bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogeography of the yucca moth Tegeticula maculata: the role of historical biogeography in reconciling high genetic structure with limited speciation
Authors: Segraves K. A. , Pellmyr O.
Published: March 2003

Tegeticula maculata is one of the most ancient and morphologically variable lineages within the yucca moths, yet has apparently undergone little diversification in comparison with much younger yucca moth lineages that have rapidly diversified. A phylogeographic approach was used to determine the number of independent lineages within T. maculata and to examine whether these patterns corresponded with morphological differences between its subspecies maculata and extranea. Phylogenetic analysis of mitochondrial DNA sequence variation indicated that the two subspecies are in separate clades, but there was also an equally deep split within subspecies maculata. There was no evidence for gene flow among regions and there was considerable substructure within clades. The phylogeographic structure of moth populations among and within subspecies can be explained in part by historical biogeographic boundaries and increasingly patchy postglacial distribution of the exclusive host plant, Hesperoyucca whipplei. Local specialization and co‐adaptation would be possible in the absence of apparent gene flow, yet gross morphological divergence is limited to the very old split between the subspecies. Sorting of ancient mitochondrial lineages followed by local genetic differentiation may explain the pattern of high genetic structure with limited speciation.
Species boundaries and genetic diversity among Hawaiian crickets of the genus Laupala identified using amplified fragment length polymorphism
Authors: Parsons Y. M. , Shaw K. L.
Published: March 2003

Crickets of the genus Laupala represent one of the many morphologically cryptic groups of insects, with the most closely related species distinguished only by the male calling song. Cryptic groups provide a challenge in determining the genetic boundaries between closely related populations and species. We have addressed the question of species boundaries in the Hawaiian cricket, Laupala, using nuclear DNA patterns sampled by the amplified fragment length polymorphism (AFLP) technique. This method has been used widely by plant researchers to facilitate the rapid assessment of genetic diversity in very closely related species and varieties. The AFLP technique is simple and robust, can be applied to any organism, and overcomes problems associated with cost, development time, information content and reproducibility that can plague other marker systems. Our results support previously hypothesized taxonomic relationships among sympatric populations and suggest close genetic relationships among allopatric, conspecific populations.
Insights into recently fragmented vole populations from combined genetic and demographic data
Authors: Tallmon D. A. , Draheim H. M. , Mills L. Scott , Allendorf F. W.
Published: March 2003

We combined demographic and genetic data to evaluate the effects of habitat fragmentation on the population structure of the California red‐backed vole (Clethrionomys californicus). We analysed variation in the mitochondrial DNA (mtDNA) control region and five nuclear microsatellite loci in small samples collected from two forest fragments and an unfragmented control site in 1990–91. We intensively sampled the same forest fragments and two different control sites in 1998 and 1999. Vole abundances fluctuated greatly at sizes below 50 individuals per fragment. Fragment populations had significantly lower mtDNA allelic diversity than controls, but not nuclear heterozygosity or numbers of alleles. The use of only trapping and/or mtDNA marker data would imply that fragment populations are at least partially isolated and vulnerable to inbreeding depression. In contrast, the abundance estimates combined with microsatellite data show that small fragment populations must be linked to nearby forests by high rates of migration. These results provide evidence for the usefulness of combining genetic and demographic data to understand nonequilibrium population structure in recently fragmented habitats.
Community genetics in the Northwestern Atlantic intertidal
Authors: Wares J. P.
Published: March 2003

Our ability to make inferences about the processes acting upon a biological system can be dramatically improved through integration of information from other fields. In particular, this is true of the field of phylogeography, a discipline that attempts to describe geographical variation in species using neutral genetic diversity as a correlate of time. Through comparisons of information from multiple species, as well as background information about the abiotic environment and how it has changed over time, we should be able to reassemble many of the forces that were important in assembling the communities and community interactions found in a given region. Here I review the information available for coastal species in the northwestern Atlantic, and argue that an integration of ecological, genetic, geological and oceanographic information can illustrate emergent patterns of community genetics.
Extreme microallopatric divergence in a cichlid species from Lake Malawi
Authors: Rico C. , Turner G. F.
Published: March 2003

We demonstrate significant population structuring on an extremely small spatial scale between adjacent demes of a Lake Malawi haplochromine cichlid species of the mbuna group, Pseudotropheus callainos, separated by only 35 m of habitat discontinuity. This substantiates further the notion that intralacustrine allopatric divergence may help to explain the high level of species richness of the mbuna in comparison to other Malawian cichlids, as well as of the Malawian haplochromines as a whole.
Molecular analysis of the Pleistocene history of Saxifraga oppositifolia in the Alps
Authors: Holderegger R. , Stehlik I. , Abbott R. J.
Published: March 2003

