bluuebunny/crossref_metadata_2025 huggingface dataset:
The Midas cichlid species complex: incipient sympatric speciation in Nicaraguan cichlid fishes?
Authors: BARLUENGA MARTA , MEYER AXEL
Published: December 2004

Sympatric speciation is a contentious concept, although theoretical models as well as empirical evidence support its relevance in evolutionary biology. The Midas cichlid species complex (Amphilophus citrinellus,labiatus,zaliosus) from several crater lakes in Nicaragua fits several of the key characteristics of a sympatric speciation model. In particular, inA. citrinellus(i) strong assortative mating on the basis of colour polymorphism and (ii) ecological differentiation based on morphological polymorphisms involving the feeding apparatus and body shape might both be mechanisms of incipient speciation. Seven microsatellite markers and mtDNA control region sequences [836 base pairs (bp)] were used to study the population genetic structure of 519 specimens of Midas cichlid populations from the two Great Lakes Managua and Nicaragua, and three crater lakes in Nicaragua, Central America. The three named species of the species complex occupy different ecological niches, are morphologically distinct and can be distinguished genetically. We uncovered allopatric genetic differentiation of populations ofA. citrinellusfrom different lakes and distant locations within Lake Managua and, more interestingly, incipient genetic differentiation of several sympatric populations based on colouration (inA. citrinellusandA. labiatus) but not on the morphology of the pharyngeal jaws (inA. citrinellus). Sexual selection and assortative mating might be the driven forces of diversification within named species. The Midas cichlid species complex in Nicaragua is an excellent model system for the study of the incipient stages of adaptation, speciation and the formation of species flocks.
Inverse relationship betweenFSTand microsatellite polymorphism in the marine fish, walleye pollock (Theragra chalcogramma): implications for resolving weak population structure
Authors: O'REILLY P. T. , CANINO M. F. , BAILEY K. M. , BENTZEN P.
Published: December 2004

Microsatellites have proved to be useful for the detection of weak population structure in marine fishes and other species characterized by large populations and high gene flow. None the less, uncertainty remains about the net effects of the particular mutational properties of these markers, and the wide range of locus polymorphism they exhibit, on estimates of differentiation. We examined the effect of varying microsatellite polymorphism on the magnitude of observed differentiation in a population survey of walleye pollock,Theragra chalcogramma. Genetic differentiation at 14 microsatellite loci among six putative populations from across the North Pacific Ocean and Bering Sea was weak but significant on large geographical scales and conformed to an isolation‐by‐distance pattern. A negative relationship was found between locus variability and the magnitude of estimated population subdivision. Estimates ofFSTdeclined with locus polymorphism, resulting in diminished power to discriminate among samples, and we attribute this loss to the effects of size homoplasy. This empirical result suggests that mutation rates of some microsatellite loci are sufficiently high to limit resolution of weak genetic structure typical of many marine fishes.
Infiltration by alien predators into invertebrate food webs in Hawaii: a molecular approach
Authors: SHEPPARD S. K. , HENNEMAN M. L. , MEMMOTT J. , SYMONDSON W. O. C.
Published: December 2004

Alien invertebrate predators have been introduced to Hawaii to control pests, particularly in lowland areas where most crops are grown. We developed techniques for assessing the impact of these predators on native food webs in relatively pristine upland areas where, it was hypothesized, few lowland predators might be found. Predator densities were assessed along transects within the Alakaii Swamp on Kaua’i. The most numerous alien biocontrol agents found were Halmus chalybeus (Coccinellidae), a species known to feed on Lepidoptera eggs. Laboratory experiments were conducted using two genera of endemic Lepidoptera, Scotorythra and Eupithecia (Geometridae), that are of considerable conservation value, the former because of its recent speciation across Hawaii, the latter for its unique predatory larvae. Techniques were developed for detecting Lepidoptera DNA within the guts of alien predators using prey‐specific PCR primers. General primers amplified fragments of the mitochondrial cytochrome oxidase I gene from beetles and Lepidoptera. The sequences were aligned and used successfully to design target‐specific primers for general detection of the remains of Geometridae and for particular species, including Scotorythra rara and Eupithecia monticolans. DNA fragments amplified were short [140–170 base pairs (bp)], optimizing detection periods following prey ingestion. Trials using the introduced biocontrol agent Curinus coeruleus (Coccinellidae) demonstrated detection of Lepidoptera DNA fragments = 151 bp in 85–100% of beetles after 24 h digestion of an early instar larva. This study provides a framework for future use of molecular gut analysis in arthropod conservation ecology and food web research with considerable potential for quantifying threats to endemic species in Hawaii and elsewhere.
Strong genetic clines and geographical variation in gene flow in the rocky intertidal barnacleBalanus glandula
Authors: SOTKA ERIK E. , WARES JOHN P. , BARTH JOHN A. , GROSBERG RICHARD K. , PALUMBI STEPHEN. R.
Published: December 2004

