bluuebunny/crossref_metadata_2025 huggingface dataset:
Confirmation of low genetic diversity and multiple breeding females in a social group of Eurasian badgers from microsatellite and field data
Authors: Domingo‐Roura X. , Macdonald D. W. , Roy M. S. , Marmi J. , Terradas J. , Woodroffe R. , Burke T. , Wayne R. K.
Published: March 2003

The Eurasian badger (Meles meles) is a facultatively social carnivore that shows only rudimentary co‐operative behaviour and a poorly defined social hierarchy. Behavioural evidence and limited genetic data have suggested that more than one female may breed in a social group. We combine pregnancy detection by ultrasound and microsatellite locus scores from a well‐studied badger population from Wytham Woods, Oxfordshire, UK, to demonstrate that multiple females reproduce within a social group. We found that at least three of seven potential mothers reproduced in a group that contained 11 reproductive age females and nine offspring. Twelve primers showed variability across the species range and only five of these were variable in Wytham. The microsatellites showed a reduced repeat number, a significantly higher number of nonperfect repeats, and moderate heterozygosity levels in Wytham. The high frequency of imperfect repeats and demographic phenomena might be responsible for the reduced levels of variability observed in the badger.
Widespread occurrence of a domestic dog mitochondrial DNA haplotype in southeastern US coyotes
Authors: Adams J. R. , Leonard J. A. , Waits L. P.
Published: March 2003

Sequence analysis of the mitochondrial DNA control region from 112 southeastern US coyotes (Canis latrans) revealed 12 individuals with a haplotype closely related to those in domestic dogs. Phylogenetic analyses grouped this new haplotype in the dog/grey wolf (Canis familiaris/Canis lupus) clade with 98% bootstrap support. These results demonstrate that a male coyote hybridized with a female dog, and female hybrid offspring successfully integrated into the coyote population. The widespread distribution of this haplotype from Florida to West Virginia suggests that the hybridization event occurred long ago before the southeastern USA was colonized by coyotes. However, it could have occurred in the southeastern USA before the main front of coyotes arrived in the area between male coyotes released for sport and a local domestic dog. The introgression of domestic dog genes into the southeastern coyote population does not appear to have substantially affected the coyote's genetic, morphological, or behavioural integrity. However, our results suggest that, contrary to previous reports, hybridization can occur between domestic and wild canids, even when the latter is relatively abundant. Therefore, hybridization may be a greater threat to the persistence of wild canid populations than previously thought.
Mitochondrial haplotype distribution, seed dispersal and patterns of postglacial expansion of ponderosa pine
Authors: Johansen A. D. , Latta R. G.
Published: March 2003

Maternally inherited mtDNA in a secondary contact zone of ponderosa pine revealed a cline less than 10 km wide — much narrower than previously described. A survey of 76 populations gave no evidence either of intermixing or of a mosaic contact zone. Such sharp contact zones are consistent with diffusive range expansion, rather than long distance colonization. However, evidence for long distance seed dispersal events was found in two populations where haplotypes were observed far from their main area of occurrence. The results suggest a small number of long distance colonists with diffusive dispersal from these centres.
Mitochondrial phylogeography of the Woodmouse (Apodemus sylvaticus) in the Western Palearctic region
Authors: Michaux J. R. , Magnanou E. , Paradis E. , Nieberding C. , Libois R.
Published: March 2003

We sequenced 965 base pairs of the mitochondrial DNA cytochrome b from 102 woodmice (Apodemus sylvaticus) collected from 40 European localities. The aims of the study were to answer the following questions. (i) Did the Mediterranean peninsulas play a role as refuge for woodmice? (ii) Is genetic variability of A. sylvaticus higher in the Mediterranean region compared with northern Europe? (iii) Are the patterns of the postglacial colonization of Europe by woodmice similar to those presently recognized for other European species? The results provide a clear picture of the impact of the Quaternary glaciations on the genetic and geographical structure of the woodmouse. Our analyses indicate a higher genetic variability of woodmice in the Mediterranean peninsulas compared to northern Europe, suggesting a role of the former as refuge regions for this small mammal. An original pattern of postglacial colonization is proposed where the Iberian and southern France refuge populations colonized almost all European regions. The Sicilian population appears to be very differentiated and highly variable. This emphasizes the importance of this island as a ‘hot spot’ for the intraspecific genetic diversity of the woodmouse. Finally, woodmice in North Africa originated from southwestern Europe, most probably as a result of a recent anthropogenic introduction.
Genetic divergence between cave and surface populations of Astyanax in Mexico (Characidae, Teleostei)
Authors: Strecker U. , Bernatchez L. , Wilkens H.
Published: March 2003

