bluuebunny/crossref_metadata_2025 huggingface dataset:
Population structure of the large Japanese field mouse,Apodemus speciosus(Rodentia: Muridae), in suburban landscape, based on mitochondrial D‐loop sequences
Authors: HIROTA TADAO , HIROHATA TETSUO , MASHIMA HIROSHI , SATOH TOSHIYUKI , OBARA YOSHIAKI
Published: December 2004

Genetic structure of the large Japanese field mouse populations in suburban landscape of West Tokyo, Japan was determined using mitochondrial DNA control region sequence. Samples were collected from six habitats linked by forests and green tract along the Tama River, and from two forests segregated by urban areas from those continuous habitats. Thirty‐five haplotypes were detected in 221 animals. Four to eight haplotypes were found within each local population belonging to the continuous landscape. Some haplotypes were shared by two or three adjacent local populations. On the other hand, two isolated habitats were occupied by one or two indigenous haplotypes. Significant genetic differentiation between all pairs of local populations, except for one pair in the continuous habitats, was found by analysis of molecular variance (amova). The geographical distance between habitats did not explain the large variance of pairwiseFST‐values among local populations.FST‐values between local populations segregated by urban areas were higher than those between local populations in the continuous habitat, regardless of geographical distance. The results of this study demonstrated quantitatively that urban areas inhibit the migration ofApodemus speciosus, whereas a linear green tract along a river functions as a corridor. Moreover, it preserves the metapopulation structure ofA. speciosusas well as the corridors in suburban landscape.
Estimation of genetically effective breeding numbers using a rejection algorithm approach
Authors: RAMAKRISHNAN UMA , STORZ JAY F. , TAYLOR BARBARA L. , LANDE RUSSELL
Published: December 2004

Polygynous mating results in nonrandom sampling of the adult male gamete pool in each generation, thereby increasing the rate of genetic drift. In principle, genetic paternity analysis can be used to infer the effective number of breeding males (Nebm). However, this requires genetic data from an exhaustive sample of candidate males. Here we describe a new approach to estimate Nebm using a rejection algorithm in association with three statistics: Euclidean distance between the frequency distributions of maternally and paternally inherited alleles, average number of paternally inherited alleles and average gene diversity of paternally inherited alleles. We quantify the relationship between these statistics and Nebm using an individual‐based simulation model in which the male mating system varied continuously between random mating and extreme polygyny. We evaluate this method using genetic data from a natural population of highly polygynous fruit bats (Cynopterous sphinx). Using data in the form of mother–offspring genotypes, we demonstrate that estimates of Nebm are very similar to independent estimates based on a direct paternity analysis that included data on candidate males. Our method also permits an evaluation of uncertainty in estimates of Nebm and thus facilitates inferences about the mating system from genetic data. Finally, we investigate the sensitivity of our method to sample size, model assumptions, adult population size and the mating system. These analyses demonstrate that the rejection algorithm provides accurate estimates of Nebm across a broad range of demographic scenarios, except when the true Nebm is high.
Molecular markers reveal extensive intraspecific below‐ground overlap of silver fir fine roots
Authors: BRUNNER I. , RUF M. , LÜSCHER P. , SPERISEN C.
Published: December 2004

Fine roots are expected to be important determinants of plant competition, but very little is known about the extent of root system overlap. Here, we describe the application of two highly variable plastide microsatellites to study the fine root distribution of tree individuals in a silver fir forest. We demonstrate that the spread of fine roots exceeds the width of above‐ground parts, and that fine root overlaps among neighbouring trees are extensive both laterally and in depth. This approach will help to improve models of below‐ground competition and will facilitate estimations of fine root biomass and thus of below‐ground C pools.
Neutral genetic variation among wild North American populations of the weedy plant Arabidopsis thaliana is not geographically structured
Authors: JØRGENSEN S. , MAURICIO R.
Published: December 2004

We investigated neutral genetic variation within and among 53 wild‐collected populations of the weedy annual plant, Arabidopsis thaliana, in North America, using amplified fragment length polymorphism (AFLP) markers. A. thaliana is thought to have been introduced to North America from Eurasia by humans; such an introduction might be expected to leave a clear geographical signal in the genetic data. To detect such patterns, we sampled populations at several hierarchical geographical levels. We collected individuals from populations in two areas of the Southeast and one in the Midwest, as well as individuals from populations in the Pacific Northwest and Northeast. To estimate within‐population variation, we sampled eight individuals from each of six populations in the Southeast and Midwest. Among all 95 individuals analysed, we detected 131 polymorphic AFLP fragments. We found no evidence for continental or regional diversification. Individuals sampled from Midwestern and Southeastern populations intermingled in a neighbour‐joining tree, and Mantel tests conducted within the Midwestern and Southeastern regions as well as the full data set failed to detect any significant relationship between geographical and genetic distance. These results mirror those found for most global surveys of neutral genetic variability in A. thaliana. Surprisingly, we detected substantial amounts of neutral genetic variability within populations. The levels of genetic variation within populations, coupled with the nongeographical nature of divergence among populations, are consistent with contemporary gene flow and point to a complex and dynamic population history of A. thaliana in North America.
Prey selection by linyphiid spiders: molecular tracking of the effects of alternative prey on rates of aphid consumption in the field
Authors: HARWOOD JAMES D. , SUNDERLAND KEITH D. , SYMONDSON WILLIAM O. C.
Published: December 2004

