bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogeography and population structure of an ecotonal marsupial,Bettongia tropica, determined using mtDNA and microsatellites
Authors: Pope L. C. , Estoup A. , Moritz C.
Published: March 2003

The northern bettong,Bettongia tropica, is an endangered species of Potoroidae with a restricted distribution in the wet tropics of north Queensland, Australia. The species is only found within a thin strip of sclerophyll forest along the western margin of rainforest. This tight association with rainforest boundaries is predicted to have resulted in population isolation as rainforest contracted during the Pleistocene, though some have proposed that the northern bettong was not present in the wet tropics until the late Pleistocene. The dispersal ability of the species, and of the family, is not known. This study examined gene flow among populations within areas of continuous habitat complemented by a broader analysis of phylogeography. Individuals trapped at each of the four known regions (one region was subsampled at three different sites), were sequenced for 547 base pairs of the mitochondrial DNA (mtDNA) control region and typed for seven microsatellite loci. The mtDNA phylogeny showed congruence with a biogeographical hypothesis, a relatively deep split suggesting historical isolation in separate northern and southern refugia. The two divergent clades were both present within the Lamb Range, indicating an expansion from these refuges and subsequent admixture at one site. mtDNA allele frequencies indicated relatively limited gene flow within the Lamb Range over distances as short as nine km. Tests of population divergence using microsatellites (FSTand assignment tests) strongly supported this result. A molecular signal indicative of a recent bottleneck was unexpectedly detected in one of the Lamb Range subpopulations. This lead us to examine the behaviour of the statistics used in this bottleneck test under a linear stepping‐stone model with varying migration rates. We found that it may be more difficult to detect molecular signatures for recent bottlenecks under conditions of very low migration rates than for isolated populations and, conversely, that ‘false’ bottleneck signatures may be observed at higher migration rates. The Lamb RangeFSTestimate clearly fell within the category of potentially ‘false’ bottleneck signals. Despite relatively limited gene flow, evidence for asymmetric dispersal suggests more complicated population dynamics than a simple linear stepping‐stone model.
Colonization, extinction, and phylogeographic patterning in a freshwater crustacean
Authors: Cox Andrea J. , Hebert Paul D. N.
Published: March 2003

Phylogeographic analyses have revealed the importance of Pleistocene vicariance events in shaping the distribution of genetic diversity in freshwater fishes. However, few studies have examined the patterning of variation in freshwater organisms with differing dispersal syndromes and life histories. The present investigation addresses this gap, examining the phylogeography of Sida crystallina, a species whose production of diapausing eggs capable of passive dispersal was thought to constrain its regional genetic differentiation. By contrast, the present analysis has revealed deep allozyme and cytochrome oxidase I mitochondrial DNA divergence between populations from North America and Europe. Moreover, North American populations are separated into four allopatric assemblages, whose distribution suggests their derivation from different Pleistocene refugia. These lineages show higher haplotype diversity and deeper sequence divergence than those of any fish from temperate North America. Its distinctive life history traits have evidently sheltered lineages of Sida from extinction, contributing to a remarkably comprehensive and high resolution phylogeographic record.
Molecular ecology of social behaviour: analyses of breeding systems and genetic structure
Authors: Ross Kenneth G.
Published: March 2003

Molecular genetic studies of group kin composition and local genetic structure in social organisms are becoming increasingly common. A conceptual and mathematical framework that links attributes of the breeding system to group composition and genetic structure is presented here, and recent empirical studies are reviewed in the context of this framework. Breeding system properties, including the number of breeders in a social group, their genetic relatedness, and skew in their parentage, determine group composition and the distribution of genetic variation within and between social units. This group genetic structure in turn influences the opportunities for conflict and cooperation to evolve within groups and for selection to occur among groups or clusters of groups. Thus, molecular studies of social groups provide the starting point for analyses of the selective forces involved in social evolution, as well as for analyses of other fundamental evolutionary problems related to sex allocation, reproductive skew, life history evolution, and the nature of selection in hierarchically structured populations. The framework presented here provides a standard system for interpreting and integrating genetic and natural history data from social organisms for application to a broad range of evolutionary questions.
Using phylogeographic analyses of gene trees to test species status and processes
Authors: Templeton Alan R.
Published: March 2003