A long‐standing issue in marine biology is identifying spatial scales at which populations of sessile adults are connected by planktonic offspring. We examined the genetic continuity of the acorn barnacleBalanus glandula, an abundant member of rocky intertidal communities of the northeastern Pacific Ocean, and compared these genetic patterns to the nearshore oceanography described by trajectories of surface drifters. Consistent with its broad dispersal potential, barnacle populations are genetically similar at both mitochondrial (cytochrome oxidase I) and nuclear (elongation factor 1‐alpha) loci across broad swaths of the species’ range. In central California, however, there is a striking genetic cline across 475 km of coastline between northern and southern populations. These patterns indicate that gene flow within central California is far more restricted spatially than among other populations. Possible reasons for the steep cline include the slow secondary introgression of historically separated populations, a balance between diversifying selection and dispersal, or some mix of both. Geographic trajectories of oceanic drifters closely parallel geographical patterns of gene flow. Drifters placed to the north (Oregon; ∼44°N) and south (Santa Barbara, California; ∼34° N) of the cline disperse hundreds of kilometres within 40 days, yet over the long‐term their trajectories never overlapped. The lack of communication between waters originating in Oregon and southern California probably helps to maintain strong genetic differentiation between these regions. More broadly, the geographical variation in gene flow implies that focusing on species‐level averages of gene flow can mask biologically important variance within species which reflects local environmental conditions and historical events.
Variation of female frequency and cytoplasmic male‐sterility gene frequency among natural gynodioecious populations of wild radish (Raphanus sativus L.)
Authors: MURAYAMA K. , YAHARA T. , TERACHI T.
Published: December 2004

In gynodioecious plant populations, sex determination often involves both cytoplasmic male‐sterility (CMS) genes and specific nuclear genes that restore male function. How gynodioecy is maintained under the joint dynamics of CMS and restorer genes remains controversial. Although many theoretical models deal with interactions between CMS genes and restorer genes with sexual phenotypes and predict changes in their frequencies, it is difficult to observe the frequencies because no molecular markers have been established for either CMS or restorer genes in well‐studied gynodioecious plants. This is the first report of the frequency of a CMS gene determined using a molecular marker in natural populations of a gynodioecious plant. Using a set of CMS gene‐specific polymerase chain reaction primers, we compared female and CMS gene frequencies in 18 natural populations of Raphanus sativus. Female frequency was relatively low, ranging from 0 to 0.21. In contrast, the CMS gene frequency was highly variable among populations, ranging from 0 to 1. Estimated restorer gene frequency seemed less variable than observed CMS gene frequency, probably due to higher gene flow than in the CMS gene. Genetic drift may play a role in maintaining high variability of the CMS gene, although other possibilities are not excluded.
Effects of hydrographic barriers on population genetic structure of the sea star Coscinasterias muricata (Echinodermata, Asteroidea) in the New Zealand fiords
Authors: PERRIN CÉCILE , WING STEPHEN R. , ROY MICHAEL S.
Published: December 2004

New Zealand's 14 deep‐water fiords possess persistent salinity stratification and mean estuarine circulation that may serve to isolate populations of marine organisms that have a dispersal larval phase. In order to investigate this idea, we analysed the population structure of the sea star Coscinasterias muricata using a mitochondrial DNA marker. Genetic differentiation among populations of C. muricata was analysed using 366 base pairs of mtDNA D‐loop. We compared populations from the fiords with several others sampled from around New Zealand. At a macro‐geographical scale (> 1000 km), restricted gene flow between the North and South Islands was observed. At a meso‐geographical scale (10–200 km), significant population structure was found among fiords and between fiords and open coast. The pattern of population genetic structure among the fiords suggests a secondary contact between a northern population and a southern one, separated by a contact or mixing zone. These populations may have diverged by the effects of random genetic drift and population isolation as a consequence of the influence of estuarine circulation on dispersal. In northern Fiordland, genetic structure approximated an isolation by distance model. However, the pattern in genetic differences suggests that distance alone cannot explain the most divergent populations and that fiord hydrography may increase the effect of genetic drift within populations in the fiords. Finally, our study indicates that populations within the fiords underwent recent rapid expansion, followed most probably by genetic drift due to a lack of gene flow among the fiords.
The diminutive supercolony: the Argentine ants of the southeastern United States
Authors: BUCZKOWSKI GRZEGORZ , VARGO EDWARD L. , SILVERMAN JULES
Published: December 2004

Native to Argentina and Brazil, the Argentine ant (Linepithema humile) is an invasive species that has become established on six continents and many oceanic islands. In several parts of its introduced range, including the western United States, southern Europe and Chile, the Argentine ant is unicolonial, forming extensive supercolonies. We examined population genetic structure and intercolony aggression in two regions of the introduced range of this species in the United States: California and the southeastern United States. Our results show that the southeastern L. humile population has high genotypic variability and strong intercolony aggression relative to the California population. In the California population, intercolony aggression was absent and 23 alleles were found across seven polymorphic microsatellite loci. However, in the Southeast, aggression between colonies was high and 47 alleles were present across the same seven loci in an equal number of colonies. We suggest that distinctly different colonization patterns for California and the Southeast may be responsible for the striking disparity in the genetic diversity of introduced populations. Southeastern colonies may have descended from multiple, independent introductions from the native range, undergoing a bottleneck at each introduction. In contrast, the California supercolony may have originated from one or more colonies inhabiting the southeastern United States, thus experiencing a double bottleneck. The differences in present‐day distribution patterns between California and the Southeast may be due to the combined effect of two factors: lower winter temperatures in the Southeast and/or competition with another successful and widely distributed ant invader, the fire ant Solenopsis invicta.
Hidden Floridian biodiversity: mitochondrial and nuclear gene trees reveal four cryptic species within the scorched mussel,Brachidontes exustus, species complex
Authors: LEE T. , Ó FOIGHIL D.
Published: December 2004