A molecular approach, using aphid‐specific monoclonal antibodies, was used to test the hypothesis that alternative prey can affect predation on aphids by linyphiid spiders. These spiders locate their webs in cereal crops within microsites where prey density is high. Previous work demonstrated that of two subfamilies of Linyphiidae, one, the Linyphiinae, is web‐dependent and makes its webs at sites where they were more likely to intercept flying insects plus those (principally aphids) falling from the crop above. The other, the Erigoninae, is less web‐dependent, making its webs at ground level at sites with higher densities of ground‐living detritivores, especially Collembola. The guts of the spiders were analysed to detect aphid proteins using enzyme‐linked immunosorbent assay (ELISA). Female spiders were consuming more aphid than males of both subfamilies and female Linyphiinae were, as predicted, eating more aphid than female Erigoninae. Rates of predation on aphids by Linyphiinae were related to aphid density and were not affected by the availability of alternative prey. However, predation by the Erigoninae on aphids was significantly affected by Collembola density. Itinerant Linyphiinae, caught away from their webs, contained the same concentration of aphid in their guts as web‐owners. However, nonweb‐owning Erigoninae, living away from Collembola aggregations at web‐sites, contained significantly higher concentrations of aphid. For both subfamilies there was evidence of a disproportionate increase in predation on aphids once Collembola populations had declined. It was concluded that nonaphid prey, by helping to maintain spiders in the field, can significantly affect predation on aphids.
Spatial and population genetic structure of microsatellites in white pine
Authors: MARQUARDT PAULA E. , EPPERSON BRYAN K.
Published: December 2004

We evaluated the population genetic structure of seven microsatellite loci for old growth and second growth populations of eastern white pine (Pinus strobus). From each population, located within Hartwick Pines State Park, Grayling, Michigan, USA, 120–122 contiguous trees were sampled for genetic analysis. Within each population, genetic diversity was high and inbreeding low. When comparing these populations, there is a significant, but small (less than 1%), genetic divergence between populations. Spatial distance between populations or timber harvest at the second growth site were reasonable explanations for the observed minor differences in allele frequencies between populations. Spatial autocorrelation analysis suggested that, for the old growth population, weak positive structuring at 15 m fits the isolation by distance model for a neighbourhood size of about 100 individuals. In comparison, genotypes were randomly distributed in the second growth population. Thus, logging may have decreased spatial structuring at the second growth site, suggesting that management practices may be used to alter natural spatial patterns. In addition, the amount of autocorrelation in the old growth population appears to be lower for some of the microsatellites, suggesting higher numbers of rare alleles and that higher mutation rates may have directly affected spatial statistics by reducing structure.
Chloroplast DNA variation in the Quercus affinis–Q. laurina complex in Mexico: geographical structure and associations with nuclear and morphological variation
Authors: GONZÁLEZ‐RODRÍGUEZ A. , BAIN J. F. , GOLDEN J. L. , OYAMA K.
Published: December 2004

The geographical distribution of chloroplast DNA (cpDNA) variation in 39 populations of two hybridizing Mexican red oaks, Quercus affinis and Q. laurina, was investigated using polymerase chain reaction‐restriction fragment length polymorphism (PCR–RFLP). Six haplotypes were identified. Of these, two (H1 and H4), separated by four mutations, had high frequencies (58 and 23% of the individuals, respectively) and were present across the whole geographical range of both species, often co occurring in the same populations. The other four haplotypes were rare, geographically restricted, and are probably derived from the two frequent haplotypes. Latitudinal or other clinal patterns in diversity levels or haplotype composition of populations were not apparent. The pattern of haplotype distribution was characterized by some mosaicism, with contrasting populations often situated in proximity. Average within‐population diversity (hS = 0.299) and population differentiation (GST = 0.499) were, respectively, higher and lower than values reported in previous studies of oak species. There was evidence for phylogeographical structure, as indicated by NST (0.566) being significantly higher than GST. Haplotypic variation was largely species‐independent, although some very weak associations were detected between haplotypes H1 and H4 and morphological and nuclear molecular variation correspondingly characterizing Q. affinis and Q. laurina. These oaks probably did not experience a marked restriction to one or a few particular subregions of their present range during the last glacial cycle. It is more likely that substantial populations persisted throughout several episodes of climatic change, but experienced recurrent latitudinal and altitudinal migrations which may have caused the widespread distribution of haplotypes H1 and H4 and frequent intermixing of populations.
Strong fungal specificity and selectivity for algal symbionts in Florida scrubCladonialichens
Authors: YAHR REBECCA , VILGALYS RYTAS , DEPRIEST PAULA T.
Published: December 2004

