bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of transgenic Bt corn litter on the earthwormLumbricus terrestris
Authors: Zwahlen C. , Hilbeck A. , Howald R. , Nentwig W.
Published: December 2004

A 200‐day study was carried out to investigate the impact of transgenicBacillus thuringiensis(Bt) corn on immature and adultLumbricus terrestrisin the field and in the laboratory. Another objective of this study was to develop test methods that could be used for standard testing of the impact of transgenic plants on different earthworm species in the field and in the laboratory. For this purpose two different experiments were involved, a laboratory experiment with adultL. terrestrisand a field experiment with immatureL. terrestris. No lethal effects of transgenic Bt corn on immature and adult earthworms were observed. ImmatureL. terrestrisin the field had a very similar growth pattern when fed either (Bt+) or (Bt−) corn litter. No significant differences in relative weights of (Bt+) and (Bt−) corn‐fed adultL. terrestriswere observed during the first 160 days of the laboratory trial, but after 200 days adultL. terrestrishad a significant weight loss of 18% of their initial weight when fed (Bt+) corn litter compared to a weight gain of 4% of the initial weight of (Bt−) corn‐fed earthworms. Further studies are necessary to see whether or not this difference in relative weight was due to the Bt toxin or other factors discussed in the study. Degradation of Cry1Ab toxin in corn residues was significantly slower in the field than at 10 °C in the laboratory. Enzyme‐linked immunosorbent assay results indicated that earthworms in both experiments were exposed to the Bt toxin throughout the whole experimental time.
Natural selection for salt tolerance quantitative trait loci (QTLs) in wild sunflower hybrids: Implications for the origin ofHelianthus paradoxus, a diploid hybrid species
Authors: Lexer C. , Welch M. E. , Durphy J. L. , Rieseberg L. H.
Published: December 2004

For a new diploid or homoploid hybrid species to become established, it must diverge ecologically from parental genotypes. Otherwise the hybrid neospecies will be overcome by gene flow or competition. We initiated a series of experiments designed to understand how the homoploid hybrid species,Helianthus paradoxus, was able to colonize salt marsh habitats, when both of its parental species (H. annuus×H. petiolaris) are salt sensitive. Here, we report on the results of a quantitative trait locus (QTL) analysis of mineral ion uptake traits and survivorship in 172 BC2hybrids betweenH. annuusandH. petiolaristhat were planted inH. paradoxussalt marsh habitat in New Mexico. A total of 14 QTLs were detected for mineral ion uptake traits and three for survivorship. Several mineral ion QTLs mapped to the same position as the survivorship QTLs, confirming previous studies, which indicated that salt tolerance inHelianthusis achieved through increased Ca uptake, coupled with greater exclusion of Na and related mineral ions. Of greater general significance was the observation that QTLs with effects in opposing directions were found for survivorship and for all mineral ion uptake traits with more than one detected QTL. This genetic architecture provides an ideal substrate for rapid ecological divergence in hybrid neospecies and offers a simple explanation for the colonization of salt marsh habitats byH. paradoxus. Finally, selection coefficients of +0.126, −0.084 and −0.094 for the three survivorship QTLs, respectively, are sufficiently large to account for establishment of new, homoploid hybrid species.
Adaptation at specific loci. VII. Natural selection, dispersal and the diversity of molecular–functional variation patterns among butterfly species complexes (Colias: Lepidoptera, Pieridae)
Authors: Watt W. B. , Wheat C. W. , Meyer E. H. , Martin J.‐F.
Published: December 2004

Natural genetic variants at the phosphoglucose isomerase, PGI, gene differ in spatial patterning of their polymorphism among species complexes ofColiasbutterflies in North America. In both lowland and alpine complexes, molecular–functional properties of the polymorphic genotypes can be used to predict genotype‐specific adult flight performances and resulting large genotypic differences in adult fitness components. In the lowland species complex, there is striking uniformity of PGI polymorph frequencies at a number of sites across the American West; this fits with earlier findings of strong, similar differences in fitness components over this range. In an alpine complex,Colias meadiishows similar uniformity of PGI frequencies within habitat types, either montane steppe or alpine tundra, over several hundred kilometres in the absence of dispersal. At the same time, large shifts (10–20%) in frequency of the most common alleles occur between steppe and tundra populations, whether these are isolated or, as in some cases, are in contact and exchange many dispersing adults each generation. Data on male mating success of commonC. meadiiPGI genotypes in steppe and tundra show heterozygote advantage in both habitat types, with shifts in relative homozygote disadvantage between habitats which are consistent with observed frequency differences. Nonadaptive explanations for this situation are rejected, and alternative, thermal‐ecology‐based adaptive hypotheses are proposed for later experimental test. These findings show that strong local selection may dominate dispersal as an evolutionary agent, whether or not dispersal is present, and that selection may often be the major force promoting ‘cohesion’ of species over long distances. This case offers new opportunities for integrating studies of molecular structure and function with ecological aspects of natural selection in the wild, both within and among species.
Genetics of drought adaptation in Arabidopsis thaliana: I. Pleiotropy contributes to genetic correlations among ecological traits
Authors: Mckay J. K. , Richards J. H. , Mitchell‐Olds T.
Published: December 2004