A study of genetic diversity at microsatellite loci and the mitochondrial DNA (mtDNA) cytochrome b gene was carried out to assess genetic relationships among four Mexican cave (Pachon, Sabinos, Tinaja, Chica) and four surface populations of Astyanax fasciatus (Characidae) from northeast Mexico and the Yucatan. With the exception of Chica, the cave populations were all characterized by extremely low microsatellite variability, which most likely resulted from bottleneck events. Population analyses of the microsatellite data indicated no measurable levels of gene flow between all cave and surface populations (FST > 0.0707). Phylogenetic analyses of mtDNA data showed that only two cave populations — Sabinos and Tinaja — group together to the exclusion of surface populations. From the microsatellite data these cave populations cluster with the Pachon cave fish population. The mtDNA thus appears to have been replaced in Pachon because of introgressive hybridization. It is likely that these three cave populations have descended from a surface ancestor in common with current surface populations, rather than evolving recently from one of the extant surface populations. Like Pachon, the Chica population clustered with the surface populations according to mtDNA data, but was not clearly associated with either the surface or the other cave populations according to the microsatellite data. Our data indicate that the Chica population evolved recently from a surface population, and subsequently hybridized with a phylogenetically older cave population. In conclusion, both the microsatellite and mtDNA data suggest multiple origins of cave populations and the Chica and Sabinos/Tinaja/Pachon were founded after at least two independent invasions from surface populations.
Pollen dispersal of tropical trees (Dinizia excelsa: Fabaceae) by native insects and African honeybees in pristine and fragmented Amazonian rainforest
Authors: Dick Christopher W. , Etchelecu Gabriela , Austerlitz Frédéric
Published: March 2003

Tropical rainforest trees typically occur in low population densities and rely on animals for cross‐pollination. It is of conservation interest therefore to understand how rainforest fragmentation may alter the pollination and breeding structure of remnant trees. Previous studies of the Amazonian tree Dinizia excelsa (Fabaceae) found African honeybees (Apis mellifera scutellata) as the predominant pollinators of trees in highly disturbed habitats, transporting pollen up to 3.2 km between pasture trees. Here, using microsatellite genotypes of seed arrays, we compare outcrossing rates and pollen dispersal distances of (i) remnant D. excelsa in three large ranches, and (ii) a population in undisturbed forest in which African honeybees were absent. Self‐fertilization was more frequent in the disturbed habitats (14%, n = 277 seeds from 12 mothers) than in undisturbed forest (10%, n = 295 seeds from 13 mothers). Pollen dispersal was extensive in all three ranches compared to undisturbed forest, however. Using a twogener analysis, we estimated a mean pollen dispersal distance of 1509 m in Colosso ranch, assuming an exponential dispersal function, and 212 m in undisturbed forest. The low effective density of D. excelsa in undisturbed forest (∼0.1 trees/ha) indicates that large areas of rainforest must be preserved to maintain minimum viable populations. Our results also suggest, however, that in highly disturbed habitats Apis mellifera may expand genetic neighbourhood areas, thereby linking fragmented and continuous forest populations.
Inbreeding depression influences genet size distribution in a marine angiosperm
Authors: Hämmerli A. , Reusch T. B. H.
Published: March 2003

Although inbreeding depression is a major genetic phenomena influencing individual fitness, it is difficult to measure in wild populations. An alternative approach is to correlate heterozygosity, measured using highly polymorphic markers, with a fitness‐correlated trait. In clonal plants, genet size is predicted to be fitness correlated. Here we test the prediction that the genet size distribution of the marine clonal plant Zostera marina (eelgrass) is influenced by inbreeding depression. We used nine polymorphic microsatellite markers to access the fine scale clonal structure and to measure individual heterozygosity within 4 plots (each corresponds to 256 m2, sampled at 1‐m intervals) in two populations along the German Baltic Coast. The same plots were also sampled for flowering and vegetative shoots to obtain estimates for sexual reproductive output at the level of the genetic individual. We found substantial differences in the genet size distribution between the two populations that may be explained by different disturbance frequency. In both populations, clone size was significantly positively correlated with the total number of flowering shoots, indicating that larger clones have a higher reproductive output. Individual heterozygosity was significantly positively associated with clone size. The effect was much stronger in Falkenstein (low disturbance) than in Maasholm (high disturbance). The results indicate that in a low disturbance population the relatively outbred clones occupy a higher proportion of the available space, possibly because they outcompete relatively inbred neighbours.
Differences in chromosome number and genome rearrangements in the genus Brucella
Authors: Jumas‐Bilak Estelle , Michaux‐Charachon Sylvie , Bourg Gisèle , O'Callaghan David , Ramuz Michel
Published: March 2003

We have studied the genomic structure and constructed the SpeI, PacI and I‐CeuI restriction maps of the four biovars of the pathogenic bacterium Brucella suis. B. suis biovar 1 has two chromosomes of 2.1 Mb and 1.15 Mb, similar to those of the other Brucella species: B. melitensis, B. abortus, B. ovis and B. neotomae. Two chromosomes were also observed in the genome of B. suis biovars 2 and 4, but with sizes of 1.85 Mb and 1.35 Mb, whereas only one chromosome with a size of 3.1 Mb was found in B. suis biovar 3. We show that the differences in chromosome size and number can be explained by rearrangements at chromosomal regions containing the three rrn genes. The location and orientation of these genes confirmed that these rearrangements are due to homologous recombination at the rrn loci. This observation allows us to propose a scheme for the evolution of the genus Brucella in which the two chromosome‐containing strains can emerge from an hypothetical ancestor with a single chromosome, which is probably similar to that of B. suis biovar 3. As the genus Brucella is certainly monospecific, this is the first time that differences in chromosome number have been observed in strains of the same bacterial species.
Using the yeast two‐hybrid system to identify human epithelial cell proteins that bind gonococcal Opa proteins: intracellular gonococci bind pyruvate kinase via their Opa proteins and require host pyruvate for growth
Authors: Williams John M. , Chen Gi‐Chung , Zhu Li , Rest Richard F.
Published: March 2003