Symbiosis is a major theme in the history of life and can be an important force driving evolution. However, across symbioses, it is difficult to tease apart the mechanisms that structure the interactions among potential partners. We used genetic similarity and frequency‐based methods to qualitatively and quantitatively examine the patterns of association among several co‐occurringCladonialichen fungi and their algal photobionts in six disjunct Florida scrub sites. The patterns of association were described by the degree of specificity, i.e. the phylogenetic range of associated partners, and of selectivity, i.e. the frequency of association among partners. Six fungal species associated with only one algal internal transcribed spacer clade, with the remaining two fungi being associated with two algal clades. In all cases, the fungi associated in unequal frequencies with the observed algal photobiont genotypes within those clades — suggesting that both specificity and selectivity were higher than expected. Fungal species can be grouped into three significantly different specificity classes: photobiont specialists, intermediates and generalists. In contrast to the pronounced specificity for photobionts among fungal species, the different Florida scrub sites do not harbour distinct photobiont pools, and differential photobiont availability cannot explain the patterning of lichen associations at this spatial scale. Therefore, we conclude that fungal specificity and selectivity for algal photobionts are major factors in determining the local composition of symbiotic partnerships.
Quantifying genotyping errors in noninvasive population genetics
Authors: BROQUET THOMAS , PETIT ERIC
Published: December 2004

The use of noninvasively collected samples greatly expands the range of ecological issues that may be investigated through population genetics. Furthermore, the difficulty of obtaining reliable genotypes with samples containing low quantities of amplifiable DNA may be overcome by designing optimal genotyping schemes. Such protocols are mainly determined by the rates of genotyping errors caused by false alleles and allelic dropouts. These errors may not be avoided through laboratory procedure and hence must be quantified. However, the definition of genotyping error rates remains elusive and various estimation methods have been reported in the literature. In this paper we proposed accurate codification for the frequencies of false alleles and allelic dropouts. We then reviewed other estimation methods employed in hair‐ or faeces‐based population genetics studies and modelled the bias associated with erroneous methods. It is emphasized that error rates may be substantially underestimated when using an erroneous approach. Genotyping error rates may be important determinants of the outcome of noninvasive studies and hence should be carefully computed and reported.
Major histocompatibility complex and microsatellite variation in two populations of wild gorillas
Authors: LUKAS D. , BRADLEY B. J. , NSUBUGA A. M. , DORAN‐SHEEHY D. , ROBBINS M. M. , VIGILANT L.
Published: December 2004

In comparison to their close relatives the chimpanzees and humans, very little is known concerning the amount and structure of genetic variation in gorillas. Two species of gorillas are recognized and while the western gorillas number in the tens of thousands, only several hundred representatives of the mountain gorilla subspecies of eastern gorillas survive. To analyse the possible effects of these different population sizes, this study compares the variation observed at microsatellite and major histocompatibility complex (MHC) loci in samples of wild western and mountain gorillas, collected using a sampling scheme that targeted multiple social groups within defined geographical areas. Noninvasive samples proved a viable source of DNA for sequence analysis of the second exon of the DRB loci of the MHC. Observed levels of variation at the MHC locus were similar between the two gorilla species and were comparable to those in other primates. Comparison of results from analysis of variation at multiple microsatellite loci found only a slight reduction in heterozygosity for the mountain gorillas despite the relatively smaller population size.
The autolysin Ami contributes to the adhesion of Listeria monocytogenes to eukaryotic cells via its cell wall anchor
Authors: Milohanic Eliane , Jonquières Renaud , Cossart Pascale , Berche Patrick , Gaillard Jean‐Louis
Published: December 2004

Adherence of pathogenic microorganisms to the cell surface is a key event during infection. We have previously reported the characterization of Listeria monocytogenes transposon mutants defective in adhesion to eukaryotic cells. One of these mutants had lost the ability to produce Ami, a 102 kDa autolytic amidase with an N‐terminal catalytic domain and a C‐terminal cell wall‐anchoring domain made up of repeated modules containing the dipeptide GW (‘GW modules’). We generated ami null mutations by plasmid insertion into L. monocytogenes strains lacking the invasion proteins InlA (EGDΔinlA), InlB (EGDΔinlB) or both (EGDΔinlAB). These mutants were 5–10 times less adherent than their parental strains in various cell types. The adhesion capacity of the mutants was restored by complementation with a DNA fragment encoding the Ami cell wall‐anchoring domain fused to the Ami signal peptide. The cell‐binding activity of the Ami cell wall‐anchoring domain was further demonstrated using the purified polypeptide. Growth of the ami null mutants constructed in EGD and EGDΔinlAB backgrounds was attenuated in the livers of mice inoculated intravenously, indicating a role for Ami in L. monocytogenes virulence. Adhesive properties have recently been reported in the non‐catalytic domain of two other autolysins, Staphylococcus epidermidis AtlE and Staphylococcus saprophyticus Aas. Interestingly, we found that these domains were also composed of repeated GW modules. Thus, certain autolysins appear to promote bacterial attachment by means of their GW repeat domains. These molecules may contribute to the colonization of host tissues by Gram‐positive bacteria.
Generalized approach to the regulation and integration of gene expression
Authors: Oh Jeong‐Il , Kaplan Samuel
Published: December 2004