We examined patterns of genetic variance and covariance in two traits (i) carbon stable isotope ratio δ13C (dehydration avoidance) and (ii) time to flowering (drought escape), both of which are putative adaptations to local water availability. Greenhouse screening of 39 genotypes of Arabidopsis thaliana native to habitats spanning a wide range of climatic conditions, revealed a highly significant positive genetic correlation between δ13C and flowering time. Studies in a range of C3 annuals have also reported large positive correlations, suggesting the presence of a genetically based trade‐off between mechanisms of dehydration avoidance (δ13C) and drought escape (early flowering). We examined the contribution of pleiotropy by using a combination of mutant and near‐isogenic lines to test for positive mutational covariance between δ13C and flowering time. Ecophysiological mutants generally showed variation in δ13C but not flowering time. However, flowering time mutants generally demonstrated pleiotropic effects consistent with natural variation. Mutations that caused later flowering also typically resulted in less negative δ13C and thus probably higher water use efficiency. We found strong evidence for pleiotropy using near‐isogenic lines of FRIGIDA and FLOWERING LOCUS C, cloned loci known to be responsible for natural variation in flowering time. These data suggest the correlated evolution of δ13C and flowering time is explained in part by the fixation of pleiotropic alleles that alter both δ13C and time to flowering.
Statistical phylogeography: methods of evaluating and minimizing inference errors
Authors: Templeton Alan R.
Published: December 2004

Nested clade phylogeographical analysis (NCPA) has become a common tool in intraspecific phylogeography. To evaluate the validity of its inferences, NCPA was applied to actual data sets with 150 strong a priori expectations, the majority of which had not been analysed previously by NCPA. NCPA did well overall, but it sometimes failed to detect an expected event and less commonly resulted in a false positive. An examination of these errors suggested some alterations in the NCPA inference key, and these modifications reduce the incidence of false positives at the cost of a slight reduction in power. Moreover, NCPA does equally well in inferring events regardless of the presence or absence of other, unrelated events. A reanalysis of some recent computer simulations that are seemingly discordant with these results revealed that NCPA performed appropriately in these simulated samples and was not prone to a high rate of false positives under sampling assumptions that typify real data sets. NCPA makes a posteriori use of an explicit inference key for biological interpretation after statistical hypothesis testing. Alternatives to NCPA that claim that biological inference emerges directly from statistical testing are shown in fact to use an a priori inference key, albeit implicitly. It is argued that the a priori and a posteriori approaches to intraspecific phylogeography are complementary, not contradictory. Finally, cross‐validation using multiple DNA regions is shown to be a powerful method of minimizing inference errors. A likelihood ratio hypothesis testing framework has been developed that allows testing of phylogeographical hypotheses, extends NCPA to testing specific hypotheses not within the formal inference key (such as the out‐of‐Africa replacement hypothesis of recent human evolution) and integrates intra‐ and interspecific phylogeographical inference.
Inbreeding in a natural population of Euodynerus foraminatus (Hymenoptera: Vespidae), a solitary wasp with single‐locus complementary sex determination
Authors: Stahlhut Julie K. , Cowan David P.
Published: December 2004

The solitary wasp Euodynerus foraminatus has single‐locus complementary sex determination (sl‐CSD), which is normally incompatible with inbreeding because it increases the production of sterile or inviable diploid males. Previous field observations of E. foraminatus have suggested that high levels of sibling mating are present in this species. However, conclusions about inbreeding and its genetic consequences could be flawed if based solely upon behavioural observations. Through microsatellite DNA genotyping of 102 E. foraminatus females in southwest Michigan, we estimate that between 55% and 77% of the matings in this population take place between siblings, but the frequency of diploid males is lower than expected. Our data suggest that a mixture of inbreeding and outbreeding persists in E. foraminatus despite the presence of sl‐CSD.
Gene flow in the endophyteNeotyphodiumand implications for coevolution withFestuca arizonica
Authors: Sullivan T. J. , Faeth S. H.
Published: December 2004

Arizona fescue (Festuca arizonica) often harbours asymptomatic, asexual endophytic fungi from the genusNeotyphodium. In agronomic grasses,Neotyphodiumendophytes are often credited with a wide range of mutualistic benefits to its host many of which are related to fungal production of alkaloids for herbivore deterrence.Neotyphodiumin the native grass Arizona fescue, however, usually produces alkaloids at levels too low to deter herbivores, and in general, does not behave mutualistically. This study uses microsatellite markers to examine rates of gene flow among four Arizona populations ofNeotyphodium. Haplotypic diversity was generally low; only one population contained more than two haplotypes. Haplotypes carrying multiple loci for some or all of the microsatellite loci were also found, indicating a vegetative hybridization event betweenNeotyphodiumand the grass choke pathogen from the genusEpichloë. Gene flow betweenNeotyphodiumpopulations is very low, and likely much lower than the pollen mediated gene flow of its host. These differing rates of gene flow are predicted to create trait mismatching between endophyte and host and may explain the low, or lack of, alkaloid production byNeotyphodiumin Arizona fescue and other native grass species.
‘What's larvae got to do with it?’ Disparate patterns of post‐glacial population structure in two benthic marine gastropods with identical dispersal potential
Authors: Marko P. B.
Published: December 2004