A recent circumpolar survey of chloroplast DNA (cpDNA) haplotypes identified Pleistocene glacial refugia for the Arctic‐Alpine Saxifraga oppositifolia in the Arctic and, potentially, at more southern latitudes. However, evidence for glacial refugia within the ice sheet covering northern Europe during the last glacial period was not detected either with cpDNA or in another study of S. oppositifolia that surveyed random amplified polymorphic DNA (RAPD) variation. If any genotypes survived in such refugia, they must have been swamped by massive postglacial immigration of periglacial genotypes. The present study tested whether it is possible to reconstruct the Pleistocene history of S. oppositifolia in the European Alps using molecular methods. Restriction fragment length polymorphism (RFLP) analysis of cpDNA of S. oppositifolia, partly sampled from potential nunatak areas, detected two common European haplotypes throughout the Alps, while three populations harboured two additional, rare haplotypes. RAPD analysis confirmed the results of former studies on S. oppositifolia; high within, but low among population genetic variation and no particular geographical patterning. Some Alpine populations were not perfectly nested in this common gene pool and contained private RAPD markers, high molecular variance or rare cpDNA haplotypes, indicating that the species could possibly have survived on ice‐free mountain tops (nunataks) in some parts of the Alps during the last glaciation. However, the overall lack of a geographical genetic pattern suggests that there was massive immigration of cpDNA and RAPD genotypes by seed and pollen flow during postglacial times. Thus, the glacial history of S. oppositifolia in the Alps appears to resemble closely that suggested previously for the species in northern Europe.
Genetic differentiation of the dengue vector, Aedes aegypti (Ho Chi Minh City, Vietnam) using microsatellite markers
Authors: Huber K. , Le Loan L. , Hoang T. H. , Ravel S. , Rodhain F. , Failloux A.‐B.
Published: March 2003

Dengue haemorrhagic fever emerged in the 1950s and has become a major public health concern in most Asian countries. In Vietnam, little is known about the intraspecific variation of the vector and its consequences on vectorial capacity. Here we report the use of microsatellite markers to differentiate Aedes aegypti populations in Ho Chi Minh City, a typical, overcrowded Asian city. Six microsatellite loci, with 5–14 alleles per locus, were scored in 20 mosquito samples collected in 1998 in Ho Chi Minh City. We found substantial differentiation among Ae. aegypti populations from the outskirts, whereas populations from the centre of the city showed less differentiation. These results are consistent with the hypothesis that populations of Ae. aegypti in central Ho Chi Minh City are panmictic because there are abundant larval breeding sites and an abundance of humans for adults to feed upon. In contrast, populations on the outskirts become differentiated largely through the processes of genetic drift because larval breeding sites are not as abundant. These findings implicate human activities associated with urbanization, as factors shaping the genetic structure of Ae. aegypti populations.
Post‐glacial colonization of northwestern North America by the forest‐associated American marten (Martes americana, Mammalia: Carnivora: Mustelidae)
Authors: Stone Karen D. , Flynn Rodney W. , Cook Joseph A.
Published: March 2003

Phylogeographic patterns were used to assess intraspecific diversification of American martens (Martes americana). Within martens, two morphological groups (americanaandcaurina) have been recognized, though the level of distinction between them has been debated. We examined mitochondrial cytochromebgene haplotypes from 680 martens to explore the colonization history of the Pacific Northwest and found two clades that correspond to the morphological groups. The widespreadamericanaclade extends from interior Alaska south to Montana and eastward to Newfoundland and New England (i.e. northwestern, north‐central and northeastern North America). Thecaurinaclade occurs in western North America, minimally extending from Admiralty Island (southeastern Alaska) south to Oregon and Wyoming. Our data indicated two colonization events for the Pacific Northwest (one by members of each clade) and were consistent with the persistence of populations throughout past glacial periods in eastern and western refugia. Due to vegetational and geological history following the past deglaciation, we hypothesize that martens of thecaurinaclade spread along the North Pacific Coast, and into southeastern Alaska, earlier than martens of theamericanaclade. Mismatch distributions for theamericanaclade were indicative of populations that recently experienced demographic expansion, while mismatch distributions for thecaurinaclade suggested that populations were at equilibrium. These clades are reciprocally monophyletic and distinctive (interclade divergence ranged from 2.5 to 3.0% (uncorrectedp), whereas, intraclade divergence was < 0.7%), and two regions of sympatry have been identified. Genetic signatures of past admixture in hybrid zones may have been extinguished during subsequent glacial periods when ranges contracted. This recurrent pattern of relatively restricted western, or Pacific coastal, lineages and more widespread eastern, or interior continental, lineages exists across broad taxonomic groups and suggests a shared biogeographical history.
Contrasting patterns of divergence in quantitative traits and neutral DNA markers: analysis of clinal variation
Authors: Storz Jay F.
Published: March 2003