The volume of electron flow through the cbb3 branch of the electron transport chain and the redox state of the quinone pool generate signals that regulate photosynthesis gene expression in Rhodobacter sphaeroides. An inhibitory signal is generated at the level of the catalytic subunit of the cbb3 cytochrome c oxidase and is transduced through the membrane‐localized PrrC polypeptide to the PrrBA two‐component activation system, which controls the expression of most of the photosynthesis genes in response to O2. The redox state of the quinone pool is monitored by the redox‐active AppA antirepressor protein, which determines the functional state of the PpsR repressor protein. The antirepressor/repressor system as well as a modulator of AppA function, TspO, together with FnrL and PrrA stringently control photopigment gene expression. These regulatory elements, together with spectral complex‐specific assembly factors, control the ultimate cellular levels and composition of the photosynthetic membrane.
Three‐component‐mediated serotype conversion in Pseudomonas aeruginosa by bacteriophage D3
Authors: Newton Gregory J. , Daniels Craig , Burrows Lori L. , Kropinski Andrew M. , Clarke Anthony J. , Lam Joseph S.
Published: December 2004

Bacteriophage D3 is capable of lysogenizing Pseudomonas aeruginosa PAO1 (serotype O5), converting the O‐antigen from O5 to O16 and O‐acetylating the N‐acetylfucosamine moiety. To investigate the mechanism of lysogenic conversion, a 3.6 kb fragment from the D3 genome was isolated capable of mediating serotypic conversion identical to the D3 lysogen strain (AK1380). The PAO1 transformants containing this 3.6 kb of D3 DNA exhibited identical lipopolysaccharide (LPS) banding patterns to serotype O16 in silver‐stained SDS–PAGE gels and displayed reactivity to an antibody specific for O‐acetyl groups. Further analysis led to the identification of three open reading frames (ORFs) required for serotype conversion: an α‐polymerase inhibitor (iap); an O‐acetylase (oac); and a β‐polymerase (wzyβ). The α‐polymerase inhibitor (Iap) is capable of inhibiting the assembly of the serotype‐specific O5 B‐band LPS and allows the phage‐encoded β‐polymerase (Wzyβ) to form new β‐linked B‐band LPS. The D3 phage also alters the LPS by the addition of O‐acetyl groups to the FucNAc residue in the O‐antigen repeat unit by the action of the D3 O‐acetylase (Oac). These three components form a simple yet elegant system by which bacteriophage D3 is capable of altering the surface of P. aeruginosa PAO1.
Chromosomal insertion and excision of a 30 kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila
Authors: Lüneberg Edeltraud , Mayer Barbara , Daryab Neda , Kooistra Oliver , Zähringer Ulrich , Rohde Manfred , Swanson John , Frosch Matthias
Published: December 2004

We recently described the phase‐variable expression of a virulence‐associated lipopolysaccharide (LPS) epitope in Legionella pneumophila. In this study, the molecular mechanism for phase variation was investigated. We identified a 30 kb unstable genetic element as the molecular origin for LPS phase variation. Thirty putative genes were encoded on the 30 kb sequence, organized in two putative opposite transcription units. Some of the open reading frames (ORFs) shared homologies with bacteriophage genes, suggesting that the 30 kb element was of phage origin. In the virulent wild‐type strain, the 30 kb element was located on the chromosome, whereas excision from the chromosome and replication as a high‐copy plasmid resulted in the mutant phenotype, which is characterized by alteration of an LPS epitope and loss of virulence. Mapping and sequencing of the insertion site in the genome revealed that the chromosomal attachment site was located in an intergenic region flanked by genes of unknown function. As phage release could not be induced by mitomycin C, it is conceivable that the 30 kb element is a non‐functional phage remnant. The protein encoded by ORF T on the 30 kb plasmid could be isolated by an outer membrane preparation, indicating that the genes encoded on the 30 kb element are expressed in the mutant phenotype. Therefore, it is conceivable that the phenotypic alterations seen in the mutant depend on high‐copy replication of the 30 kb element and expression of the encoded genes. Excision of the 30 kb element from the chromosome was found to occur in a RecA‐independent pathway, presumably by the involvement of RecE, RecT and RusA homologues that are encoded on the 30 kb element.
Dominant‐negative mutants of prgX: evidence for a role for PrgX dimerization in negative regulation of pheromone‐inducible conjugation
Authors: Bae Taeok , Dunny Gary M.
Published: December 2004

PrgX negatively regulates prgQ transcriptional readthrough in the pheromone‐inducible enterococcal conjugative plasmid pCF10. We isolated and characterized 13 dominant‐negative prgX mutants, all of which mapped in either the N‐ or the C‐terminus of PrgX. In all mutants, the in vivo level of Qa RNA, an antisense RNA to prgQ RNA, was greatly reduced. When oligomerization of PrgX was tested with a phage lambda cI repressor fusion system, the oligomerization domain was found to be between amino acid residues 78 and 280. When histidine‐tagged PrgX (His‐PrgX) was purified by nickel column chromatography from a strain also expressing PrgX, PrgX was co‐purified with His‐PrgX. Although PrgX was expressed at a much higher level than His‐PrgX, an approximately equal amount of PrgX was co‐purified. Pheromone induction greatly decreased the co‐purification of PrgX. Based on these data, we propose that both the N‐ and the C‐terminal domains of PrgX are required for PrgX positive autoregulation and for the repression of prgQ transcription readthrough. In vivo, PrgX exists as a dimer, and dimerization is mediated by the central region of PrgX.
Mycobacterial FurA is a negative regulator of catalase–peroxidase gene katG
Authors: Zahrt Thomas C. , Song Jian , Siple Jessica , Deretic Vojo
Published: December 2004