A gene tree is an evolutionary reconstruction of the genealogical history of the genetic variation found in a sample of homologous genes or DNA regions that have experienced little or no recombination. Gene trees have the potential of straddling the interface between intra‐ and interspecific evolution. It is precisely at this interface that the process of speciation occurs, and gene trees can therefore be used as a powerful tool to probe this interface. One application is to infer species status. The cohesion species is defined as an evolutionary lineage or set of lineages with genetic exchangeability and/or ecological interchangeability. This species concept can be phrased in terms of null hypotheses that can be tested rigorously and objectively by using gene trees. First, an overlay of geography upon the gene tree is used to test the null hypothesis that the sample is from a single evolutionary lineage. This phase of testing can indicate that the sampled organisms are indeed from a single lineage and therefore a single cohesion species. In other cases, this null hypothesis is not rejected due to a lack of power or inadequate sampling. Alternatively, this null hypothesis can be rejected because two or more lineages are in the sample. The test can identify lineages even when hybridization and lineage sorting occur. Only when this null hypothesis is rejected is there the potential for more than one cohesion species. Although all cohesion species are evolutionary lineages, not all evolutionary lineages are cohesion species. Therefore, if the first null hypothesis is rejected, a second null hypothesis is tested that all lineages are genetically exchangeable and/or ecologically interchangeable. This second test is accomplished by direct contrasts of previously identified lineages or by overlaying reproductive and/or ecological data upon the gene tree and testing for significant transitions that are concordant with the previously identified lineages. Only when this second null hypothesis is rejected is a lineage elevated to the status of cohesion species. By using gene trees in this manner, species can be identified with objective, a priori criteria with an inference procedure that automatically yields much insight into the process of speciation. When one or more of the null hypotheses cannot be rejected, this procedure also provides specific guidance for future work that will be needed to judge species status.
Mitochondrial DNA variation and GIS analysis confirm a secondary origin of geographical variation in the bushcricketEphippiger ephippiger(Orthoptera: Tettigonioidea), and resurrect two subspecies
Authors: Ritchie Michael G. , Kidd David M. , Gleason Jennifer M.
Published: March 2003

Geographic variation within species can originate through selection and driftin situ(primary variation) or from vicariant episodes (secondary variation). Most patterns of subspecific variation within European flora and fauna are thought to have secondary origins, reflecting isolation in refugia during Quaternary ice ages. The bushcricketEphippiger ephippigerhas an unusual pattern of geographical variability in morphology, behaviour and allozymes in southern France, which has been interpreted as reflecting recent primary origins rather than historical isolation. Re‐analysis of this variation using Geographical Information Systems (GIS) suggests a possible zone of hybridization within a complex pattern of geographical variation. Here we produce a genetic distance matrix from restriction fragment length polymorphism (RFLP) bandsharing of an approximately 4.5 kb fragment of mitochondrial DNA (mtDNA), and compare this with predictions resulting from the GIS analysis. The mtDNA variation supports a postglacial origin of geographical variation. Partial Mantel test comparisons of genetic distances with matrices of geographical distance, relevant environmental characteristics and possible refugia show refugia to be the best predictors of genetic distance. There is no evidence to support isolation by distance. However, environmental contrasts do explain significant variation in genetic distance after allowing for the effect of refugial origin. Also, a neighbour‐joining tree has a major division separating eastern and western forms. We conclude that the major source of variation within the species is historical isolation in glacial refugia, but that dispersal, hybridization and selection associated with environmental features has influenced patterns of mtDNA introgression. At least two valid subspecies can be defined.
Temporal and spatial dynamics of a parapatric boundary between two Australian reptile ticks
Authors: Bull C. Michael , Burzacott Dale
Published: March 2003

Two tick species Aponomma hydrosauri and Amblyomma limbatum that infest large reptiles have an abrupt parapatric boundary near Mt Mary in South Australia. A previous model has suggested that the boundary is maintained at population density troughs resulting from habitat heterogeneity along a gradual environmental gradient. This paper describes the dynamics of the boundary on three transects over 17 years, 1982–98. Over the last seven years of that period there has been a significant increase in rainfall. At the same time, the boundary position has moved 1–2 km on the transects, with the more mesic adapted Ap. hydrosauri advancing into the distribution of the more xeric adapted Amb. limbatum. Also over the same time the density of ticks on lizards in regions flanking the boundary zone has increased for Ap. hydrosauri and decreased for Amb. limbatum. These results suggest that the environmental gradient has been altered, perhaps by increased rainfall, to favour Ap. hydrosauri, which has been able to colonize more successfully across the density troughs and extend its distribution.
Population subdivision in marine environments: the contributions of biogeography, geographical distance and discontinuous habitat to genetic differentiation in a blennioid fish, Axoclinus nigricaudus
Authors: Riginos C. , Nachman M. W.
Published: March 2003

The relative importance of factors that may promote genetic differentiation in marine organisms is largely unknown. Here, contributions to population structure from a biogeographic boundary, geographical distance and the distribution of suitable habitat were investigated in Axoclinus nigricaudus, a small subtidal rock‐reef fish, throughout its range in the Gulf of California. A 408‐bp fragment of the mitochondrial control region was sequenced from 105 individuals. Variation was significantly partitioned between 28 of 36 possible combinations of population pairs. Phylogenetic analyses, hierarchical analyses of variance and a modified Mantel test substantiated a major break between two putative biogeographic regions. This genetic discontinuity coincides with an abrupt change in ecological characteristics, including temperature and salinity, but does not coincide with known oceanographic circulation patterns or any known historic barriers. There was an overall relationship of increasing genetic distance with increasing geographical distance between population pairs, in a manner consistent with isolation‐by‐distance. A significant habitat‐by‐geographical‐distance interaction term indicated that, for a given geographical distance, populations separated by discontinuous habitat (sand) are more distinct genetically than are populations separated by continuous habitat (rock). In addition, populations separated by deep open waters were more genetically distinct than populations separated by continuous habitat (rock). These results indicate that levels of genetic differentiation among populations of A. nigricaudus cannot be explained by a single factor, but are due to the combined influences of biogeography, geographical distance and availability of suitable habitat.
Comparative phylogeography of the two pink salmon broodlines: an analysis based on a mitochondrial DNA genealogy
Authors: Churikov D. , Gharrett A. J.
Published: March 2003