Neisseria gonorrhoeae opacity‐associated (Opa) proteins are a family of outer membrane proteins involved in gonococcal adherence to and invasion of human cells. We wanted to identify additional roles for Opa in the infectious process and used the yeast two‐hybrid system to identify human epithelial cell proteins that interact with Opa proteins. Although this system has been used successfully to identify many types of interacting proteins, it has not been used to screen a human cell cDNA library for binding partners of a prokaryotic outer membrane protein. Therefore, we were also interested in exploring the versatility of the yeast two‐hybrid system in identifying bacteria–host interactions. Using OpaP from strain F62SF as bait, we screened a HeLa cell cDNA library for Opa‐interacting proteins (OIPs). We identified five different OIPs, designated OIP1–OIP5, two of which are homologous to human proteins — thyroid hormone receptor interacting protein (TRIP6) and pyruvate kinase isoenzyme M2 (PK). In the studies presented here, we investigated the interaction between Opa proteins and PK in more depth. Opa–PK interactions were confirmed by in vitro and in vivo assays independent of the yeast two‐hybrid system. Escherichia coli expressing six different Opa proteins from gonococcal strain FA1090 all bound more PK than Opa‐negative E. coli in in vitro binding assays. Using anti‐PK antibody and fluorescence microscopy, we showed that human epithelial cell PK co‐localizes with intracellular Opa+ gonococci and E. coli expressing Opa proteins. Using a mutant of N. gonorrhoeae unable to grow on pyruvate or lactate, it appears that intracellular pyruvate is essential for gonococcal growth and survival. These results suggest a novel mechanism in bacterial pathogenesis, i.e. the requirement for direct molecular interaction with a host metabolic enzyme (PK) for the acquisition of an essential intracellular carbon source and growth substrate (pyruvate). These results demonstrate that the yeast two‐hybrid system is a valuable tool for identifying biologically relevant interactions between bacteria and host proteins, providing valuable leads for further investigations into novel mechanisms of bacterial pathogenesis.
Tn4451 from Clostridium perfringens is a mobilizable transposon that encodes the functional Mob protein, TnpZ
Authors: Crellin Paul K. , Rood Julian I.
Published: March 2003

The 6.3 kb Clostridium perfringens transposon Tn4451 encodes a 50 kDa protein, TnpZ, which has amino acid sequence similarity to a group of plasmid mobilization and recombination proteins that comprise the Mob/Pre family. Members of this family interact with an upstream palindromic sequence called an RSA site, and an RSA‐like sequence has been identified upstream of the tnpZ gene. In Escherichia coli, in the presence of a chromosomally integrated derivative of the broad‐host‐range IncP plasmid, RP4, TnpZ was able to promote plasmid mobilization in cis and was able to function in trans to enable the mobilization of a co‐resident plasmid carrying an RSA site. It was also able to mediate the conjugative transfer of plasmids from E. coli to C. perfringens. Site‐directed mutagenesis of two bases within the RSA site resulted in a significant reduction in mobilization frequency, demonstrating that the RSA site is required for efficient plasmid mobilization. TnpZ is the only Mob/Pre protein known to be associated with a transposable genetic element, and Tn4451 is the first mobilizable but non‐self‐transmissible transposon to be identified from a Gram‐positive bacterium.
Targeting of Yersinia Yop proteins into the cytosol of HeLa cells: one‐step translocation of YopE across bacterial and eukaryotic membranes is dependent on SycE chaperone
Authors: Lee Vincent T. , Anderson Deborah M. , Schneewind Olaf
Published: March 2003

Pathogenic Yersiniae adhere to and kill macrophages by targeting some of their Yop proteins into the eukaryotic cytosol. There is debate about whether YopE targeting proceeds as a direct translocation of polypeptide between cells or in two distinct steps, each requiring specific signals for YopE secretion across the bacterial envelope and for translocation into the eukaryotic cytosol. Here, we used the selective solubilization of the eukaryotic plasma membrane with digitonin to measure Yop targeting during Yersinia infections of HeLa cells. YopE, YopH, YopM and YopN were found in the eukaryotic cytosol but not in the extracellular medium. When bound to SycE chaperone in the Yersinia cytoplasm, YopE residues 1–100 are necessary and sufficient for the targeting of hybrid neomycin phosphotransferase. Electron microscopic analysis failed to detect an extracellular intermediate of YopE targeting, suggesting a one‐step translocation mechanism.
Identification of an additional member of the secretin superfamily of proteins in Pseudomonas aeruginosa that is able to function in type II protein secretion
Authors: Martínez Alejandro , Ostrovsky Paula , Nunn David N.
Published: March 2003