In marine environments, many species have apparently colonized high latitude regions following the last glacial maximum (LGM) yet lack a life‐history stage, such as a free‐living larva, that is clearly capable of long‐distance dispersal. Two hypotheses can explain the modern high latitude distributions of these marine taxa: (1) survival in northern refugia during the LGM or (2) rapid post‐glacial dispersal by nonlarval stages. To distinguish these two scenarios, I characterized the genetic structure of two closely related northeastern Pacific gastropods that lack planktonic larvae but which have distributions extending more than 1000 km north of the southern limit of glaciers at the LGM. Despite having identical larval dispersal potential, these closely related species exhibit fundamentally different patterns of genetic structure. InNucella ostrina, haplotype diversity among northern populations (British Columbia and Alaska) is low, no pattern of isolation by distance exists and a coalescent‐based model of population growth indicates that during the LGM population size was reduced to less than 35% of its current size. In the congeneric and often sympatricN. lamellosa, northern populations harbour a diversity of ancient private haplotypes, significant evidence of isolation by distance exists and regional subdivision was found between northern (Alaska) and southern (southern British Columbia, Washington and Oregon) populations. Estimates of coalescent parameters indicate only a modest reduction in population size during the LGM and that northern and southern populations ofN. lamellosasplit ∼50 Kyr before the LGM. The patterns are consistent with the hypothesis thatN. ostrinarecently reinvaded the northeastern Pacific butN. lamellosasurvived the LGM in a northern refuge. A comparison of similar studies in this region indicates that depleted levels of genetic variation at high latitudes — evidence suggestive of recent colonization from a southern refuge — is more common among intertidal species that live relatively high on the shore, where exposure times to cold stress in air are longer than for species living lower on the shore. These data suggest that for some faunas, ecological differences between taxa may be more important than larval dispersal potential in determining species’ long‐term biogeographical responses to climate change.
Phylogeographical patterns of genetic divergence and speciation in African mole‐rats (Family: Bathyergidae)
Authors: Faulkes C. G. , Verheyen E. , Verheyen W. , Jarvis J. U. M. , Bennett N. C.
Published: December 2004

African mole‐rats are subterranean Hystricomorph rodents, distributed widely throughout sub‐Saharan Africa, and displaying a range of social and reproductive strategies from solitary dwelling to the ‘insect‐like’ sociality of the naked mole‐rat, Heterocephalus glaber. Both molecular systematic studies of Rodentia and the fossil record of bathyergids indicate an ancient origin for the family. This study uses an extensive molecular phylogeny and mitochondrial cytochrome b and 12s rRNA molecular clocks to examine in detail the divergence times, and patterns of speciation of the five extant genera in the context of rift valley formation in Africa. Based on a value of 40–48 million years ago (Myr) for the basal divergence of the family (Heterocephalus), we estimate divergence times of 32–40 Myr for Heliophobius, 20–26 Myr for Georychus/Bathyergus and 12–17 Myr for Cryptomys, the most speciose genus. While early divergences may have been independent of rifting, patterns of distribution of later lineages may have been influenced directly by physical barriers imposed by the formation of the Kenya and Western Rift, and indirectly by accompanying climatic and vegetative changes. Rates of chromosomal evolution and speciation appear to vary markedly within the family. In particular, the genus Cryptomys appears to have undergone an extensive radiation and shows the widest geographical distribution. Of the two distinct clades within this genus, one exhibits considerable karyotypic variation while the other does not, despite comparatively high levels of sequence divergence between some taxa. These different patterns of speciation observed both within the family and within the genus Cryptomys may have been a result of environmental changes associated with rifting.
Population genetics of the potentially invasive African fruit fly species,Ceratitis rosaandCeratitis fasciventris(Diptera: Tephritidae)
Authors: Baliraine F. N. , Bonizzoni M. , Guglielmino C. R. , Osir E. O. , Lux S. A. , Mulaa F. J. , Gomulski L. M. , Zheng L. , Quilici S. , Gasperi G. , Malacrida A. R.
Published: December 2004

A set of 10 microsatellite markers was used to survey the levels of genetic variability and to analyse the genetic aspects of the population dynamics of two potentially invasive pest fruit fly species,Ceratitis rosaandC. fasciventris,in Africa. The loci were derived from the closely related species,C. capitata. The degree of microsatellite polymorphism inC. rosaandC. fasciventriswas extensive and comparable to that ofC. capitata. InC. rosa, the evolution of microsatellite polymorphism in its distribution area reflects the colonization history of this species. The mainland populations are more polymorphic than the island populations. Low levels of differentiation were found within the Africa mainland area, while greater levels of differentiation affect the islands.Ceratitis fasciventrisis a central–east African species. The microsatellite data over the Uganda/Kenya spatial scale suggest a recent expansion and possibly continuing gene flow within this area. The microsatellite variability data fromC. rosaandC. fasciventris, together with those ofC. capitata, support the hypothesis of an east African origin of theCeratitisspp.
Strong population structure in the marine sponge Crambe crambe (Poecilosclerida) as revealed by microsatellite markers
Authors: Duran S. , Pascual M. , Estoup A. , Turon X.
Published: December 2004

