bluuebunny/crossref_metadata_2025 huggingface dataset:
Clonal diversity and structure within a population of the pondweed Potamogeton pectinatus foraged by Bewick's swans
Authors: Hangelbroek H. H. , Ouborg N. J. , Santamaría L. , Schwenk K.
Published: March 2003

Clonal diversity within plant populations is affected by factors that influence genet (clone) survival and seed recruitment, such as resource availability, disturbance, seed dispersal mechanism, propagule predation and the age of the population. Here we studied a population of Potamogeton pectinatus, a pseudo‐annual aquatic macrophyte. Within populations reproduction appears to be mainly asexually through subterranean propagules (tubers), while recruitment via seeds is believed to be relatively unimportant. RAPD markers were used to analyse clonal diversity and genetic variation within the population. Ninety‐seven genets were identified among 128 samples taken from eight plots. The proportion of distinguishable genets (0.76) and Simpson's diversity index (0.99) exhibited high levels of clonal diversity compared to other clonal plants. According to an analysis of molecular variance (amova) most genetic variation occurred between individuals within plots (93–97%) rather than between plots (8–3%). These results imply that sexual reproduction plays an unexpectedly important role within the population. Nevertheless, autocorrelation statistics revealed a spatial genetic structure resulting from clonal growth. In contrast to genetic variation, clonal diversity was affected by several ecological factors. Water depth and silt content had direct negative effects on clonal diversity. Tuber predation by Bewick's swans had an unexpected indirect negative effect on clonal diversity through reducing the tuber‐bank biomass in spring, which on its turn was positively correlated to clonal diversity. The disturbance by swans, therefore, did not enhance seed recruitment and thus clonal diversity; on the contrary, heavily foraged areas are probably more prone to stochastic loss of genets leading to reduced clonal diversity.
Patterns of molecular genetic variation among African elephant populations
Authors: Comstock Kenine E. , Georgiadis Nicholas , Pecon‐Slattery Jill , Roca Alfred L. , Ostrander Elaine A. , O'Brien Stephen J. , Wasser Samuel K.
Published: March 2003

The highly threatened African elephants have recently been subdivided into two species, Loxodonta africana (savannah or bush elephant) and L. cyclotis (forest elephant) based on morphological and molecular studies. A molecular genetic assessment of 16 microsatellite loci across 20 populations (189 individuals) affirms species level genetic differentiation and provides robust genotypic assessment of species affiliation. Savannah elephant popula‐tions show modest levels of phylogeographic subdivision based on composite microsatellite genotype, an indication of recent population isolation and restricted gene flow between locales. The savannah elephants show significantly lower genetic diversity than forest elephants, probably reflecting a founder effect in the recent history of the savannah species.
Spatial pattern of chloroplast DNA variation of Cyclobalanopsis glauca in Taiwan and East Asia
Authors: Huang Sophie S. F. , Hwang Shih‐Ying , Lin Tsan‐Piao
Published: March 2003

This study examined the spatial pattern of chloroplast DNA (cpDNA) variation in Cyclobalanopsis glauca (Thunb. ex Murray) Oerst. (Fagaceae) in 140 trees from Taiwan (25 populations), Japan (three), Ryukyus (two), Hong Kong (one) and Mainland China (one). By sequencing three cpDNA intergenic spacer fragments using universal primers (trnT‐trnL, trnV‐trnM, including the trnV intron, and petG‐trnP), we found a total of 1980 bp and 15 polymorphic sites. Among them, 12 sites were caused by point mutation, and three resulted from insertion. This gives rise to a total of 13 cpDNA haplotypes. The level of differentiation among the populations studied is relatively high (GST = 0.612). Two ancestral haplotypes (A and B) are distributed widely in East Asia. Interestingly, all the derived cpDNA variations are found only in Taiwan but not in other areas. The Central Mountain Ridge (CMR) of Taiwan creates an unsurpassed barrier to the east–west gene flow of C. glauca. Among the populations on the west of CMR, only three separated populations, Yangmingshan, Wushe and Chinshuiying, have high haplotype diversity, each consisting of sister haplotypes all mutated from the same ancestral haplotype. Thus, they have probably originated from de novo mutation after the last glaciation. This inference agrees with the observation that no spatial autocorrelation existed on the west side. Two unrelated dominant lineages on the east of the CMR (haplotypes D and F) showed significant spatial genetic structure. Estimate of NST – GST was –0.090 and differed significantly from zero. Thus at the local scale, the phylogeographical component of the genetic structure is significant on the east of the CMR. Accompanied by published palynological records of the last glaciation, this study suggests the possibility that these two types were colonized northward from the southeastern part of Taiwan. ‘Star‐like’ genealogy is characterized, with all the haplotypes coalescing rapidly and as a general outcome of population expansion (Page & Holmes 1998). A neutrality test also suggested a demographic expansion recovered from a bottleneck. We therefore inferred that the southeastern part of Taiwan might be a potential refugium for C. glauca.
Does natural selection promote population divergence? A comparative analysis of population structure using amplified fragment length polymorphism markers and quantitative traits
Authors: Steinger T. , Haldimann P. , Leiss K. A. , Müller‐Schärer H.
Published: March 2003