The well‐documented Floridian ‘Gulf/Atlantic’ marine genetic disjunction provides an influential example of vicariant cladogenesis along a continental coastline for major elements of a diverse nearshore fauna. We are engaged in a two‐part study that aims to place this disjunction into a regional Caribbean Basin phylogenetic perspective using the scorched musselBrachidontes exustusas an exemplar. Our first step, documented here, is to thoroughly characterize the genetic structure of Floridian scorched mussel populations using mitochondrial (mt) and nuclear markers. Both sets of markers recovered the expected disjunction involving sister clades distributed on alternate flanks of peninsular Florida and lineage‐specific mt molecular clocks placed its origin in the Pliocene. The two sister clades had distinct population genetic profiles and the Atlantic clade appears to have experienced an evolutionarily recent bottleneck, although plots of the relative estimates ofNthrough time are consistent with its local persistence through the last Ice Age Maximum. Our primary novel result, however, was the discovery that the Gulf/Atlantic disjunction represents but one of three cryptic, nested genetic discontinuities represented in Floridian scorched mussel populations. The most pronounced phylogenetic split distinguished the Gulf and Atlantic sister clades from two additional nested cryptic sister clades present in samples taken from the southern Florida tropical marine zone. Floridian populations ofB. exustusare composed of four cryptic taxa, a result consistent with the hypothesis that the Gulf/Atlantic disjunction in this morphospecies is but one of multiple latent regional genetic breakpoints.
Genetic diversity and population structure of the commercially harvested sea urchinParacentrotus lividus(Echinodermata, Echinoidea)
Authors: DURAN SANDRA , PALACÍN CRUZ , BECERRO MIKEL A. , TURON XAVIER , GIRIBET GONZALO
Published: December 2004

The population structure of the edible Atlanto‐Mediterranean sea urchinParacentrotus lividusis described by analysing sequence variation in a fragment of the mitochondrial gene cytochromecoxidase subunit I in 127 individuals from 12 localities across south‐west Europe. The study revealed high levels of genetic diversity but low levels of genetic structure, suggesting a large degree of gene flow between populations and panmixis within each, the Mediterranean and Atlantic basins. However, we found significant genetic differentiation between the two basins probably due to restricted gene flow across the geographical boundary imposed by the area of the Strait of Gibraltar. Populations ofP. lividusappeared to have experienced a recent demographic expansion in the late Pleistocene. We provide new evidence on the population structure of this commercial species, predicting a healthy stock of this sea urchin on the Mediterranean and Atlantic coasts.
Variation in pollen dispersal between years with different pollination conditions in a tropical emergent tree
Authors: KENTA T. , ISAGI Y. , NAKAGAWA M. , YAMASHITA M. , NAKASHIZUKA T.
Published: December 2004

We examined differences in pollen dispersal efficiency between 2 years in terms of both spatial dispersal range and genetic relatedness of pollen in a tropical emergent tree, Dipterocarpus tempehes. The species was pollinated by the giant honeybee (Apis dorsata) in a year of intensive community‐level mass‐flowering or general flowering (1996), but by several species of moths in a year of less‐intensive general flowering (1998). We carried out paternity analysis based on six DNA microsatellite markers on a total of 277 mature trees forming four spatially distinct subpopulations in a 70 ha area, and 147 and 188 2‐year‐old seedlings originating from seeds produced in 1996 and 1998 (cohorts 96 and 98, respectively). Outcrossing rates (0.93 and 0.96 for cohorts 96 and 98, respectively) did not differ between years. Mean dispersal distances (222 and 192 m) were not significantly different between the 2 years but marginally more biased to long distance in 1996. The mean relatedness among cross‐pollinated seedlings sharing the same mothers in cohort 96 was lower than that in cohort 98. This can be attributed to the two facts that the proportion of intersubpopulations pollen flow among cross‐pollination events was marginally higher in cohort 96 (44%) than in cohort 98 (33%), and that mature trees within the same subpopulations are genetically more related to each other than those between different subpopulations. We conclude that D. tempehes maintained effective pollen dispersal in terms of outcrossing rate and pollen dispersal distance in spite of the large difference in foraging characteristics between two types of pollinators. In terms of pollen relatedness, however, a slight difference was suggested between years in the level of biparental inbreeding.
Lower fitness of hatchery and hybrid rainbow trout compared to naturalized populations in Lake Superior tributaries
Authors: MILLER L. M. , CLOSE T. , KAPUSCINSKI A. R.
Published: December 2004

