bluuebunny/crossref_metadata_2025 huggingface dataset:
The decline of Steller sea lionsEumetopias jubatusin Alaska: a review of the nutritional stress hypothesis
Authors: TRITES A. W. , DONNELLY C. P.
Published: December 2004

1. The decline of Steller sea lionsEumetopias jubatusin the Gulf of Alaska and Aleutian Islands between the late 1970s and 1990s may have been related to reduced availability of suitable prey. Many studies have shown that pinnipeds and other mammals suffering from nutritional stress typically exhibit reduced body size, reduced productivity, high mortality of pups and juveniles, altered blood chemistry and specific behavioural modifications.

2. Morphometric measurements of Steller sea lions through the 1970s and 1980s in Alaska indicate reduced body size. Reduced numbers of pups born and an apparent increase in juvenile mortality rates also appear to be nutritionally based. Blood chemistry analyses have further shown that Steller sea lions in the Gulf of Alaska and Aleutian Islands area exhibited signs of an acute phase reaction, or immune reaction, in response to unidentified physical and/or environmental stress. Behavioural studies during the 1990s have not noted any changes that are indicative of an overall shortage in the quantity of prey available to lactating female sea lions.

3. The data collected in Alaska are consistent with the hypothesis that Steller sea lions in the declining regions were nutritionally compromised because of the relative quality of prey available to them (chronic nutritional stress), rather than because of the overall quantity of fishper se(acute nutritional stress). This is further supported by captive studies that indicate the overall quality of prey that has been available to Steller sea lions in the declining population could compromise the health of Steller sea lions and hinder their recovery.
Cryptic species, cryptic endosymbionts, and geographical variation in chemical defences in the bryozoan Bugula neritina
Authors: Mcgovern Tamara M. , Hellberg Michael E.
Published: December 2004

Molecular markers often offer the only means to discriminate between species and to elucidate the specificity of many community interactions, both of which are key to the understanding of ecological patterns. Western Atlantic populations of the bryozoan Bugula neritina vary in the palatability of their larvae to predators: individuals south of Cape Hatteras produce chemical deterrents to fish predators that are absent in more northern individuals. We use mitochondrial cytochrome oxidase c subunit I (COI) sequences to show that the differences in palatability between populations correlate with the geographical distributions of two cryptic species within B. neritina. Furthermore, these cryptic species differ in their associations with bacteria that may confer chemical resistance to predation. Small subunit rRNA primers specific to a subset of γ‐proteobacteria amplified only the bacterium Endobugula sertula from the southern cryptic species. Endobugula sertula produces a family of chemical compounds (bryostatins) that may deter predators of its animal host. In contrast, the same primers amplified an array of γ‐proteobacteria from the unprotected northern cryptic bryozoan species, but never E. sertula. In combination, these findings suggest that the geographical variation in palatability observed in the larvae of B. neritina is not the result of local adaptation of a single species to regions of differing predation pressure, but rather results from the comparison of cryptic species that differ in the presence or absence of a bacterium that may provide protection against predators. The ability to identify the cryptic Bugula species and their differing relationships with bacterial associates provides an example of the important role molecular techniques may play in addressing ecological questions.
Differential cycles of range contraction and expansion in European high mountain plants during the Late Quaternary: insights from Pritzelago alpina (L.) O. Kuntze (Brassicaceae)
Authors: Kropf Matthias , Kadereit Joachim W. , Comes Hans Peter
Published: December 2004

Nuclear DNA sequence variation of the internal transcribed spacer (ITS) and amplified fragment length polymorphisms (AFLPs) were used to illuminate the evolutionary history of Pritzelago alpina, a herbaceous perennial of (sub)alpine to nival habitats of the European high mountains. Maximum likelihood analysis of ITS sequences of P. alpina, Hornungia petraea and Hymenolobus procumbens (the ‘Pritzelago alliance’) resolved P. alpina and H. petraea as sister taxa. ITS divergence estimates support an origin for P. alpina in the Late Tertiary, while intraspecific diversification started in the Late Quaternary (0.4–0.9 million years ago). AFLP analysis of 76 individuals of P. alpina, representing 24 localities across its entire west–east distribution, identified four mountain lineages in Cantabria, the Pyrenees, (south‐) western Alps, and northeastern Alps/Tatras/Carpathians. In an analysis of molecular variance (amova), 14.3% of the total variation derived from this separation. However, relationships among these lineages remained unresolved in neighbour‐joining and principal co‐ordinates analyses, suggesting a population history of near simultaneous vicariance. Comparison with our previous ITS/AFLP study of Anthyllis montana (Fabaceae) indicates that the two co‐distributed but altitudinally differentiated plant species exhibit temporally concordant but spatially discordant patterns of genetic variation. Moreover, levels of AFLP divergence were significantly lower in P. alpina than in the submediterranean, lower‐elevation A. montana. Together, these data are consistent with a ‘displacement refugia model’, which predicts that European mountain plant species associated with lower‐ and upper‐elevation habitats had a different cycle of range contraction into (long‐term) glacial and (short‐term) interglacial refugia, respectively.
Hierarchical spatial structure of genetically variable nucleopolyhedroviruses infecting cyclic populations of western tent caterpillars
Authors: Cooper Dawn , Cory Jenny S. , Myers Judith H.
Published: December 2004