In several bacteria, the catalase–peroxidase gene katG is under positive control by oxyR, a transcriptional regulator of the peroxide stress response. The Mycobacterium tuberculosis genome also contains sequences corresponding to oxyR, but this gene has been inactivated in the tubercle bacillus because of the presence of multiple mutations and deletions. Thus, M. tuberculosis katG and possibly other parts of the oxidative stress response in this organism are either not regulated or are controlled by a factor different from OxyR. The mycobacterial FurA is a homologue of the ferric uptake regulator Fur and is encoded by a gene located immediately upstream of katG. Here, we examine the possibility that FurA regulates katG expression. Inactivation of furA on the Mycobacterium smegmatis chromosome, a mycobacterial species that also lacks an oxyR homologue, resulted in derepression of katG, concomitant with increased resistance of the furA mutant to H2O2. In addition, M. smegmatis furA::Kmr was more sensitive to the front‐line antituberculosis agent isonicotinic acid hydrazide (INH) compared with the parental furA+ strain. The phenotypic manifestations were specific, as the mutant strain did not show altered sensitivity to organic peroxides, and both H2O2 and INH susceptibility profiles were complemented by the wild‐type furA+ gene. We conclude that FurA is a second regulator of oxidative stress response in mycobacteria and that it negatively controls katG. In species lacking a functional oxyR, such as M. tuberculosis and M. smegmatis, FurA appears to be a dominant regulator affecting mycobacterial physiology and intracellular survival.
Phosphatidylcholine levels in Bradyrhizobium japonicum membranes are critical for an efficient symbiosis with the soybean host plant
Authors: Minder Alexander C. , De Rudder Karel E. E. , Narberhaus Franz , Fischer Hans‐Martin , Hennecke Hauke , Geiger Otto
Published: December 2004

Phosphatidycholine (PC), the major membrane phospholipid in eukaryotes, is found in only some bacteria including members of the family Rhizobiaceae. For this reason, it has long been speculated that rhizobial PC might be required for a successful interaction of rhizobia with their legume host plants in order to allow the formation of nitrogen‐fixing root nodules. A major pathway for PC formation in prokaryotes involves a threefold methylation of the precursor phosphatidylethanolamine (PE). Here, we report on the isolation of a Bradyrhizobium japonicum gene (pmtA) encoding the phospholipid N‐methyltransferase PmtA. Upon expression of the bradyrhizobial pmtA gene in Escherichia coli, predominantly monomethylphosphatidylethanolamine was formed from PE. PmtA‐deficient B. japonicum mutants still produced low levels of PC by a second methylation pathway. The amount of PC formed in such mutants (6% of total phospholipids) was greatly decreased compared with the wild type (52% of total phospholipids). Root nodules of soybean plants infected with B. japonicum pmtA mutants showed a nitrogen fixation activity of only 18% of the wild‐type level. The interior colour of the nodules was beige instead of red, suggesting decreased amounts of leghaemoglobin. Moreover, ultrastructure analysis of these nodules demonstrated a greatly reduced number of bacteroids within infected plant cells. These data suggest that the biosynthesis of wild‐type amounts of PC are required to allow for an efficient symbiotic interaction of B. japonicum with its soybean host plant.
Anaerobic acquisition of [4Fe 4S] clusters by the inactive FNR(C20S) variant and restoration of activity by second‐site amino acid substitutions
Authors: Ralph E. T. , Scott C. , Jordan P. A. , Thomson A. J. , Guest J. R. , Green J.
Published: December 2004

The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia. The anaerobic incorporation of oxygen‐sensitive [4Fe 4S] clusters promotes dimerization, which in turn enhances DNA binding. Four potential iron ligands (C20, C23, C29 and C122) are essential for normal FNR activity in vivo. Three FNR variants (C20S, C23G and C29G) retained the ability to incorporate oxygen‐sensitive [4Fe 4S] clusters and to bind target DNA with essentially unimpaired affinity, suggesting that their failure to function normally in vivo resides at a later stage in the signal transduction pathway. The C122 variant failed to assemble iron–sulphur clusters and to bind DNA. Second‐site substitutions that partially restore activity to FNR(C20S) were generated by error‐prone polymerase chain reaction and were located in the dimer interface, in the activating regions (AR1, 2 or 3) or close to C122. Substitutions at E47, R48, E123, I124, E127 or T128 allowed the extent of the FNR AR2 surface to be defined. Only one revertant, FNR(C20S Y69F G149S), specifically corrected the C20S defect. It was concluded that [4Fe 4S] cluster acquisition, dimerization and DNA binding are not sufficient to confer transcription regulatory activity on FNR: the iron–sulphur cluster must also be correctly liganded in order to establish effective activating contacts between FNR and RNA polymerase.
Expression of the Bacillus subtilis sulphonate–sulphur utilization genes is regulated at the levels of transcription initiation and termination
Authors: Van Der Ploeg Jan R. , Barone Mario , Leisinger Thomas
Published: December 2004