Clinal variation in quantitative traits is often attributed to the effects of spatially varying selection. However, identical patterns can be produced by the interplay between purely stochastic processes (i.e. drift in combination with spatially restricted gene flow). One means of distinguishing between adaptive and nonadaptive causes of geographical variation is to compare relative levels of between‐population divergence in quantitative traits and neutral DNA markers. Such comparisons can be used to test whether levels of trait divergence attributable to additive genetic effects (as measured byQST) exceed null expectations based on the level of divergence at neutral marker loci (as measured byFST). The purpose of this study was to use an approach based on ‘QSTvs.FST’ contrasts to test for evidence of diversifying selection on body size of an Indian fruit bat,Cynopterus sphinx(Chiroptera: Pteropodidae). Specifically, relative levels of between‐population divergence in body size and microsatellite DNA markers were compared to assess whether the observed pattern of clinal size variation could be explained by a neutral model of isolation by distance.QSTfor body size was calculated using unbiased estimators of within‐ and between‐population variance of principal component scores. The association between body size variation and geographical/environmental distance was tested using pairwise and partial matrix correspondence tests (MCTs). Independent variables (representing causal hypotheses) were constructed as between‐locality distance matrices. The effects of neutral genetic divergence were assessed by including a matrix of pairwiseFSTas an independent variable. Partial MCTs revealed highly significant associations between phenotypic divergence (QST) and both geographical and environmental distance, even when the effects of neutral genetic divergence (FST) were partialled out. Results of the tests confirmed that migration–drift equilibrium is not a sufficient explanation for the latitudinal pattern of clinal size variation inC. sphinx. The geographical patterning of pairwiseQSTis most likely attributable to spatially varying selection and/or the direct influence of latitudinally ordered environmental effects.
Correlation between requirement for SecA during export and folding properties of precursor polypeptides
Authors: De Cock Hans , Randall Linda L.
Published: March 2003

The structural complexity of a ligand in association with the molecular chaperones SecB and SecA was investigated using three species of precursor maltose‐binding protein, which differ in their stability as a result of an amino acid substitution in each that affects the rate of folding of the polypeptide. In the presence of high concentrations of both SecB and SecA, the precursors were translocated in vitro with indistinguishable kinetics. However, when SecA was limiting, the translocation was more rapid for precursor species, which had lower stability in the native state relative to the stability of the wild‐type precursor. We propose that, when in complex with SecB, precursors can form an element of tertiary structure and that these tertiary contacts are blocked when SecA is bound.
A competence regulon in Streptococcus pneumoniae revealed by genomic analysis
Authors: Campbell Elizabeth A. , Choi Sook Yung , Masure H. Robert
Published: March 2003

Transformation in bacteria is the uptake and incorporation of exogenous DNA into a cell's genome. Several species transform naturally during a regulated state defined as competence. Genetic elements in Streptococcus pneumoniae induced during transformation were identified by combining a genetic screen with genomic analysis. Six loci were discovered that composed a competence‐induced regulon. These loci shared a consensus promoter sequence and encoded proteins, some of which were similar to proteins involved in DNA processing during transformation in other bacteria. Each locus was induced during competence and essential for genetic transformation.
Identification of novel staphylococcal virulence genes byin vivoexpression technology
Authors: Lowe Adrian M. , Beattie David T. , Deresiewicz Robert L.
Published: March 2003

We have appliedin vivoexpression technology (IVET) to the study of staphylococcal virulence. Using a promoter trap that relies on genetic recombination as a reporter of gene expression, we identified 45 staphylococcal genes that are induced during infection in a murine renal abscess model. Of these, only six were known previously; 11 others have homology to known non‐staphylococcal genes. The known staphylococcal genes includeagrA, part of a key locus regulating numerous virulence products, and a glycerol ester hydrolase, which may enhance staphylococcal survival in abscesses. We constructed 11 strains containing mutations in previously unknownivigenes. Of these strains, seven were significantly attenuated in virulence compared with the wild‐type parent. The mutagenizedivigenes may encode novel staphylococcal virulence factors.
Analyses of the cag pathogenicity island of Helicobacter pylori
Authors: Akopyants Natalia S. , Clifton Sandra W. , Kersulyte Dangeruta , Crabtree Jean E. , Youree Bryan E. , Reece C. Adonis , Bukanov Nick O. , Drazek E. Susan , Roe Bruce A. , Berg Douglas E.
Published: March 2003

Most strains of Helicobacter pylori from patients with peptic ulcer disease or intestinal‐type gastric cancer carry cagA, a gene that encodes an immunodominant protein of unknown function, whereas many of the strains from asymptomatically infected persons lack this gene. Recent studies showed that the cagA gene lies near the right end of a ≈37 kb DNA segment (a pathogenicity island, or PAI) that is unique to cagA+ strains and that the cag PAI was split in half by a transposable element insertion in the reference strain NCTC11638. In complementary experiments reported here, we also found the same cag PAI, and sequenced a 39 kb cosmid clone containing the left ‘cagII’ half of this PAI. Encoded in cagII were four proteins each with homology to four components of multiprotein complexes of Bordetella pertussis (‘Ptl’), Agrobacterium tumefaciens (‘Vir’), and conjugative plasmids (‘Tra’) that help deliver pertussis toxin and T (tumour inducing) and plasmid DNA, respectively, to target eukaryotic or prokaryotic cells, and also homologues of eukaryotic proteins that are involved in cytoskeletal structure. To the left of cagII in this cosmid were genes for homologues of HslU (heat‐shock protein) and Era (essential GTPase); to the right of cagII were homologues of genes for a type I restriction endonuclease and ion transport functions. Deletion of the cag PAI had no effect on synthesis of the vacuolating cytotoxin, but this deletion and several cag insertion mutations blocked induction of synthesis of proinflammatory cytokine IL‐8 in gastric epithelial cells. Comparisons among H. pylori strains indicated that cag PAI gene content and arrangement are rather well conserved. We also identified two genome rearrangements with end‐points in the cag PAI. One, in reference strain NCTC11638, involved IS605, a recently described transposable element (as also found by others). Another rearrangement, in 3 of 10 strains tested (including type strain NCTC11637), separated the normally adjacent cagA and picA genes and did not involve IS605. Our results are discussed in terms of how cag‐encoded proteins might help trigger the damaging inflammatory responses in the gastric epithelium and possible contributions of DNA rearrangements to genome evolution.
In vivo studies on the positive control function of NifA: a conserved hydrophobic amino acid patch at the central domain involved in transcriptional activation
Authors: González Víctor , Olvera Leticia , Soberón Xavier , Morett Enrique
Published: March 2003