The cyclic population dynamics of western tent caterpillars, Malacosoma californicum pluviale, are associated with epizootics of a nucleopolyhedrovirus, McplNPV. Given the dynamic fluctuations in host abundance and levels of viral infection, host resistance and virus virulence might be expected to change during different phases of the cycle. As a first step in determining if McplNPV virulence and population structure change with host density, we used restriction fragment length polymorphism (RFLP) analysis to examine the genetic diversity of McplNPV infecting western tent caterpillar populations at different spatial scales. Thirteen dominant genetic variants were identified in 39 virus isolates (individual larvae) collected from field populations during one year of low host density, and another distinct variant was discovered among nine additional isolates in two subsequent years of declining host density. The distribution of these genetic variants was not random and indicated that the McplNPV population was structured at several spatial levels. A high proportion of the variation could be explained by family grouping, which suggested that isolates collected within a family were more likely to be the same than isolates compared among populations. Additionally, virus variants from within populations (sites) were more likely to be the same than isolates collected from tent caterpillar populations on different islands. This may indicate that there is limited mixing of virus among tent caterpillar families and populations when host population density is low. Thus there is potential for the virus to become locally adapted to western tent caterpillar populations in different sites. However, no dominant genotype was observed at any site. Whether and how selection acts on the genetically diverse nucleopolyhedrovirus populations as host density changes will be investigated over the next cycle of tent caterpillar populations.
Patterns of molecular evolution and diversification in a biodiversity hotspot: the California Floristic Province
Authors: Calsbeek Ryan , Thompson John N. , Richardson James E.
Published: December 2004

The California Floristic Province harbours more endemic plant and animal taxa and more identifiable subspecies than any other area of comparable size in North America. We present evidence that physical historical processes have resulted in congruent patterns of genetic diversity over the past 2–10 million years. Using a molecular clock approach we show that diversification and establishment of spatial genetic structure across six taxonomic groups coincide with the putative age of California's mountain ranges and aridification in the region. Our results demonstrate the importance of geographical barriers and climatological events to species diversification and the overall geographical structure of biodiversity. These results should facilitate conservation efforts in this biodiversity hotspot for taxa whose population genetic structure is still unknown and may suggest the potential utility of this approach in regional conservation planning efforts.
Testing Pleistocene refugia theory: phylogeographical analysis of Desmognathus wrighti, a high‐elevation salamander in the southern Appalachians
Authors: Crespi Erica J. , Rissler Leslie J. , Browne Robert A.
Published: December 2004

During the colder climates of the Pleistocene, the ranges of high‐elevation species in unglaciated areas may have expanded, leading to increased gene flow among previously isolated populations. The phylogeography of the pygmy salamander, Desmognathus wrighti, an endemic species restricted to the highest mountain peaks of the southern Appalachians, was examined to test the hypothesis that the range of D. wrighti expanded along with other codistributed taxa during the Pleistocene. Analyses of genetic variation at 14 allozymic loci and of the 12S rRNA gene in the mtDNA genome was conducted on individuals sampled from 14 population isolates throughout the range of D. wrighti. In contrast to the genetic patterns of many other high‐elevation animals and plants, genetic distances derived from both molecular markers showed significant isolation by distance and genetic structuring of populations, suggesting long‐term isolation of populations. Phylogeographical analyses revealed four genetically distinct population clusters that probably remained fragmented during the Pleistocene, although there was also evidence supporting recent gene flow among some population groups. Support for isolation by distance is rare among high‐elevation species in unglaciated areas of North and Middle America, although not uncommon among Plethodontid Salamanders, and this pattern suggests that populations of D. wrighti did not expand entirely into suitable habitat during the Pleistocene. We propose that intrinsic barriers to dispersal, such as species interactions with other southern Appalachian plethodontid salamanders, persisted during the Pleistocene to maintain the fragmented distribution of D. wrighti and allow for significant genetic divergence of populations by restricting gene flow.
Diverging patterns of mitochondrial and nuclear DNA diversity in subarctic black spruce: imprint of a founder effect associated with postglacial colonization
Authors: Gamache Isabelle , Jaramillo‐Correa Juan Pablo , Payette Serge , Bousquet Jean
Published: December 2004