Divergent natural selection is considered an important force in plant evolution leading to phenotypic differentiation between populations exploiting different environments. Extending an earlier greenhouse study of population differentiation in the selfing annual plantSenecio vulgaris, we estimated the degree of population divergence in several quantitative traits related to growth and life history and compared these estimates with those based on presumably neutral molecular markers (amplified fragment length polymorphisms; AFLPs). This approach allowed us to disentangle the effects of divergent selection from that of other evolutionary forces (e.g. genetic drift). Five populations were examined from each of two habitat types (ruderal and agricultural habitats). We found a high proportion of total genetic variance to be among populations, both for AFLP markers (φST = 0.49) and for quantitative traits (range ofQST: 0.26–0.77). There was a strong correlation between molecular and quantitative genetic differentiation between pairs of populations (Mantel'sr = 0.59). However, estimates of population differentiation in several quantitative traits exceeded the neutral expectation (estimated from AFLP data), suggesting that divergent selection contributed to phenotypic differentiation, especially between populations from ruderal and agricultural habitats. Estimates of within‐population variation in AFLP markers and quantitative genetic were poorly correlated, indicating that molecular marker data may be of limited value to predict the evolutionary potential of populations ofS. vulgaris.
Phylogeny, paraphyly and ecological adaptation of the colour and pattern in the Anolis roquet complex on Martinique
Authors: Thorpe R. S. , Stenson A. G.
Published: March 2003

Martinique is an environmentally heterogeneous island with a complex geological history. It is occupied by a solitary anole, Anolis roquet, showing marked geographical variation in colour and other features. Phylogenetic analysis of a segment (1 kb) of the mitochondrial cytochrome b gene across the Anolis roquet series in the southern Lesser Antilles and at 63 localities of Anolis roquet in Martinique indicate that A. roquet is paraphyletic as A. extremus (Barbados) is nested within the Martinique populations. Moreover, divergent phylogenetic lineages exist within Martinique (max. 10.6% uncorrected pairwise), and these lineages are closely associated with the geological history of this complex island. However, objective quantification of the spectroradiometric analysis of hue by delta analysis, together with analysis of the colour pattern, indicate that they are primarily determined by adaptation to environmental conditions, irrespective of these phylogenetic lineages. There is remarkable convergence in hue and pattern in both extreme xeric (dark chevrons on a dull, generally grey/brown, background), and montane conditions (black reticulation and non‐UV white spots on a bright, saturated green background). Moreover, parallel trends occur between Martinique and other Lesser Antillean anoles, which further argues for adaptation (increase in green saturation in montane areas and higher levels of UV on the dewlap of some Atlantic forms). As an exception, there are two specific situations where anoles from different lineages look different. These are (i) in the low‐altitude regions of the northwest where the northwestern and central lineages make contact, and (ii) in the far south of the island where the southern and central lineages meet.
Dispersal barriers and isolation among deep‐sea mussel populations (Mytilidae: Bathymodiolus) from eastern Pacific hydrothermal vents
Authors: Won Y. , Young C. R. , Lutz R. A. , Vrijenhoek R. C.
Published: March 2003

Deep‐sea hydrothermal vent species are widely dispersed among habitat islands found along the global mid‐ocean ridge system. We examine factors that affect population structure, gene flow and isolation in vent‐endemic mussels of the genus Bathymodiolus from the eastern Pacific Ocean. Mussels were sampled from localities including the Galapagos Rift (GAR, 0°48′ N; 86°10′ W) and the East Pacific Rise (EPR, 13° N to 32° S latitude) across a maximum distance of 4900 km. The sampled range crossed a series of topographical features that interrupt linear aspects of the ridge system, and it encompassed regions of strong cross‐axis currents that could impede along‐axis dispersal of mussel larvae. Examinations of mitochondrial DNA sequences and allozyme variation revealed significant barriers to gene flow along the ridge axis. All populations from the GAR and EPR from 13° N to 11° S were homogeneous genetically and appeared to experience unimpeded high levels of interpopulational gene flow. In contrast, mussels from north and south of the Easter Microplate were highly divergent (4.4%), possibly comprising sister‐species that diverged after formation of the microplate ≈ 4.5 Ma. Strong cross‐axis currents associated with inflated bathymetry of the microplate region may reinforce isolation across this region.
Large extent of mitochondrial DNA transfer fromOreochromis aureustoO. niloticusin West Africa
Authors: Rognon X. , Guyomard R.
Published: March 2003

Introgressive hybridization has an important evolutionary significance in terms of gene diversity and speciation. Among the major groups of vertebrates, fish show a strong propensity to hybridize. In order to highlight the possible occurrence of gene flow between two tilapia species,Oreochromis niloticusandO. aureus, a comparison of allozyme and mitochondrial DNA (mtDNA) polymorphism was performed on sympatric and allopatric populations of these two species. Nuclear data were congruent with the morphological identification ofO. niloticusandO. aureuspopulations. In opposition, the mtDNA analysis resulted in two strictly differentiated groups which did not follow the morphological and nuclear DNA classification. The first group consisted of East AfricanO. niloticuspopulations and the second included all theO. aureuspopulations and the West AfricanO. niloticuspopulations. Moreover, in some cases, the same sequences were detected in both species. These data strongly support a differential introgression of mtDNA fromO. aureustoO. niloticusinvolving all the West African area. This work points out the risk of misinterpretation of mtDNA or nuclear DNA data when only one single class of marker is used.
Paternity success and the direction of sexual selection in a field population of a semelparous marsupial, Antechinus agilis
Authors: Kraaijeveld‐Smit F. J. L. , Ward S. J. , Temple‐Smith P. D.
Published: March 2003