Over most of their natural northern Pacific Ocean range, pink salmon (Oncorhynchus gorbuscha) spawn in a habitat that was repeatedly and profoundly affected by Pleistocene glacial advances. A strictly two‐year life cycle of pink salmon has resulted in two reproductively isolated broodlines, which spawn in alternating years and evolved as temporal replicates of the same species. To study the influence of historical events on phylogeographical and population genetic structure of the two broodlines, we first reconstructed a fine‐scale mtDNA haplotype genealogy from a sample of 80 individuals and then determined the geographical distribution of the major genealogical assemblages for 718 individuals sampled from nine Alaskan and eastern Asian even‐ and nine odd‐year pink salmon populations. Analysis of restriction site states in seven polymerase chain reaction (PCR)‐amplified mtDNA regions (comprising 97% of the mitochondrial genome) using 13 endonucleases resolved 38 haplotypes, which clustered into five genealogical lineages that differed from 0.065 to 0.225% in net sequence divergence. The lineage sorting between broodlines was incomplete, which suggests a recent common ancestry. Within each lineage, haplotypes exhibited star‐like genealogies indicating recent population growth. The depth of the haplotype genealogy is shallow (∼0.5% of nucleotide sequence divergence) and probably reflects repeated decreases in population size due to Pleistocene glacial advances. Nested clade analysis (NCA) of geographical distances showed that the geographical distribution observed for mitochondrial DNA (mtDNA) haplotypes resulted from alternating influences of historical range expansions and episodes of restricted dispersal. Analyses of molecular variance showed weak geographical structuring of mtDNA variation, except for the strong subdivision between Asian and Alaskan populations within the even‐year broodline. The genetic similarities observed among and within geographical regions probably originated from postglacial recolonizations from common sources rather than extensive gene flow. The phylogeographical and population genetic structures differ substantally between broodlines. This can be explained by stochastic lineage sorting in glacial refugia and perhaps different recolonization routes in even‐ and odd‐year broodlines.
An exceptional case of historical outbreeding in African sable antelope populations
Authors: Pitra Christian , Hansen Anders J. , Lieckfeldt Dietmar , Arctander Peter
Published: March 2003

Empirical investigations of intraspecific outbreeding and subsequent introgressive hybridization in natural populations are rare, particularly among conspecific populations of large mammals. Using mitochondrial DNA data [partial control region (496 basepairs — bp) and cytochrome b gene (343 bp) sequences analysed from 95 individuals representing 17 sampling locations scattered through the African miombo (Brachystegia) woodland ecosystem] and phylogeographical statistical procedures (gene genealogy, nested cladistic and admixture proportion analyses), we (i) give a detailed dissection of the geographical genetic structure of Hippotragus niger; (ii) infer the processes and events potentially involved in the population history; and (iii) trace extensive introgressive hybridization in the species. The present‐day sable antelope population shows a tripartite pattern of genetic subdivision representing West Tanzanian, Kenya/East Tanzanian and Southern Africa locations. Nested clade analysis revealed that past allopatric fragmentation, caused probably by habitat discontinuities associated with the East African Rift Valley system, together with intermediary episodic long‐distance colonization and restricted, recurrent gene flow have played an predominant role in shaping the extent of maternal genetic diversity (10.4%) and population structure. An extensive (average rate of admixture = 20.0%), but geographically circumscribed and unidirectional hybridization event in the past was inferred, resulting in an extreme (the highest discovered so far in mammals) intraspecific difference of 18.2% among morphologically monotypic sable antelopes from West Tanzania. The results are used to provide an evolutionary framework within which taxonomic implications and conservation decisions can be evaluated.
Exploring the potential of life‐history key innovation: brook breeding in the radiation of the Malagasy treefrog genus Boophis
Authors: Vences M. , Andreone F. , Glaw F. , Kosuch J. , Meyer A. , Schaefer H.‐C. , Veith M.
Published: March 2003