Different categories of molecular markers have been used so far to study the population structure of sponges. However, these markers often did not have the resolution power to address precisely questions on structuring processes, especially at the intrapopulational level. In this study we show that microsatellites fulfil these expectations, allowing a fine description of population structure at different geographical scales in the marine sponge Crambe crambe. Specimens were collected in 11 locations, representing most of the Atlanto‐Mediterranean range of the species, and were analysed at six loci. As expected for a sessile invertebrate with lecitotrophic larvae, high levels of between‐population structure were found (FST = 0.18) and a significant isolation‐by‐distance pattern was observed. A strong genetic structure was also found within sampled sites (FIS = 0.21) that may be explained by several factors including inbreeding, selfing and the Wahlund effect. In spite of a sampling design planned to avoid the sampling of clones, genotypically identical individuals for the six loci were found in some locations. The significance of these potential clones is discussed and their effect on the observed pattern of population structure assessed. Patterns of allelic distribution within populations suggest the possibility of a recent colonization of the Atlantic range from the Mediterranean Sea.
Mutagenesis of the Neisseria gonorrhoeae porin reduces invasion in epithelial cells and enhances phagocyte responsiveness
Authors: Bauer Frank J. , Rudel Thomas , Stein Markus , Meyer Thomas F.
Published: December 2004

Porin (PorB), the major outer membrane protein of Neisseria gonorrhoeae, has been implicated in pathogenesis previously. However, the fact that porin deletion mutants are not viable has complicated investigations. Here, we describe a method of manipulating the porin gene site‐specifically. N. gonorrhoeae MS11, which harbours the porB1B (P.1B) porin allele, was used to generate mutants carrying deletions in the surface loops 1 and 5. An 11‐amino‐acid deletion in loop 1 impaired Opa50‐dependent invasion into human Chang epithelial cells, whereas loop 5 deletion exhibited no apparent phenotype. In a second approach, the complete gonococcal porB1B was replaced by the porBNla gene of Neisseria lactamica. Such mutants were unable to induce efficient uptake by epithelial cells but induced an enhanced respiratory response in HL60 phagocytic cells. The increased respiratory burst was accompanied by an enhanced phagocytic uptake of the mutant compared with the wild‐type strain. Our data extend previous evidence for multiple central functions of PorB in the infection process.
Genetics of Methanococcus: possibilities for functional genomics in Archaea
Authors: Tumbula Debra L. , Whitman William B.
Published: December 2004

Although the genomic sequences of a number of Archaea have been completed in the last three years, genetic systems in the sequenced organisms are absent. In contrast, genetic studies of the mesophiles in the archaeal genus Methanococcus have become commonplace following the recent developments of antibiotic resistance markers, DNA transformation methods, reporter genes, shuttle vectors and expression vectors. These developments have led to investigations of the transcription of the genes for hydrogen metabolism, nitrogen fixation and flagellin assembly. These genetic systems can potentially be used to analyse the genomic sequence of the hyperthermophile Methanococcus jannaschii, addressing questions of its physiology and the function of its many uncharacterized open reading frames. Thus, the sequence of M. jannaschii can serve as a starting point for gene isolation, while in vivo genetics in the mesophilic methanococci can provide the experimental systems to test the predictions from genomics.
Comparative analysis of prototype two‐component systems with either bifunctional or monofunctional sensors: differences in molecular structure and physiological function
Authors: Alves Rui , Savageau Michael A.
Published: December 2004

Signal transduction by a traditional two‐component system involves a sensor protein that recognizes a physiological signal, autophosphorylates and transfers its phosphate, and a response regulator protein that receives the phosphate, alters its affinity toward specific target proteins or DNA sequences and causes change in metabolic activity or gene expression. In some cases the sensor protein, when unphosphorylated, has a positive effect upon the rate of dephosphorylation of the regulator protein (bifunctional sensor), whereas in other cases it has no such effect (monofunctional sensor). In this work we identify structural and functional differences between these two designs. In the first part of the paper we use sequence data for two‐component systems from several organisms and homology modelling techniques to determine structural features for response regulators and for sensors. Our results indicate that each type of reference sensor (bifunctional and monofunctional) has a distinctive structural feature, which we use to make predictions regarding the functionality of other sensors. In the second part of the paper we use mathematical models to analyse and compare the physiological function of systems that differ in the type of sensor and are otherwise equivalent. Our results show that a bifunctional sensor is better than a monofunctional sensor both at amplifying changes in the phosphorylation level of the regulator caused by signals from the sensor and at attenuating changes caused by signals from small phosphodonors. Cross‐talk to or from other two‐component systems is better suppressed if the transmitting sensor is monofunctional, which is the more appropriate design when such cross‐talk represents pathological noise. Cross‐talk to or from other two‐component systems is better amplified if the transmitting sensor is bifunctional, which is the more appropriate design when such cross‐talk represents a physiological signal. These results provide a functional rationale for the selection of each design that is consistent with available experimental evidence for several two‐component systems.
Functional regulation of the Listeria monocytogenes bacteriophage A118 holin by an intragenic inhibitor lacking the first transmembrane domain
Authors: Vukov Nataša , Moll Isabella , Bläsi Udo , Scherer Siegfried , Loessner Martin J.
Published: December 2004

