bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic structure of Anopheles gambiae (Diptera: Culicidae) in São Tomé and Príncipe (West Africa): implications for malaria control
Authors: Pinto J. , Donnelly M. J. , Sousa C. A. , Gil V. , Ferreira C. , Elissa N. , Do Rosário V. E. , Charlwood J. D.
Published: March 2003

The impact of a vector eradication programme, conducted in the 1980s, on Anopheles gambiae populations from the islands of São Tomé and Príncipe, was evaluated by microsatellite DNA analysis. Significant genetic differentiation was observed within and between the two islands and between the islands and a population from Gabon, suggesting a degree of isolation between them. Large estimates of long‐term Ne suggested that the control programme did not affect the effective population size of the vector. Heterozygosity tests were also not consistent with a recent bottleneck.
Glacial‐induced altitudinal migrations inArmeria(Plumbaginaceae) inferred from patterns of chloroplast DNA haplotype sharing
Authors: Gutiérrez Larena B. , Fuertes Aguilar J. , Nieto Feliner G.
Published: March 2003

In contrast to northern European areas where large‐scale migrations occurred to recolonize territories after glacial periods, species in southern regions survived and diverged without large geographical displacements. As a result of the importance of orography in much of the southern areas, such displacements must have involved populations ascending or descending mountains. The present study provides support for glacial‐induced altitudinal migrations from chloroplast phylogeographic patterns inArmeria(Plumbaginaceae) in southeast Spain. One hundred and five sequences of thetrnL‐Fspacer were obtained from seven species. Fifteen different haplotypes were recognized, their genealogy was inferred, and associations with geography were explored using nested clade analysis. Seven instances were detected in which the same haplotype is shared by two or three species within a particular massif. In all the cases, at least one of the species involved displayed different haplotypes in other areas; in most, the haplotype shared is predominant either in one of the species involved or in the massif. These patterns of haplotype sharing strongly suggest horizontal transfer between species. In one of the massifs (Sierra Nevada) the three species involved in haplotype sharing (A. splendens,A. filicaulisssp.nevadensis,A. villosassp.bernisii) occur at markedly different altitudinal belts. It is argued that altitudinal migrations within the contraction–expansion model provide the best explanation for the current pattern, and that at least in one case it resulted in the formation of a new hybrid taxon,A. filicaulisssp.nevadensis.
Diversity and geographic distribution of secondary endosymbiotic bacteria in natural populations of the pea aphid,Acyrthosiphon pisum
Authors: Tsuchida Tsutomu , Koga Ryuichi , Shibao Harunobu , Matsumoto Tadao , Fukatsu Takema
Published: March 2003

In addition to the essential intracellular symbiotic bacteriumBuchnera, several facultative endosymbiotic bacteria called collectively secondary symbionts (S‐symbionts) have been identified from the pea aphidAcyrthosiphon pisum. We conducted an extensive and systematic survey of S‐symbionts in Japanese local populations ofA. pisumusing a specific PCR detection technique. Five S‐symbionts ofA. pisum, PASS, PAUS, PABS,RickettsiaandSpiroplasma, and two facultative endosymbionts universally found in various insects,WolbachiaandArsenophonus, were targeted. Of 119 isofemale strains originating from 81 localities, 66.4% of the strains possessed either of four S‐symbionts: PASS (38.7%); PAUS (16.0%);Rickettsia(8.4%); andSpiroplasma(3.4%), while 33.6% of the strains contained onlyBuchnera. PABS,WolbachiaandArsenophonuswere not detected from the Japanese strains ofA. pisum. In order to understand intra‐ and interpopulational diversity of S‐symbiont microbiota in detail, 858 insects collected from 43 localities were examined for infection with the four S‐symbionts. It was demonstrated that different S‐symbionts coexist commonly in the same local populations, but double infections with two S‐symbionts were rarely detected. Notably, the S‐symbionts exhibited characteristic geographical distribution patterns: PASS at high frequencies all over Japan; PAUS at high frequencies mainly in the northeastern part of Japan; andRickettsiaandSpiroplasmaat low frequencies sporadically in the southwestern part of Japan. These results indicate that the geographical distribution and infection frequency of the S‐symbionts, in particular PAUS, might be affected by environmental and/or historical factors. Statistical analyses suggested that the distribution of PAUS infection might be related to host plant species, temperature and precipitation.
Multilocus population structure of Tapesia yallundae in Washington State
Authors: Douhan G. W. , Peever T. L. , Murray T. D.
Published: March 2003

Population genetic structure of the fungal wheat pathogen Tapesia yallundae in Washington State was determined using genetically characterized amplified fragment length polymorphic (AFLP) markers and mating‐type (MAT1‐1 or MAT1‐2). Segregation and linkage relationships among 164 AFLP markers and MAT were analysed using 59 progeny derived from an in vitro cross. Alleles at 158 AFLP loci and the mating‐type locus segregated in a 1:1 ratio. Ten unlinked markers were chosen to determine genetic and genotypic diversity and to test the hypothesis of random mating and population differentiation among five subpopulations of T. yallundae representative of the geographical distribution of wheat production in eastern Washington. Among 228 isolates collected, overall gene diversity was high (h = 0.425) and a total of 91 unique multilocus genotypes (MLG) were identified, with 32 MLG occurring at least twice. The overall population genetic structure was consistent with random mating based on the segregation of mating‐type, index of association (IA), parsimony tree length permutation test (PTLPT) and genotypic diversity analyses. However, clonal genotypes were found within each subpopulation and were also distributed among the five subpopulations. No significant differences in allele frequencies were found among the five subpopulations for all 10 loci based on contingency table analysis (G2) and Wier & Cockerham's population differentiation statistic θ (θ = −0.008, P = 0.722). T. yallundae appears to consist of a large homogeneous population throughout eastern Washington with both sexual and asexual reproduction contributing to the observed population genetic structure despite no report of sexual fruiting bodies of T. yallundae occurring under natural field conditions.
Intraspecific phylogeography of the slender madtom: the complex evolutionary history of the Central Highlands of the United States
Authors: Hardy M. E. , Grady J. M. , Routman E. J.
Published: March 2003