The treefrog genus Boophis is one of the most species‐rich endemic amphibian groups of Madagascar. It consists of species specialized to breeding in brooks (48 species) and ponds (10 species). We reconstructed the phylogeny of Boophis using 16S ribosomal DNA sequences (558 bp) from 27 species. Brook‐breeders were monophyletic and probably derived from an ancestral pond‐breeding lineage. Pond‐breeders were paraphyletic. The disparity in diversification among pond‐breeders and brook‐breeders was notable among endemic Malagasy frogs, although it was not significant when considering Boophis alone. Sibling species which have different advertisement calls but are virtually indistinguishable by morphology were common among brook‐breeders; genetic divergence between these species was high (modal 8% total pairwise divergence). Substitution rates in brook‐breeding species were significantly higher than in pond‐breeders. Speciation of pond‐breeders may be hindered by their usually more synchronous reproduction and a higher vagility which enhances gene flow, while a higher potential of spatial segregation and speciation may exist along brooks.
Natural selection and the genetic differentiation of coastal and Arctic populations of the Atlantic cod in northern Norway: a test involving nucleotide sequence variation at the pantophysin (PanI) locus
Authors: Pogson Grant H. , Fevolden Svein‐Erik
Published: March 2003

To examine the role of contemporary selection in maintaining significant allele frequency differences at the pantophysin (PanI) locus among populations of the Atlantic cod,Gadus morhua, in northern Norway, we sequenced 127PanIAalleles sampled from six coastal and two Barents Sea populations. The distributions of variable sites segregating within thePanIAallelic class were then compared among the populations. Significant differences were detected in the overall frequencies ofPanIAalleles among populations within coastal and Arctic regions that was similar in magnitude to heterogeneity in the distributions of polymorphic sites segregating within thePanIAallelic class. The differentiation observed at silent sites in thePanIAallelic class contradicts the predicted effects of widescale gene flow and suggests that postsettlement selection acting on cohorts cannot be responsible for the genetic differences described between coastal and Arctic populations. Our results suggest that the marked differences observed between coastal and Arctic populations ofG. morhuain northern Norway at thePanI locus reflect the action of recent diversifying selection and that populations throughout the region may be more independent than suggested by previous studies.
DNA‐binding characteristics of the Escherichia coli CytR regulator: a relaxed spacing requirement between operator half‐sites is provided by a flexible, unstructured interdomain linker
Authors: Jørgensen C. I. , Kallipolitis B. H. , Valentin‐Hansen P.
Published: March 2003

The Escherichia coli CytR regulator belongs to the LacI family of sequence‐specific DNA‐binding proteins and prevents CRP‐mediated transcription in the CytR regulon. Unlike the other members of this protein family, CytR binds with only modest affinity to its operators and transcription repression thus relies on the formation of nucleoprotein complexes with the cAMP–CRP complex. Moreover, CytR exhibits a rotational and translational flexibility in operator binding that is unprecedented in the LacI family. In this report we examined the effect of changing the spacing between CytR half‐operators on CytR regulation in vivo and on CytR binding in vitro. Maximum repression was seen with the short spacing variants: repression peaks when the half‐operators lie on the same face of the DNA helix. Repression was retained for most spacing variants with centre separations of half‐operators ≤ 3 helical turns. Our data confirm and extend the view that CytR is a highly flexible DNA binder that can adapt many different conformations for co‐operative binding with CRP. Furthermore, limited proteolysis of radiolabelled CytR protein showed that the interdomain linker connecting the DNA binding domains and the core part of CytR does not become structured upon DNA binding. We conclude that CytR does not use hinge α‐helices for minor groove recognition. Rather, CytR possesses a highly flexible interdomain linker that allows it to form complexes with CRP at promoters with quite different architecture.
Regulation of expression of ribosomal protein L‐21 genes of Entamoeba histolytica and E. dispar is at the post‐transcriptional level
Authors: Moshitch‐Moshkovitch S. , Petter R. , Levitan A. , Stolarsky T. , Mirelman D.
Published: March 2003

Two genes, EhgLE3 and Ehg34, encoding the ribosomal protein L21 (rp‐L21) were identified and characterized from Entamoeba histolytica. Their coding regions are highly conserved, but their flanking regions differ significantly. Analogous genes (EdgLE3 and Edg34) were characterized in E. dispar. The two rp‐L21 copies are transcribed at similar levels in the two parasites. However, their relative binding to the polyribosomal complex during active translation is different. In E. histolytica, binding of EhgLE3 transcripts to the polyribosomes is significantly higher in comparison with that of Ehg34 transcripts, whereas in E. disparthe binding pattern is inverse. The importance of each of the rp‐L21 flanking regions to gene translation was investigated by constructing hybrid plasmids containing the CAT reporter gene flanked by rp‐L21 flanking regions. The plasmids were stably transfected into E. histolytica and E. dispar, and CAT mRNA and enzymatic activity levels were determined. All plasmids promoted transcription of CAT. Yet, in E. histolytica, high levels of CAT activity were observed only when gLE3 upstream regions flanked CAT. In contrast, in E. dispar, high levels of CAT activity were observed when g34 upstream regions flanked CAT. The downstream regions showed no significant effect on CAT translation.
Targeted gene deletion of Leishmania major genes encoding developmental stage‐specific leishmanolysin (GP63)
Authors: Joshi Phalgun B. , Sacks David L. , Modi Govind , McMaster W. Robert
Published: March 2003