We have documented an early life survival advantage by naturalized populations of anadromous rainbow trout Oncorhynchus mykiss over a more recently introduced hatchery population and outbreeding depression resulting from interbreeding between the two strains. We tested the hypothesis that offspring of naturalized and hatchery trout, and reciprocal hybrid crosses, survive equally from fry to age 1+ in isolated reaches of Lake Superior tributary streams in Minnesota. Over the first summer, offspring of naturalized females had significantly greater survival than offspring of hatchery females in three of four comparisons (two streams and 2 years of stocking). Having an entire naturalized genome, not just a naturalized mother, was important for survival over the first winter. Naturalized offspring outperformed all others in survival to age 1+ and hybrids had reduced, but intermediate, survival relative to the two pure crosses. Averaging over years and streams, survival relative to naturalized offspring was 0.59 for hybrids with naturalized females, 0.37 for the reciprocal hybrids, and 0.21 for hatchery offspring. Our results indicate that naturalized rainbow trout are better adapted to the conditions of Minnesota's tributaries to Lake Superior so that they outperform the hatchery‐propagated strain in the same manner that many native populations of salmonids outperform hatchery or transplanted fish. Continued stocking of the hatchery fish may conflict with a management goal of sustaining the naturalized populations.
Replication mutations differentially enhance RecA‐dependent and RecA‐independent recombination between tandem repeats in Bacillus subtilis
Authors: Bruand Claude , Bidnenko Vladimir , Ehrlich S. Dusko
Published: December 2004

We have studied DNA recombination between 513 bp tandem direct repeats present in a kanamycin resistance gene inserted in the Bacillus subtilis chromosome. Tandem repeat deletion was not significantly affected by a recA mutation. However, recombination was stimulated by mutations in genes encoding replication proteins, including the primosomal proteins DnaB, DnaD and the DnaG primase, the putative DNA polymerase III subunits PolC, DnaN and DnaX, as well as the DNA polymerase DnaE. Hyper‐recombination was found to be dependent on RecA in the dnaE, dnaN and dnaX mutants, whereas the dnaG and dnaD mutants stimulated recombination independently of RecA. Altogether, these data show that both RecA‐dependent and RecA‐independent mechanisms contribute to recombination between tandem repeats in B. subtilis and that both types of recombination are stimulated by replication mutations.
Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis
Authors: Laun Peter , Pichova Alena , Madeo Frank , Fuchs Jörg , Ellinger Adolf , Kohlwein Sepp , Dawes Ian , Fröhlich Kai‐Uwe , Breitenbach Michael
Published: December 2004

Recently, we and others have shown that genetic and environmental changes that increase the load of yeast cells with reactive oxygen species (ROS) lead to a shortening of the life span of yeast mother cells. Deletions of yeast genes coding for the superoxide dismutases or the catalases, as well as changes in atmospheric oxygen concentration, considerably shortened the life span. The presence of the physiological antioxidant glutathione, on the other hand, increased the life span of yeast cells. Taken together, these results pointed to a role for oxygen in the yeast ageing process. Here, we show by staining with dihydrorhodamine that old yeast mother cells isolated by elutriation, but not young cells, contain ROS that are localized in the mitochondria. A relatively large proportion of the old mother cells shows phenotypic markers of yeast apoptosis, i.e. TUNEL (TdT‐mediated dUTP nick end labelling) and annexin V staining. Although it has been shown previously that apoptosis in yeast can be induced by a cdc48 allele, by expressing pro‐apoptotic human cDNAs or by stressing the cells with hydrogen peroxide, we are now showing a physiological role for apoptosis in unstressed but aged wild‐type yeast mother cells.
A novel group I intron‐encoded endonuclease specific for the anticodon region of tRNAfMet genes
Authors: Bonocora Richard P. , Shub David A.
Published: December 2004

Open reading frames (ORFs) are frequently inserted into group I self‐splicing introns. These ORFs encode either maturases that are required for splicing of the intron or DNA endonucleases that promote intron mobility. A self‐splicing intron in the tRNAfMet gene of Synechocystis PCC 6803, which has been proposed to have moved laterally within the cyanobacteria, contains an ORF that is unrelated to known intron‐encoded endonucleases or maturases. Here, using an in vitro transcription–translation system, we show that this intronic ORF encodes a double‐strand DNA endonuclease, I‐Ssp6803I. I‐Ssp6803I cleaves each strand of the intronless tRNAfMet gene adjacent to the anticodon triplet leaving 3 bp 3′ extensions and has no activity at intron–exon boundaries. Using an in vitro cleavage assay and scanning deletion mutants of the intronless target site, the minimal recognition site was determined to be a partially palindromic 20 bp region encompassing the entire anticodon stem and loop of the tRNAfMet gene. I‐Ssp6803I represents a novel intron‐encoded DNA endonuclease and is the first example of a chromosomally encoded group I intron endonuclease in bacteria.
Role of the Bacillus subtilis fatty acid desaturase in membrane adaptation during cold shock
Authors: Weber Michael H. W. , Klein Wolfgang , Müller Lin , Niess Ulf M. , Marahiel Mohamed A.
Published: December 2004