A number of different biogeographical studies of the Central Highlands of the United States have yielded conflicting area cladograms. We estimate the mtDNA phylogeny of populations of the slender madtom, Noturus exilis, a clear‐water stream catfish. The goal is to compare population relationships to those reported in previous studies that used upland, stream‐dwelling vertebrates. A region of the NADH dehydrogenase subunit 4 gene, along with adjacent tRNAs, was sequenced for population samples from 21 different Central Highlands rivers. Sequence difference among 39 haplotypes ranged from 0.1% to 4.8%. Most haplotypes were restricted to specific rivers and mapped well onto geography. Slender madtoms from different drainages contained mostly monophyletic groups of haplotypes genetically divergent from haplotypes found in other drainages, although a few haplotypes were found in well‐separated drainages. The area cladogram for the slender madtom was not similar to any of the other cladograms for other species and species groups from the area. We discuss a variety of methodological and biological reasons for the discordance, and suggest that some of the discrepancies may be resolved by the sequencing of multiple genes per species. We recommend that more, and more extensive, intraspecific phylogeography studies should be conducted for species living in the Central Highlands rivers.
Complex sociogenetic organization and reproductive skew in a primitively eusocial sweat bee,Lasioglossum malachurum, as revealed by microsatellites
Authors: Paxton Robert J. , Ayasse Manfred , Field Jeremy , Soro Antonella
Published: March 2003

The sweat bees (Family Halictidae) are a socially diverse taxon in which eusociality has arisen independently numerous times. The obligate, primitively eusocialLasioglossum malachurum, distributed widely throughout Europe, has been considered the zenith of sociality within halictids. A single queen heads a colony of smaller daughter workers which, by mid‐summer, produce new sexuals (males and gynes), of which only the mated gynes overwinter to found new colonies the following spring. We excavated successfully 18 nests during the worker‐ and gyne‐producing phases of the colony cycle and analysed each nest's queen and either all workers or all gynes using highly variable microsatellite loci developed specifically for this species. Three important points arise from our analyses. First, queens are facultatively polyandrous (queen effective mating frequency: range 1–3, harmonic mean 1.13). Second, queens may head colonies containing unrelated individuals (n= 6 of 18 nests), most probably a consequence of colony usurpation during the early phase of the colony cycle before worker emergence. Third, nonqueen's workers may, but the queen's own workers do not, lay fertilized eggs in the presence of the queen that successfully develop into gynes, in agreement with so‐called ‘concession’ models of reproductive skew.
Male production in stingless bees: variable outcomes of queen–worker conflict
Authors: Tóth Eva , Strassmann Joan E. , Nogueira‐Neto Paulo , Imperatriz‐Fonseca Vera L. , Queller David C.
Published: March 2003

The genetic structure of social insect colonies is predicted to affect the balance between cooperation and conflict. Stingless bees are of special interest in this respect because they are singly mated relatives of the multiply mated honeybees. Multiple mating is predicted to lead to workers policing each others’ male production with the result that virtually all males are produced by the queen, and this prediction is borne out in honey bees. Single mating by the queen, as in stingless bees, causes workers to be more related to each others’ sons than to the queen's sons, so they should not police each other. We used microsatellite markers to confirm single mating in eight species of stingless bees and then tested the prediction that workers would produce males. Using a likelihood method, we found some worker male production in six of the eight species, although queens produced some males in all of them. Thus the predicted contrast with honeybees is observed, but not perfectly, perhaps because workers either lack complete control or because of costs of conflict. The data are consistent with the view that there is ongoing conflict over male production. Our method of estimating worker male production appears to be more accurate than exclusion, which sometimes underestimates the proportion of males that are worker produced.
The use of AFLP to find an informative SNP: genetic differences across a migratory divide in willow warblers
Authors: Bensch Staffan , Åkesson Susanne , Irwin Darren E.
Published: March 2003

We used the amplified fragment length polymorphism (AFLP) method to obtain genetic markers distinguishing two subspecies of willow warblersPhylloscopus trochilusthat have different migratory behaviours but are not differentiated in mitochondrial DNA or at several microsatellite loci. With the inverse‐polymerase chain reaction (PCR) approach we converted a dominant AFLP‐marker to a codominant single nucleotide polymorphism (SNP). Across Scandinavia we typed 621 birds at the SNP locus AFLP‐WW1 and we found a sigmoid change in allele frequencies centred around 62 degrees latitude. North of the latitudinal cline was a west‐east cline. Both clines are narrower than one would expect from dispersal distances in willow warblers, which suggests that these are maintained by selection. The latitudinal cline at the locus AFLP‐WW1 is paralleled by changes in several other traits, all of which might be maintained by a single selective force. The most plausible selection factor that we have identified is selection against hybrids because of inferior migratory behaviour. The selective force maintaining the east–west cline is less obvious. We discuss alternatives to the selection scenario, involving colonization history and asymmetric gene flow.
Mitochondrial DNA variation in Indo‐Pacific populations of the giant tiger prawn, Penaeus monodon
Authors: Benzie J. A. H. , Ballment E. , Forbes A. T. , Demetriades N. T. , Sugama K. , Haryanti  , Moria S.
Published: March 2003