Antechinus agilis is a small sexually size dimorphic marsupial with a brief annual mating period of 2–3 weeks. All males die after this period, and females give birth to up to 10 young. Mating is thought to be promiscuous, however, there is no field data to confirm this. Using microsatellites, we investigated paternity patterns over two seasons in a wild population. Male weight was significantly positively related to the number of females fertilized and with the number of offspring sired, in both years. Furthermore, selection gradients indicated selection for larger males. Both results suggest that size dimorphism in A. agilis can be explained by sexual selection for larger males. The proportion of offspring sired within litters, did not relate to male size. Therefore, larger males are more successful through higher mating access, not through their sperm outcompeting that of smaller males. As expected from their known ranging behaviour, the number of offspring within litters left unassigned to a father did not depend on the grid location of the mother. Female size did not differ between successful reproducing and unsuccessful females. However, females that weaned offspring had larger heads than females that did not wean offspring. Males did not ‘prefer’ mating with larger females, nor did assortative mating occur. From our results, the mating system of A. agilis is clearly promiscuous. Selection for larger males occurred in both years, even though in one year the operational sex ratio was highly female biased, suggesting that the potential reproductive rate is a better predictor of the direction of sexual selection in A. agilis.
Genetic variation within and among fragmented populations of lesser prairie‐chickens (Tympanuchus pallidicinctus)
Authors: Van Den Bussche Ronald A. , Hoofer Steven R. , Wiedenfeld David A. , Wolfe Donald H. , Sherrod Steve K.
Published: March 2003

As a result of recurrent droughts and anthropogenic factors, the range of the lesser prairie‐chicken (Tympanuchus pallidicinctus) has contracted by 92% and the population has been reduced by approximately 97% in the past century, resulting in the smallest population size and most restricted geographical distribution of any North American grouse. We examined genetic variation through DNA sequence analysis of 478 base pairs of the mitochondrial genome and by assaying allelic variation at five microsatellite loci from lesser prairie‐chickens collected on 20 leks in western Oklahoma and east‐central New Mexico. Traditional population genetic analyses indicate that lesser prairie‐chickens maintain high levels of genetic variation at both nuclear and mitochondrial loci. Although some genetic structuring among lesser prairie‐chicken leks was detected within Oklahoma and New Mexico for both nuclear and mitochondrial loci, high levels of differentiation were detected between Oklahoma and New Mexico populations. Nested‐clade analysis of mitochondrial haplotypes revealed that both historic and contemporary processes have influenced patterns of haplotype distributions and that historic processes have most likely led to the level of differentiation found between the Oklahoma and New Mexico populations.
Competence‐specific induction of recA is required for full recombination proficiency during transformation in Streptococcus pneumoniae
Authors: Mortier‐Barrière Isabelle , De Saizieu Antoine , Claverys Jean‐Pierre , Martin Bernard
Published: March 2003

Transcriptional activation of the recA gene of Streptococcus pneumoniae was previously shown to occur at competence. A 5.7 kb recA‐specific transcript that contained at least two additional genes, cinA and dinF, was identified. We now report the complete characterization of the recA operon and investigation of the role of the competence‐specific induction of recA. The 5.7 kb competence‐specific recA transcript is shown to include lytA, which encodes the pneumococcal autolysin, a protein previously shown to contribute to virulence of S. pneumoniae. Uncoupling (denoted Ind−) of recA and/or the downstream genes was achieved through the placement of transcription terminators within the operon, either upstream or downstream of recA. Prevention of the competence‐specific induction of recA severely affected spontaneous transformation. Transformation efficiencies of recA+ (Ind−) and of wild‐type cells were compared under various conditions and with different donor DNA. Chromosomal transformation was reduced 17‐ (chromosomal donor) to 45‐fold (recombinant plasmid donor), depending on the donor DNA, and plasmid establishment was reduced 129‐fold. Measurement of uptake of radioactively labelled donor DNA in transformed cells in parallel with scoring for transformants (chromosomal donor) revealed normal uptake, but a 21‐fold reduction in recombination in a recA+ (Ind−) strain, indicating that the transformation defect was primarily in recombination. Strikingly enough, a much larger (460‐fold) reduction in recombination was observed for the shortest homologous donor fragment used (878 nucleotides long). Possible interpretations of the observation that basal RecA appears unable to promote efficient recombination whatever the number and the length of donor fragments taken up are proposed. The role of recA induction is discussed in view of the potential contribution of transformation to genome plasticity in this pathogen.
A chloride‐inducible acid resistance mechanism in Lactococcus lactis and its regulation
Authors: Sanders Jan Willem , Leenhouts Kees , Burghoorn Jan , Brands Jan Roel , Venema Gerard , Kok Jan
Published: March 2003