In our attempt to understand the cold shock response of Bacillus subtilis, we report on the role of the B. subtilis fatty acid desaturase (FA‐D) Des during membrane adaptation to low temperatures and demonstrate its importance during cold shock. A des null mutant was constructed and analysed in comparison with its parental strain. Growth studies and large‐scale comparative fatty acid (FA) analysis revealed a severe cold‐sensitive phenotype of the des deletion mutant during the absence of isoleucine and showed that four unsaturated fatty acid (UFA) species differing in length, branching pattern and position of the double bond are synthesized in B. subtilis JH642 but not in the des null mutant. Apart from the lack of UFA synthesis, the FA‐D deletion strain showed a dramatically altered saturated fatty acid (SFA) profile at the onset of the stationary growth phase in the presence of exogenous isoleucine sources. Expression of des integrated in trans at the amyE locus of the des deletion strain not only cured the cold‐sensitive phenotype observed for the des mutant but allowed much better growth than in strain JH642 after a shift from 37°C to 15°C. These results show that, during cold shock adaptation, des expression can completely replace the isoleucine‐dependent, long‐term, FA branching adaptation mechanism. We conclude that the crucial aspect in cold adaptation of the cytoplasmic membrane is not its specific molecular composition but rather its physical status in terms of its fluidity.
Catabolite repression mediated by the CcpA protein in Bacillus subtilis: novel modes of regulation revealed by whole‐genome analyses
Authors: Moreno Matthew S. , Schneider Barbara L. , Maile Randal R. , Weyler Walter , Saier Milton H.
Published: December 2004

Previous studies have shown that the CcpA protein of Bacillus subtilis is a major transcription factor mediating catabolite repression. We report here whole‐transcriptome analyses that characterize CcpA‐dependent, glucose‐dependent gene expression and correlate the results with full‐genome computer analyses of DNA binding (CRE) sites for CcpA. The data obtained using traditional approaches show good agreement with those obtained using the transcriptome approach. About 10% of all genes in B. subtilis are regulated > 3× by glucose, with repressed genes outnumbering activated genes three to one. Eighty per cent of these genes depend on CcpA for regulation. Classical approaches have provided only evidence for CcpA‐mediated, glucose‐dependent activation or repression. We show here that CcpA also mediates glucose‐independent activation or repression, and that glucose may alter either the direction or the intensity of either effect. Computer analyses revealed the presence of CRE sites in most operons subject to CcpA‐mediated glucose repression, but not in those subject to glucose activation, suggesting that either secondary transcription factors regulate the latter genes or activation by CcpA involves a dissimilar binding site. Operons encoding the constituents of ABC‐type transporters that are subject to CcpA‐mediated glucose regulation show two distinct patterns: either all genes in the operon are regulated in parallel (the minor class) or the gene encoding the extracytoplasmic solute‐binding receptor is preferentially regulated (the major class). Genes subject to CcpA‐independent catabolite repression are primarily concerned with sporulation. Several transcription factors were identified that are themselves regulated by CcpA at the transcriptional level. Representative data with functionally characterized genes are presented to illustrate the novel findings. The comprehensive transcriptome data are available on our website: www.biology.uesd.edu/~MSAIER/regulation/.
Dominant active Rac and dominant negative Rac revert the dominant active Ras phenotype in Colletotrichum trifolii by distinct signalling pathways
Authors: Chen Changbin , Dickman Martin B.
Published: December 2004

The small G‐protein superfamily is an evolutionarily conserved group of GTPases that regulate diverse signalling pathways including pathways for growth and development in eukaryotes. Previously, we showed that dominant active mutation in the unique Ras gene (DARas) of the fungal phytopathogen Colletotrichum trifolii displays a nutrient‐dependent phenotype affecting polarity, growth and differentiation. Signalling via the MAP kinase pathway is significantly impaired in this mutant as well. Here we describe the cloning and functional characterization of Rac (Ct‐Rac1), a member of the Rho family of G proteins. Ct‐Rac1 expression is downregulated by DARas under limiting nutrition. Co‐expression of DARas with dominant active Rac (DARac) stimulates MAPK activation and restores the wild‐type phenotype. Inhibition of MAPK activation suppresses phenotypic restoration suggesting Rac‐mediated MAPK activation is responsible for reversion to the wild‐type phenotype. We also examined the role of reactive oxygen species (ROS) in these genetic backgrounds. The DARas mutant strain generates high levels of ROS as determined by DCFH‐DA fluorescence. Co‐expression with DNRac decreases ROS generation to wild‐type levels and restores normal fungal growth and development. Pretreatment of DARas with antioxidants or a cytosolic phospholipase A2 inhibitor also restores the wild‐type phenotype. These findings suggest that Ras‐mediated ROS generation is dependent on a Rac–cPLA2‐linked signalling pathway. Taken together, this study provides evidence that Rac functions to restore the hyphal morphology of DARas by regulating MAPK activation and intracellular ROS generation.
Loss of the mono‐allelic control of the VSG expression sites during the development of Trypanosoma brucei in the bloodstream
Authors: Amiguet‐Vercher Amelia , Pérez‐Morga David , Pays Annette , Poelvoorde Philippe , Van Xong Huang , Tebabi Patricia , Vanhamme Luc , Pays Etienne
Published: December 2004

Transcription of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei occurs in a single of multiple polycistronic expression sites (ESs). Analysis of RNA from proliferative long slender (LS) bloodstream forms demonstrated that initiation of transcription occurs in different ESs, but inefficient RNA processing and elongation is linked to RNA polymerase arrest in all except one unit at a time. The pattern of ES transcripts was analysed during the transformation of dividing LS forms into quiescent short stumpy (SS) forms. The results demonstrated that the mono‐allelic control allowing preferential RNA production from a given ES stops during this process. Accordingly, the steady‐state ES transcripts, particularly the VSG mRNA, were strongly reduced. However, transcripts from the beginning of different ESs were still synthesized, and in vitro run‐on transcription analysis indicated that RNA polymerase was still fully associated with the promoter‐proximal half of the ‘active’ ES. Analysis of transcripts from two central tandem genes confirmed the existence of a residual decreasing transcriptional gradient in the ‘active’ ES of SS forms. Thus, in these forms the RNA polymerase of the ES is inactivated in situ. This inactivation is accompanied by a strong overall reduction of nuclear DNA transcription. Although cAMP is involved in the LS to SS transformation, no direct effect of cAMP was observed on the VSG ES control.
Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence
Authors: Cabanes Didier , Dussurget Olivier , Dehoux Pierre , Cossart Pascale
Published: December 2004