Pseudomonas aeruginosa is a prolific exporter of virulence factors and contains three of the four protein secretion systems that have been described in Gram‐negative bacteria. The P. aeruginosa type II general secretory pathway (GSP) is used to export the largest number of proteins from this organism, including lipase, phospholipase C, alkaline phosphatase, exotoxin A, elastase and LasA. Although these exoproteins contain no sequence similarity, they are specifically and efficiently transported by the secretion apparatus. Bacterial homologues of XcpQ (GspD), the only outer membrane component of this system, have been proposed to play the role of gatekeeper, by presumably interacting and recognizing the exported substrates to allow their passage through the outer membrane. While determining the phenotype of non‐polar deletions in each of the xcp genes, we have shown that a deletion of the P. aeruginosa strain K xcpQ does not completely abolish protein secretion. As the proposed function of XcpQ should be requisite for secretion, we searched for additional factors that could carry out this role. A cosmid DNA library from a PAK strain deleted for xcpP‐Z was tested for its ability to increase protein secretion by screening for enhanced growth on lipid agar, a medium that selects for the secretion of lipase. In this manner, we have identified an XcpQ homologue, XqhA, that is solely responsible for the residual export observed in a ΔxcpQ strain, although it is not required for efficient secretion in wild‐type P. aeruginosa. We have also demonstrated that this protein is capable of recognizing all of the exoproteins of P. aeruginosa, arguing against the proposed role of members of the secretin family as determinants of specificity.
Characterization and splicing in vivo of a Sinorhizobium meliloti group II intron associated with particular insertion sequences of the IS630‐Tc1/IS3 retroposon superfamily
Authors: Martínez‐Abarca Francisco , Zekri Sanae , Toro Nicolás
Published: March 2003

By sequence analysis of Sinorhizobium meliloti strain GR4 plasmid pRmeGR4b, we have identified a group II intron named RmInt1 inserted within the insertion sequence ISRm2011‐2 of the IS630‐Tc1/IS3 retroposon superfamily. Like some other group II introns, RmInt1 possesses, in addition to the structurally conserved ribozyme core, an open reading frame (ORF) with homology to reverse transcriptases. Using a T7 expression system in Escherichia coli, we show that the intron is active in splicing in vivo and that splicing efficiency requires the intron‐encoded ORF, which suggests that the putative intron encoded protein has a maturase function. DNA hybridization studies indicate that intron RmInt1 is widespread within S. meliloti native populations and appears to be mostly located within this IS element. Nevertheless, some S. meliloti strains harbour one copy of RmInt1 at a different location. DNA sequence analysis of the 5′ exon of one of these heterologous intron insertion sites revealed the presence of a putative IS element closely related to insertion sequence ISRm2011‐2. The intron‐binding sites (IBS1 and IBS2 motifs) are conserved, although a transition of a G→A in the IBS1 has occurred. Our results demonstrate an association of intron RmInt1 with particular insertion sequences of the IS630‐Tc1/IS3 retroposon superfamily that may have ensured the spread and maintenance of this group II intron in S. meliloti.
A nonameric core sequence is required upstream of the LYS genes of Saccharomyces cerevisiae for Lys14p‐mediated activation and apparent repression by lysine
Authors: Becker Bénédicte , Feller André , El Alami Mohamed , Dubois Evelyne , Piérard André
Published: March 2003

The expression of the structural genes for lysine (LYS) biosynthesis is controlled by a pathway‐specific regulation mediated by the transcriptional activator Lys14 in the presence of α‐aminoadipate semialdehyde, an intermediate of the pathway acting as a co‐inducer. Owing to end product inhibition of the first step of the pathway, excess lysine reduces the production of the co‐inducer and causes apparent repression of the LYS genes. Analysis of LYS promoters and insertions within an heterologous reporter gene have allowed the characterization of an upstream activating element (UASLYS) able to confer Lys14‐ and α‐aminoadipate semialdehyde‐dependent activation as well as apparent repression by lysine to another yeast gene. This DNA motif is present as one or several copies in the promoters of at least six LYS genes. The consensus sequence derived from the comparison of the UASLYS showing the highest activation capacities comprises the nonameric core sequence TCCRNYGGA. The RNY sequence of the 3 bp spacer as well as the presence of flanking AT‐rich regions on both sides of the core sequence appear essential for optimal activation. Further evidence that this element is the target of Lys14p was provided by the demonstration that Lys14p binds to UASLYSin vitro. The binding is independent of the presence of the co‐inducer and is not affected by lysine. It depends on the integrity of the putative Zn(II)2Cys6 binuclear cluster contained in the Lys14p.
The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus
Authors: Lindsay Jodi A. , Ruzin Alexey , Ross Hope F. , Kurepina Natasha , Novick Richard P.
Published: March 2003

Tst, the gene for toxic shock syndrome toxin‐1 (TSST‐1), is part of a 15.2 kb genetic element in Staphylococcus aureus that is absent in TSST‐1‐negative strains. The prototype, in RN4282, is flanked by a 17 nucleotide direct repeat and contains genes for a second possible superantigen toxin, a Dichelobacter nodosus VapE homologue and a putative integrase. It is readily transferred to a recA− recipient, and it always inserts into a unique chromosomal copy of the 17 nucleotide sequence in the same orientation. It is excised and circularized by staphylococcal phages φ13 and 80α and replicates during the growth of the latter, which transduces it at very high frequency. Because of its site and orientation specificity and because it lacks other identifiable phage‐like genes, we consider it to be a pathogenicity island (PI) rather than a transposon or a defective phage. The tst element in RN4282, near tyrB, is designated SaPI1. That in RN3984 in the trp region is only partially homologous to SaPI1 and is excised by phage 80 but not by 80α. It is designated SaPI2. These PIs are the first in any Gram‐positive species and the first for which mobility has been demonstrated. Their mobility may be responsible for the spread of TSST‐1 production among S. aureus strains.
Archaea and the cell cycle
Authors: Bernander Rolf
Published: March 2003