We have dissected the functional properties of the holin encoded by Listeria monocytogenes bacteriophage A118. Native hol118 was cloned into λΔSthf, devoid of the S holin, and tested in an E. coli background. Surprisingly, it caused very late cell lysis, beginning at 80 min after induction. Immunological analyses demonstrated that Hol118 appears in the cytoplasmic membrane shortly after infection. The hol118 gene features a dual start motif similar to λ S. Therefore, different N‐terminally modified Hol118 variants were tested. However, in contrast to λ S, inactivation of AUG‐1 or AUG‐2 showed no significant influence on lysis timing. In addition, Hol118‐mediated lysis could not be triggered by energy poisons, indicating a functional regulation different from that of S. Toeprinting assays on hol118 mRNA revealed an unexpected translational start codon (AUG‐3) at nucleotide position 40. We demonstrated by in vitro and in vivo approaches that the predicted Hol118(83) product is actually produced together with the full‐length polypeptide. However, although the truncated holin lacking its first transmembrane domain appeared in the cytoplasmic membrane, it was shown to be functionally deficient and unable to support λ R‐mediated lysis. In contrast, specific mutations introduced to abolish translation initiation at AUG‐3 drastically accelerated lysis, pointing to an inhibitor function of Hol118(83). This hypothesis was supported by the observation that hol118(83) inhibited holin function when expressed in trans. A deviation from the λ S paradigm is proposed, which represents a new model of holin functional regulation: the intragenic, in frame translated Hol118(83) product, which is devoid of its first transmembrane domain, acts as a functional inhibitor and constitutes a key part of the lysis clock of A118. Presence of the dominant inhibitor function also explains the long latent period of A118, where the onset of lysis takes about 70 min, more than twice the time needed by λ.
Dimerization of the quorum sensing regulator RhlR: development of a method using EGFP fluorescence anisotropy
Authors: Ventre Isabelle , Ledgham Fouzia , Prima Valérie , Lazdunski Andrée , Foglino Maryline , Sturgis James N.
Published: December 2004

Of considerable interest in the biology of pathogenic bacteria are the mechanisms of intercellular signalling that can lead to the formation of persistent infections. In this article, we have examined the intracellular behaviour of a Pseudomonas aeruginosa quorum sensing regulator RhlR believed to be important in this process. We have further examined the modulation of this behaviour in response to various auto‐inducers. For these measurements, we have developed an assay based on the fluorescence anisotropy of EGFP fusion proteins that we use to measure protein–protein interactions in vivo. We show that the transcriptional regulator, RhlR, expressed as an EGFP fusion protein in Escherichia coli, forms a homodimer. This homodimer can be dissociated into monomers by the auto‐inducer N‐(3‐oxododecanoyl)‐l‐homoserine lactone (3O‐C12‐HSL) whereas N‐(butanoyl)‐l‐homoserine lactone (C4‐HSL) has little effect. These observations are of particular interest as RhlR modulation of gene expression depends on the presence of C4‐HSL, whereas 3O‐C12‐HSL modulates the expression of genes regulated by LasR. These observations thus provide a framework for understanding the regulatory network that links the various different QS regulators in P. aeruginosa. Furthermore, the technique we have developed should permit the study of numerous protein/protein or protein/nucleic acid interactions in vivo and so shed light on natural protein function.
TupA, the Penicillium marneffei Tup1p homologue, represses both yeast and spore development
Authors: Todd Richard B. , Greenhalgh Jennifer R. , Hynes Michael J. , Andrianopoulos Alex
Published: December 2004

Fungal pathogenesis is frequently associated with dimorphism – morphological changes between yeast and filamentous forms. Penicillium marneffei, an opportunistic human pathogen, exhibits temperature‐dependent dimorphism, with growth at 25°C as filamentous multinucleate hyphae switching at 37°C to uninucleate yeast cells associated with intracellular pathogenesis. The filamentous hyphae also undergo asexual development generating uninucleate spores, the infectious propagules. Both processes require a switch to coupled nuclear and cell division. Homologous regulators, including Tup1p/GROUCHO‐related WD40 repeat transcription factors, control dimorphism in Candida albicans and asexual development in Aspergillus nidulans. Unlike these fungi, P. marneffei has both developmental programmes allowing examination of common and programme‐specific controls. We show that deletion of tupA, the P. marneffei TUP1 homologue, confers reduced filamentation and inappropriate yeast morphogenesis at 25°C, in stark contrast to constitutive filamentation observed when C. albicans TUP1 is deleted. Deletion of tupA also confers premature brlA‐dependent asexual development, unlike reduced asexual development in the corresponding A. nidulans rcoA deletion mutant. Furthermore, the A. nidulans rcoA deletion mutant is self‐sterile, and we show that tupA from P. marneffei, which lacks an apparent sexual cycle, complements both the asexual and sexual development phenotypes. Therefore, TupA coordinates cell fate by promoting filamentation and repressing both spore and yeast morphogenetic programmes.
Anticipating an alkaline stress through the Tor phosphorelay system in Escherichia coli
Authors: Bordi Christophe , Théraulaz Laurence , Méjean Vincent , Jourlin‐Castelli Cécile
Published: December 2004