High‐latitude ecotonal populations at the species margins may exhibit altered patterns of genetic diversity, resulting from more or less recent founder events and from bottleneck effects in response to climate oscillations. Patterns of genetic diversity were investigated in nine populations of the conifer black spruce (Picea mariana[Mill.] BSP.) in northwestern Québec, Canada, using seed‐dispersed mitochondrial (mt) DNA and nuclear (nc) DNA. mtDNA diversity (mitotypes) was assessed at three loci, and ncDNA diversity was estimated for nine expressed sequence tag polymorphism (ESTP) loci. Sampling included populations from the boreal forest and the southern and northern subzones of the subarctic forest‐tundra, a fire‐born ecotone. For ncDNA, populations from all three vegetation zones were highly diverse with little population differentiation (θN = 0.014); even the northernmost populations showed no loss of rare alleles. Patterns of mitotype diversity were strikingly different: within‐population diversity and population differentiation were high for boreal forest populations [expected heterozygosity per locus (HE) = 0.58 and θM = 0.529], but all subarctic populations were fixed for a single mitotype (HE = 0). This lack of variation suggests a founder event caused by long‐distance seed establishment during postglacial colonization, consistent with palaeoecological data. The estimated movement of seeds alone (effective number of migrants per generation, NmM < 2) was much restricted compared to that estimated from nuclear variants, which including pollen movement (NmN > 17). This could account for the conservation of a founder imprint in the mtDNA of subarctic black spruce. After reduction, presumably in the early Holocene, the diversity in ncDNA would have been replenished rapidly by pollen‐mediated gene flow, and maintained subsequently through vegetative layering during the current cooler period covering the last 3000 years.
Negative cross‐talk between salicylate‐ and jasmonate‐mediated pathways in the Wassilewskija ecotype of Arabidopsis thaliana
Authors: Traw M. B. , Kim J. , Enright S. , Cipollini D. F. , Bergelson J.
Published: December 2004

Plants often respond to attack by insect herbivores and necrotrophic pathogens with induction of jasmonate‐dependent resistance traits, but respond to attack by biotrophic pathogens with induction of salicylate‐dependent resistance traits. To assess the degree to which the jasmonate‐ and salicylate‐dependent pathways interact, we compared pathogenesis‐related protein activity and bacterial performance in four mutant Arabidopsis thaliana lines relative to their wild‐type backgrounds. We found that two salicylate‐dependent pathway mutants (cep1, nim1‐1) exhibited strong effects on the growth of the generalist biotrophic pathogen, Pseudomonas syringae pv. tomato, whereas two jasmonate‐dependent pathway mutants (fad3‐2fad7‐2fad8, jar1‐1) did not. Leaf peroxidase and exochitinase activity were negatively correlated with bacterial growth, whereas leaf polyphenol oxidase activity and trypsin inhibitor concentration were not. Interestingly, leaf total glucosinolate concentration was positively correlated with bacterial growth. In the same experiment, we also found that application of jasmonic acid generally increased leaf peroxidase activity and trypsin inhibitor concentration in the mutant lines. However, the cep1 mutant, shown previously to overexpress salicylic acid, exhibited no detectable biological or chemical responses to jasmonic acid, suggesting that high levels of salicylic acid may have inhibited a plant response. In a second experiment, we compared the effect of jasmonic acid and/or salicylic acid on two ecotypes of A. thaliana. Application of salicylic acid to the Wassilewskija ecotype decreased bacterial growth. However, this effect was not observed when both salicylic acid and jasmonic acid were applied, suggesting that jasmonic acid negated the beneficial effect of salicylic acid. Collectively, our results confirm that the salicylate‐dependent pathway is more important than the jasmonate‐dependent pathway in determining growth of P. syringae pv. tomato in A. thaliana, and suggest important negative interactions between these two major defensive pathways in the Wassilewskija ecotype. In contrast, the Columbia ecotype exhibited little evidence of negative interactions between the two pathways, suggesting intraspecific variability in how these pathways interact in A. thaliana.
Nucleotide diversity at two phytochrome loci along a latitudinal cline in Pinus sylvestris
Authors: García‐Gil M. R. , Mikkonen M. , Savolainen O.
Published: December 2004

Forest tree species provide many examples of well‐studied adaptive differentiation, where the search for the underlying genes might be possible. In earlier studies and in our common conditions in a greenhouse, northern populations set bud earlier than southern ones. A difference in latitude of origin of one degree corresponded to a change of 1.4 days in number of days to terminal bud set of seedlings. Earlier physiological and ecological genetics work in conifers and other plants have suggested that such variation could be governed by phytochromes. Nucleotide variation was examined at two phytochrome loci (PHYP and PHYO, homologues of the Arabidopsis thaliana PHYB and PHYA, respectively) in three populations: northern Finland, southern Finland and northern Spain. In our samples of 12–15 sequences (2980 and 1156 base pairs at the two loci) we found very low nonsynonymous variation; π was 0.0003 and 0.0002 at PHYP and PHYO loci, respectively. There was no functional differentiation between populations at the photosensory domains of either locus. The overall silent variation was also low, only 0.0024 for the PHYP locus. The low estimates of silent variation are consistent with the estimated low synonymous substitution rates between Pinus sylvestris and Picea abies at the PHYO locus. Despite the low level of nucleotide variation, haplotypic diversity was relatively high (0.42 and 0.41 for fragments of 1156 nucleotides) at the two loci.
Evidence for the adaptive evolution of the carbon fixation generbcLduring diversification in temperature tolerance of a clade of hot spring cyanobacteria
Authors: Miller S. R.
Published: December 2004