Sequence similarity data suggest that archaeal chromosome replication is eukaryotic in character. Putative nucleoid‐processing proteins display similarities to both eukaryotic and bacterial counterparts, whereas cell division may occur through a predominantly bacterial mechanism. Insights into the organization of the archaeal cell cycle are therefore of interest, not only for understanding archaeal biology, but also for investigating how components from the other two domains interact and work in concert within the same cell; in addition, archaea may have the potential to provide insights into eukaryotic initiation of chromosome replication.
The RcsB–RcsC regulatory system of Salmonella typhi differentially modulates the expression of invasion proteins, flagellin and Vi antigen in response to osmolarity
Authors: Arricau Nathalie , Hermant Daniel , Waxin Hervé , Ecobichon Chantal , Duffey Paul S. , Popoff Michel Y.
Published: March 2003

Entry into intestinal epithelial cells is an essential feature in the pathogenicity of Salmonella typhi, which causes typhoid fever in humans. This process requires intact motility and secretion of the invasion‐promoting Sip proteins, which are targets of the type III secretion machinery encoded by the inv, spa and prg loci. During our investigations into the entry of S. typhi into cultured epithelial cells, we observed that the secretion of Sip proteins and flagellin was impaired in Vi‐expressing strains. We report here that the production of Sip proteins, flagellin and Vi antigen is differentially modulated by the RcsB–RcsC regulatory system and osmolarity. This regulation occurs at both transcriptional and post‐translational levels. Under low‐osmolarity conditions, the transcription of iagA, invF and sipB genes is negatively controlled by the RcsB regulator, which probably acts in association with the viaB locus‐encoded TviA protein. The cell surface‐associated Vi polysaccharide, which was maximally produced under these growth conditions, prevented the secretion of Sip proteins and flagellin. As the NaCl concentration in the growth medium was increased, transcription of iagA, invF and sipB was found to be markedly increased, whereas transcription of genes involved in Vi antigen biosynthesis was greatly reduced. The expression of iagA, whose product is involved in invF and sipB transcription, occurred selectively during the exponential growth phase and was maximal in the presence of 300 mM NaCl. At this osmolarity, large amounts of Sips and flagellin were secreted in culture supernatants. As expected from these results, and given the essential role of Sip proteins and motility in entry, RcsB and osmolarity modulated the invasive capacity of S. typhi. Together, these findings might reflect the adaptive response of S. typhi to the environments encountered during the different stages of pathogenesis.
Crl stimulates RpoS activity during stationary phase
Authors: Pratt Leslie A. , Silhavy Thomas J.
Published: March 2003

RpoS, an alternative primary sigma factor, has been shown to be regulated at multiple levels, including transcription, translation and protein stability. Here, we present evidence that suggests that RpoS is regulated at yet another level by the product of the crl gene. The crl gene was first thought to encode the major curlin subunit of curli (curli are surface structures that are induced by growth into stationary phase under conditions of low osmolarity and low temperature). Later, it was determined that crl actually contributes in a positive fashion to stimulate transcription of csgBA, the true locus encoding for the major subunit of curli. RpoS is also required for normal stationary‐phase induction of csgBA. We found that lesions in crl, like lesions in rpoS, cause increased transcription of ompF during stationary phase. Taken together, these observations prompted us to analyse the effects of crl on an additional RpoS‐regulated phenomenon. We found that a crl null allele influences expression of RpoS‐regulated genes in a fashion similar to an rpoS null allele. Genetic evidence suggests that crl and rpoS function in a single pathway and that Crl functions upstream, or in concert with, RpoS. Although the effects of Crl on RpoS‐regulated genes is entirely dependent on the integrity of RpoS, the presence of a crl null allele does not decrease the level of RpoS protein. Thus, we propose that Crl stimulates the activity of the RpoS regulon by stimulating RpoS activity during stationary phase.
Timing, self‐control and a sense of direction are the secrets of multicopy plasmid stability
Authors: Summers David
Published: March 2003

Multicopy plasmids of Escherichia coli are distributed randomly at cell division and, as long as copy number remains high, plasmid‐free cells arise only rarely. Copy number variation is minimized by plasmid‐encoded control circuits, and the limited data available suggest that deviations are corrected efficiently under most circumstances. However, plasmid multimers confuse control circuits, leading to copy number depression. To make matters worse, multimers out‐replicate monomers and accumulate clonally within the culture, creating a subpopulation of cells with a significantly increased rate of plasmid loss. Multimers of natural multicopy plasmids, such as ColE1, are resolved to monomers by a site‐specific recombination system (Xer‐cer ) whose activity is limited to intramolecular recombination. Recombination requires the heterodimeric XerCD recombinase plus two accessory proteins (ArgR and PepA), which activate recombination and prevent intermolecular events. Evidence is accumulating that Xer‐cer recombination is relatively slow, and there is a risk that cells might divide before multimer resolution is complete. The Rcd transcript encoded within cer may solve this problem by preventing the division of multimer‐containing cells. Working in concert, the triumvirate of copy number control, multimer resolution and cell division control achieve an extremely high fidelity of plasmid maintenance.
Molecular cloning and characterization of a Candida albicans gene coding for cytochrome c haem lyase and a cell wall‐related protein
Authors: Cervera Ana M. , Gozalbo Daniel , McCreath Kenneth J. , Gow Neil A. R. , Martínez José P. , Casanova Manuel
Published: March 2003