The torCAD operon encoding the TMAO reductase respiratory system is induced in the presence of TMAO by the two‐component regulatory system TorS/TorR. The TorS sensor detects TMAO and transphosphorylates the TorR response regulator via a four‐step phosphorelay. Once phosphorylated, TorR activates expression of the torCAD structural operon. In order to identify new genes regulated by the Tor regulatory system, we performed a genome‐wide transcriptional analysis by using the DNA array technology. We identified seven new transcriptional units whose expression is modulated by the TorS/TorR phosphorelay system. One unit, tnaLAB, is positively regulated whereas the other six, gadA, gadBC, hdeAB, hdeD, yhiE and yhiM, are negatively regulated by this system. Interestingly, the products of some of these units seem to play a role in the survival of E. coli in conditions of extreme pH. The TnaA tryptophanase has been proposed to counteract alkaline stress, whereas the GadA and GadB glutamate decarboxylases and the HdeA and HdeB proteins are involved in the defence against acid stress. Our hypothesis is that the TorS/TorR phosphorelay triggers alkaline‐stress defence to limit alkalinization resulting from the reduction of TMAO in alkaline TMA by the Tor respiratory system. The fact that a ΔtnaLAB mutant showed a dramatic decrease in survival as a result of TMAO respiration is in agreement with such a model. As regulation of these genes by the TorS/TorR system does not depend on pH modification but rather on the presence of TMAO, we propose that E. coli anticipates alkalinization of the medium due to TMA production by base‐resistance gene activation and acid‐resistance gene repression.
The Candida albicans Cdr2p ATP‐binding cassette (ABC) transporter confers resistance to caspofungin
Authors: Schuetzer‐Muehlbauer Manuela , Willinger Birgit , Krapf Gerd , Enzinger Sabine , Presterl Elisabeth , Kuchler Karl
Published: December 2004

Multidrug resistance may pose a serious problem to antifungal therapy. The Candida albicans Cdr2p is one of two ATP‐binding cassette (ABC) transporters mediating antifungal resistance in vivo through increased drug efflux. Echinocandins such as caspofungin represent the newest class of antifungals that target cell wall synthesis. We show here by agar plate resistance assays that cross‐resistant clinical isolates of C. albicans display high minimal inhibitory concentrations (MICs) to caspofungin when compared with a sensitive ATCC reference strain. Northern analysis and immunoblotting indicate that these isolates also show high levels of CDR1 and CDR2 expression. To determine a possible contribution of Cdr1p or Cdr2p to caspofungin resistance, we have functionally expressed Cdr1p and Cdr2p in appropriate recipient strains of the yeast Saccharomyces cerevisiae. Yeast cells expressing Cdr1p or Cdr2p exhibit cross‐resistance to established antifungal drugs such as azoles and terbinafine. However, Cdr2p and, to a much lesser extent, Cdr1p confer caspofungin hyper‐resistance when expressed in yeast. Likewise, Cdr2p confers caspofungin resistance when constitutively overexpressed in a drug‐sensitive C. albicans strain. We therefore propose that Cdr2p may contribute to clinical candin resistance. Finally, our data suggest that cross‐resistance phenotypes of clinical isolates are the consequence of distinct mechanisms that may operate simultaneously.
The Haemophilus influenzae HMW1 adhesin is glycosylated in a process that requires HMW1C and phosphoglucomutase, an enzyme involved in lipooligosaccharide biosynthesis
Authors: Grass Susan , Buscher Amy Z. , Swords W. Edward , Apicella Michael A. , Barenkamp Stephen J. , Ozchlewski Neil , St Geme Joseph W.
Published: December 2004

Non‐typeable Haemophilus influenzae is a common respiratory pathogen and an important cause of morbidity in humans. The non‐typeable H. influenzae HMW1 and HMW2 adhesins are related proteins that mediate attachment to human epithelial cells, an essential step in the pathogenesis of disease. Secretion of these adhesins requires accessory proteins called HMW1B/HMW2B and HMW1C/HMW2C. In the present study, we investigated the specific function of HMW1C. Examination of mutant constructs demonstrated that HMW1C influences both the size and the secretion of HMW1. Co‐immunoprecipitation and yeast two‐hybrid assays revealed that HMW1C interacts with HMW1 and forms a complex in the cytoplasm. Additional experiments and homology analysis established that HMW1C is required for glycosylation of HMW1 and may have glycotransferase activity. The glycan structure contains galactose, glucose and mannose and appears to be generated in part by phosphoglucomutase, an enzyme important for lipooligosaccharide biosynthesis. In the absence of glycosylation, HMW1 is partially degraded and is efficiently released from the surface of the organism, resulting in reduced adherence. Based on these results, we conclude that glycosylation is a prerequisite for HMW1 stability. In addition, glycosylation appears to be essential for optimal HMW1 tethering to the bacterial surface, which in turn is required for HMW1‐mediated adherence, thus revealing a novel mechanism by which glycosylation influences cell–cell interactions.
Chlamydia trachomatis type III secretion: evidence for a functional apparatus during early‐cycle development
Authors: Fields K. A. , Mead D. J. , Dooley C. A. , Hackstadt T.
Published: December 2004