The Bacillus subtilis ssuBACD gene cluster is required for the utilization of aliphatic sulphonates as sulphur sources. The regulation of expression of the ssu genes was studied in constructs carrying chromosomal transcriptional fusions of the ssu promoter region to lacZ. When sulphate or cystine served as sulphur sources, expression of the ssu genes was repressed. A putative terminator located between the promoter and the start of the ssuB gene partially overlaps a putative antiterminator. Removal of both the antiterminator and the terminator led to a decrease in the repression ratio of about 10‐fold, but not to constitutive expression. Replacement of the ssu promoter by the Pspac promoter led to decreased expression of the ssu genes, but not to loss of repression by sulphate and cystine. Thus, the repression exerted by sulphate and cystine resulted from regulation at the level of both transcription initiation and transcription termination. O‐acetyl‐l‐serine, a precursor of cysteine, served as effector molecule in both regulation systems.
A two‐component system that controls the expression of pneumococcal surface antigen A (PsaA) and regulates virulence and resistance to oxidative stress in Streptococcus pneumoniae
Authors: McCluskey J. , Hinds J. , Husain S. , Witney A. , Mitchell T. J.
Published: December 2004

Recent genomic‐based studies have identified 13 two‐component signal transduction systems (TCS) in Streptococcus pneumoniae. Bacterial TCSs are important for regulating expression of bacterial genes, including those which are important to the virulence of pathogenic bacteria. We have used virulence assays together with microarray analysis to investigate the importance of pneumococcal TCS04 in the virulence and gene regulation of this pathogen. Deletion mutants of the response regulator of TCS04, rr04, were examined in three independent pneumococcal strains representing three different pneumococcal serotypes. Analysis of the virulence of the three strains enabled us to identify a serotype‐specific attenuation of virulence due to deletion of rr04. Microarray comparison of the transcriptional profiles of the wild‐type strains with the rr04 mutants allowed us to determine which transcriptional changes were occurring in the rr04 mutants. Virulence‐associated changes were demonstrated in the attenuated strain with significant downregulation of a previously determined virulence locus, psaB, psaC and psaA.
The principal sigma factor sigA mediates enhanced growth of Mycobacterium tuberculosis in vivo
Authors: Wu Shiping , Howard Susan T. , Lakey David L. , Kipnis Andre , Samten Buka , Safi Hassan , Gruppo Veronica , Wizel Benjamin , Shams Homayoun , Basaraba Randall J. , Orme Ian M. , Barnes Peter F.
Published: December 2004

The ability of Mycobacterium tuberculosis to grow in macrophages is central to its pathogenicity. We found previously that the widespread 210 strain of M. tuberculosis grew more rapidly than other strains in human macrophages. Because principal sigma factors influence virulence in some bacteria, we analysed mRNA expression of the principal sigma factor, sigA, in M. tuberculosis isolates during growth in human macrophages. Isolates of the 210 strain had higher sigA mRNA levels and higher intracellular growth rates, compared with other clinical strains and the laboratory strain H37Rv. SigA was also upregulated in the 210 isolate TB294 during growth in macrophages, compared with growth in broth. In contrast, H37Rv sigA mRNA levels did not change under these conditions. Overexpression of sigA enhanced growth of recombinant M. tuberculosis in macrophages and in lungs of mice after aerosol infection, whereas recombinant strains expressing antisense transcripts to sigA showed decreased growth in both models. In the presence of superoxide, sense sigA transformants showed greater resistance than vector controls, and the antisense sigA transformant did not grow. We conclude that M. tuberculosis sigA modulates the expression of genes that contribute to virulence, enhancing growth in human macrophages and during the early phases of pulmonary infection in vivo. This effect may be mediated in part by increased resistance to reactive oxygen intermediates.
Chromosomal fragmentation in dUTPase‐deficient mutants of Escherichia coli and its recombinational repair
Authors: Kouzminova Elena A. , Kuzminov Andrei
Published: December 2004

Recent findings suggest that DNA nicks stimulate homologous recombination by being converted into double‐strand breaks, which are mended by RecA‐catalysed recombinational repair and are lethal if not repaired. Hyper‐rec mutants, in which DNA nicks become detectable, are synthetic‐lethal with recA inactivation, substantiating the idea. Escherichia coli dut mutants are the only known hyper‐recs in which presumed nicks in DNA do not cause inviability with recA, suggesting that nicks stimulate homologous recombination directly. Here, we show that dut recA mutants are synthetic‐lethal; specifically, dut mutants depend on the RecBC–RuvABC recombinational repair pathway that mends double‐strand DNA breaks. Although induced for SOS, dut mutants are not rescued by full SOS induction if RecA is not available, suggesting that recombinational rather than regulatory functions of RecA are needed for their viability. We also detected chromosomal fragmentation in dut rec mutants, indicating double‐strand DNA breaks. Both the synthetic lethality and chromosomal fragmentation of dut rec mutants are suppressed by preventing uracil excision via inactivation of uracil DNA‐glycosylase or by preventing dUTP production via inactivation of dCTP deaminase. We suggest that nicks become substrates for recombinational repair after being converted into double‐strand DNA breaks.
Translocation of Escherichia coli RNA polymerase against a protein roadblock in vivo highlights a passive sliding mechanism for transcript elongation
Authors: Mosrin‐Huaman Christine , Turnbough Charles L. , Rahmouni A. Rachid
Published: December 2004