The eubacterial enhancer‐binding proteins activate transcription by binding to distant sites and, simultaneously, contacting the RNA polymerase σ54 promoter complex (Eσ54). The positive control function is located at the central domain of these proteins, but it is not know which specific region has the determinants for the interaction with Eσ54. Here, we present genetic evidence that a small region of hydrophobic amino acids, previously denominated C3, at the central domain of Bradyrhizobium japonicum NifA is involved in positive control. We obtained 26 missense mutants along this conserved region. Among these, only strains expressing the NifAF307→Y and NifAA310→S mutant proteins retained some of the transcriptional activity (< 20%), whereas those carrying NifAE298→D and NifAT308→S had very low but detectable activity (< 1.0%). The rest of the NifA mutants did not induce any measurable transcriptional activity. When expressed in the presence of wild‐type NifA, the great majority of the mutants displayed a dominant phenotype, suggesting that their oligomerization determinants were not altered. In vivo dimethyl‐sulphate footprinting experiments for a subset of the NifA mutants showed that they were still able to bind specifically to DNA. Analysis of intragenic supressors highlight the functional role of a hydroxyl group at position 308 to activate transcription.
Genetic advances for studying Mycobacterium tuberculosis pathogenicity
Authors: Pelicic Vladimir , Reyrat Jean‐Marc , Gicquel Brigitte
Published: March 2003

Tuberculosis remains the greatest cause of death worldwide because of a single pathogen. Despite its importance, the genetic basis of the pathogenicity of Mycobacterium tuberculosis remains poorly understood, mainly because the most productive investigative approach, molecular genetic analysis, has been severely hampered by a lack of efficient tools. However, significant advances, including the development of methods for inactivating genes and studying their expression with reporter genes, have been recently made. This progress may lead to opportunities for developing new vaccines and antituberculous drugs. The aim of this review is to examine the present state of the art in mycobacterial molecular genetics and pinpoint some expected or promising areas for future research.
Microbial magnesium transport: unusual transporters searching for identity
Authors: Smith Ronald L. , Maguire Michael E.
Published: March 2003

Mg2+ is unique among biological cations because of its charge density and solution chemistry. This is abundantly reflected in its transport systems, studied primarily in Salmonella typhimurium. The constitutively expressed CorA transport system is the primary Mg2+ influx pathway for the Bacteria and the Archaea. Its structure of a large N‐terminal soluble periplasmic domain with three transmembrane segments at the C‐terminus is unique among membrane carriers, and its protein sequence bears no resemblance to other known proteins. The MgtE transport system can also mediate Mg2+ uptake, but whether this is its primary function is not known. MgtE also lacks homology to other known proteins. In contrast, the MgtA and MgtB Mg2+ transport systems of enteric bacteria are P‐type ATPases by sequence homology, mediating Mg2+ influx with, rather than against, the Mg2+ electrochemical gradient. They are closely related to mammalian Ca2+‐ATPases. Expression of MgtA and MgtB is under the control of the PhoPQ two‐component regulatory system, important in bacterial virulence. In S. typhimurium, MgtB is encoded by a two‐gene operon mgtCB ; the function of the MgtC protein is unknown, and it lacks close homologues. The ligand for the PhoQ membrane sensor kinase is Mg2+ and, at decreased extracellular Mg2+ concentrations, transcription of mgtA and mgtCB are enormously induced. All three genes are also induced upon S. typhimurium invasion of epithelial or macrophage cells. Mutation of these genes has no effect on invasion efficiency, but an insertion in mgtC renders S. typhimurium essentially avirulent in the mouse. The physiological roles of the known Mg2+ transport systems are not yet completely defined. Nonetheless, the singular sequence and apparent structure of the CorA and MgtE transport proteins, the complex regulation of MgtA, MgtB and MgtC and their involvement in pathogenesis suggests that further study will be rewarding.
Genetic uncoupling of the dsRNA‐binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III — the effect of dsRNA binding on gene expression
Authors: Dasgupta S. , Fernandez L. , Kameyama L. , Inada T. , Nakamura Y. , Pappas A. , Court D. L.
Published: March 2003