Previously, a promoter was identified in Lactococcus lactis that is specifically induced by chloride. Here, we describe the nucleotide sequence and functional analysis of two genes transcribed from this promoter, gadC and gadB. GadC is homologous to putative glutamate‐γ‐aminobutyrate antiporters of Escherichia coli and Shigella flexneri and contains 12 putative membrane‐spanning domains. GadB shows similarity to glutamate decarboxylases. A L. lactis gadB mutant and a strain that is unable to express both gadB and gadC was more sensitive to low pH than the wild type when NaCl and glutamate were present. Expression of gadCB in L. lactis in the presence of chloride was increased when the culture pH was allowed to decrease to low levels by omitting buffer from the medium, while glutamate also stimulated gadCB expression. Apparently, these genes encode a glutamate‐dependent acid resistance mechanism of L. lactis that is optimally active under conditions in which it is needed to maintain viability. Immediately upstream of the chloride‐dependent gadCB promoter Pgad, a third gene encodes a protein (GadR) that is homologous to the activator Rgg from Streptococcus gordonii. gadR expression is chloride and glutamate independent. A gadR mutant did not produce the 3 kb gadCB mRNA that is found in wild‐type cells in the presence of NaCl, indicating that GadR is an activator of the gadCB operon.
Synthesis of Escherichia coli O9a polysaccharide requires the participation of two domains of WbdA, a mannosyltransferase encoded within the wb* gene cluster
Authors: Kido Nobuo , Sugiyama Tsuyoshi , Yokochi Takashi , Kobayashi Hidemitsu , Okawa Yoshio
Published: March 2003

WbdA (previously MtfA) is one of the mannosyltransferases encoded within the Escherichia coli O9a wb* gene cluster. It is composed of two domains of similar size, connected by an α‐helix chain. Elimination of the C‐terminal half by transposon insertion or gene deletion caused synthesis of an altered structural O‐polysaccharide consisting only of α‐1,2‐linked mannose. O9a polysaccharide synthesis was restored by the C‐terminal half of WbdA in trans. No membrane incorporation of mannose from GDP mannose was observed in a strain carrying only the gene for truncated WbdA. For mannose incorporation, it was necessary to introduce both wbdB and wbdC genes into the strain. Therefore, it is likely that the N‐terminal half of truncated WbdA synthesizes the altered O‐polysaccharide together with other mannosyltransferases which participate in the initial reactions of the O9a polysaccharide synthesis. Both N‐ and C‐terminal domains of WbdA are required for the synthesis of the complete E. coli O9a polysaccharide. The chi sequence location between the two domains and homology plot analyses of the wbdA and the WbdA protein suggested that the wbdA gene might have arisen by fusion of two independent genes.
Simple sequence repeats in the Helicobacter pylori genome
Authors: Saunders Nigel J. , Peden John F. , Hood Derek W. , Moxon E. Richard
Published: March 2003

We describe an integrated system for the analysis of DNA sequence motifs within complete bacterial genome sequences. This system is based around ACeDB, a genome database with an integrated graphical user interface; we identify and display motifs in the context of genetic, sequence and bibliographic data. Tomb et al. (1997) previously reported the identification of contingency genes in Helicobacter pylori through their association with homopolymeric tracts and dinucleotide repeats. With this as a starting point, we validated the system by a search for this type of repeat and used the contextual information to assess the likelihood that they mediate phase variation in the associated open reading frames (ORFs). We found all of the repeats previously described, and identified 27 putative phase‐variable genes (including 17 previously described). These could be divided into three groups: lipopolysaccharide (LPS) biosynthesis, cell‐surface‐associated proteins and DNA restriction/modification systems. Five of the putative genes did not have obvious homologues in any of the public domain sequence databases. The reading frame of some ORFs was disrupted by the presence of the repeats, including the alpha(1‐2) fucosyltransferase gene, necessary for the synthesis of the Lewis Y epitope. An additional benefit of this approach is that the results of each search can be analysed further and compared with those from other genomes. This revealed that H. pylori has an unusually high frequency of homopurine:homopyrimidine repeats suggesting mechanistic biases that favour their presence and instability.
Antagonistic effects of dual PTS‐catalysed phosphorylation on the Bacillus subtilis transcriptional activator LevR
Authors: Martin‐Verstraete Isabelle , Charrier Véronique , Stülke Jörg , Galinier Anne , Erni Bernhard , Rapoport Georges , Deutscher Josef
Published: March 2003

LevR, which controls the expression of the lev operon of Bacillus subtilis, is a regulatory protein containing an N‐terminal domain similar to the NifA/NtrC transcriptional activator family and a C‐terminal domain similar to the regulatory part of bacterial anti‐terminators, such as BglG and LicT. Here, we demonstrate that the activity of LevR is regulated by two phosphoenolpyruvate (PEP)‐dependent phosphorylation reactions catalysed by the phosphotransferase system (PTS), a transport system for sugars, polyols and other sugar derivatives. The two general components of the PTS, enzyme I and HPr, and the two soluble, sugar‐specific proteins of the lev‐PTS, LevD and LevE, form a signal transduction chain allowing the PEP‐dependent phosphorylation of LevR, presumably at His‐869. This phosphorylation seems to inhibit LevR activity and probably regulates the induction of the lev operon. Mutants in which His‐869 of LevR has been replaced with a non‐phosphorylatable alanine residue exhibited constitutive expression from the lev promoter, as do levD or levE mutants. In contrast, PEP‐dependent phosphorylation of LevR in the presence of only the general components of the PTS, enzyme I and HPr, regulates LevR activity positively. This phosphorylation most probably occurs at His‐585. Mutants in which His‐585 has been replaced with an alanine had lost stimulation of LevR activity and PEP‐dependent phosphorylation by enzyme I and HPr. This second phosphorylation of LevR at His‐585 is presumed to play a role in carbon catabolite repression.
Use of time‐lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis
Authors: Webb Chris D. , Graumann Peter L. , Kahana Jason A. , Teleman Aurelio A. , Silver Pamela A. , Losick Richard
Published: March 2003

