mga regulon, resulting in similar levels of emmX expression. Although SAM2 exhibits a very low level of adherence to and invasion of HEp‐2 and HaCaT cells, a SAM2‐derived strain expressing M6 protein adheres to and invades both cell types. In contrast, the isogenic strain expressing M18 protein adheres to both cell types, but invades with a very low efficiency. A strain expressing M3 protein adheres to both types of cells, but its invasion of HEp‐2 cells is serum dependent. A GAS strain expressing M6 protein does not compete with the isogenic strain expressing M18 protein for adherence to or invasion of HaCaT cells. We conclude that M proteins of different serotypes recognize different repertoires of receptors on the surfaces of eukaryotic cells.