The obligate intracellular bacterium Chlamydia trachomatis occupies a parasitophorous vacuole termed an inclusion. During its intracellular developmental cycle, C. trachomatis maintains this intracellular niche, presumably by expressing a type III secretion system, which deploys a set of host cell‐interactive proteins including inclusion membrane‐localized proteins termed Incs. Some Incs are expressed and secreted by 2 h (early cycle) after infection, whereas the expression of type III‐specific genes is not detectable until 6–12 h (mid‐cycle). To resolve this paradox, we investigated the presence of a type III apparatus on elementary bodies (EBs) that might function early in infection. We demonstrate the existence of the type III secretory apparatus by matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) and immunoblot analyses of purified EB extracts. Immunoblots using polyclonal antibodies specific for the core apparatus component CdsJ identified this protein in both EB and reticulate body (RB) extracts. Furthermore, CdsJ‐specific signals were detected by immunoblot of whole infected‐culture extracts and by indirect immunofluorescence of infected monolayers at times before the detection of cdsJ‐specific message. Finally, expression of IncC, expressed by 2 h after infection during C. trachomatis infections, in Yersinia pseudotuberculosis resulted in its secretion via the Yersinia type III apparatus. Based on these data, we propose a model in which type III secretion pores are present on EBs and mediate secretion of early Incs and possible additional effectors. Mid‐cycle expression of type III genes would then replenish secretion apparatus on vegetative RBs and serve as a source of secretion pores for subsequently formed EBs.
Identification of a novel type IV pilus gene cluster required for gastrointestinal colonization of Citrobacter rodentium
Authors: Mundy Rosanna , Pickard Derek , Wilson Rebecca K. , Simmons Cameron P. , Dougan Gordon , Frankel Gad
Published: December 2004

Citrobacter rodentium is used as an in vivo model system for clinically significant enteric pathogens such as enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens all colonize the lumen side of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation. In order to identify genes required for the colonization of A/E‐forming pathogens, a library of signature‐tagged transposon mutants of C. rodentium was constructed and screened in mice. Of the 576 mutants tested, 14 were attenuated in their ability to colonize the descending colon. Of these, eight mapped to the locus of enterocyte effacement (LEE), which is required for the formation of A/E lesions, underlying the importance of this mechanism for pathogenesis. Another mutant, P5H2, was found to have a transposon insertion in an open reading frame that has strong similarity to type IV pilus nucleotide‐binding proteins. The region flanking the transposon insertion was sequenced, identifying a cluster of 12 genes that encode the first described pilus of C. rodentium (named colonization factor Citrobacter, CFC). The proteins encoded by cfc genes have identity to proteins of the type IV COF pilus of enterotoxigenic E. coli (ETEC), the toxin co‐regulated pilus of Vibrio cholerae and the bundle‐forming pilus of EPEC. A non‐polar mutation in cfcI, complementation of this strain with wild‐type cfcI and complementation of strain P5H2 with wild‐type cfcH confirmed that these genes are required for colonization of the gastrointestinal tract by C. rodentium. Thus, CFC provides a convenient model to study type IV pilus‐mediated pathogen–host interactions under physiological conditions in the natural colonic environment.
Signalling early developmental events in two highly diverged Streptomyces species
Authors: Chater Keith F. , Horinouchi Sueharu
Published: December 2004

We review three main aspects of extracellular signalling in the initiation of aerial mycelium formation in two phylogenetically distant streptomycetes, S. coelicolor A3(2) and S. griseus: (1) γ ‐butyrolactones; (2) a complex cascade of mostly undefined signals; and (3) progress towards defining an integrating endpoint of all this signalling. Although apparent orthologues of many of the genes involved are found in both species, some of the connectivities are different. Moreover, some of the genes involved in signalling have diverged more rapidly than known housekeeping genes. We propose that that this may be an important aspect of speciation, and that the differences in gene interactions may reflect the diverse soil microecologies to which different streptomycetes are adapted.
RNA‐modifying machines in archaea
Authors: Omer Arina D. , Ziesche Sonia , Decatur Wayne A. , Fournier Maurille J. , Dennis Patrick P.
Published: December 2004

It has been known for nearly half a century that coding and non‐coding RNAs (mRNA, and tRNAs and rRNAs respectively) play critical roles in the process of information transfer from DNA to protein. What is both surprising and exciting, are the discoveries in the last decade that cells, particularly eukaryotic cells, contain a plethora of non‐coding RNAs and that these RNAs can either possess catalytic activity or can function as integral components of dynamic ribonucleoprotein machines. These machines appear to mediate diverse, complex and essential processes such as intron excision, RNA modification and editing, protein targeting, DNA packaging, etc. Archaea have been shown to possess RNP complexes; some of these are authentic homologues of the eukaryotic complexes that function as machines in the processing, modification and assembly of rRNA into ribosomal subunits. Deciphering how these RNA‐containing machines function will require a dissection and analysis of the component parts, an understanding of how the parts fit together and an ability to reassemble the parts into complexes that can function in vitro. This article summarizes our current knowledge about small‐non‐coding RNAs in Archaea, their roles in ribosome biogenesis and their relationships to the complexes that have been identified in eukaryotic cells.
Bordetella bronchiseptica PagP is a Bvg‐regulated lipid A palmitoyl transferase that is required for persistent colonization of the mouse respiratory tract
Authors: Preston Andrew , Maxim Elizabeth , Toland Elinor , Pishko E. Jane , Harvill Eric T. , Caroff Martine , Maskell Duncan J.
Published: December 2004