Surveys of mitochondrial DNA (mtDNA) variation in the giant tiger prawn, Penaeus monodon, using restriction fragment length polymorphisms have provided the first clear evidence that the Indo‐West Pacific region is a site of accumulation of genetic diversity rather than a site of origin of genetic diversity. No haplotyes were found in common between a group of five southeast African populations and a group of five Australian (including Western Australia) and three southeast Asian populations. The dominant haplotype was different in the Australian and southeast Asian population groups. Genetic diversity (π) was greatest in Indonesia (π averaged 0.05), less in the Philippines and Australia (π averaged 0.01), and markedly less in the southeast African and the West Australian populations (π averaged 0.003). The high diversity of the southeast Asian populations resulted from the occurrence in those populations of a set of haplotypes found only in southeast Asia but derived from the southeast African haplotypes. These genetic variants therefore evolved in the Indian Ocean and later migrated into the Indo‐West Pacific region. Low genetic variation in the geographically marginal populations in southeast Africa and Western Australia is considered to be the result of bottlenecks, but mismatch distributions suggest that large population sizes have been maintained in Indonesian populations for long periods.
Bacterial diversity in maize rhizospheres: conclusions on the use of genetic profiles based on PCR‐amplified partial small subunit rRNA genes in ecological studies
Authors: Schmalenberger Achim , Tebbe Christoph C.
Published: March 2003

A cultivation‐independent approach based on polymerase chain reaction (PCR)‐amplified partial small subunit rRNA genes and genetic profiling by single‐strand conformation polymorphism (SSCP) was used to characterize the bacterial diversity inhabiting the rhizosphere of maize plants grown on an agricultural field. The community structures of two cultivars, a genetically engineered and a nonengineered variety, different herbicide regimes and soil tillage were compared with each other at two sampling dates. SSCP‐profiles were generated with DNA from bacterial cell consortia with primers hybridizing to evolutionarily highly conserved rRNA gene regions. On silver‐stained gels, each profile consisted of approx. 50 distinguishable bands. Similarity analyses of patterns recorded by digital image analyses could not detect any difference between cultivars or treatments that was greater than the variability between replicates. A total of 54 sequences recovered from different bands were identified and grouped into operational taxonomical units (OTUs). Surprisingly, only five of 40 OTUs contained sequences of both samplings. Three different bands from a profile were selected to test whether this small overlap was due to an incomplete recovery of sequences. From a faint band, two different OTUs were found when 12 clones were analysed, and from two strong bands 24 and 22 OTUs were detected from a total of 26 and 36 clones, respectively. The OTUs belonged to phylogenetically different groups of bacteria. Gene probes that were developed to target different bands of the profiles, however, indicated in Southern blot analyses that patterns between treatments, replicates and samplings, and even from two different growing seasons were highly conserved. Our study demonstrates that community profiles can consist of more sequences than detectable by staining and that gene probes in Southern blot can be a useful control to investigate the composition of microbial communities by genetic profiles.
Tracing the geographical origin of Megastigmus transvaalensis (Hymenoptera: Torymidae): an African wasp feeding on a South American plant in North America
Authors: Scheffer Sonja J. , Grissell E. E.
Published: March 2003

Determining the geographical origin of an introduced organism can be critical to understanding or managing a non‐native species, but is often difficult when the organism is small or inconspicuous. We used a phylogeographical approach to identify the region of endemism and determine the geographical origin of world populations of the seed‐feeding wasp Megastigmus transvaalensis (Hussey). This wasp feeds on African Rhus species and South American Schinus species in various locations around the world. Because it is present both in Africa and in South America, it is unclear whether the wasp was originally an African Rhus‐feeder that has begun feeding on Schinus or a South American Schinus‐feeder that has started feeding on Rhus. Phylogenetic analysis of 800 bp of mitochondrial cytochrome oxidase I sequence data found extensive variation and phylogeographical structure within African M. transvaalensis. Specimens from other locations around the world were all identical in COI sequence and were phylogenetically nested within the African samples. We conclude that M. transvaalensis was originally an African Rhus‐feeder that readily attacks Schinus. We evaluate potential pathways of introduction of this wasp to the New World, and we discuss implications of our results for biocontrol efforts against invasive Schinus populations.
A molecular genetic analysis of the communal nesting of the ostrich (Struthio camelus)
Authors: Kimwele C. N. , Graves J. A.
Published: March 2003

The ostrich breeding system is complex and unique; communal clutches are laid by several females, although only one female, the major female, and the resident territorial male provide parental care. More eggs are laid in the nest than can be incubated and the major female ejects surplus eggs from the incubated central clutch. Microsatellite markers were used to analyse the parentage of communal nests in Nairobi National Park. This revealed that major females contributed a disproportionate number of fertile eggs to the central, incubated clutch and that multiple paternity and maternity within a nest were common; 68.9% of all incubated eggs on a nest were not parented by both the resident territorial male and the major female of that nest. All the males fertilized eggs on the clutches of neighbouring males. Unexpectedly, every major female with her own nest was also simultaneously a minor female with incubated eggs on neighbouring clutches. The relatedness between females laying in the same nest was not significantly different from the population average and significantly less than that between chicks hatched from the same nest.
Associations among cytoplasmic molecular markers, gender, and components of fitness in Silene vulgaris, a gynodioecious plant
Authors: Mccauley D. E. , Olson M. S.
Published: March 2003