Listeria monocytogenes is an opportunistic food‐borne human and animal pathogen. Several surface proteins expressed by this intracellular pathogen are critical for the infectious process. By in silico analysis we compared the surface protein repertories of L. monocytogenes and of the non‐pathogenic species Listeria innocua and identified a gene encoding a surface protein of L. monocytogenes absent in L. innocua. This gene that we named aut encodes a protein (Auto) of 572 amino acids containing a signal sequence, a N‐terminal autolysin domain and a C‐terminal cell wall‐anchoring domain made up of four GW modules. We show here that the aut gene is expressed independently of the virulence gene regulator PrfA and encodes a surface protein with an autolytic activity. We provide evidence that Auto is required for entry of L. monocytogenes into cultured non‐phagocytic eukaryotic cells. The low invasiveness of an aut deletion mutant correlates with its reduced virulence following intravenous inoculation of mice and oral infection of guinea pigs. During infection, the autolytic activity of Auto may also be critical. Auto appears thus as a novel type of L. monocytogenes virulence factor.
IHF is the limiting host factor in transposition of Pseudomonas putida transposon Tn4652 in stationary phase
Authors: Ilves Heili , Hõrak Rita , Teras Riho , Kivisaar Maia
Published: December 2004

Transpositional activity of mobile elements is not constant. Conditional regulation of host factors involved in transposition may severely change the activity of mobile elements. We have demonstrated previously that transposition of Tn4652 in Pseudomonas putida is a stationary phase‐specific event, which requires functional sigma S (Ilves et al., 2001, J Bacteriol 183: 5445–5448). We hypothesized that integration host factor (IHF), the concentration of which is increased in starving P. putida, might contribute to the transposition of Tn4652 as well. Here, we demonstrate that transposition of Tn4652 in stationary phase P. putida is essentially limited by the amount of IHF. No transposition of Tn4652 occurs in a P. putida ihfA‐defective strain. Moreover, overexpression of IHF results in significant enhancement of transposition compared with the wild‐type strain. This indicates that the amount of IHF is a bottleneck in Tn4652 transposition. Gel mobility shift and DNase I footprinting studies revealed that IHF is necessary for the binding of transposase to both transposon ends. In vitro, transposase can bind to inverted repeats of transposon only after the binding of IHF. The results obtained in this study indicate that, besides sigma S, IHF is another host factor that is implicated in the elevation of transposition in stationary phase.
Molecular architecture of the Cryptococcus neoformans capsule
Authors: Gates Marcellene A. , Thorkildson Peter , Kozel Thomas R.
Published: December 2004

Many microbes are surrounded by phagocytosis‐inhibiting capsules. We took advantage of the large size of the polysaccharide capsule of the pathogenic yeast Cryptococcus neoformans to examine capsular architecture and the relationship between molecular architecture and the interaction of the capsule with potentially opsonic serum proteins. Our experimental design used complementary approaches in which (i) assessment of permeability to macromolecules of different Stokes radii; (ii) determination of the binding of Fab fragments of anticapsular antibodies as a measure of matrix density; (iii) capsular deconstruction by treatment with dimethyl sulphoxide; and (iv) evaluation of capsule plasticity, were used to probe the molecular structure of the capsule. The results showed that the capsule is a matrix with a variable porosity that increases with distance from the cell wall. A high density of the matrix at the capsule interior prevents penetration of large macromolecules to sites near the cell wall. In contrast, the capsular edge that is the interface with phagocytes presents capsular polysaccharide in a very low density that exhibits considerable plasticity and permeability to macromolecules. Notably, the capsule of yeast cells harvested from infected tissue showed a greater matrix density than yeast cells grown in vitro under capsule induction conditions.
A basic‐region helix–loop–helix protein‐encoding gene (devR) involved in the development of Aspergillus nidulans
Authors: Tüncher André , Reinke Hans , Martic Goran , Caruso Maria Louise , Brakhage Axel A.
Published: December 2004