Determining the molecular basis of enzyme adaptation is central to understanding the evolution of environmental tolerance but is complicated by the fact that not all amino acid differences between ecologically divergent taxa are adaptive. Analysing patterns of nucleotide sequence evolution can potentially guide the investigation of protein adaptation by identifying candidate codon sites on which diversifying selection has been operating. Here, I test whether there is evidence for molecular adaptation of the carbon fixation generbcLfor a clade of hot spring cyanobacteria in the genusSynechococcusthat has diverged in thermotolerance. Amino acid replacements duringSynechococcusradiation have resulted in an increase in the number of hydrophobic residues in the RbcLs of more thermotolerant strains. A similar increase in hydrophobicity has been observed for many thermostable proteins. Maximum likelihood models which allow for heterogeneity among codon sites in the ratio of nonsynonymous to synonymous nucleotide substitutions estimated a class of amino acid sites as a target of positive selection. Depending on the model, a single amino acid site that interacts with a flexible element involved in the opening and closing of the active site was estimated with either low or moderate support to be a member of this class. Site‐directed mutagenesis approaches are being explored in order to directly test its adaptive significance.
Population genetics of tandem trypsin inhibitor genes in Arabidopsis species with contrasting ecology and life history
Authors: Clauss M. J. , Mitchell‐Olds T.
Published: December 2004

Duplicated genes are important in the evolution and ecology of plant‐defences because herbivore and pathogen attack can be countered via functional diversification at two levels: among duplicated loci and within loci. We explore molecular sequence variation for two members of a defence‐related gene family, Arabidopsis thaliana trypsin inhibitors (ATTI), in A. thaliana and a closely related species, A. lyrata subspp. petraea. A worldwide sample of the inbreeding annual A. thaliana had less genetic variation at two ATTI loci (πTOTAL ≤ 0.0006) than observed previously at other functional loci. A significant excess of high frequency derived alleles in the signal sequence and 5′ UTR of ATTI2 was consistent with a model of positive selection. However, demographic processes such as population subdivision and expansion, both likely to have occurred in A. thaliana during the last 10 000 years, can also give rise to similar deviations from neutrality. A single population of A. lyrata subspp. petraea in Germany had up to an order of magnitude more standing genetic variation at ATTI loci than the species‐wide sample of A. thaliana. Although the level of variability for ATTI1 and ATTI2 within this single population was similar to, or even greater than, observed species‐wide diversity for other loci in A. lyrata, there was little evidence to reject an equilibrium neutral model. A spatially explicit sample of 87 A. lyrata subspp. petraea individuals detected outbreeding (FIS = −0.16; FIT = −0.15) but little population subdivision (FST = 0.006) in this self‐incompatible perennial herb. Genetic differences between Arabidopsis species were consistent with, but not fully explained by, divergence in ecology and life history. Diversification appears to have occurred in different functional domains for the tandemly duplicated ATTI1 and ATTI2 genes; the majority of fixed replacements in ATTI1 surround the enzyme binding site of the mature protein, whereas in ATTI2 most functional evolutionary change is located in the signal peptide. This pattern is consistent with a hypothesis of subfunctionalization in trypsin inhibitory function.
The phylogeography of human viruses
Authors: Holmes Edward C.
Published: December 2004

Viruses, especially those with RNA genomes, represent ideal organisms to study the dynamics of microevolutionary change. In particular, their rapid rate of nucleotide substitution means that the epidemiological processes that shape their diversity act on the same time‐scale as mutations are fixed in viral populations. Consequently, the branching structure of virus phylogenies provides a unique insight into spatial and temporal dynamics. Herein, I describe the key processes in virus phylogeography. These are generally associated with the relative rates of dispersal among populations and virus‐host codivergence (vicariance), and the division between acute (short‐term) and persistent (long‐term) infections. These processes will be illustrated by important human viruses — HIV, dengue, rabies, polyomavirus JC and human papillomavirus – which display varying spatial and temporal structures and virus‐host relationships. Key research questions for the future will also be established.
The incomplete natural history of mitochondria
Authors: Ballard J. William O. , Whitlock Michael C.
Published: December 2004

Mitochondrial DNA (mtDNA) has been used to study molecular ecology and phylogeography for 25 years. Much important information has been gained in this way, but it is time to reflect on the biology of the mitochondrion itself and consider opportunities for evolutionary studies of the organelle itself and its ecology, biochemistry and physiology. This review has four sections. First, we review aspects of the natural history of mitochondria and their DNA to show that it is a unique molecule with specific characteristics that differ from nuclear DNA. We do not attempt to cover the plethora of differences between mitochondrial and nuclear DNA; rather we spotlight differences that can cause significant bias when inferring demographic properties of populations and/or the evolutionary history of species. We focus on recombination, effective population size and mutation rate. Second, we explore some of the difficulties in interpreting phylogeographical data from mtDNA data alone and suggest a broader use of multiple nuclear markers. We argue that mtDNA is not a sufficient marker for phylogeographical studies if the focus of the investigation is the species and not the organelle. We focus on the potential bias caused by introgression. Third, we show that it is not safe to assumea priorithat mtDNA evolves as a strictly neutral marker because both direct and indirect selection influence mitochondria. We outline some of the statistical tests of neutrality that can, and should, be applied to mtDNA sequence data prior to making any global statements concerning the history of the organism. We conclude with a critical examination of the neglected biology of mitochondria and point out several surprising gaps in the state of our knowledge about this important organelle. Here we limelight mitochondrial ecology, sexually antagonistic selection, life‐history evolution including ageing and disease, and the evolution of mitochondrial inheritance.
Spatial structure and genetic diversity of two tropical tree species with contrasting breeding systems and different ploidy levels
Authors: Ng Kevin K. S. , Lee S. L. , Koh C. L.
Published: December 2004