Bordetella bronchiseptica lipopolysaccharide (LPS) expression varies depending on growth conditions, regulated by the Bvg system. A B. bronchiseptica pagP homologue was identified that is required for Bvg‐mediated modification of the lipid A core region of LPS that occurs on switching from the Bvg– to the Bvg+ phase. Structural analysis demonstrated that the lipid A of a B. bronchiseptica pagP mutant differed from wild‐type lipid A by the absence of a palmitate group in secondary acylation at the C3′ position. The putative pagP promoter drove the expression of a green fluorescent protein (GFP) reporter gene in a Bvg‐regulated fashion. These data suggest that B. bronchiseptica pagP encodes a Bvg‐regulated lipid A palmitoyl transferase that mediates modification of the lipid A as part of the overall Bvg‐mediated adaptation of this organism to changing environmental conditions. We also show that pagP is not required for the initial colonization of the mouse respiratory tract by B. bronchiseptica, but is required for persistence of the organism within this organ.
3D structure of EspA filaments from enteropathogenic Escherichia coli
Authors: Daniell Sarah J. , Kocsis Eva , Morris Edward , Knutton Stuart , Booy Frank P. , Frankel Gad
Published: December 2004

The type III secretion system (TTSS) is a modular apparatus assembled by many pathogenic Gram‐negative bacteria and is designed to translocate proteins through the bacterial cell wall into the eukaryotic host cell. The conserved components of the TTSS comprise stacks of rings spanning the inner and outer bacterial membrane and a narrow, needle‐like structure projecting outwards. The TTSS of enteropathogenic E. coli is unique in that one of the translocator proteins, EspA, polymerizes to form an extension to the needle complex which interacts with the host cell. In this study we present the 3D structure of EspA filaments to c. 26 Å resolution determined from electron micrographs of negatively stained preparations by image processing. The structure comprises a helical tube with a diameter of 120 Å enclosing a central channel of 25 Å diameter through which effector proteins may be transported. The subunit arrangement corresponds to a one‐start helix with 28 subunits present in five turns of the helix and an axial rise of 4.6 Å per subunit. This is the first report of a 3D structure of a filamentous extension to the TTSS.
The RepA and RepB autorepressors and TraR play opposing roles in the regulation of a Ti plasmid repABC operon
Authors: Pappas Katherine M. , Winans Stephen C.
Published: December 2004

The replicator regions of the Ti plasmids of Agrobacterium tumefaciens belong to the repABC family of replication and partitioning systems, members of which are widely distributed among alpha proteobacteria. In the region upstream of the octopine‐type Ti plasmid repABC operon, three promoters were recently shown to be activated by the LuxR‐type regulator TraR. Activation of these promoters by TraR led to enhanced rep gene expression and increased Ti plasmid copy number. Here we describe a fourth promoter, designated P4. This promoter lies directly upstream of repA and is not regulated by TraR. The promoter was localized by subcloning and demonstrated to be strongly autorepressed. RepA is the major cis‐acting autorepressor of this promoter, though RepB enhanced repression and was essential for RepA‐mediated repression in trans. Purified RepA bound to an approximately 70‐nucleotide operator site overlapping the P4 promoter and extending well downstream. Binding affinity was increased by adenosine di‐ and tri‐phosphates and also by purified RepB. Activation of P1, P2, and P3 enhanced the activity of P4, suggesting that P4 somehow communicates with the upstream promoters. These findings demonstrate that both autoinduction and autorepression play critical and opposing roles in regulating repABC expression and hence in the replication, stability and copy number of the Ti plasmid.
A lytic transglycosylase homologue, PleA, is required for the assembly of pili and the flagellum at the Caulobacter crescentus cell pole
Authors: Viollier Patrick H. , Shapiro Lucy
Published: December 2004

Two distinct protein complexes, the flagellum and the pilus biogenesis machinery, are asymmetrically assembled at one pole of the Caulobacter predivisional cell. Cell division yields dissimilar daughter cells: a stalked cell and a swarmer cell that assembles several pili at the flagellated cell pole. Strains bearing mutations in the pleA gene are pililess and non‐flagellated. The PleA protein contains a region that is similar to a peptidoglycan‐hydrolytic active site, and a point mutation at this site in PleA results in the loss of flagellum and pili biogenesis. PleA was found to be required for the insertion of the outer membrane pilus secretion channel at the cell pole and for the accumulation of the PilA pilin subunit. PleA is also required for the assembly of substructures of the flagellar basal body hook complex that are located in or traverse the peptidoglycan layer. These results argue that PleA facilitates the assembly of envelope‐spanning structures at the cell pole. In support of this, PleA was found to be present only during a short interval in the cell cycle that coincides with the assembly of the flagellum and the pilus secretion apparatus.