We describe the use of time‐lapse fluorescence microscopy to visualize the movement of the DNA replication origin and terminus regions on the Bacillus subtilis chromosome during the course of the cell cycle. The origin and terminus regions were tagged with a cassette of tandem lac operator repeats and visualized through the use of a fusion of the green fluorescent protein to the LacI repressor. We have discovered that origin regions abruptly move apart towards the cell poles during a brief interval of the cell cycle. This movement was also seen in the absence of cell wall growth and in the absence of the product of the parB homologue spo0J. The origin regions moved apart an average distance of 1.4 μm in an 11 min period of abrupt movement, representing an average velocity of 0.17 μm min−1. and reaching a maximum velocity of greater than 0.27 μm min−1. The terminus region also exhibited a striking pattern of movement but not as far or a rapid as the origin region. These results provide evidence for a mitotic‐like motor that is responsible for segregation of the origin regions of the chromosomes.
The expression of the secreted aspartyl proteinases Sap4 to Sap6 from Candida albicans in murine macrophages
Authors: Borg‐von Zepelin M. , Beggah S. , Boggian K. , Sanglard D. , Monod M.
Published: March 2003

Medically important yeasts of the genus Candida secrete aspartyl proteinases (Sap), which are of particular interest as virulence factors. Six closely related gene sequences, SAP1 to SAP6, for secreted proteinases are present in Candida albicans. The methylotrophic yeast Pichia pastoris was chosen as an expression system for preparing substantial amounts of each Sap isoenzyme. Interestingly, Sap4, Sap5 and Sap6, which have not yet been detected in C. albicans cultures in vitro, were produced as active recombinant enzymes. Different Sap polyclonal antibodies were raised in rabbits and tested before further application by enzyme‐linked immunosorbent assay (ELISA) against each recombinant Sap. Two antisera recognized only Sap4 to Sap6. Using these antisera, together with sap null mutants obtained by targeted mutagenesis, we could demonstrate a high production of Sap4, Sap5 and Sap6 by C. albicans cells after phagocytosis by murine peritoneal macrophages. Furthermore, a Δsap4,5,6 null mutant was killed 53% more effectively after contact with macrophages than the wild‐type strain. These results support a role for Sap4 to Sap6 in pathogenicity.
Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism
Authors: Leuthner Birgitta , Leutwein Christina , Schulz Henk , Hörth Patric , Haehnel Wolfgang , Schiltz Emile , Schägger Hermann , Heider Johann
Published: March 2003

Toluene is anoxically degraded to CO2 by the denitrifying bacterium Thauera aromatica. The initial reaction in this pathway is the addition of fumarate to the methyl group of toluene, yielding benzylsuccinate as the first intermediate. We purified the enzyme catalysing this reaction, benzylsuccinate synthase (EC 4.1.99‐), and studied its properties. The enzyme was highly oxygen sensitive and contained a redox‐active flavin cofactor, but no iron centres. The native molecular mass was 220 kDa; four subunits of 94 (α), 90 (α′), 12 (β) and 10 kDa (γ) were detected on sodium dodecyl sulphate (SDS) gels. The N‐terminal sequences of the α‐ and α′‐subunits were identical, suggesting a C‐terminal degradation of half of the α‐subunits to give the α′‐subunit. The composition of native enzyme therefore appears to be α2β2γ2. A 5 kb segment of DNA containing the genes for the three subunits of benzylsuccinate synthase was cloned and sequenced. The masses of the predicted gene products correlated exactly with those of the subunits, as determined by electrospray mass spectrometry. Analysis of the derived amino acid sequences revealed that the large subunit of the enzyme shares homology to glycyl radical enzymes, particularly near the predicted radical site. The highest similarity was observed with pyruvate formate lyases and related proteins. The radical‐containing subunit of benzylsuccinate synthase is oxygenolytically cleaved at the site of the glycyl radical, producing the α′‐subunit. The predicted cleavage site was verified using electrospray mass spectrometry. In addition, a gene coding for an activating protein catalysing glycyl radical formation was found. The four genes for benzylsuccinate synthase and the activating enzyme are organized as a single operon; their transcription is induced by toluene. Synthesis of the predicted gene products was achieved in Escherichia coli in a T7‐promotor/polymerase system.
Effects of the Escherichia coli DNA‐binding protein H‐NS on rRNA synthesis in vivo
Authors: Afflerbach Henning , Schröder Oliver , Wagner Rolf
Published: March 2003

The Escherichia coli DNA‐binding protein H‐NS is known to interact specifically with the upstream region of ribosomal RNA transcription units, where it causes transcriptional repression in vitro. Here, we present results demonstrating the effect of H‐NS on rRNA transcription in vivo. rRNA synthesis rates were compared in cells that differ in the expression of functional H‐NS or FIS molecules. We could show that in the absence of H‐NS derepression of rRNA synthesis occurs at low growth rates. During the cell cycle H‐NS is responsible for the rapid shut‐off of rRNA synthesis at the end of the exponential phase. As it is known for FIS‐dependent activation, the inhibitory function of H‐NS is specific for P1, the first of the tandem rRNA promoters. The effect of H‐NS on rRNA synthesis was further assessed under stress conditions. While under osmotic upshift the reduction in rRNA synthesis is clearly H‐NS‐dependent, no such influence could be detected at cold shock. Determination of the cellular ppGpp concentrations revealed that H‐NS does not mediate its function via alterations in the synthesis of the global effector ppGpp.
Nucleoside diphosphate kinase: role in bacterial growth, virulence, cell signalling and polysaccharide synthesis
Authors: Chakrabarty A.M.
Published: March 2003