The major surface glycoprotein of Leishmania major is a zinc metalloproteinase of 63 kDa referred to as leishmanolysin or GP63, which is encoded by a family of seven genes. Targeted gene replacement was used to delete gp63 genes 1–6 encoding the highly expressed promastigote and constitutively expressed GP63. In the L. major homozygous mutants deficient in gp63 genes 1–6, there was no expression of GP63 as detected by reverse transcription–polymerase chain reaction (RT–PCR) or fluorescent staining in promastigotes from the procyclic stage (logarithmic growth phase). The remaining L. major gP63 gene 7 was shown to be developmentally regulated, as it was expressed exclusively in infectious metacyclic stage (late stationary growth phase) promastigotes and in lesion amastigotes. The gp63 genes 1–6‐deficient mutants showed increased sensitivity to complement‐mediated lysis. The sensitivity to lysis was greater in procyclics than in metacyclics when compared with the equivalent wild‐type stages. Increased resistance of the mutant metacyclic promastigotes correlated with the expression of gp63 gene 7 and was restored to the same levels as wild‐type promastigotes by transfection with gp63 gene 1. Thus, expression of GP63 is clearly involved in conferring resistance to complement‐mediated lysis. The L. major GP63 1–6 mutants were capable of infecting mouse macrophages and differentiating into amastigotes. Similar levels of infection and subsequent intracellular survival were observed when mouse macrophages were infected in vitro with wild type, GP63 1–6 mutants and mutants transfected with gp63 gene 1. The GP63 1–6 mutants were capable of lesion formation in BALB/c mice and, thus, gp63 genes 1–6 do not play a role in the survival of the parasite within mouse macrophages. The role of gp63 genes 1–6 in parasite development within the sandfly vector was studied. GP63 1–6 mutants grew normally in the blood‐engorged midgut of both Phlebotomus argentipes and P. papatasi. However, both wild‐type and mutant promastigotes were lost after 2 days' growth in P. papatasi. The complete developmental pathway in P. argentipes was observed for wild‐type promastigotes, GP63 1–6 mutants and mutants transfected with gp63 gene 1. Normal stage differentiation from amastigotes to procyclics, to nectomonads, to haptomonads and to infectious metacyclics was observed. Thus, the highly expressed promastigote forms of GP63, encoded by gp63 genes 1–6, do not appear to be required for nutrient utilization in the bloodmeal during the early stages of development in the sandfly or for midgut attachment and further development. gp63 1–6 genes do, however, play a major protective role against complement‐mediated lysis when promastigotes are introduced into the mammalian host.
Genetic analysis of the stationary phase‐induced mcb operon promoter in Escherichia coli
Authors: Mao Weimin , Siegele Deborah A.
Published: March 2003

A combination of deletion analysis and random mutagenesis was used to identify regulatory elements in Pmcb, the stationary phase‐induced promoter of the mcb operon. Our results indicate that Pmcb is controlled by at least three different factors, two previously identified and at least one unknown factor, which act at four different sites in the promoter. Sequences between −344 and −164 upstream of the transcriptional start site were required for wild‐type levels of mcb transcription in stationary phase. More dramatic reductions in both exponential and stationary phase expression were observed when sequences from −164 to −54 were deleted. Point mutations located between −105 and −138 decreased both exponential and stationary phase expression. All but one of these mutations decreased OmpR‐dependent activation of Pmcb transcription. EmrR, also known as MprA, acts directly or indirectly at sequences downstream of −54 to repress Pmcb. A minimal promoter containing sequences from −34 to +79 was still induced ≥10‐fold in stationary phase. Point mutations within this region identified sequences at −8, −11, −30, −31 and −32 as important for Pmcb activity. These bases are in the gearbox sequence, present in Pmcb and several other stationary phase‐induced Escherichia coli promoters.
Consequences of the loss of O‐linked glycosylation of meningococcal type IV pilin on piliation and pilus‐mediated adhesion
Authors: Marceau Michael , Forest Katrina , Béretti Jean‐Luc , Tainer John , Nassif Xavier
Published: March 2003