Current models for transcription elongation infer that RNA polymerase (RNAP) moves along the template by a passive sliding mechanism that takes advantage of random lateral oscillations in which single basepair sliding movements interconvert the elongation complex between pre‐ and post‐translocated states. Such passive translocational equilibrium was tested in vivo by a systematic change in the templated NTP that is to be incorporated by RNAP, which is temporarily roadblocked by the lac repressor. Our results show that, under these conditions that hinder the forward movement of the polymerase, the elongation complex is able to extend its RNA chain one nucleotide further when the incoming NTP is a kinetically favoured substrate (i.e. low Km). The addition of an extra nucleotide destabilizes the repressor–operator roadblock leading to an increase in transcriptional readthrough. Similar results are obtained when the incoming NTPs are less kinetically favoured substrates (i.e. high Kms) by specifically increasing their intracellular concentrations. Altogether, these in vivo data are consistent with a passive sliding model in which RNAP forward translocation is favoured by NTP binding. They also suggest that fluctuations in the intracellular NTP pools may play a key role in gene regulation at the transcript elongation level.
Localization and activity of multidrug resistance protein 1 in the secretory pathway of Leishmania parasites
Authors: Dodge Matthew A. , Waller Ross F. , Chow Larry M. C. , Zaman Muhammad M. , Cotton Leanne M. , McConville Malcolm J. , Wirth Dyann F.
Published: December 2004

Upregulation of the multidrug resistance protein 1 (LeMDR1) in the protozoan parasite, Leishmania enriettii, confers resistance to hydrophobic drugs such as vinblastine, but increases the sensitivity of these parasites to the mitochondrial drug, rhodamine 123. In order to investigate the mechanism of action of LeMDR1, the subcellular localization of green fluorescent protein (GFP)‐tagged versions of LeMDR1 and the fate of the traceable‐fluorescent LeMDR1 substrate calcein AM were examined in both Leishmania mexicana and L. enriettii LeMDR1 –/– and overexpressing cell lines. The LeMDR1‐GFP chimera was localized by fluorescence microscopy to a number of secretory and endocytic compartments, including the Golgi apparatus, endoplasmic reticulum (ER) and a multivesicular tubule (MVT)‐lysosome. Pulse–chase labelling experiments with calcein AM suggested that the Golgi and ER pools, but not the MVT‐lysosome pool, of LeMDR1 were active in pumping calcein AM out of the cell. Cells labelled with calcein AM under conditions that slow vesicular transport (low temperature and stationary growth) inhibited export and resulted in the accumulation of fluorescent calcein in both the Golgi and the mitochondria. We propose that LeMDR1 substrates are pumped into secretory compartments and exported from the parasite by exocytosis. Accumulation of MDR substrates in the ER can result in alternative transport to the mitochondrion, explaining the reciprocal sensitivity of drug‐resistant Leishmania to vinblastine and rhodamine 123.
The H2‐sensing complex of Ralstonia eutropha: interaction between a regulatory [NiFe] hydrogenase and a histidine protein kinase
Authors: Buhrke Thorsten , Lenz Oliver , Porthun Antje , Friedrich Bärbel
Published: December 2004

Two [NiFe] hydrogenases enable the proteobacterium Ralstonia eutropha H16 to grow on molecular hydrogen as the sole energy source. A third [NiFe] hydrogenase (RH) acts as an H2 sensor in a multiple component signal transduction chain that controls hydrogenase gene transcription. The RH forms a dimeric heterodimer (HoxBC)2 in which HoxC contains the H2‐sensing active site and HoxB the electron‐transferring components including an organic, not yet identified redox cofactor. This oligomer forms a tight complex with the histidine protein kinase HoxJ. Both the sensor and the kinase were analysed by mutagenesis for functional domains that are instrumental in H2 signal transmission. A mutant deleted for a C‐terminal peptide of 55 amino acids in HoxB lost its H2‐sensing ability but still catalysed H2 oxidation. The mutant protein failed to form the dimeric heterodimer and a complex with HoxJ. The organic redox cofactor was no longer detectable in the truncated sensor. H2 sensing was also abolished by deletion of the PAS domain of HoxJ, indicating that this domain is involved in signal transduction. A truncated version of HoxJ consisting of only the input domain of the kinase was still capable of forming a complex with the RH. Mass determination of the purified HoxJ protein revealed that the kinase forms a homotetramer. The unique oligomeric structure of the H2‐sensing complex with respect to its regulatory function is discussed.
Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system
Authors: Livny Jonathan , Friedman David I.
Published: December 2004