It has been suggested that the dynamics of chloroplast DNA (cpDNA) or mitochondrial DNA (mtDNA) genetic markers used in studies of plant populations could be influenced by natural selection acting elsewhere in the genome. This could be particularly true in gynodioecious plants if cpDNA or mtDNA genetic markers are in gametic disequilibrium with genes responsible for sex expression. In order to investigate this possibility, a natural population of the gynodioecious plant Silene vulgaris was used to study associations among mtDNA haplotype, cpDNA haplotype, sex and some components of fitness through seed. Individuals were sampled for mtDNA and cpDNA haplotype as determined using restriction fragment length polymorphism (RFLP) methods, sex (female or hermaphrodite), fruit number, fruit set, seeds/fruit and seed germination. The sex of surviving germinating seeds was also noted. All individuals in the population fell into one of two cytoplasmic categories, designated haplotypes f and g by a unique electrophoretic signature in both the mtDNA and cpDNA. The subset of the population carrying haplotype g included a significantly higher proportion of females when compared with the sex ratio of the subset carrying the f haplotype. Haplotype g had a significantly higher fitness when measured by fruit number, fruit set and seeds/fruit, whereas haplotype f had significantly higher fitness when measured by seed germination. Offspring of individuals carrying haplotype g included a significantly greater proportion of females when compared with offspring of individuals carrying the f haplotype. Other studies of gynodioecious plants have shown that females generally have higher fitness through seed than hermaphrodites, but in this study not all fitness differences between haplotypes could be predicted from differences in haplotype‐specific sex ratio alone. Rather, some differences in haplotype‐specific fitness were due to differences in fitness between individuals of the same sex, but carrying different haplotypes. The results are discussed with regard to the potential for hitchhiking selection to influence the dynamics of the noncoding regions used to designate the cpDNA and mtDNA haplotypes.
Population genetics of a successful invader: the marsh frog Rana ridibunda in Britain
Authors: Zeisset Inga , Beebee Trevor J. C.
Published: March 2003

We investigated the genetic outcome of successful invasion by an alien species, the marsh frog Rana ridibunda, in Britain. Twelve adults translocated from Hungary into Kent (Romney) in 1935 resulted rapidly in a large localized population. A further successful translocation in 1973 from Romney to Sussex (Lewes), together with other range extensions, provided an opportunity to test bottleneck effects during colonization events. Romney and Lewes frogs had similar genetic diversities to those in Hungary at 14 random amplified polymorphic DNA marker (RAPD) and five microsatellite loci. The introduced populations were, however, differentiated genetically from each other and from a reference population in Hungary. Fitness assessments (larval growth and survival) revealed no differences between the Lewes and Romney populations. Despite starting with few founders, significant bottleneck effects on R. ridibunda in Britain were therefore undetectable, presumably because population expansions were rapid immediately after the translocations.
Analysis of the gene cluster involved in production and immunity of the peptide antibiotic AS‐48 in Enterococcus faecalis
Authors: Martínez‐Bueno Manuel , Valdivia Eva , Gálvez Antonio , Coyette Jacques , Maqueda Mercedes
Published: March 2003

A region of 7.8 kb of the plasmid pMB2 from Enterococcus faecalis S‐48 carrying the information necessary for production and immunity of the peptide antibiotic AS‐48 has been cloned and sequenced. It contains the as‐48A structural gene plus five open reading frames (as‐48B, as‐48C, as‐48C1, as‐48D and as‐48D1 ). Besides As‐48D, all the predicted gene products are basic hydrophobic proteins with potential membrane‐spanning domains (MSDs). None of them shows any homology with protein sequences stored in databanks, except for As‐48D, which shows similarity to the C‐terminal domain of ABC transporters and contains a highly conserved ATP‐binding site. The gene products of as‐48B, as‐48C, as‐48C1 and as‐48D are thought to be involved in AS‐48 production and secretion. The only gene able to provide resistance to AS‐48 by itself is as‐48D1. Immunity also seems to be enhanced at least by the products of as‐48B, as‐48C1 and as‐48D genes. Transcription analysis using probes derived from the different ORFs revealed two large (3.5 and 2.7 kb) mRNAs, suggesting that the different genes are organized in two constitutive operons.
The Salmonella typhimurium tyrosine phosphatase SptP is translocated into host cells and disrupts the actin cytoskeleton
Authors: Fu Yixin , Galán Jorge E.
Published: March 2003