Immunoscreening of a Candida albicans cDNA library with a monoclonal antibody (mAb 4C12) recognizing an epitope present in high‐molecular‐weight mannoprotein (HMWM) components specific for the mycelial cell walls (a 180 kDa component and a polydispersed 260 kDa species) resulted in the isolation of the gene CaCYC3 encoding for cytochrome c haem lyase (CCHL). The CaCYC3 gene was transcribed preferentially in mycelial cells in which two mRNA transcripts of 0.8 and 1 kb were found. The nucleotide and the deduced amino acid sequences of this gene displayed 45% homology and 46% identity, respectively, to the Saccharomyces cerevisiae CYC3 gene and shared common features with other reported genes encoding for CCHL. The CaCYC3 gene restored the respiratory activity when transformed in a S. cerevisiae cyc3 − mutant strain. A C. albicans CYC3 null mutant was constructed after sequential disruption using the hisG ::URA3 ::hisG (‘ura‐blaster’) cassette. Null mutant cells were unable to use lactate as a sole carbon source and had a reduced ability to form germ tubes. Western immunoblotting analysis of subcellular fractions from wild‐type and null mutant strains demonstrated the presence of two gene products, a 33 kDa mitochondrial protein and a 40 kDa cell wall‐associated moiety reacting with antibodies against CCHL, in both yeast cells and germ tubes. mAb 4C12 still reacted with the CaCYC3 null mutant (by immunofluorescence and immunoblotting) but showed an altered pattern of immunoreactivity against cell wall HMWM species, indicating a relationship between these moieties and the CaCYC3 gene products. The results suggest that the CaCYC3 gene encodes two proteins, one targeted to the mitochondria and the other to the cell wall.
The Myxococcus xanthus lipopolysaccharide O‐antigen is required for social motility and multicellular development
Authors: Bowden M. Gabriela , Kaplan Heidi B.
Published: March 2003

The gliding bacterium Myxococcus xanthus aggregates to form spore‐filled fruiting bodies when nutrients are limiting. Defective fruiting‐body formation and sporulation result from mutations in the sasA locus, which encodes the wzm wzt wbgA (formerly rfbABC) lipopolysaccharide (LPS) O‐antigen biosynthesis genes. Mutants carrying these same sasA mutations are defective in social motility and form small glossy colonies. We report here that the developmental and motility phenotypes of four mutants each containing different Tn5 insertions in LPS O‐antigen biosynthesis genes are similar to those of the original sasA locus mutants. All of the LPS O‐antigen mutants tested exhibited defective developmental aggregation and sporulated at only 0.02–15% of the wild‐type level. In addition, all of the LPS O‐antigen mutants were determined by genetic analyses to be wild type for adventurous motility and defective in social motility, indicating that the LPS O‐antigen is necessary for normal development and social motility. The two previously identified cell‐surface components required for social motility, type IV pili and the protein‐associated polysaccharide material termed fibrils, were detected on the surfaces of all of the LPS O‐antigen mutants. This indicates that LPS O‐antigen is a third cell‐surface component required for social motility.
Stimulus response coupling in bacterial chemotaxis: receptor dimers in signalling arrays
Authors: Levit Mikhail N. , Liu Yi , Stock Jeffry B.
Published: March 2003

In the Escherichia coli chemotaxis system, a family of chemoreceptors in the cytoplasmic membrane binds stimulatory ligands and regulates the activity of an associated histidine kinase CheA to modulate swimming behaviour and thereby cause a net migration towards attractants and away from repellents. The chemoreceptors themselves have been shown to be predominantly dimeric, but in the presence of the kinase CheA plus an adapter protein, CheW, much higher order structures have been observed. Recent results indicate that transmembrane signalling occurs within receptor clusters rather than through isolated dimers. We propose that the mechanism involves receptor arrays where binding of ligands at the outside surface of the membrane affects lateral packing interactions that cause perturbations in the organization of the signalling array at the opposing surface of the membrane. Results with receptor chimeras as well as findings with tyrosine kinase receptors suggest that this mechanism may represent a common theme in membrane receptor function.
Opa binding to cellular CD66 receptors mediates the transcellular traversal of Neisseria gonorrhoeae across polarized T84 epithelial cell monolayers
Authors: Wang Jun , Gray‐Owen Scott D. , Knorre Alexander , Meyer Thomas F. , Dehio Christoph
Published: March 2003

We have analysed the capacity of the 11 phase‐variable, opacity‐associated (Opa) proteins encoded by Neisseria gonorrhoeae MS11 to mediate traversal across polarized monolayers of the human colonic carcinoma T84 cell line. Gonococci expressing either the heparan sulphate proteoglycan (HSPG) binding Opa protein (Opa50) or no Opa protein (Opa−) did not interact with the apical pole of T84 monolayers, whereas the 10 variant Opa proteins previously shown to bind CD66 receptors were found to mediate efficient gonococcal adherence and transepithelial traversal. Consistent with this, T84 cells were shown by reverse transcriptase–polymerase chain reaction (RT–PCR) and immunoblotting to co‐express CD66a (BGP), CD66c (NCA) and CD66e (CEA). The recruitment of CD66 receptors by Opa‐expressing gonococci indicates their involvement in mediating adherence to the surface of T84 cells, and these bacterial interactions could be inhibited completely using polyclonal antibodies cross‐reacting with all of the CD66 proteins co‐expressed on T84 cells. Consistent results were obtained when Opa proteins were expressed in Escherichia coli, suggesting that the Opa–CD66 interaction is sufficient to mediate bacterial traversal. Transcytosis of Opa‐expressing N. gonorrhoeae or E. coli did not disrupt the barrier function of infected monolayers, as indicated by a sustained transepithelial electrical resistance (TEER) throughout the course of infection, and confocal laser scanning and electron microscopy both suggest a transcellular rather than a paracellular route of traversal across the monolayers. Parallels between the results seen here and previous work done with organ cultures confirm that T84 monolayers provide a valid model for studying neisserial interactions with the mucosal surface, and suggest that CD66 receptors contribute to this process in vivo.
The complete nucleotide sequence of φCTX, a cytotoxin‐converting phage of Pseudomonas aeruginosa: implications for phage evolution and horizontal gene transfer via bacteriophages
Authors: Nakayama Keisuke , Kanaya Shigehiko , Ohnishi Makoto , Terawaki Yoshiro , Hayashi Tetsuya
Published: March 2003