RNase III, a double‐stranded RNA‐specific endonuclease, is proposed to be one of Escherichia coli 's global regulators because of its ability to affect the expression of a large number of unrelated genes by influencing post‐transcriptional control of mRNA stability or mRNA translational efficiency. Here, we describe the phenotypes of bacteria carrying point mutations in rnc, the gene encoding RNase III. The substrate recognition and RNA‐processing properties of mutant proteins were analysed in vivo by measuring expression from known RNase III‐modulated genes and in vitro from the proteins' binding and cleavage activities on known double‐stranded RNA substrates. Our results show that although the point mutation rnc70 exhibited all the usual rnc null‐like phenotypes, unlike other mutations, it was dominant over the wild‐type allele. Multicopy expression of rnc70 could suppress a lethal phenotype of the wild‐type rnc allele in a certain genetic background; it could also inhibit the RNase III‐mediated activation of λN gene translation by competing for the RNA‐binding site of the wild‐type endonuclease. The mutant protein failed to cleave the standard RNase III substrates in vitro but exhibited an affinity for double‐stranded RNA when passed through poly(rI):poly(rC) columns. Filter binding and gel‐shift assays with purified Rnc70 showed that the mutant protein binds to known RNase III mRNA substrates in a site‐specific manner. In vitro processing reactions with purified enzyme and labelled RNA showed that the in vivo dominant effect of the mutant enzyme over the wild‐type was not necessarily caused by formation of mixed dimers. Thus, the rnc70 mutation generates a mutant RNase III with impaired endonucleolytic activity but without blocking its ability to recognize and bind double‐stranded RNA substrates.
Interplay between global regulators of Escherichia coli : effect of RpoS, Lrp and H‐NS on transcription of the gene osmC
Authors: Bouvier Jean , Gordia Sylvie , Kampmann Gabriele , Lange Roland , Hengge‐Aronis Regine , Gutierrez Claude
Published: March 2003

The transcription of the osmC gene of Escherichia coli is regulated as a function of the phase of growth. It is induced during the decelerating phase, before entry into stationary phase. osmC expression is directed by two overlapping promoters, osmCp1 and osmCp2. osmCp2 is mainly transcribed by E‐σs, the RNA polymerase using the σs (RpoS) sigma factor, and is responsible for the growth phase regulation. Transcription from osmCp1 is independent of σs. The leucine‐responsive protein (Lrp) has been shown to bind the osmC promoter region in band shift experiments. In vivo analysis using osmC–lacZ transcriptional fusions demonstrated that Lrp affects the expression of both promoters. It represses the transcription of osmCp1 and activates the transcription of osmCp2 by E‐σs. An absence of Lrp results in an increase in the amount of RpoS during exponential growth in minimal medium. The nucleoid‐associated protein H‐NS also represses osmC transcription from both promoters. However, this happens through different mechanisms. The effect on osmCp2 is probably mediated by the increase in σs concentration in the cytoplasm of hns− mutants, while the effect on osmCp1 is independent of σs. No binding of H‐NS to the promoter region DNA could be detected, indicating that the effect on osmCp1 could also be indirect.
Development of competence in Streptococcus pneumoniae: pheromone autoinduction and control of quorum sensing by the oligopeptide permease
Authors: Alloing Geneviève , Martin Bernard , Granadel Chantal , Claverys Jean‐Pierre
Published: March 2003

Competence for genetic transformation in the human pathogen Streptococcus pneumoniae is a transient physiological property. A competence‐stimulating peptide, CSP, was recently identified as the processed product of the comC gene. As conflicting results have been reported regarding CSP autoinduction, we monitored the CSP‐induced expression of comCDE in derivatives of strain R6 using comC::lacZ fusions. Autoinduction was demonstrated in this genetic background. The kinetics of CSP‐induced transcription of comCDE and of a late competence‐induced (cin) operon were compared. While the comCDE mRNA level was highest 5 min after CSP addition then decreased, maximal cin expression required 10 min exposure to CSP. Transformation frequencies paralleled cin expression. After 20 min exposure to CSP, both mRNAs disappeared almost completely, providing evidence for an intrinsic mechanism for shutting off CSP signal transduction. Investigation of spontaneous competence development in mixed cultures indicated that transformation of wild‐type cells was delayed in the presence of CSP non‐producers, consistent with a direct role of CSP in quorum sensing. The effect of varying inoculum size on the timing of competence development was investigated. While competence developed in wild‐type cultures at a similar critical density, about OD550 = 0.15, a mutant lacking the three oligopeptide‐binding lipoproteins transformed at a 50‐fold reduced cell density. The latter effect was mimicked in a strain harbouring a duplication of comC. Altogether, these results suggest that CSP does not accumulate passively in pneumoccal cultures, but that comCDE basal expression can be modulated.
Binding of the purified integron DNA integrase IntI1 to integron‐ and cassette‐associated recombination sites
Authors: Collis Christina M. , Kim Mi‐Jurng , Stokes H. W. , Hall Ruth M.
Published: March 2003