Basic‐region helix–loop–helix (bHLH) proteins form an interesting class of eukaryotic transcription factors often involved in developmental processes. Here, a so far unknown bHLH protein‐encoding gene of the filamentous ascomycete Aspergillus nidulans was isolated and designated devR for regulator of development. Deletion of devR revealed that the gene is non‐essential for vegetative growth. However, the deletion mutant produced wrinkled colonies, a yellow pigment and did not form conidia on minimal agar plates. Conidiophore development was initiated normally, and colonies produced conidiophores with metulae and phialides. However, the phialides continued to grow filamentously and produced a second conidiophore with a vesicle at its end. The addition of KCl (0.6 M) to the medium suppressed the knock‐out phenotype. The ΔdevR phenotype resembled that of a mutation in the tcsA gene encoding a histidine kinase domain and a response regulator domain. Here, we generated a tcsA deletion mutant. In a ΔtcsA strain, a DevR–Egfp protein fusion was detected in the cytoplasm, whereas in the wild type, the protein fusion was exclusively located in the nuclei, indicating that TcsA is required for nuclear localization of DevR. devR mRNA steady‐state levels were similar in sporulating and vegetatively growing mycelia, and independent of a functional brlA gene. Moreover, under all conditions tested, self‐crossing of the ΔdevR mutant strain was never observed. Taken together, devR encodes a bHLH regulatory protein that is part of the tcsA signal transduction network and required for development under standard growth conditions.
Zinc is a key factor in controlling alternation of two types of L31 protein in theBacillus subtilisribosome
Authors: Nanamiya Hideaki , Akanuma Genki , Natori Yousuke , Murayama Rikinori , Kosono Saori , Kudo Toshiaki , Kobayashi Kazuo , Ogasawara Naotake , Park Seung‐Moon , Ochi Kozo , Kawamura Fujio
Published: December 2004

We have analysed changes in the composition of ribosomal proteins during cell growth inBacillus subtilis. Ribosome fractions were prepared fromB. subtiliscells at different phases of growth and were separated by radical‐free and highly reducing (RFHR) two‐dimensional polyacrylamide gel electrophoresis. We identified 50 ribosomal proteins, including two paralogues of L31 protein (RpmE and YtiA). Although the ribosome fraction extracted from exponentially growing cells contained RpmE protein, this protein disappeared during the stationary phase. In contrast, YtiA was detected in the ribosome fraction extracted after the end of exponential growth. Expression of theytiAgene encoding YtiA was found to be negatively controlled by Zur, a zinc‐specific transcriptional repressor that controls zinc transport operons. Analysis by inductively coupled plasma mass spectrometry (ICP‐MS) indicated that RpmE contains one zinc ion per molecule of protein. In addition, mutagenesis of therpmEgene encoding RpmE revealed that Cys‐36 and Cys‐39, located within a CxxC motif, are required not only for binding zinc but also for the accumulation of RpmE in the cell. Taken together, these results indicate that zinc plays an essential role in the alternation between two types of L31 protein in the ribosome ofB. subtilis.
Has1p, a member of the DEAD‐box family, is required for 40S ribosomal subunit biogenesis in Saccharomyces cerevisiae†
Authors: Emery Bertrand , De La Cruz Jesús , Rocak Sanda , Deloche Olivier , Linder Patrick
Published: December 2004

The Has1 protein, a member of the DEAD‐box family of ATP‐dependent RNA helicases in Saccharomyces cerevisiae, has been found by different proteomic approaches to be associated with 90S and several pre‐60S ribosomal complexes. Here, we show that Has1p is an essential trans‐acting factor involved in 40S ribosomal subunit biogenesis. Polysome analyses of strains genetically depleted of Has1p or carrying a temperature‐sensitive has1‐1 mutation show a clear deficit in 40S ribosomal subunits. Analyses of pre‐rRNA processing by pulse‐chase labelling, Northern hybridization and primer extension indicate that these strains form less 18S rRNA because of inhibition of processing of the 35S pre‐rRNA at the early cleavage sites A0, A1 and A2. Moreover, processing of the 27SA3 and 27SB pre‐rRNAs is delayed in these strains. Therefore, in addition to its role in the biogenesis of 40S ribosomal subunits, Has1p is required for the optimal synthesis of 60S ribosomal subunits. Consistent with a role in ribosome biogenesis, Has1p is localized to the nucleolus. On sucrose gradients, Has1p is associated with a high‐molecular‐weight complex sedimenting at positions equivalent to 60S and pre‐60S ribosomal particles. A mutation in the ATP‐binding motif of Has1p does not support growth of a has1 null strain, suggesting that the enzymatic activity of Has1p is required in ribosome biogenesis. Finally, sequence comparisons suggest that Has1p homologues exist in all eukaryotes, and we show that a has1 null strain can be fully complemented by the Candida albicans homologue.
Gene expression patterns during swarming in Salmonella typhimurium: genes specific to surface growth and putative new motility and pathogenicity genes
Authors: Wang Qingfeng , Frye Jonathan G. , McClelland Michael , Harshey Rasika M.
Published: December 2004

Swarming is a specialized form of surface motility displayed by several flagellated bacterial genera, which shares features with other surface phenomenon such as biofilm formation and host invasion. Swarmer cells are generally more flagellated and longer than vegetative cells of the same species propagated in liquid media, and move within an encasement of polysaccharide ‘slime’. Signals and signalling pathways controlling swarm cell differentiation are largely unknown. In order to test whether there is a genetic programme specific to swarming, we have determined global gene expression profiles of Salmonella typhimurium over an 8 h time course during swarming, and compared the microarray data with a similar time course of growth in liquid media as well as on harder agar where the bacteria do not swarm. Our data show that bacteria growing on the surface of agar have a markedly different physiology from those in broth, as judged by differential regulation of nearly one‐third of the functional genome. The large number of genes showing surface‐specific upregulation included those for lipopolysaccharide synthesis, iron metabolism and type III secretion. Although swarming‐specific induction of flagellar gene expression was not generally apparent, genes for iron metabolism were strongly induced specifically on swarm agar. Surface‐dependent regulation of many virulence genes suggests that growth on an agar surface could serve as a model for gene expression during the initial stages of host infection. Based on cluster analysis of distinctive expression patterns, we report here the identification of putative new genes involved in motility and virulence.
clpC and clpP1P2 gene expression in Corynebacterium glutamicum is controlled by a regulatory network involving the transcriptional regulators ClgR and HspR as well as the ECF sigma factor σH
Authors: Engels Sabine , Schweitzer Jens‐Eric , Ludwig Carsten , Bott Michael , Schaffer Steffen
Published: December 2004