φCTX is a cytotoxin‐converting phage isolated from Pseudomonas aeruginosa. In this study, we determined the complete nucleotide sequence of the φCTX phage genome. The precise genome size was 35 538 bp with 21 base 5′‐extruding cohesive ends. Forty‐seven open reading frames (ORFs) were identified on the φCTX genome, including two previously identified genes, ctx and int. Among them, 15 gene products were identified in the phage particle by protein microsequencing. The most striking feature of the φCTX genome was an extensive homology with the coliphage P2 and P2‐related phages; more than half of the ORFs (25 ORFs) had marked homology to P2 genes with 28.9–65.8% identity. The gene arrangement on the genome was also highly conserved for the two phages, although the G+C content and codon usage of most φCTX genes were similar to those of the host P. aeruginosa chromosome. In addition, φCTX was found to share several common features with P2, including the morphology, non‐inducibility, use of lipopolysaccharide core oligosaccharide as receptor and Ca2+‐dependent receptor binding. These findings indicate that φCTX is a P2‐like phage well adapted to P. aeruginosa, and provide clear evidence of the intergeneric spread and evolution of bacteriophages. Furthermore, comparative analysis of genome structures of φCTX, P2 and other P2 relatives revealed the presence of several hot‐spots where foreign DNAs, including the cytotoxin gene, were inserted. They appear to be deeply concerned in the acquisition of various genes that are horizontally transferred by bacteriophage infection.
ToxR co‐operative interactions are not modulated by environmental conditions or periplasmic domain conformation
Authors: Dziejman Michelle , Kolmar Harald , Fritz Hans‐Joachim , Mekalanos John J.
Published: March 2003

ToxR is a transmembrane regulatory protein that controls virulence gene expression in Vibrio cholerae. Previous experiments using λ repressor–ToxR chimeric proteins and a λ repressor‐controlled reporter system (OR1 PR–lacZY ) established that ToxR sequences can effectively dimerize the amino‐terminal domain of λ repressor in Escherichia coli. However, in E. coliToxR does not respond to environmental signals that control virulence gene expression in V. cholerae. Here, we report the results of experiments designed to test whether environmental signals that modulate virulence gene expression in V. cholerae also modulate a monomer to dimerization transition of λ–ToxR chimeras. When the OR1 PR–lacZY reporter fusion and chimeric proteins were transferred to V. cholerae, we unexpectedly found that λ–ToxR chimeras did not dimerize significantly. Interestingly, experiments evaluating the ability of λ–ToxR proteins to form tetramers in E. coli suggested that λ–ToxR dimers could act co‐operatively. Using a redesigned reporter system containing multiple λ operator sites (OR1 OR2 OR3 PR–lacZY ), we found that λ–ToxR could dimerize quite efficiently in V. cholerae. These data imply that multiple DNA binding sites might enhance the ability of ToxR to dimerize in V. cholerae and suggest that ToxR dimers might be capable of co‐operative interactions. However, we failed to correlate a monomer–dimer transition of the λ–ToxR chimeras with changes in virulence gene expression in response to environmental signals in V. cholerae. Finally, because of conflicting results in the literature, the importance of membrane localization of ToxR and dimerization of the ToxR periplasmic domain was re‐evaluated. This was accomplished by measuring the ability of various chimeric proteins to activate toxin gene expression in both E. coli and V. cholerae. These assays suggest that, in V. choleraedeletion of the transmembrane domain has a profound effect on ToxR activity, although it is not an absolute requirement when ToxR is dimerized by a heterologous domain. In addition, we noted differences in chimeric protein activity when expressed in E. coli and V. cholerae. A construct substituting the monomeric MalE domain for the periplasmic domain of ToxR was unable to activate a ctx::lacZ reporter fusion in E. coli. Although the addition of leucine zipper sequences to this construct resulted in enhanced activity of the chimera in E. coli, both chimeras were able to produce wild‐type levels of toxin in V. cholerae. These data support the notion that dimerization of ToxR stimulates its activity as a transcriptional activator in E. coli. In V. cholerae, however, we present data that do not demonstrate a correlation between dimerization of the periplasmic domain and ToxR activity.
A genetic analysis of the function of LuxO, a two‐component response regulator involved in quorum sensing in Vibrio harveyi
Authors: Freeman Jeremy A. , Bassler Bonnie L.
Published: March 2003