Nucleoside diphosphate kinase (Ndk) is an important enzyme that generates nucleoside triphosphates (NTPs) or their deoxy derivatives by terminal phosphotransfer from an NTP such as ATP or GTP to any nucleoside diphosphate or its deoxy derivative. As NTPs, particularly GTP, are important for cellular macromolecular synthesis and signalling mechanisms, Ndk plays an important role in bacterial growth, signal transduction and pathogenicity. Specific examples of the role of Ndk in regulating growth, NTP formation and cell surface polysaccharide synthesis in two respiratory tract pathogens, Pseudomonas aeruginosa and Mycobacterium tuberculosis, are discussed.
Tandem genes of Chlamydia psittaci that encode proteins localized to the inclusion membrane
Authors: Bannantine J. P. , Rockey D. D. , Hackstadt T.
Published: March 2003

Chlamydiae are obligate intracellular bacteria that replicate within a non‐acidified vacuole, termed an inclusion. To identify chlamydial proteins that are unique to the intracellular phase of the life cycle, a lambda expression library of Chlamydia psittaci DNA was differentially screened with convalescent antisera from infected guinea pigs and antisera directed at formalin‐fixed purified chlamydial elementary bodies (EBs). One library clone was identified that harboured two open reading frames (ORFs) with coding potential for similar‐sized proteins of ≈20 kDa. These proteins were subsequently termed IncB and IncC. Sequencing of the cloned insert revealed a strong Escherichia coli‐like promoter sequence immediately upstream of incB and a 36 nt intergenic region between the ORFs. Sequence analysis of the region upstream of incB and incC revealed two ORFs that had strong homologies to an amino acid transporter and a sodium‐dependent transporter. Immunoblotting with antisera directed at IncB or IncC demonstrated that these proteins are present in C. psittaci‐infected HeLa cells but are absent or below the level of detection in purified EBs. Reverse transcriptase‐polymerase chain reactions provided evidence that incB and incC are transcribed in an operon. Immunofluorescence microscopy demonstrated that IncB and IncC are each localized to the inclusion membrane of infected cells. No primary sequence similarity is evident between IncA, IncB or IncC, but each contains a large hydrophobic domain of similar size and character as in IncA. Analysis of the recently completed C. trachomatis serovar D genome database has revealed C. trachomatis ORFs encoding homologues to incB and incC, indicating that these genes are conserved among the chlamydiae.
The pha gene cluster of Rhizobium meliloti involved in pH adaptation and symbiosis encodes a novel type of K+ efflux system
Authors: Putnoky Péter , Kereszt Attila , Nakamura Tatsunosuke , Endre Gabriella , Grosskopf Erich , Kiss Péter , Kondorosi Ádám
Published: March 2003

The fix‐2 mutant of Rhizobium meliloti affected in the invasion of alfalfa root nodules (Inf−/Fix−) is K+ sensitive and unable to adapt to alkaline pH in the presence of K+. Using directed Tn5 mutagenesis, we delimited a 6 kb genomic region in which mutations resulted in both Inf−/Fix− and K+‐sensitive phenotypes. In this DNA region, seven open reading frames (ORFs) were identified and the corresponding genes were designated phaA, B, C, D, E, F and G. The putative PhaABC proteins exhibit homology to the subunits of a Na+/H+ antiporter from an alkalophilic Bacillus strain. Moreover, PhaA and PhaD also show similarity to the ND5 and ND4 subunits of the proton‐pumping NADH:ubiquinone oxidoreductase respectively. Computer analysis suggests that all seven proteins are highly hydrophobic with several possible transmembrane domains. Some of these domains were confirmed by generating active alkaline phosphatase fusions. Ion transport studies on phaA mutant cells revealed a defect in K+ efflux at alkaline pH after the addition of a membrane‐permeable amine. These results suggest that the pha genes of R. meliloti encode for a novel type of K+ efflux system that is involved in pH adaptation and is required for the adaptation to the altered environment inside the plant.
Symbiotic implications of type III protein secretion machinery in Rhizobium
Authors: Viprey Virginie , Del Greco Aixa , Golinowski Wladyslaw , Broughton William J. , Perret Xavier
Published: March 2003

The symbiotic plasmid of Rhizobium sp. NGR234 carries a cluster of genes that encodes components of a bacterial type III secretion system (TTSS). In both animal and plant pathogens, the TTSS is an essential component of pathogenicity. Here, we show that secretion of at least two proteins (y4xL and NolX) is controlled by the TTSS of NGR234 and occurs after the induction with flavonoids. Polar mutations in two TTSS genes, rhcN and the nod‐box controlled regulator of transcription y4xI, block the secretion of both proteins and strongly affect the ability of NGR234 to nodulate a variety of tropical legumes including Pachyrhizus tuberosus and Tephrosia vogelii.
Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors
Authors: Bsat Nada , Herbig Andrew , Casillas‐Martinez Lilliam , Setlow Peter , Helmann John D.
Published: March 2003