Analyses of the spatial distribution pattern, spatial genetic structure and of genetic diversity were carried out in two tropical tree species with contrasting breeding systems and different ploidy levels using a 50‐ha demographic plot in a lowland dipterocarp forest in Peninsular Malaysia. Shorea leprosula is a diploid and predominantly outcrossed species, whereas S. ovalis ssp. sericea is an autotetraploid species with apomictic mode of reproduction. Genetic diversity parameters estimated for S. leprosula using microsatellite were consistently higher than using allozyme. In comparisons with S. leprosula and other tropical tree species, S. ovalis ssp. sericea also displayed relatively high levels of genetic diversity. This might be explained by the lower pressure of genetic drift due to tetrasomic inheritance, and for autotetraploids each locus can accommodate up to four different alleles and this allows maintenance of more alleles at individual loci. The observed high levels of genetic diversity in S. ovalis ssp. sericea can also be due to a random retention of more heterogeneous individuals in the past, and the apomictic mode of reproduction might be an evolutionary strategy, which allows the species to maintain high levels of genetic diversity. The spatial distribution pattern analyses of both species showed significant levels of aggregation at small and medium but random distribution at the big diameter‐class. The decrease in magnitude of spatial aggregation from small‐ to large‐diameter classes might be due to compensatory mortality during recruitment and survival under competitive thinning process. Spatial genetic structure analyses for both species revealed significant spatial genetic structure for short distances in all the three diameter‐classes. The magnitude of spatial genetic structure in both species was observed to be decreasing from smaller‐ to larger‐diameter classes. The high spatial genetic structuring observed in S. ovalis ssp. sericea at the small‐diameter class is due primarily to limited seed dispersal and apomictic mode of reproduction. The similar observation in S. leprosula, however, can be explained by limited seed and pollen dispersal, which supports further the fact that the species is pollinated by weak fliers, mainly of Thrips and Megalurothrips in the lowland dipterocarp forest.
Moment estimation of population diversity and genetic distance from data on recessive markers*
Authors: Hill W. G. , Weir B. S.
Published: December 2004

A moment‐based method for estimating a measure of population diversity, θ or Wright's FST, is given for dominant markers such as amplified fragment length polymorphisms (AFLPs) or RAPDs in noninbred populations. Basic assumptions are that there is random mating, Hardy–Weinberg equilibrium, linkage equilibrium, no mutation from common ancestor and equally distant populations. It is based on the variances between and within populations of genotype frequencies, whereas previously moment methods for dominant markers have been indirect in that they have been based on first estimating allele frequencies and then using the variances of those frequencies. The use of genotype frequencies directly appears to be more robust. Approximate sampling errors of the estimates are given. Methods are extended to estimate genetic distances and their sampling errors. The AFLP data from samples of breeds of pig are used for illustration.
Two distinct regions on the surface of an RNA‐binding domain are crucial for RNase E function
Authors: Diwa Alexis A. , Jiang Xunqing , Schapira Matthieu , Belasco Joel G.
Published: December 2004

Despite its importance for RNA processing and degradation in Escherichia coli, little is known about the structure of RNase E or its mechanism of action. We have modelled the three‐dimensional structure of an essential amino‐terminal domain of RNase E on the basis of its sequence homology to the S1 family of RNA‐binding domains. Each of the five surface‐exposed aromatic residues and most of the 14 basic residues of this RNase E domain were replaced with alanine to determine their importance for RNase E function. All the surface residues essential for cell growth and feedback regulation of RNase E synthesis mapped to one end of the domain. In vitro assays indicate that these essential residues fall into two functionally distinct groups that form discrete clusters on opposite faces of the S1 domain. One group, comprising Phe‐57, Phe‐67 and Tyr‐112, is of general importance for the ribonuclease activity of RNase E, whereas the other group, comprising Lys‐37 and Tyr‐60, is entirely dispensable for catalytic activity in vitro. The side‐chains of two residues previously identified as sites of temperature‐sensitive mutations lie buried directly beneath the surface region defined by Phe‐57, Phe‐67 and Tyr‐112, which probably enhances RNase E activity by making a crucial contribution to the binding of substrate RNAs. In contrast to the S1 domain, an arginine‐rich RNA‐binding domain in the carboxyl half of RNase E appears to have a more peripheral role in RNase E function, as it is not required for feedback regulation, cell growth or ribonuclease activity.
Aminoacyl‐tRNA synthesis in archaea: different but not unique
Authors: Prætorius‐Ibba Mette , Ibba Michael
Published: December 2004