The ATP‐dependent protease Clp plays important roles in the cell's protein quality control system and in the regulation of cellular processes. In Corynebacterium glutamicum, the levels of the proteolytic subunits ClpP1 and ClpP2 as well as of the corresponding mRNAs were drastically increased upon deletion of the clpC gene, coding for a Clp ATPase subunit. We identified a regulatory protein, designated ClgR, binding to a common palindromic sequence motif in front of clpP1P2 as well as of clpC. Deletion of clgR in the ΔclpC background completely abolished the increased transcription of both operons, indicating that ClgR activates transcription of these genes. ClgR activity itself is probably controlled via ClpC‐dependent regulation of its stability, as ClgR is only present in ΔclpC and not in wild‐type cells, whereas the levels of clgR mRNA are comparable in both strains. clpC, clpP1P2 and clgR expression is induced upon severe heat stress, however, independently of ClgR. Identification of the heat‐responsive transcriptional start sites in front of these genes revealed the presence of sequence motifs typical for σECF‐dependent promoters. The ECF sigma factor σH could be identified as being required for transcriptional activation of clpC, clpP1P2 and clgR in response to severe heat stress. A second heat‐responsive but σH‐independent promoter in front of clgR could be identified that is subject to negative regulation by the transcriptional repressor HspR. Taken together, these results show that clpC and clpP1P2 expression in C. glutamicum is subject to complex regulation via both independent and hierarchically organized pathways, allowing for the integration of multiple environmental stimuli. Both the ClgR‐ and σH‐dependent regulation of clpC and clpP1P2 expression appears to be conserved in other actinomycetes.
Characterization of HOG1 homologue, CpMK1, from Cryphonectria parasitica and evidence for hypovirus‐mediated perturbation of its phosphorylation in response to hypertonic stress
Authors: Park Seung‐Moon , Choi Eun‐Sil , Kim Myoung‐Ju , Cha Byeong‐Jin , Yang Moon‐Sik , Kim Dae‐Hyuk
Published: December 2004

We examined the biological function of cpmk1, which encodes a MAPK of Cryphonectria parasitica, and its regulation by mycovirus. Sequence comparisons revealed that cpmk1 had highest homology with osm1, a hog1‐homologue from Magnaporthe grisea. A growth defect was observed in the cpmk1‐null mutant under hyperosmotic conditions, indicating that cpmk1 functionally belongs to a hog1 subfamily. Immunoblot analyses indicated that the CpMK1 pathway was affected specifically in hyperosmotic conditions by the hypovirus CHV1‐EP713. Moreover, the virus‐infected hypovirulent UEP1 strain also exhibited severe osmosensitivity compared to the virus‐free isogenic strain EP155/2, thus providing additional evidence for viral regulation of cpmk1 in response to a hypertonic stress. Besides osmosensitivity, disruption of cpmk1 resulted in several, but not all, hypovirulence‐associated changes, such as reduced pigmentation, conidiation, laccase production and cryparin expression. However, the cpmk1‐null mutant exhibited an increased accumulation of pheromone gene transcripts. Virulence assays of the cpmk1‐null mutant revealed reduced canker area, but not as severe as that of UEP1. These results suggest that mycoviruses modulate the MAPK and thereby provoke the aberrant expression of target genes, some of which are likely to be implicated in viral symptom development.
Thiol peroxidase is critical for virulence and resistance to nitric oxide and peroxide in the fungal pathogen, Cryptococcus neoformans
Authors: Missall Tricia Ann , Pusateri Mary Ellen , Lodge Jennifer K.
Published: December 2004

Cryptococcus neoformans is a fungal pathogen most commonly causing meningitis in immunocompromised patients. Current therapies are inadequate, and novel antifungal targets are needed. We have identified by proteomics two thiol peroxidases that are differentially expressed at 37°C, the temperature of the mammalian host. Consistent with their antioxidant role, we show that the genes encoding these thiol‐specific antioxidants, TSA1 and TSA3, are transcriptionally induced when C. neoformans is exposed to hydrogen peroxide. Genome sequence analysis of C. neoformans revealed a third thiol peroxidase, TSA4. We constructed single, double and triple mutants of the thiol peroxidase genes through homologous recombination and analysed their function by comparing the growth of these mutants with that of the wild‐type strain. The tsa1Δ mutant shows sensitivity to hydrogen peroxide and t‐butylhydroperoxide, as well as significant growth retardation at 25°C and 38.5°C. The tsa1Δ mutant is also sensitive to NO, demonstrating a link between oxidative and nitrosative stress pathways. In two mouse models of cryptococcosis, the tsa1Δ mutant is significantly less virulent.