Pili, which are assembled from protein subunits called pilin, are indispensable for the adhesion of capsulated Neisseria meningitidis (MC) to eukaryotic cells. Both MC and Neisseria gonorrhoeae (GC) pilins are glycosylated, but the effect of this modification is unknown. In GC, a galactose α‐1,3‐N‐acetyl glucosamine is O‐linked to Ser‐63, whereas in MC, an O‐linked trisaccharide is present between residues 45 and 73 of pilin. As Ser‐63 was found to be conserved in pilin variants from different strains, it was replaced by Ala in two MC variants to test the possible role of this residue in pilin glycosylation and modulation of pili function. The mutated alleles were stably expressed in MC, and the proteins they encoded migrated more quickly than the normal protein during SDS–PAGE. As controls, neighbouring Asn‐61 and Ser‐62 were replaced by an Ala with no effect on electrophoretic mobility. Silver staining of purified pilin obtained from MC after oxidation with periodic acid confirmed the loss of glycosylation in the Ser‐63→Ala pilin variants. Mass spectrometry of HPLC‐purified trypsin‐digested peptides of pilin and Ser‐63→Ala pilin confirmed that peptide 45–73 has the molecular size of a glycopeptide in the wild type. In strains producing non‐glycosylated pilin variants, we observed that (i) no truncated S pilin monomer was produced; (ii) piliation was slightly increased; and (iii) presumably as a consequence, adhesiveness for epithelial cells was increased 1.6‐ to twofold in these derivatives. In addition, pilin monomers and/or individual pilus fibres, obtained after solubilization of a crude pili preparation in a high pH buffer, were reassociated into insoluble aggregates of pili more completely with non‐glycosylated variants than with the normal pilin. Taken together, these data eliminate a major role for pilin glycosylation in piliation and subsequent pilus‐mediated adhesion, but they demonstrate that glycosylation facilitates solubilization of pilin monomers and/or individual pilus fibres.
Control of expression of LlaI restriction in Lactococcus lactis
Authors: O'sullivan Daniel J. , Klaenhammer T. R.
Published: March 2003

The plasmid encoded LlaI R/M system from Lactococcus lactis ssp. lactis consists of a bidomain methylase, with close evolutionary ties to type IIS methylases, and a trisubunit restriction complex. Both the methylase and restriction subunits are encoded on a polycistronic 6.9 kb operon. In this study, the 5′ end of the llaI 6.9 kb transcript was determined by primer extension analysis to be 254 bp upstream from the first R/M gene on the operon, llaIM. Deletion of this promoter region abolished LlaI restriction in L. lactis. Analysis of the intervening sequence revealed a 72‐amino‐acid open reading frame, designated llaIC, with a conserved ribosome binding site and helix–turn–helix domain. Overexpression of llaIC in Escherichia coli with a T7 expression vector produced the predicted protein of 8.2 kDa. Mutation and in trans complementation analyses indicated that C·LlaI positively enhanced LlaI restriction activity in vivo. Northern analysis and transcriptional fusions of the llaI promoter to a lacZ reporter gene indicated that C·LlaI did not enhance transcription of the llaI operon. Databank searches with the deduced protein sequence for llaIC revealed significant homologies to the E. coli Rop regulatory and mRNA stabilizer protein. Investigation of the effect of C·LlaI on enhancement of LlaI restriction in L. lactis revealed that growth at elevated temperatures (40°C) completely abolished any enhancement of restriction activity. These data provide molecular evidence for a mechanism on how the expression of a restriction system in a prokaryote can be drastically reduced during elevated growth temperatures, by a small regulatory protein.
PmrA–PmrB‐regulated genes necessary for 4‐aminoarabinose lipid A modification and polymyxin resistance
Authors: Gunn John S. , Lim Kheng B. , Krueger Jackie , Kim Kevin , Guo Lin , Hackett Murray , Miller Samuel I.
Published: March 2003

Antimicrobial peptides are distributed throughout the animal kingdom and are a key component of innate immunity. Salmonella typhimurium regulates mechanisms of resistance to cationic antimicrobial peptides through the two‐component systems PhoP–PhoQ and PmrA–PmrB. Polymyxin resistance is encoded by the PmrA–PmrB regulon, whose products modify the lipopolysaccharide (LPS) core and lipid A regions with ethanolamine and add aminoarabinose to the 4′ phosphate of lipid A. Two PmrA–PmrB‐regulated S. typhimurium loci (pmrE and pmrF ) have been identified that are necessary for resistance to polymyxin and for the addition of aminoarabinose to lipid A. One locus, pmrE, contains a single gene previously identified as pagA (or ugd ) that is predicted to encode a UDP‐glucose dehydrogenase. The second locus, pmrF, is the second gene of a putative operon predicted to encode seven proteins, some with similarity to glycosyltransferases and other complex carbohydrate biosynthetic enzymes. Genes immediately flanking this putative operon are also regulated by PmrA–PmrB and/or have been associated with S. typhimurium polymyxin resistance. This work represents the first identification of non‐regulatory genes necessary for modification of lipid A and subsequent antimicrobial peptide resistance, and provides support for the hypothesis that lipid A aminoarabinose modification promotes resistance to cationic antimicrobial peptides.
Reconstruction of the proteolytic pathway for use of β‐casein by Lactococcus lactis
Authors: Kunji Edmund R. S. , Fang Gang , Jeronimus‐Stratingh C. Margot , Bruins Andries P. , Poolman Bert , Konings Wil N.
Published: March 2003