Two independent quorum‐sensing systems control the expression of bioluminescence (lux) in the marine bacterium Vibrio harveyi. Each system is composed of an autoinducer (AI‐1 or AI‐2) and its cognate sensor (LuxN or LuxQ). The sensors are two‐component hybrid kinases, containing both sensor kinase domains and response regulator domains. Sensory information from the two systems is relayed by a phosphotransfer mechanism to a shared integrator protein called LuxO. LuxO is a member of the response regulator class of the two‐component family of signal transduction proteins, and LuxO acts negatively to control luminescence. In this report, missense and in frame deletion mutations were constructed in luxO that encoded proteins mimicking either the phosphorylated or the unphosphorylated form, and these mutations were introduced into the V. harveyi chromosome at the luxO locus. Phenotypical analyses of the resulting mutant V. harveyi strains indicate that the phosphorylated form of LuxO is the repressor, and that the unphosphorylated form of the protein is inactive. Analysis of the lux phenotypes of V. harveyi strains containing single and double luxN and luxQ mutations indicate that LuxN and LuxQ have two activities on LuxO. They act as LuxO protein kinases at low cell density in the absence of autoinducers, and they switch to LuxO protein phosphatases at high cell density in the presence of autoinducers. Furthermore, the timing and potency of inputs from the two systems into regulation of quorum sensing are different.
Disruption of the Toxoplasma gondii bradyzoite‐specific gene BAG1 decreases in vivo cyst formation
Authors: Zhang Yi Wei , Kim Kami , Ma Yan Fen , Wittner Murray , Tanowitz Herbert B. , Weiss Louis M.
Published: March 2003

The bradyzoite stage of the Apicomplexan protozoan parasite Toxoplasma gondii plays a critical role in maintenance of latent infection. We reported previously the cloning of a bradyzoite‐specific gene BAG1/hsp30 (previously referred to as BAG5 ) encoding a cytoplasmic antigen related to small heat shock proteins. We have now disrupted BAG1 in the T. gondii PLK strain by homologous recombination. H7, a cloned null mutant, and Y8, a control positive for both cat and BAG1, were chosen for further characterization. Immunofluorescence and Western blot analysis of bradyzoites with BAG1 antisera demonstrated expression of BAG1 in the Y8 and the PLK strain but no expression in H7. All three strains expressed a 116 kDa bradyzoite cyst wall antigen, a 29 kDa matrix antigen and the 65 kDa matrix reactive antigen MAG1. Mice inoculated with H7 parasites formed significantly fewer cysts than those inoculated with the Y8 and the PLK strains. H7 parasites were complemented with BAG1 using phleomycin selection. Cyst formation in vivo for the BAG1‐complemented H7 parasites was similar to wild‐type parasites. We therefore conclude that BAG1 is not essential for cyst formation, but facilitates formation of cysts in vivo.
Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis
Authors: Manganelli Riccardo , Dubnau Eugenie , Tyagi Sanjay , Kramer Fred Russell , Smith Issar
Published: March 2003

The ability of Mycobacterium tuberculosis to adapt to different environments in the infected host is essential for its pathogenicity. Consequently, this organism must be able to modulate gene expression to respond to the changing conditions it encounters during infection. In this paper we begin a comprehensive study of M. tuberculosis gene regulation, characterizing the transcript levels of 10 of its 13 putative sigma factor genes. We developed a real‐time RT‐PCR assay using a family of novel fluorescent probes called molecular beacons to quantitatively measure the different mRNAs. Three sigma factor genes were identified that have increased mRNA levels after heat shock, two of which also responded to detergent stress. In addition, we also identified a sigma factor gene whose mRNA increased after mild cold shock and a second that responded to conditions of low aeration.
Characterization of nra, a global negative regulator gene in group A streptococci
Authors: Podbielski Andreas , Woischnik Markus , Leonard Bettina A. B. , Schmidt Karl‐Hermann
Published: March 2003

During sequencing of an 11.5 kb genomic region of a serotype M49 group A streptococcal (GAS) strain, a series of genes were identified including nra (negative regulator of GAS). Transcriptional analysis of the region revealed that nra was primarily monocistronically transcribed. Polycistronic expression was found for the three open reading frames (ORFs) downstream and for the four ORFs upstream of nra. The deduced Nra protein sequence exhibited 62% homology to the GAS RofA positive regulator. In contrast to RofA, Nra was found to be a negative regulator of its own expression and that of the two adjacent operons by analysis of insertional inactivation mutants. By polymerase chain reaction and hybridization assays of 10 different GAS serotypes, the genomic presence of nra, rofA or both was demonstrated. Nra‐regulated genes include the fibronectin‐binding protein F2 gene (prtF2) and a novel collagen‐binding protein (cpa). The Cpa polypeptide was purified as a recombinant maltose‐binding protein fusion and shown to bind type I collagen but not fibronectin. In accordance with nra acting as a negative regulator of prtF2 and cpa, levels of attachment of the nra mutant strain to immobilized collagen and fibronectin was increased above wild‐type levels. In addition, nra was also found to regulate negatively (four‐ to 16‐fold) the global positive regulator gene, mga. Using a strain carrying a chromosomally integrated duplication of the nra 3′ end and an nra–luciferase reporter gene transcriptional fusion, nra expression was observed to reach its maximum during late logarithmic growth phase, while no significant influence of atmospheric conditions could be distinguished clearly.