The site‐specific recombinase IntI1, encoded by class 1 integrons, catalyses the integration and excision of gene cassettes by recognizing two classes of sites, the integron‐associated attI1 site and the 59‐base element (59‐be) family of sites that are associated with gene cassettes. IntI1 includes the four conserved amino acids that are characteristic of members of the integrase family, and IntI1 proteins with single amino acid substitutions at each of these positions had substantially reduced catalytic activity, consistent with this classification. IntI1 was purified as a fusion protein and shown to bind to isolated attI1 or 59‐be recombination sites. Binding to attI1 was considerably stronger than to a 59‐be. Binding adjacent to the recombination cross‐over point was not detected. A strong IntI1 binding site within attI1 was localized by both deletion and footprinting analysis to a 14 bp region 24–37 bp to the left of the recombination cross‐over point, and this region is known to be critical for recombination in vivo (Recchia et al., 1994). An imperfect (13/15) direct repeat of this region, located 41–55 bp to the left of the recombination cross‐over point, contains a weaker IntI1 binding site. Mutation of the stronger binding site showed that a single base pair change accounted for the difference in the strength of binding.
FtsK is an essential cell division protein that is localized to the septum and induced as part of the SOS response
Authors: Wang Lilin , Lutkenhaus Joe
Published: March 2003

The role of ftsK in the growth of Escherichia coli was examined by turning off its expression. This resulted in smooth filaments without constrictions, indicating that FtsK was required at an early step in septation. Consistent with this, FtsK was found to localize to the septum in 70% of the cells, indicating that it was recruited relatively early in this process. FtsK localization required the function of FtsZ and FtsA but not FtsI and FtsQ. Consistent with this, Z rings were present in FtsK‐depleted filaments. Subcellular localization of FtsK confirmed that it was a membrane protein. Only the first 202 amino acids of FtsK were essential for its role in membrane localization, cell division and viability. The expression of ftsK increased as part of the SOS response, and increased expression of ftsK conferred increased resistance to DNA damage.
Functional conservation of the effector protein translocators PopB/YopB and PopD/YopD of Pseudomonas aeruginosa and Yersinia pseudotuberculosis
Authors: Frithz‐Lindsten Elisabet , Holmström Anna , Jacobsson Lars , Soltani Mehnam , Olsson Jan , Rosqvist Roland , Forsberg Åke
Published: March 2003

Virulent Yersinia species cause systemic infections in rodents, and Y. pestis is highly pathogenic for humans. Pseudomonas aeruginosa, on the other hand, is an opportunistic pathogen, which normally infects only compromised individuals. Surprisingly, these pathogens both encode highly related contact‐dependent secretion systems for the targeting of toxins into eukaryotic cells. In Yersinia, YopB and YopD direct the translocation of the secreted Yop effectors across the target cell membrane. In this study, we have analysed the function of the YopB and YopD homologues, PopB and PopD, encoded by P. aeruginosa. Expression of the pcrGVHpopBD operon in defined translocation‐deficient mutants (yopB/yopD ) of Yersinia resulted in complete complementation of the cell contact‐dependent, YopE‐induced cytotoxicity of Y. pseudotuberculosis on HeLa cells. We demonstrated that the complementation fully restored the ability of Y. pseudotuberculosis to translocate the effector molecules YopE and YopH into the HeLa cells. Similar to YopB, PopB induced a lytic effect on infected erythrocytes. The lytic activity induced by PopB could be prevented if the erythrocytes were infected in the presence of sugars larger than 3 nm in diameter, indicating that PopB induced a pore of similar size compared with that induced by YopB. Our findings show that the contact‐dependent toxin‐targeting mechanisms of Y. pseudotuberculosis and P. aeruginosa are conserved at the molecular level and that the translocator proteins are functionally interchangeable. Based on these similarities, we suggest that the translocation of toxins such as ExoS, ExoT and ExoU by P. aeruginosa across the eukaryotic cell membrane occurs via a pore induced by PopB.
Preprotein transfer to the Escherichia coli translocase requires the co‐operative binding of SecB and the signal sequence to SecA
Authors: Fekkes Peter , De Wit Janny G. , Van Der Wolk Jeroen P. W. , Kimsey Harvey H. , Kumamoto Carol A. , Driessen Arnold J. M.
Published: March 2003

In Escherichia coli, precursor proteins are targeted to the membrane‐bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxy‐terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo, are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA‐dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (Δ8proOmpA) bearing a non‐functional signal sequence. Δ8proOmpA is not translocated across wild‐type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prlA4 strain. SecB reduces the translocation of Δ8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB–SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase.
Putative membrane components of signal transduction pathways for ambient pH regulation in Aspergillus and meiosis in Saccharomyces are homologous
Authors: Denison Steven H. , Negrete‐Urtasun Susana , Mingot José Manuel , Tilburn Joan , Mayer Wendy A. , Goel Alpa , Espeso Eduardo A. , Peñalva Miguel A. , Arst Jr Herbert N.
Published: March 2003