The Salmonella typhimurium protein tyrosine phosphatase SptP is a target of the centisome 63 type III protein secretion system. This system is essential for the interaction of these bacteria with host cells. We have shown here by a combination of biochemical and microscopy techniques that S. typhimurium directs the translocation of SptP into cultured epithelial cells. Translocation requires the function of the secreted proteins, SipB, SipC and SipD, as strains carrying mutations in any of the genes encoding these proteins fail to translocate SptP. Microinjection of purified GST–SptP into cultured cells results in the disruption of the actin cytoskeleton and the disappearance of stress fibres. These changes are reversible, as microinjected cells regain the normal appearance of their actin cytoskeleton upon prolonged incubation. Microinjection of the catalytically inactive GST–SptP(C481S) protein results in changes similar to those induced by the wild‐type toxin. Furthermore, microinjection of a fusion protein between GST and the first 285 amino acids of SptP also leads to identical disruption of the host cell actin cytoskeleton, indicating that the amino‐terminal half of SptP is sufficient to mediate this effect. However, microinjection of a fusion protein between GST and the last 259 amino acids of SptP also disrupted the normal appearance of the cytoskeleton. These results support the hypothesis that SptP is an effector protein arranged in modular domains that may co‐operate with each other to exert related functions.
Periplasmic copper–zinc superoxide dismutase protects Haemophilus ducreyi from exogenous superoxide
Authors: San Mateo Lani R. , Hobbs Marcia M. , Kawula Thomas H.
Published: March 2003

Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcer disease implicated in increased heterosexual transmission of HIV. As part of an effort to identify H. ducreyi gene products involved in virulence and pathogenesis, we created random TnphoA insertion mutations in an H. ducreyi 35 000 library cloned in Escherichia coli. Inserts encoding exported or secreted PhoA fusion proteins were characterized by DNA sequencing. One such clone encoded a Cu–Zn superoxide dismutase (SOD) enzyme. The Cu–Zn SOD was periplasmic in H. ducreyi and accounted for most of the detectable SOD activity in whole‐cell lysates of H. ducreyi grown in vitro. To investigate the function of the Cu–Zn SOD, we created a Cu–Zn SOD‐deficient H. ducreyi strain by inserting a cat cassette into the sodC gene. The wild‐type and Cu–Zn SOD null mutant strains were equally resistant to excess cytoplasmic superoxide induced by paraquat, demonstrating that the Cu–Zn SOD did not function in the detoxification of cytoplasmic superoxide. However, the Cu–Zn SOD null strain was significantly more susceptible to killing by extracellular superoxide than the wild type. This result suggests that the H. ducreyi Cu–Zn SOD may play a role in bacterial defence against oxidative killing by host immune cells during infection.
Alginate, inorganic polyphosphate, GTP and ppGpp synthesis co‐regulated in Pseudomonas aeruginosa: implications for stationary phase survival and synthesis of RNA/DNA precursors
Authors: Kim Hong‐Yeoul , Schlictman D. , Shankar Sandeep , Xie Zhidong , Chakrabarty A. M. , Kornberg A.
Published: March 2003

The regulatory protein AlgR2 in Pseudomonas aeruginosa positively regulates nucleoside diphosphate kinase (Ndk) and succinyl‐CoA synthetase, enzymes critical in nucleoside triphosphate (NTP) formation. AlgR2 positively regulates the production of alginate, GTP, ppGpp and inorganic polyphosphate (poly P). An algR2 mutant with low levels of these metabolites has them restored by introducing and overexpressing either the algR2 or the ndk gene into the algR2 mutant. Thus, Ndk is involved in the formation of these compounds and largely prevents the death of the algR2 mutant, which occurs early in the stationary phase. We demonstrate that the 12 kDa Ndk–pyruvate kinase (Pk) complex, previously shown to generate predominantly GTP instead of all the NTPs, has a low affinity for the deoxynucleoside diphosphates and cannot generate the dNTPs needed for DNA replication and cell division; this complex may thus be involved in regulating the levels of both NTPs and dNTPs that modulate cell division and survival in the stationary phase.
High‐affinity binding sites for the initiator protein DnaA on the chromosome of Escherichia coli
Authors: Roth Angelika , Messer Walter
Published: March 2003

The initiator protein DnaA of Escherichia coli binds with unusually high affinity to five regions on the chromosome, in addition to the replication origin, oriC. Using a solid‐phase DNA binding assay, in which the DNA binding C‐terminal domain of DnaA is bound via a biotin tag to magnetic beads, we could fish only fragments with these six regions from different chromosomal digests. Except for oriC, these fragments contain only one or two consensus DnaA binding sites, DnaA boxes. The distribution of these high‐affinity DnaA boxes on the chromosome is random.
Polypeptide binding of Escherichia coli FtsH (HflB)
Authors: Akiyama Yoshinori , Ehrmann Michael , Kihara Akio , Ito Koreaki
Published: March 2003

The Escherichia coli FtsH protein is a membrane‐bound and ATP‐dependent protease. In this study, we describe ATP‐dependent conformational changes in FtsH as well as a polypeptide binding ability of this protein. A 33 kDa segment of FtsH became trypsin resistant in the presence of ATP. ATP and ATPγS prevented self‐aggregation of detergent‐solubilized FtsH‐His6‐Myc at 37°C, again suggesting that the binding of ATP induces a conformational change in FtsH. Affinity chromatography showed that FtsH‐His6‐Myc can associate with denatured alkaline phosphatase (PhoA) but not with the native enzyme. Denatured PhoA also prevented the aggregation of FtsH, and these two proteins co‐sedimented through a sucrose gradient. Binding between FtsH‐His6‐Myc and detergent‐solubilized SecY was also demonstrated. Although FtsH‐bound SecY was processed further for ATP‐dependent proteolysis, FtsH‐bound PhoA was not. Thus, FtsH association with denatured PhoA is uncoupled from proteolysis. Overproduction of FtsH significantly increased the cytoplasmic localization of the PhoA moiety of a MalF–PhoA hybrid protein, in which a charged residue had been introduced into a transmembrane segment. Thus, denatured PhoA binding of FtsH may also occur in vivo.
Targeted deletion and mutational analysis of the essential (2Fe–2S) plant‐like ferredoxin in Synechocystis PCC6803 by plasmid shuffling
Authors: Poncelet Mireille , Cassier‐Chauvat Corinne , Leschelle Xavier , Bottin Hervé , Chauvat Franck
Published: March 2003