Fur (ferric uptake regulator) proteins control iron uptake in many Gram‐negative bacteria. Although Fur homologues have been identified in Gram‐positive bacteria, their roles in gene regulation are unknown. Genome sequencing has revealed three fur homologues in Bacillus subtilis: yqkL, yqfV and ygaG. We demonstrate that yqkL encodes an iron uptake repressor: both siderophore biosynthesis and transcription of ferri‐siderophore uptake genes is constitutive in the yqkL mutant. Thus, yqkL encodes a repressor that is functionally as well as structurally related to Fur. B. subtilis peroxide stress genes are induced by either H2O2 or by metal ion limitation. Previous genetic studies defined a regulatory locus, perR, postulated to encode the peroxide regulon repressor. We demonstrate that a ygaG mutant has the perR phenotype: it is highly resistant to peroxides and overexpresses catalase, alkyl hydroperoxide reductase and the DNA binding protein MrgA. Nine spontaneous perR mutations, isolated by virtue of their ability to derepress mrgA transcription in the presence of manganous ion, all contain sequence changes in the ygaG locus and can be complemented by the cloned ygaG gene. Thus, ygaG encodes the peroxide regulon repressor and is allelic with perR.
A UDP glucosyltransferase from Bacillus subtilis successively transfers up to four glucose residues to 1,2‐diacylglycerol: expression of ypfP in Escherichia coli and structural analysis of its reaction products
Authors: Jorasch Petra , Wolter Frank P. , Zähringer Ulrich , Heinz Ernst
Published: March 2003

We have isolated the ypfP gene (accession number P54166) from genomic DNA of Bacillus subtilis Marburg strain 60015 (Freese and Fortnagel, 1967) using PCR. After cloning and expression in E. coli, SDS–PAGE showed strong expression of a protein that had the predicted size of 43.6 kDa. Chromatographic analysis of the lipids extracted from the transformed E. coli revealed several new glycolipids. These glycolipids were isolated and their structures determined by nuclear magnetic resonance (NMR) and mass spectrometry. They were identified as 3‐[O‐β‐D‐glucopyranosyl‐(1→6)‐O‐β‐D‐glucopyranosyl]‐1,2‐diacylglycerol, 3‐[O‐β‐D‐glucopyranosyl‐(1→6)‐O‐β‐D‐glucopyranosyl‐(1→6)‐O‐β‐D‐glucopyranosyl]‐1,2‐diacylglycerol and 3‐[O‐β‐D‐glucopyranosyl‐(1→6)‐O‐β‐D‐glucopyranosyl‐(1→6)‐O‐β‐D‐glucopyranosyl‐(1→6)‐O‐β‐D‐glucopyranosyl]‐1,2‐diacylglycerol. The enzymatic activity expected to catalyse the synthesis of these compounds was confirmed by in vitro assays with radioactive substrates. In these assays, one additional glycolipid was formed and tentatively identified as 3‐[O‐β‐D‐glucopyranosyl]‐1,2‐diacylglycerol, which was not detected in the lipid extract of transformed cells. Experiments with some of the above‐described glycolipids as 14C‐labelled sugar acceptors and unlabelled UDP‐glucose as glucose donor suggest that the ypfP gene codes for a new processive UDP‐glucose: 1,2‐diacylglycerol‐3‐β‐D‐glucosyl transferase. This glucosyltransferase can use diacylglycerol, monoglucosyl‐diacylglycerol, diglucosyldiacylglycerol or triglucosyldiacylglycerol as sugar acceptor, which, apart from the first member, are formed by repetitive addition of a glucopyranosyl residue in β (1→6) linkage to the product of the preceding reaction.
Overexpression of MID2 suppresses the profilin‐deficient phenotype of yeast cells
Authors: Marcoux Nathaly , Bourbonnais Yves , Charest Pierre‐Mathieu , Pallotta Dominick
Published: March 2003

Profilin‐deficient Saccharomyces cerevisiae cells show abnormal growth, actin localization, chitin deposition, bud formation and cytokinesis. Previous studies have also revealed a synthetic lethality between pfy1 and late secretory mutants, suggesting a role for profilin in intracellular transport. In this work, we document further the secretion defect associated with the pfy1Δ mutant. Electron microscopic observations reveal an accumulation of glycoproteins in the bud and in the mother cell. The MATa, pfy1Δ cells mate as well as wild‐type cells, while the mating efficiency of MATα, pfy1Δ cells is reduced. Pulse‐chase experiments demonstrate an accumulation of the 19 kDa α‐factor precursor and delayed secretion of the mature α‐factor. The TGN protein Kex2p is the principal enzyme responsible for the endoproteolytic cleavage of the α‐factor precursor. An immunofluorescence detection of Kex2p shows an altered localization in pfy1Δ cells. Instead of a discrete punctate distribution, the enzyme is dispersed throughout the cytoplasm. A high‐copy‐number plasmid containing MID2, which encodes a potential transmembrane protein involved in cell cycle control, suppresses the abnormal growth, actin distribution, α‐factor maturation and the accumulation of intracellular membranous structures in pfy1Δ cells.
Morphological adaptation and inhibition of cell division during stationary phase in Caulobacter crescentus
Authors: Wortinger Mark A. , Quardokus Ellen M. , Brun Yves V.
Published: March 2003