Accurate aminoacyl‐tRNA synthesis is essential for correct translation of the genetic code in all organisms. Whereas many aspects of this process are conserved, others display a surprisingly high level of divergence from the canonical Escherichia coli model system. These differences are most pronounced in archaea where novel mechanisms have recently been described for aminoacylating tRNAs with asparagine, cysteine, glutamine and lysine. Whereas these mechanisms were initially assumed to be uniquely archaeal, both the alternative asparagine and lysine pathways have subsequently been demonstrated in numerous bacteria. Similarly, studies of the means by which archaea insert the rare amino acid selenocysteine in response to UGA stop codons have helped provide a better understanding of both archaeal and eukaryal selenoprotein synthesis. Most recently a new co‐translationally inserted amino acid, pyrrolysine, has been found in archaea although again there is some suggestion that it may also be present in bacteria. Thus, whereas archaea contain a preponderance of non‐canonical aminoacyl‐tRNA synthesis systems most are also found elsewhere albeit less frequently.
Coupling gene expression and multicellular morphogenesis during fruiting body formation in Myxococcus xanthus
Authors: Søgaard‐Andersen Lotte , Overgaard Martin , Lobedanz Sune , Ellehauge Eva , Jelsbak Lars , Rasmussen Anders Aa.
Published: December 2004

A recurring theme in morphogenesis is the coupling of the expression of genes that drive morphogenesis and the morphogenetic process per se. This coupling ensures that gene expression and morphogenesis are carried out in synchrony. Morphogenesis of the spore‐filled fruiting bodies in Myxococcus xanthus illustrates this coupling in the construction of a multicellular structure. Fruiting body formation involves two stages: aggregation of cells into mounds and the position‐specific sporulation of cells that have accumulated inside mounds. Developmental gene expression propels these two processes. In addition, gene expression in individual cells is adjusted according to their spatial position. Progress in the understanding of the cell surface‐associated C‐signal is beginning to reveal the framework of an intercellular signalling system that allows the coupling of gene expression and multicellular morphogenesis. Accumulation of the C‐signal is tightly regulated and involves transcriptional activation of the csgA gene and proteolysis of the full‐length CsgA protein to produce the shorter cell surface‐associated 17 kDa C‐signal protein. The C‐signal induces aggregation, sporulation and developmental gene expression at specific thresholds. The ordered increase in C‐signalling levels, in combination with the specific thresholds, allows the C‐signal to induce these three processes in the correct temporal order. The contact‐dependent C‐signal transmission mechanism, in turn, guarantees that C‐signalling levels reflect the spatial position of individual cells relative to other cells and, thus, allows the cells to decode their spatial position during morphogenesis. By this mechanism, individual cells can tailor their gene expression profile to one that matches their spatial position. In this scheme, the molecular device that keeps gene expression in individual cells in register with morphogenesis is the C‐signalling system, and the morphological structure, which is assessed, is the spatial position of individual cells relative to that of other cells.
C‐terminal domain mutations in ClpX uncouple substrate binding from an engagement step required for unfolding
Authors: Joshi Shilpa A. , Baker Tania A. , Sauer Robert T.
Published: December 2004

ClpX mediates ATP‐dependent denaturation of specific target proteins and disassembly of protein complexes. Like other AAA + family members, ClpX contains an αβ ATPase domain and an α‐helical C‐terminal domain. ClpX proteins with mutations in the C‐terminal domain were constructed and screened for disassembly activity in vivo. Seven mutant enzymes with defective phenotypes were purified and characterized. Three of these proteins (L381K, D382K and Y385A) had low activity in disassembly or unfolding assays in vitro. In contrast to wild‐type ClpX, substrate binding to these mutants inhibited ATP hydrolysis instead of increasing it. These mutants appear to be defective in a reaction step that engages bound substrate proteins and is required both for enhancement of ATP hydrolysis and for unfolding/disassembly. Some of these side chains form part of the interface between the C‐terminal domain of one ClpX subunit and the ATPase domain of an adjacent subunit in the hexamer and appear to be required for communication between adjacent nucleotide binding sites.
Citrobacter rodentium translocated intimin receptor (Tir) is an essential virulence factor needed for actin condensation, intestinal colonization and colonic hyperplasia in mice
Authors: Deng Wanyin , Vallance Bruce A. , Li Yuling , Puente Jose L. , Finlay B. Brett
Published: December 2004

Citrobacter rodentium infection of mice serves as a relevant small animal model to study enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) infections in man. Enteropathogenic E. coli and EHEC translocate Tir into the host cytoplasmic membrane, where it serves as the receptor for the bacterial adhesin intimin and plays a central role in actin condensation beneath the adherent bacterium. In this report, we examined the function of C. rodentium Tir both in vitro and in vivo. Similar to EPEC, C. rodentium Tir is tyrosine phosphorylated and is essential for actin condensation. Citrobacter Tir and EPEC Tir are functionally interchangeable and both require tyrosine phosphorylation to mediate actin rearrangements. In contrast, Citrobacter Tir supports actin nucleation in EHEC independent of tyrosine phosphorylation, while EHEC Tir cannot replace Citrobacter Tir for this function. This indicates that C. rodentium and EPEC use an actin nucleating mechanism different from EHEC. We also found that Tir is expressed and translocated into mouse enterocytes in vivo by C. rodentium during infections. This represents the first direct demonstration of a type III effector translocated in vivo into a natural host by any pathogen. In addition, we showed that Tir, but not its tyrosine phosphorylation, is essential for C. rodentium to colonize the large bowel and induce attaching/effacing (A/E) lesions and colonic hyperplasia in mice, and that both EPEC Tir and EHEC Tir can substitute for Citrobacter Tir for these activities in vivo. These results thus demonstrate that Tir is an essential virulence factor in this infection model. The data also show that the function of Tir tyrosine phosphorylation and its subsequent actin nucleating activity are not essential for C. rodentium colonization of the mouse gut nor for inducing A/E lesions and colonic hyperplasia, thereby uncoupling colonization and disease from actin condensation for this A/E pathogen.
Global gene expression in Escherichia coli biofilms
Authors: Schembri Mark A. , Kjærgaard Kristian , Klemm Per
Published: December 2004