The zinc finger regions of the Aspergillus nidulans PacC transcription factor, mediating regulation of gene expression by ambient pH, and the Saccharomyces cerevisiae Rim1p transcription factor, mediating control of meiosis and invasiveness, are homologous and both transcription factors undergo proteolytic processing of the C‐terminus for conversion to the functional form. In both cases, functioning of a signal transduction pathway involving several gene products is a necessary prerequisite for processing. We now show that the Aspergillus PalI pH signal transduction component is homologous to the Saccharomyces Rim9p meiotic signal transduction component throughout a region containing four hydrophobic, putative membrane‐spanning segments. This suggests that PalI might be a membrane sensor for ambient pH. Deletion of the palI gene established that the less extreme phenotype of palI mutations compared with mutations in the other five genes of the pH signalling pathway is a general feature of palI mutations.
Regulation of the Enterococcus faecalis pAD1‐related sex pheromone response: analyses of traD expression and its role in controlling conjugation functions
Authors: Do Carmo de Freire Bastos Maria , Tomita Haruyoshi , Tanimoto Koichi , Clewell Don B.
Published: March 2003

The Enterococcus faecalis haemolysin plasmid pAD1 (60 kb) confers a conjugative mating response to an octapeptide sex pheromone (cAD1) secreted by plasmid‐free strains. The response involves two plasmid‐borne regulatory determinants: traE1, whose product positively regulates all or most conjugation‐related structural genes; and traA, whose product negatively regulates traE1 by controlling transcriptional readthrough of an upstream termination site (TTS1/TTS2). TraA binds to the promoter region of iad, which encodes a pheromone‐inhibitor peptide, iAD1; and TTS1/TTS2 tightly terminates transcription arriving from this promoter during the uninduced state. A determinant, traD, appearing to encode a small peptide (23 amino acids), is located just downstream of iad and is in the opposite orientation. Transcripts of traD were identified and found to be present at a relatively high level in cells not expressing conjugation functions; the amount of RNA was greatly reduced, however, upon induction of the pheromone response. The decrease in traD RNA was not a consequence of the induced activity of TraE1, as it also occurred in a traE1 insertion mutant. A mutation in traD that would eliminate translation but that did not affect transcription had no apparent effect on the cell phenotype, indicating that RNA was likely to be the functional product. This was consistent with our finding that synthesis of traD RNA containing the translational defect was able to complement, in trans, a temperature‐sensitive traD mutation. Thus, transcription of the traD determinant is significantly involved in downregulation of the pAD1 pheromone response.
Maximal transcriptional activation by the IHF protein of Escherichia coli depends on optimal DNA bending by the activator
Authors: Engelhorn Manuel , Geiselmann Johannes
Published: March 2003

Transcriptional activation in prokaryotes can be mediated by at least two different mechanisms: direct contacts between the activator and RNA polymerase or modulation of the overall geometry of DNA. In the latter case, an activator protein that bends DNA favours contacts between the DNA upstream of the activator binding site and the back of RNA polymerase. The architectural protein integration host factor (IHF) of Escherichia coli bends DNA and activates transcription at several promoters. We have isolated mutants of IHF that maximize transcriptional activation by adjusting the bending angle of the DNA. The amino acid residues of IHF that adjust the bending angle are close to the DNA and probably make electrostatic interactions with the DNA. We show that transcriptional activation is maintained when the IHF binding site is moved further upstream or when its orientation is inverted, and we conclude from these data that direct interactions between IHF and RNA polymerase do not participate in activation. IHF acts merely by bending DNA; weaker bending leading to stronger activation. We propose that wild‐type IHF induces too strong a DNA bend (180°) for optimal interactions between DNA upstream of the IHF binding site and the back of RNA polymerase.
Roles of integrins and fibronectin in the entry of Streptococcus pyogenes into cells via protein F1
Authors: Ozeri Vered , Rosenshine Ilan , Mosher Deane F. , Fässler Reinhard , Hanski Emanuel
Published: March 2003

Entry of group A streptococcus (GAS) into cells has been suggested as an important trait in GAS pathogenicity. Protein F1, a fibronectin (Fn) binding protein, mediates GAS adherence to cells and the extracellular matrix, and efficient cell internalization. We demonstrate that the cellular receptors responsible for protein F1‐mediated internalization of GAS are integrins capable of Fn binding. In HeLa cells, bacterial entry is blocked by anti‐β1 integrin monoclonal antibody. In the mouse cell line GD25, a β1 null mutant, the αvβ3 integrin promotes GAS entry. Internalization of these cells by GAS is blocked by a peptide that specifically binds to αvβ3 integrin. In both cell lines, entry of GAS requires the occupancy of protein F1 by Fn. Neither the 29 kDa nor the 70 kDa N‐terminal fragments or the 120 kDa cell‐binding fragment of Fn promote bacterial entry. Fn‐coated beads are taken up efficiently by HeLa cells. Both the entry of GAS via protein F1 and the uptake of Fn‐coated beads are blocked by anti‐β1 antibody but are unaffected by a large excess of soluble Fn. Internalization of HeLa cells by bacteria bearing increasing amounts of prebound Fn to protein F1 reveals a sigmoidal ultrasensitive curve. These suggest that the ability of particles to interact via Fn with multiple integrin sites plays a central role in their ability to enter cells.
A gonococcal porA pseudogene: implications for understanding the evolution and pathogenicity of Neisseria gonorrhoeae
Authors: Feavers Ian M. , Maiden Martin C. J.
Published: March 2003