Amino acid auxotrophous bacteria such as Lactococcus lactis use proteins as a source of amino acids. For this process, they possess a complex proteolytic system to degrade the protein(s) and to transport the degradation products into the cell. We have been able to dissect the various steps of the pathway by deleting one or more genes encoding key enzymes/components of the system and using mass spectrometry to analyse the complex peptide mixtures. This approach revealed in detail how L. lactis liberates the required amino acids from β‐casein, the major component of the lactococcal diet. Mutants containing the extracellular proteinase PrtP, but lacking the oligopeptide transport system Opp and the autolysin AcmA, were used to determine the proteinase specificity in vivo. To identify the substrates of Opp present in the casein hydrolysate, the PrtP‐generated peptide pool was offered to mutants lacking the proteinase, but containing Opp, and the disappearance of peptides from the medium as well as the intracellular accumulation of amino acids and peptides was monitored in peptidase‐proficient and fivefold peptidase‐deficient genetic backgrounds. The results are unambiguous and firmly establish that (i) the carboxyl‐terminal end of β‐casein is degraded preferentially despite the broad specificity of the proteinase; (ii) peptides smaller than five residues are not formed in vivo ; (iii) use of oligopeptides of 5–10 residues becomes only possible after uptake via Opp; (iv) only a few (10–14) of the peptides generated by PrtP are actually used, even though the system facilitates the transport of oligopeptides up to at least 10 residues. The technology described here allows us to monitor the fate of individual peptides in complex mixtures and is applicable to other proteolytic systems.
Replication strand preference for deletions associated with DNA palindromes
Authors: Pinder David J. , Blake Catherine E. , Lindsey Janet C. , Leach David R. F.
Published: March 2003

We have isolated and sequenced a set of deletions stimulated by DNA palindromes in Escherichia coli. All of the deletions are asymmetric with respect to the parental sequence and have occurred at short direct repeats. This is consistent with deletion by strand slippage during DNA replication. The orientation of the asymmetry in such deletion products is diagnostic of the direction of the strand slippage event. It is therefore also diagnostic of its occurrence on the leading or lagging strand of the replication fork when the direction of replication is known. In all cases in which the orientation of the asymmetry could be determined with respect to DNA replication, the products were consistent with a preference for deletion on the lagging strand of the fork. The data include replication slippage in three situations: on the chromosome of E. coli, in bacteriophage λ and in high‐copy‐number pUC‐based plasmids.
Variations on a molecular switch: transport and sensory signalling by archaeal rhodopsins
Authors: Spudich John L.
Published: March 2003

The archaeal rhodopsins are a family of seven‐transmembrane‐helix, visual pigment‐like proteins found in Halobacterium salinarum and related halophilic Archaea. Two, bacteriorhodopsin (BR) and halorhodopsin (HR), are transport rhodopsins that carry out light‐driven electrogenic translocation of protons and chloride, respectively, across the cell membrane. The other two, sensory rhodopsins I and II (SRI and SRII), are phototaxis receptors that send signals to tightly bound transducer proteins that in turn control a phosphorylation cascade modulating the cell's flagellar motors. Recent progress has cast light on how nature has modified the common design of these proteins to carry out their distinctly different functions: electrogenic ion transport and non‐electrogenic signal transduction. A key shared mechanism between BR and SRII appears to be an interhelical salt bridge locked conformational switch that is released by photoisomerization of retinal. In BR disruption of the lock opens a cytoplasmic half‐channel that ensures uptake of the transported proton from the cytoplasmic side of the membrane at a critical time in the pumping cycle. Transducer‐free SRI uses the same mechanism to carry out light‐driven proton transport, but interaction with its transducer blocks the cytoplasmic half‐channel thereby interrupting the transport cycle. In SRI, transducer interaction also disrupts the salt bridge in the dark, poising the receptor in an intermediate conformation able to produce opposite signals depending on the colour of the stimulus light. A model for signalling is proposed in which the salt bridge‐controlled half‐channel is used to modulate interaction with the Htr proteins when the receptor signalling states are formed.
Are minichromosomes valid model systems for DNA replication control? Lessons learned from Escherichia coli
Authors: Asai Tsuneaki , Bates David B. , Boye Erik , Kogoma Tokio
Published: March 2003

Initiation of chromosome replication is a key event in the life cycle of any organism. Little is known, however, about the regulatory mechanisms of this vital process. Conventionally, the initiation mechanism of chromosome replication in microorganisms has been studied using plasmids in which an origin of chromosome replication has been cloned, rather than using the chromosome itself. The reason for this is that even bacterial chromosomes are so large that biochemical and genetic manipulations become difficult and cumbersome. Recently, the combination of flow cytometry and genetic methods, in which modifications of the replication origin are systematically introduced onto the chromosome, has made possible detailed studies of the molecular events involved in the control of replication initiation in Escherichia coli. The results indicate that requirements for initiation at the chromosomal origin, oriC, are drastically different from those for initiation at cloned oriC.
A novel aminopeptidase associated with the 60 kDa chaperonin in the thermophilic archaeon Sulfolobus solfataricus
Authors: Condò Ivano , Ruggero Davide , Reinhardt Richard , Londei Paola
Published: March 2003