During exponential growth, each cell cycle of the α‐purple bacterium Caulobacter crescentus gives rise to two different cell types: a motile swarmer cell and a sessile stalked cell. When cultures of C. crescentus are grown for extended periods in complex (PYE) medium, cells undergo dramatic morphological changes and display increased resistance to stress. After cultures enter stationary phase, most cells are arrested at the predivisional stage. For the first 6–8 days after inoculation, the colony‐forming units (cfu) steadily decrease from 109 cfu ml−1 to a minimum of 3 × 107 cfu ml−1 after which cells gradually adopt an elongated helical morphology. For days 9–12, the cfu of the culture increase and stabilize around 2 × 108 cfu ml−1. The viable cells have an elongated helical morphology with no constrictions and an average length of 20 μm, which is 15–20 times longer than exponentially growing cells. The level of the cell division initiation protein FtsZ decreases during the first week in stationary phase and remains at a low constant level consistent with the lack of cell division. When resuspended in fresh medium, the elongated cells return to normal size and morphology within 12 h. Cells that have returned from stationary phase proceed through the same developmental changes when they are again grown for an extended period and have not acquired a heritable growth advantage in stationary phase (GASP) compared with overnight cultures. We conclude that the changes observed in prolonged cultures are the result of entry into a new developmental pathway and are not due to mutation.
Negative regulation by RpoS: a case of sigma factor competition
Authors: Farewell Anne , Kvint Kristian , Nyström Thomas
Published: March 2003

A mutation in the Escherichia coli gene encoding the stationary phase‐inducible sigma factor (σs, RpoS) not only abolishes transcription of some genes in stationary phase, but also causes superinduction of other stationary phase‐induced genes. We have examined this phenomenon of repression by σs using as a model system the divergently transcribed stationary phase‐inducible genes, uspA and uspB. uspA is transcribed by σ70‐programmed RNA polymerase and is superinduced in an rpoS mutant, while uspB induction is σs dependent. The data suggest that the superinduction of uspA is caused by an increased amount of σ70 bound to RNA polymerase in the absence of the competing σs. Increasing the ability of σ70 to compete against σs by overproducing σ70 mimics the effect of an rpoS mutation by causing superinduction of σ70‐dependent stationary phase‐inducible genes (uspA and fadD), silencing of σs‐dependent genes (uspB, bolAp1 and fadL) and inhibiting the development of σs‐dependent phenotypes, such as hydrogen peroxide resistance in stationary phase. In addition, overproduction of σs markedly reduced stationary phase expression of a σ70‐dependent promoter. Thus, we conclude that sigma factors compete for a limiting amount of RNA polymerase during stationary phase. The implications of this competition in the passive control of promoter activity is discussed.
Protein W, a spore‐specific protein in Myxococcus xanthus, formation of a large electron‐dense particle in a spore
Authors: Otani Mieko , Kozuka Satoshi , Xu Chunying , Umezawa Chisae , Sano Keijo , Inouye Sumiko
Published: March 2003

The gene for the major spore‐specific protein, termed protein W, was cloned, and it was found that protein W is composed of 426 amino acid residues including 31% charged (133 residues) and 39% hydrophobic (166 residues) amino acids. In the protein, a motif consisting of five amino acid residues [(V/L/I)–R–E–R–(V/L/I)] is repeated 28 times, and another motif [M–M–(P/G)–Q–G] five times. Protein W is synthesized during a very late stage of development, forming a single, large electron‐dense particle (200–400 nm in diameter) inside a spore. X‐ray microanalysis of the particle revealed that it contains a high amount of phosphate in addition to calcium and magnesium. It is proposed that protein W consisting of highly charged repetitive sequences is a polyphosphate storage protein to store energy in spores. The disruption of the gene for protein W resulted in delayed fruiting body formation and a lower spore yield.
Role of lipid‐bound peptidoglycan precursors in the formation of pores by nisin, epidermin and other lantibiotics
Authors: Brötz Heike , Josten Michaele , Wiedemann Imke , Schneider Ursula , Götz Friedrich , Bierbaum Gabriele , Sahl Hans‐Georg
Published: March 2003

It is generally assumed that type A lantibiotics primarily kill bacteria by permeabilization of the cytoplasmic membrane. As previous studies had demonstrated that nisin interacts with the membrane‐bound peptidoglycan precursors lipid I and lipid II, we presumed that this interaction could play a role in the pore formation process of lantibiotics. Using a thin‐layer chromatography system, we found that only nisin and epidermin, but not Pep5, can form a complex with [14C]‐lipid II. Lipid II was then purified from Micrococcus luteus and incorporated into carboxyfluorescein‐loaded liposomes made of phosphatidylcholine and cholesterol (1:1). Liposomes supplemented with 0.05 or 0.1 mol% of lipid II did not release any marker when treated with Pep5 or epilancin K7 (peptide concentrations of up to 5 mol% were tested). In contrast, as little as 0.01 mol% of epidermin and 0.1 mol% of nisin were sufficient to induce rapid marker release; phosphatidylglycerol‐containing liposomes were even more susceptible. Controls with moenomycin‐, undecaprenol‐ or dodecaprenolphosphate‐doped liposomes demonstrated the specificity of the lantibiotics for lipid II. These results were correlated with intact cells in an in vivo model. M. luteus and Staphylococcus simulans were depleted of lipid II by preincubation with the lipopeptide ramoplanin and then tested for pore formation. When applied in concentrations below the minimal inhibitory concentration (MIC) and up to 5–10 times the MIC, the pore formation by nisin and epidermin was blocked; at higher concentrations of the lantibiotics the protective effect of ramoplanin disappeared. These results demonstrate that, in vitro and in vivo, lipid II serves as a docking molecule for nisin and epidermin, but not for Pep5 and epilancin K7, and thereby facilitates the formation of pores in the cytoplasmic membrane.