The genes encoding (2Fe–2S) plant‐like ferredoxins were studied in the widely used cyanobacterium Synechocystis PCC6803. The fedI gene (ssl0020) coding for the most abundant ferredoxin product was found to be expressed strongly as a light‐induced monocistronic transcript, whereas the other fed genes appeared to be silent (slr1828) or moderately expressed as polycistronic transcripts regulated by either light fluence (slr0150, negative control) or glucose availability (sll1382). fedI was found to be critical to Synechocystis PCC6803 viability in spite of slr0150, sll1382 or flavodoxin induction, even after the addition of glucose that compensates for the loss of photosynthesis. Nevertheless, fedI could be deleted from all chromosome copies in cells propagating a fedI gene (even of heterologous origin) on a replicating plasmid. This strain was used as the host for the subsequent introduction of fedI mutant alleles propagated on a second vector. Analysis of the fedI mutant strains generated after plasmid exchange showed that the C18–C85 disulphide bridge is not central either to the tight compaction of ferredoxin I or to its reduction by photosystem I and demonstrated that the length of the FedI carboxy terminus is important for effective PSI/FedI interactions. The plasmid‐shuffling strategy presently described has general applicability for mutational analysis of essential genes in many organisms, as it is based on promiscuous plasmids.
Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus
Authors: Quirós Luis M. , Aguirrezabalaga Ignacio , Olano Carlos , Méndez Carmen , Salas José A.
Published: March 2003

A 5.2 kb region from the oleandomycin gene cluster in Streptomyces antibioticus located between the oleandomycin polyketide synthase gene and sugar biosynthetic genes was cloned. Sequence analysis revealed the presence of three open reading frames (designated oleI, oleN2 and oleR). The oleI gene product resembled glycosyltransferases involved in macrolide inactivation including the oleD product, a previously described glycosyltransferase from S. antibioticus. The oleN2 gene product showed similarities with different aminotransferases involved in the biosynthesis of 6‐deoxyhexoses. The oleR gene product was similar to several glucosidases from different origins. The oleI, oleR and oleD genes were expressed in Streptomyces lividans. OleI and OleD intracellular proteins were partially purified by affinity chromatography in an UDP‐glucuronic acid agarose column and OleR was detected as a major band from the culture supernatant. OleI and OleD showed oleandomycin glycosylating activity but they differ in the pattern of substrate specificity: OleI being much more specific for oleandomycin. OleR showed glycosidase activity converting glycosylated oleandomycin into active oleandomycin. A model is proposed integrating these and previously reported results for intracellular inactivation, secretion and extracellular reactivation of oleandomycin.
The Yersinia Yops inhibit invasion of Listeria, Shigella and Edwardsiella but not Salmonella into epithelial cells
Authors: Mecsas Joan , Raupach Bärbel , Falkow Stanley
Published: March 2003

Yersinia virulence is dependent on the expression of plasmid‐encoded secreted proteins called Yops. After bacterial adherence to receptors on the mammalian cell membrane, several Yops are transported by a type III secretion pathway into the host cell cytoplasm. Two Yops, YopH and YopE, prevent macrophages from phagocytosing Yersinia by disrupting the host cell cytoskeleton and signal transduction pathways. In contrast to this active inhibition of phagocytosis by Yersinia, other pathogens such as Salmonella, Shigella, Listeria and Edwardsiella actively promote their entry into mammalian cells by binding to specific host surface receptors and exploiting existing cell cytoskeletal and signalling pathways. We have tested whether Yersinia Yops can prevent the uptake of these diverse invasive pathogens. We first infected epithelial cells with Yersinia to permit delivery of Yops and subsequently with an invasive pathogen. We then measured the level of bacterial invasion. Preinfection with Yersinia inhibited invasion of Edwardsiella, Shigella and Listeria, but not Salmonella. Furthermore, we found that either YopE or YopH prevented Listeria invasion, whereas only YopE prevented Edwardsiella and Shigella invasion. We correlated the inhibitory effect of the Yops with the inhibitory action of the cell‐signalling inhibitors Wortmannin, LY294002 and NDGA, and concluded that the four invasive pathogenic species enter epithelial cells using at least three distinct host cell pathways. We also speculate that YopE affects the rho pathway.
N‐terminal characterization of the Bordetella pertussis filamentous haemagglutinin
Authors: Lambert‐Buisine Corinne , Willery Eve , Locht Camille , Jacob‐Dubuisson Françoise
Published: March 2003