Members of the genus Neisseria, including the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae, express at least one member of a family of related porins. N. meningitidis is the only species known to express a second porin, the meningococcal serosubtyping antigen PorA, the most divergent member of this family. Unexpectedly, a porA gene was identified in the gonococcal genome. Both the gonococcal and meningococcal porA loci were adjacent to a homologue of the Escherichia coli greA gene, although the IS1106 element downstream of porA in some meningococci was absent in the gonococcus. Almost identical porA loci were present in four unrelated gonococcal isolates and clinical specimens from patients with gonorrhoea. Lack of PorA expression in the gonococcus resulted from mutations in the promoter region, which prevented transcription, and frameshift mutations in the coding region of the porA gene. Hybridization and amplification experiments, showing the absence of a porA gene in seven other Neisseria species, suggested that porA was acquired by a common ancestor of the gonococcus and meningococcus but inactivated in the gonococcus on speciation. This implies that, while advantageous during colonization of the upper respiratory tract, this protein has no function in, or hinders, colonization of the urogenital tract.
Heterogeneous ribosome populations are present in Plasmodium berghei during development in its vector
Authors: Thompson Joanne , Van Spaendonk Rosalina M. L. , Choudhuri Rangana , Sinden Robert E. , Janse Chris J. , Waters Andrew P.
Published: March 2003

The genome of the rodent malaria parasite, Plasmodium berghei, contains two sets of variant ribosomal RNA (rRNA) genes, termed the A and S types, that are expressed predominantly during the vertebrate and mosquito stages of the parasite's development respectively. Using in situ hybridization, we have examined the transcriptional activity of the A‐ and S‐type rRNA genes, and the switch in expression of the ribosome populations that occurs after parasite transmission to the mosquito. By detection of precursor rRNA molecules, we show that A‐type rRNA transcription is downregulated throughout development in the mosquito, whereas the initiation of S‐type rRNA transcription is linked to the proliferative phase of the oocyst. Mature A‐type rRNA persists during development of the zygote into the ookinete/young oocyst. In contrast, mature S‐type rRNA is first detectable in young oocysts and is subsequently present at high levels during further development of oocysts and sporozoites. These results demonstrate that the switch in transcription between the A‐ and S‐type rRNA genes is developmentally regulated, taking place only as the parasite begins to proliferate in the mosquito. A‐type ribosomes are therefore not only translationally active in the early stages of development in the mosquito, but are also crucial at this phase.
Cloning of two genes encoding potassium transporters inNeurospora crassaand expression of the corresponding cDNAs inSaccharomyces cerevisiae
Authors: Haro Rosario , Sainz Loreto , Rubio Francisco , Rodríguez‐Navarro Alonso
Published: March 2003

TwoNeurospora crassagenes,trk‐1andhak‐1, encode K+transporters that show sequence similarities to the TRK transporters described inSaccharomyces cerevisiaeandSchizosaccharomyces pombe, and to the HAK transporters described inSchwanniomyces occidentalisand barley. TheN. crassaTRK1 and HAK1 transporters expressed by the corresponding cDNAs in atrk1Δ trk2Δmutant ofS. cerevisiaeexhibited a high affinity for Rb+and K+. Northern blot analysis and comparison of the kinetic characteristics of the two transporters in thetrk1Δ trk2Δmutant with the kinetic characteristics of K+uptake inN. crassacells allowed TRK1 to be identified as the dominant K+transporter and HAK1 as a transporter that is only expressed when the cells are K+starved. The HAK1 transporter showed a high concentrative capacity and is identified as the K+–H+symporter described inN. crassa, whereas TRK1 might be a K+uniporter. Although the co‐existence of K+transporters of the TRK and HAK types in the same species had not been reported formerly, we discuss whether this co‐existence may be the normal situation in soil fungi.
Structure, assembly and regulation of expression of capsules in Escherichia coli
Authors: Whitfield Chris , Roberts Ian S.
Published: March 2003

Many Escherichia coli strains are covered in a layer of surface‐associated polysaccharide called the capsule. Capsular polysaccharides represent a major surface antigen, the K antigen, and more than 80 distinct K serotypes result from structural diversity in these polymers. However, not all capsules consist of K antigen. Some are due to production of an extensive layer of a polymer structurally identical to a lipopolysaccharide O antigen, but distinguished from lipopolysaccharide by the absence of terminal lipid A‐core. Recent research has provided insight into the manner in which capsules are organized on the Gram‐negative cell surface, the pathways used for their assembly, and the regulatory processes used to control their expression. A limited repertoire of capsule expression systems are available, despite the fact that the producing bacteria occupy a variety of ecological niches and possess diverse physiologies. All of the known capsule assembly systems seen in Gram‐negative bacteria are represented in E. coli, as are the majority of the regulatory strategies. Escherichia coli therefore provides a variety of working models on which studies in other bacteria are (or can be) based. In this review, we present an overview of the current molecular and biochemical models for capsule expression in E. coli. By taking into account the organization of capsule gene clusters, details of the assembly pathway, and regulatory features that dictate capsule expression, we provide a new classification system that separates the known capsules of E. coli into four distinct groups.