It is now apparent that microorganisms undergo significant changes during the transition from planktonic to biofilm growth. These changes result in phenotypic adaptations that allow the formation of highly organized and structured sessile communities, which possess enhanced resistance to antimicrobial treatments and host immune defence responses. Escherichia coli has been used as a model organism to study the mechanisms of growth within adhered communities. In this study, we use DNA microarray technology to examine the global gene expression profile of E. coli during sessile growth compared with planktonic growth. Genes encoding proteins involved in adhesion (type 1 fimbriae) and, in particular, autoaggregation (Antigen 43) were highly expressed in the adhered population in a manner that is consistent with current models of sessile community development. Several novel gene clusters were induced upon the transition to biofilm growth, and these included genes expressed under oxygen‐limiting conditions, genes encoding (putative) transport proteins, putative oxidoreductases and genes associated with enhanced heavy metal resistance. Of particular interest was the observation that many of the genes altered in expression have no current defined function. These genes, as well as those induced by stresses relevant to biofilm growth such as oxygen and nutrient limitation, may be important factors that trigger enhanced resistance mechanisms of sessile communities to antibiotics and hydrodynamic shear forces.
Phosphorylation‐mediated regulation of heat shock response in Escherichia coli
Authors: Klein Gracjana , Dartigalongue Claire , Raina Satish
Published: December 2004

Escherichia coli has two heat shock regulons under the transcriptional control of Eσ32 and EσE RNA polymerases. These polymerases control the expression of genes, the products of which are needed for correct folding of proteins in the cytoplasm and the extracytoplasm respectively. In this study, we report that mutations in a tyrosine phosphatase‐encoding gene led to decreased activity of these heat shock regulons. The activity of the tyrosine phosphatase is presumably co‐ordinated with that of a cognate kinase. We show here that mutants deleted for the phosphatase‐encoding gene accumulate phosphorylated RpoH. We find that RpoH is phosphorylated at amino acid position 260, which is located in the conserved region 4.2, and that this phosphorylation event attenuates RpoH activity as a sigma factor. The rpoH Tyr‐260Ala mutation confers a temperature‐sensitive phenotype that leads to an altered heat shock response. Additionally, we show that the antisigma factor RseA is phosphorylated at the N‐terminally located Tyr‐38 and that this phosphorylation presumably alters its binding affinity towards σE.
A plasmid‐encoded nicotinamidase (PncA) is essential for infectivity of Borrelia burgdorferi in a mammalian host
Authors: Purser Joye E. , Lawrenz Matthew B. , Caimano Melissa J. , Howell Jerrilyn K. , Radolf Justin D. , Norris Steven J.
Published: December 2004

Borrelia burgdorferi, a spirochaete that causes Lyme borreliosis, contains 21 linear and circular plasmids thought to be important for survival in mammals or ticks. Our results demonstrate that the gene BBE22 encoding a nicotinamidase is capable of replacing the requirement for the 25 kb linear plasmid lp25 during mammalian infection. Transformation of B. burgdorferi lacking lp25 with a shuttle vector containing the lp25 gene BBE22 (pBBE22) restored infectivity in mice to a level comparable to that of wild‐type Borrelia. This complementation also restored the growth and host adaptation of lp25–B. burgdorferi in dialysis membrane chambers (DMCs) implanted in rats. A single Cys to Ala conversion at the putative active site of BBE22 abrogated the ability of pBBE22 to re‐establish infectivity or growth in DMCs. Additional Salmonella typhimurium complementation studies and enzymatic analysis demonstrated that the BBE22 gene product has nicotinamidase activity and is most probably required for the biosynthesis of NAD. These results indicate that some plasmid‐encoded products fulfil physiological functions required in the enzootic cycle of pathogenic Borrelia.
A novel sRNA component of the carbon storage regulatory system of Escherichia coli
Authors: Weilbacher Thomas , Suzuki Kazushi , Dubey Ashok K. , Wang Xin , Gudapaty Seshigirao , Morozov Igor , Baker Carol S. , Georgellis Dimitris , Babitzke Paul , Romeo Tony
Published: December 2004