The major adhesin of Bordetella pertussis, filamentous haemagglutinin (FHA), is produced and secreted at high levels by the bacterium. Mature FHA derives from a large precursor, FhaB, that undergoes several post‐translational maturations. In this work, we demonstrate by site‐directed mutagenesis that the N‐terminal signal peptide of FHA is composed of 71 amino acids, including a 22‐residue‐long ‘N‐terminal extension’ sequence. This sequence, although highly conserved in various other secretory proteins, does not appear to play an essential part in FHA secretion, as shown by deletion mutagenesis. The entire N‐terminal signal region of FhaB is removed in the course of secretion by proteolytic cleavage at a site that corresponds to a Lep signal peptidase recognition sequence. After this maturation, the N‐terminal glutamine residue is modified to a pyroglutamate residue. This modification is not crucial for heparin binding, haemagglutination or secretion. Interestingly, however, the modification is absent from Escherichia coli secreted FHA derivatives. In addition, it is dependent in B. pertussis on the presence of all three cysteines contained in the signal peptide of FhaB. These observations suggest that it does not occur spontaneously but perhaps requires a specific enzymatic machinery.
A regulatory cascade involving AarG, a putative sensor kinase, controls the expression of the 2′‐N‐acetyltransferase and an intrinsic multiple antibiotic resistance (Mar) response in Providencia stuartii
Authors: Rather Philip N. , Paradise Michael R. , Parojcic Milica M. , Patel Shilpa
Published: March 2003

A recessive mutation, aarG1, has been identified that resulted in an 18‐fold increase in the expression of β‐galactosidase from an aac(2′)–lacZ fusion. Transcriptional fusions and Northern blot analysis demonstrated that the aarG1 allele also resulted in a large increase in the expression of aarP, a gene encoding a transcriptional activator of aac(2′)‐Ia. The effects of aarG1 on aac(2′)‐Ia expression were mediated by aarP‐dependent and ‐independent mechanisms. The aarG1 allele also resulted in a multiple antibiotic resistance (Mar) phenotype, which included increased chloramphenicol, tetracycline and fluoroquinolone resistance. This Mar phenotype also resulted from aarP‐dependent and ‐independent mechanisms. Sequence analysis of the aarG locus revealed the presence of two open reading frames, designated aarR and aarG, organized in tandem. The putative AarR protein displayed 75% amino acid identity to the response regulator PhoP, and the AarG protein displayed 57% amino acid identity to the sensor kinase PhoQ. The aarG1 mutation, a C to T substitution, resulted in a threonine to isoleucine substitution at position 279 (T279I) in the putative sensor kinase. The AarG product was functionally similar to PhoQ, as it was able to restore wild‐type levels of maganin resistance to a Salmonella typhimurium phoQ mutant. However, expression of the aarP and aac(2′)‐Ia genes was not significantly affected by the levels of Mg2+ or Ca2+, suggesting that aarG senses a signal other than divalent cations.
The Mycoplasma hominis vaa gene displays a mosaic gene structure
Authors: Boesen Thomas , Emmersen Jeppe , Jensen Lise T. , Ladefoged Søren A. , Thorsen Poul , Birkelund Svend , Christiansen Gunna
Published: March 2003

Mycoplasma hominis contains a variable adherence‐associated (vaa) gene. To classify variants of the vaa genes, we examined 42 M. hominis isolates by PCR, DNA sequencing and immunoblotting. This uncovered the existence of five gene categories. Comparison of the gene types revealed a modular composition of the Vaa proteins. The proteins constituted a conserved N‐terminal part followed by a varying number of interchangeable cassettes encoding approximately 110 amino acids with conserved sequence boxes flanking the cassettes. The interchangeable cassettes showed a high mutual homology and a conserved leucine zipper motif. The smallest product contained only one cassette and the largest five. Additionally, two types of stop mutations caused by substitutions resulting in the expression of truncated Vaa proteins were observed. Our results expand the known potential of the Vaa system in generating antigen variation.
FadR, transcriptional co‐ordination of metabolic expediency
Authors: Cronan, Jr John E. , Subrahmanyam Satyanarayana
Published: March 2003

FadR is an Escherichia coli transcriptional regulator that optimizes fatty acid metabolism in response to exogenously added fatty acids. Many bacteria grow well on long‐chain fatty acids as sole carbon source, but at the expense of consuming a useful structural material. Exogenous fatty acids are readily incorporated into membrane phospholipids in place of the acyl chains synthesized by the organism, and phospholipids composed of any of a large variety of exogenously derived acyl chains make biologically functional membranes. It would be wasteful for bacteria to degrade fatty acids to acetyl‐CoA and then use this acetyl‐CoA to synthesize the same (or functionally equivalent) fatty acids for phospholipid synthesis. This line of reasoning suggests that bacteria might shut down endogenous fatty acid synthesis on the addition of long‐chain fatty acids to the growth medium. Moreover, this shutdown could be closely coupled to fatty acid degradation, such that a bacterial cell would use a portion of the exogenous fatty acid for phospholipid synthesis while degrading the remainder to acetyl‐CoA. To a degree, the bacterium could both have its cake (the acyl chains for phospholipid synthesis) and eat it (to form acetyl‐CoA). This scenario turns out to be true in E. coli. The key player in this regulatory gambit is FadR, a transcription factor that acts both as a repressor of the fatty acid degradation and as an activator of fatty acid biosynthesis.
Roles in dimerization and blue light photoresponse of the PAS and LOV domains of Neurospora crassa white collar proteins
Authors: Ballario Paola , Talora Claudio , Galli Daniela , Linden Hartmut , Macino Giuseppe
Published: March 2003