Shiga toxin (Stx) genes in Stx producing Escherichia coli (STEC) are encoded in prophages of the λ family, such as H‐19B. The subpopulation of STEC lysogens with induced prophages has been postulated to contribute significantly to Stx production and release. To study induced STEC, we developed a selectable in vivo expression technology, SIVET, a reporter system adapted from the RIVET system. The SIVET lysogen has a defective H‐19B prophage encoding the TnpR resolvase gene downstream of the phage PR promoter and a cat gene with an inserted tet gene flanked by targets for the TnpR resolvase. Expression of resolvase results in excision of tet, restoring a functional cat gene; induced lysogens survive and are chloramphenicol resistant. Using SIVET we show that: (i) approximately 0.005% of the H‐19B lysogens are spontaneously induced per generation during growth in LB. (ii) Variations in cellular physiology (e.g. RecA protein) rather than in levels of expressed repressor explain why members of a lysogen population are spontaneously induced. (iii) A greater fraction of lysogens with stx encoding prophages are induced compared to lysogens with non‐Stx encoding prophages, suggesting increased sensitivity to inducing signal(s) has been selected in Stx encoding prophages. (iv) Only a small fraction of the lysogens in a culture spontaneously induce and when the lysogen carries two lambdoid prophages with different repressor/operators, 933W and H‐19B, usually both prophages in the same cell are induced.
IsdA of Staphylococcus aureus is a broad spectrum, iron‐regulated adhesin
Authors: Clarke Simon R. , Wiltshire Michael D. , Foster Simon J.
Published: December 2004

As an important facet of host–pathogen interaction, Staphylococcus aureus has the ability to adhere to human extracellular matrix (ECM) components via a range of surface proteins. Here we have shown that IsdA has broad‐spectrum ligand‐binding activity, including fibrinogen and fibronectin. Mapping studies revealed a distinct domain responsible for ligand binding. This domain is present in a number of iron‐regulated proteins of S. aureus and in other Gram‐positive organisms. The isdA gene is only expressed in iron‐limited conditions under the control of Fur and not in standard laboratory media. Such conditions occur in serum in vitro and during infection. Whole cell binding and clumping assays revealed that when the bacteria are grown under iron‐limited conditions, IsdA constitutes a physiologically relevant adhesin to both fibrinogen and fibronectin. Thus for S. aureus, iron is an important marker for the host environment, to which the bacterium responds by differential regulation of at least one element of its adhesive strategy.
A putative G protein‐coupled receptor negatively controls sexual development in Aspergillus nidulans
Authors: Han Kap‐Hoon , Seo Jeong‐Ah , Yu Jae‐Hyuk
Published: December 2004

G protein‐coupled receptors (GPCRs) are key components of heterotrimeric G protein‐mediated signalling pathways that detect environmental signals and confer rapid cellular responses. To broaden our understanding of signalling mechanisms in the filamentous fungus Aspergillus nidulans, intensive analyses of the Aspergillus nidulans genome have been carried out and nine genes (gprA∼gprI) that are predicted to encode seven transmembrane spanning GPCRs have been identified. Six of nine putative GPCRs have been disrupted and the gprD gene was found to play a central role in coordinating hyphal growth and sexual development. Deletion of gprD (ΔgprD) causes extremely restricted hyphal growth, delayed conidial germination and uncontrolled activation of sexual development resulting in a small colony covered by sexual fruiting bodies. Genetic studies indicate that GprD may not signal through the FadA (Gα)‐protein kinase A (PKA) pathway. Elimination of sexual development rescues both growth and developmental abnormalities caused by ΔgprD, suggesting that the primary role of GprD is to negatively regulate sexual development. This is supported by the fact that environmental conditions inhibiting sexual development suppress growth defects of the ΔgprD mutant. We propose that the GprD‐mediated signalling cascade negatively regulates sexual development, which is required for proper proliferation of A. nidulans.
Induction of the sufA operon encoding Fe‐S assembly proteins by superoxide generators and hydrogen peroxide: involvement of OxyR, IHF and an unidentified oxidant‐responsive factor
Authors: Lee Joon‐Hee , Yeo Won‐Sik , Roe Jung‐Hye
Published: December 2004

A promoter (sufAp), inducible by various oxidants, directs transcription of the sufABCDSE operon encoding an alternative Fe‐S cluster assembly system in Escherichia coli. Superoxide generators and H2O2 induced expression of sufA–lacZ even in ΔsoxRS and ΔoxyR mutants, suggesting participation of an additional regulator(s) in oxidant induction of the sufA operon. Through deletion and linker scanning mutagenesis, we found three cis‐acting oxidant‐responsive elements (OREs). ORE‐I lies between −236 and −197 nucleotides from the transcription start site, overlapping extensively with the OxyR binding site reported previously. ORE‐II (−156 to −127) was found to be the site of IHF action. ORE‐III (−56 to −35) had no predictable binding sites for known regulators. Gel mobility shift assays with a 50 bp DNA probe containing ORE‐III revealed the presence of an ORE‐III‐specific factor that binds only when cells are treated with oxidants. S1 mapping analysis revealed that phenazine methosulphate (PMS) and H2O2 induced sufA expression by more than 40‐fold. In a ΔoxyR mutant, sufA was still induced more than 10‐fold. Fur, a ferric uptake regulator that negatively regulates this operon in response to iron availability, did not mediate the oxidant induction. Deletion of the suf operon caused cells to be more sensitive to superoxide‐generating agents without affecting sensitivity to H2O2. From these results, we propose that the oxidant induction of the sufA operon is mediated through OxyR, IHF, plus an unidentified oxidant‐responsive factor, and that the suf gene products are needed to defend cells against oxidative stress caused by superoxide generators.