Small untranslated RNAs (sRNAs) perform a variety of important functions in bacteria. The 245 nucleotide sRNA of Escherichia coli, CsrC, was discovered using a genetic screen for factors that regulate glycogen biosynthesis. CsrC RNA binds multiple copies of CsrA, a protein that post‐transcriptionally regulates central carbon flux, biofilm formation and motility in E. coli. CsrC antagonizes the regulatory effects of CsrA, presumably by sequestering this protein. The discovery of CsrC is intriguing, in that a similar sRNA, CsrB, performs essentially the same function. Both sRNAs possess similar imperfect repeat sequences (18 in CsrB, nine in CsrC), primarily localized in the loops of predicted hairpins, which may serve as CsrA binding elements. Transcription of csrC increases as the culture approaches the stationary phase of growth and is indirectly activated by CsrA via the response regulator UvrY. Because CsrB and CsrC antagonize CsrA activity and depend on CsrA for their synthesis, a csrB null mutation causes a modest compensatory increase in CsrC levels and vice versa. Homologues of csrC are apparent in several Enterobacteriaceae. The regulatory and evolutionary implications of these findings are discussed.
Structure and genotypic plasticity of the Campylobacter fetus sap locus
Authors: Tu Zheng‐Chao , Wassenaar Trudy M. , Thompson Stuart A. , Blaser Martin J.
Published: December 2004

The Campylobacter fetus surface layer proteins (SLPs), encoded by five to nine sapA homologues, are major virulence factors. To characterize the sapA homologues further, a 65.9 kb C. fetus genomic region encompassing the sap locus from wild‐type strain 23D was completely sequenced and analysed; 44 predicted open reading frames (ORFs) were recognized. The 53.8 kb sap locus contained eight complete and one partial sapA homologues, varying from 2769 to 3879 bp, sharing conserved 553–2622 bp 5′ regions, with partial sharing of 5′ and 3′ non‐coding regions. All eight sapA homologues were expressed in Escherichia coli as antigenic proteins and reattached to the surface of SLP– strain 23B, indicating their conserved function. Analysis of the sap homologues indicated three phylogenetic groups. Promoter‐specific polymerase chain reactions (PCRs) and sapA homologue‐specific reverse transcription (RT)‐PCRs showed that the unique sapA promoter can potentially express all eight sapA homologues. Reciprocal DNA recombination based on the 5′ conserved regions can involve each of the eight sapA homologues, with frequencies from 10−1 to 10−3. Intragenic recombination between sapA7 and sapAp8, mediated by their conserved regions with a 10−1−10−2 frequency, allows the formation of new sap homologues. As divergent SLP C‐termini possess multiple antigenic sites, their reciprocal recombination behind the unique sap promoter leads to continuing antigenic variation.
The pH signalling transcription factor PacC controls virulence in the plant pathogen Fusarium oxysporum
Authors: Caracuel Zaira , Roncero M. Isabel G. , Espeso Eduardo A. , González‐Verdejo Clara I. , García‐Maceira Fe I. , Di Pietro Antonio
Published: December 2004

Gene expression in fungi by ambient pH is regulated via a conserved signalling cascade whose terminal component is the zinc finger transcription factor PacC/Rim1p. We have identified a pacC orthologue in the vascular wilt pathogen Fusarium oxysporum that binds the consensus 5′‐GCCAAG‐3′ sequence and is proteolytically processed in a similar way to PacC from Aspergillus nidulans. pacC transcript levels were elevated in F. oxysporum grown in alkaline conditions and almost undetectable at extreme acidic growth conditions. PacC+/– loss‐of‐function mutants displayed an acidity‐mimicking phenotype resulting in poor growth at alkaline pH, increased acid protease activity and higher transcript levels of acid‐expressed polygalacturonase genes. Reintroduction of a functional pacC copy into a pacC+/– mutant restored the  wild‐type phenotype. Conversely, F. oxysporum merodiploids carrying a dominant activating pacCc allele had increased pacC transcript and protein levels and displayed an alkalinity‐mimicking phenotype with reduced acid phosphatase and increased alkaline protease activities. PacC+/– mutants were more virulent than the wild‐type strain in root infection assays with tomato plants, whereas pacCc strains were significantly reduced in virulence. We propose that F. oxysporum PacC acts as a negative regulator of virulence to plants, possibly by preventing transcription of acid‐expressed genes important for infection.
Regulatory and functional co‐operation of flagella and type 1 pili in adhesive and invasive abilities of AIEC strain LF82 isolated from a patient with Crohn's disease
Authors: Barnich Nicolas , Boudeau Jérôme , Claret Laurent , Darfeuille‐Michaud Arlette
Published: December 2004

Genetic determinants that co‐operate with type 1 pili to mediate invasion were sought for in adherent‐invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease. Two mutants selected for their impaired ability to invade epithelial cells carried insertions of a TnphoA transposon within genes of the flagellar regulon. An isogenic mutant LF82‐ΔfliC deleted for the flagellin‐encoding gene did not adhere, did not invade and, surprisingly, expressed only a few type 1 pili. Type 1 pili downregulation resulted from a preferential switch towards the off‐position of the invertible DNA element located upstream of the fim operon. This was also correlated with a decrease in the flagellar regulator flhDC mRNA levels, suggesting that the transcriptional regulator FlhD2C2 could control type 1 pili expression directly or indirectly. Transformation with a cloned fim operon allowed bypass of the type 1 pili downexpression in the LF82‐ΔfliC mutant. Thus, we showed that flagella play a direct role in the adhesion process via active motility. In addition to downregulating type 1 pili expression, flagella also play an undefined role in strain LF82 invasion, which is not restricted to motility or flagellar structure, but could be related to co‐ordinate expression of invasive determinants.