The genes coding for white collar‐1 and white collar‐2 (wc‐1 and wc‐2 ) have been isolated previously, and their products characterized as Zn‐finger transcription factors involved in the control of blue light‐induced genes. Here, we show that the PAS dimerization domains present in both proteins enable the WC‐1 and WC‐2 proteins to dimerize in vitro. Homodimers and heterodimers are formed between the white collar (WC) proteins. A computer analysis of WC‐1 reveals a second domain, called LOV, also identified in NPH1, a putative blue light photoreceptor in plants and conserved in redox‐sensitive proteins and in the phytochromes. The WC‐1 LOV domain does not dimerize with canonical PAS domains, but it is able to self‐dimerize. The isolation of three blind wc‐1 strains, each with a single amino acid substitution only in the LOV domain, reveals that this region is essential for blue light responses in Neurospora. The demonstration that the WC‐1 proteins in these LOV mutants are still able to self‐dimerize suggests that this domain plays an additional role, essential in blue light signal transduction.
Identification of a novel gene involved in pilin glycosylation in Neisseria meningitidis
Authors: Jennings Michael P. , Virji Mumtaz , Evans Debbie , Foster Virginia , Srikhanta Yogitha N. , Steeghs Liana , Van Der Ley Peter , Moxon E. Richard
Published: March 2003

The pili of Neisseria meningitidis are a key virulence factor, being major adhesins of this capsulate organism that contribute to specificity for the human host. Recently it has been reported that meningococcal pili are post‐translationally modified by the addition of an O‐linked trisaccharide, Gal (β1–4) Gal (α1–3) 2,4‐diacetimido‐2,4,6‐trideoxyhexose. Using a set of random genomic sequences from N. meningitidis strain MC58, we have identified a novel gene homologous to a family of glycosyltransferases. A plasmid clone containing the gene was isolated from a genomic library of N. meningitidis strain MC58 and its nucleotide sequence determined. The clone contained a complete copy of the gene, here designated pglA (pilin glycosylation). Insertional mutations were constructed in pglA in a range of meningococcal strains with well‐defined lipopolysaccharide (LPS) or pilin‐linked glycan structures to determine whether pglA had a role in the biosynthesis of these molecules. There was no alteration in the phenotype of LPS from pglA mutant strains as judged by gel migration and the binding of monoclonal antibodies. In contrast, decreased gel migration of the pilin subunit molecules of pglA mutants was observed, which was similar to the migration of pilins of galE mutants of same strains, supporting the notion that pglA is a glycosyltransferase involved in the biosynthesis of the pilin‐linked trisaccharide structure. The pglA mutation, like the galE mutation reported previously, had no effect on pilus‐mediated adhesion to human epithelial or endothelial cells. Pilin from pglA mutants were unable to bind to monospecific antisera recognizing the Gal (β1–4) Gal structure, suggesting that PglA is a glycosyltransferase involved in the addition of galactose of the trisaccharide substituent of pilin.
Downregulation of Escherichia coli yfiD expression by FNR occupying a site at −93.5 involves the AR1‐containing face of FNR
Authors: Green Jeffrey , Baldwin Mandy L. , Richardson Joanne
Published: March 2003

The promoter of the FNR‐activated yfiD gene of Escherichia coli has an unusual architecture because it contains two FNR sites, an arrangement usually associated with FNR‐mediated repression. Investigation of yfiD promoter derivatives with altered FNR sites revealed that occupation of the far upstream FNR site (FNR II) downregulated expression, despite the presence of a FNR dimer activating expression from the promoter proximal site (FNR I). Transcript mapping by primer extension, and mutagenesis of potential −10 elements, indicated that yfiD expression is driven from a single FNR‐dependent promoter with FNR sites at −40.5 (FNR I) and −93.5 (FNR II). However, yfiD mRNA is processed in stationary‐phase cultures independently of rne, rpoS, ihfA and fis to yield transcripts lacking 12 and 21 bases from their respective 5′ ends. Single amino acid substitutions (G74→C, F92→S, A95→P, R184→P, P188→A or L193→P) in the surface of FNR that contains activating region 1 (AR1 contacts the α‐subunit of RNA polymerase to promote transcription activation) reduced the inhibitory effect of FNR at FNR II, indicating that this region of the protein may have a role in repression as well as activation. The FNR variant F92→S was notable because, although it activated transcription of yfiD (two FNR sites), it was unable to activate transcription from model Class I and II promoters, which contain only a single FNR site.
hpaA mutants of Xanthomonas campestris pv. vesicatoria are affected in pathogenicity but retain the ability to induce host‐specific hypersensitive reaction
Authors: Huguet Elisabeth , Hahn Karoline , Wengelnik Kai , Bonas Ulla
Published: March 2003

Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper and tomato plants. We reported previously that the main hrp (hypersensitive reaction and pathogenicity) gene cluster in X. c. pv. vesicatoria contains six transcription units, designated hrpA to hrpF. We present here the sequence of the hrpD operon and an analysis of non‐polar mutants in each of the six genes. Three genes, hrcQ, hrcR and hrcS, are predicted to encode conserved components of type III protein secretion systems in plant and mammalian pathogenic bacteria. For hrpD5 and hrpD6, homologues have only been found in Ralstonia solanacearum. Interestingly, the hrpD operon contains one gene, hpaA (for hrp‐associated), which is specifically required for disease development. hpaA mutants are affected in pathogenicity, but retain in part the ability to induce avirulence gene‐mediated, host‐specific hypersensitive reaction (HR). In addition, HpaA was found to contain two functional nuclear localization signals, which are important for the interaction with the plant. We propose that HpaA is an effector protein that may be translocated into the host cell via the Hrp secretion pathway.