The chaperonins are high‐molecular‐weight protein complexes having a characteristic double‐ring toroidal shape; they are thought to aid the folding of denatured or newly synthesized polypeptides. These proteins exist as two functionally similar but distantly related families, one including the bacterial and organellar chaperonins and the other (termed the CCT‐TRiC family) including the chaperonins of the Archaea and the eukaryotes. The CCT‐TRiC chaperonins, particularly their archeal members, are less well known than their bacterial counterparts, and their main cellular function is still doubtful. In this work, we report that the chaperonin of the thermophilic archaeon Sulfolobus solfataricus interacts with several polypeptides other than the two subunits that constitute the 18‐mer double‐ring structure. We have cloned and sequenced the gene encoding one 90 kDa chaperonin‐associated protein and have shown, using biochemical assays, that the product is an enzyme belonging to the family of zinc‐dependent aminopeptidases. The Sulfolobus protein shows maximal homology to eukaryotic (yeast and mouse) aminopeptidases. It contains a leucine zipper motif and can be phosphorylated by an unidentified kinase present in the cell extracts. The possible significance of an association between an aminopeptidase and a chaperonin is discussed.
Transcription of rpoH, encoding the Escherichia coli heat‐shock regulator σ32, is negatively controlled by the cAMP‐CRP/CytR nucleoprotein complex
Authors: Kallipolitis Birgitte H. , Valentin‐Hansen Poul
Published: March 2003

In Escherichia coli, the rpoH gene encoding the essential heat‐shock regulator σ32, is expressed in a complex manner. Transcription occurs from four promoters (P1, P3, P4 and P5) and is modulated by several factors including (i) two σ factors (σ70 and σE); (ii) the global regulator CRP; and (iii) the DnaA protein. Here, a further dissection of the rpoH regulatory region has revealed that an additional transcription control exists that appears to link rpoH expression to nucleoside metabolism. The cAMP–CRP complex and the CytR anti‐activator bind co‐operatively to the promoter region forming a repression complex that overlaps the σE‐dependent P3 promoter and the σ70‐dependent P4 and P5 promoters. During steady‐state growth conditions with glycerol as the carbon and energy source, transcription from P3, P4 and P5 is reduced ≈threefold by CytR, whereas transcription from the upstream promoter, P1, appears to be unaffected. Furthermore, in strains that slightly overproduce CytR, transcription from P3, P4 and P5 is reduced even further (≈10‐fold), and repression can be fully neutralized by the addition of the inducer cytidine to the growth medium. In the induced state, P4 is the strongest promoter and, together with P3 and P5, it is responsible for most rpoH transcription (65–70%). At present, CytR has been shown to ‘fine tune’ transcription of two genes (rpoH and ppiA) that are connected with protein‐folding activities. These findings suggest that additional assistance in protein folding is required under conditions in which CytR is induced (i.e. in the presence of nucleosides).
Initial binding of Shiga toxin‐producing Escherichia coli to host cells and subsequent induction of actin rearrangements depend on filamentous EspA‐containing surface appendages
Authors: Ebel Frank , Podzadel Till , Rohde Manfred , Kresse Andreas U. , Krämer Sylvia , Deibel Christina , Guzmán Carlos A. , Chakraborty Trinad
Published: March 2003

Shiga toxin‐producing Escherichia coli (STEC) induce so‐called attaching and effacing lesions that enable the tight adherence of these pathogens to the gut epithelium. All of the genes necessary for this process are present in the locus of enterocyte effacement, which encodes a type III secretion system, the secreted Esp proteins and the surface protein intimin. In this study we sequenced the espA gene of STEC, generated and characterized a corresponding deletion mutant and raised EspA‐specific monoclonal antibodies to analyse the functional role of this protein during infection. EspA was detected in often filament‐like structures decorating all bacteria that had attached to HeLa cells. These appendages were especially prominent on bacteria that had not yet induced the formation of actin pedestals, indicating that they mediate the initial contact of STEC to their target cells. Consistently, a deletion of the espA gene completely abolished the capacity of such STEC mutants to bind to HeLa cells and to induce actin rearrangements. Surface appendages similar to those described in this study are also formed by Pseudomonas syringae and may represent a structural element common to many bacterial pathogens that deliver proteins into their target cells via a type III secretion system.
Participation of IHF and a distant UP element in the stimulation of the phage λ PL promoter
Authors: Giladi Hilla , Koby Simi , Prag Gali , Engelhorn Manuel , Geiselmann Johannes , Oppenheim Amos B.
Published: March 2003

We have previously identified a UP element in the phage λ PL promoter, centred at position −90 from the transcription start site. Integration host factor (IHF), a heterodimeric DNA‐binding and ‐bending protein, binds upstream of the λ PL promoter in a region overlapping the UP element. Stimulation of transcription by IHF requires an intact αCTD and affects the initial binding of RNA polymerase to the promoter. We propose a model for the stimulation of PL by IHF in which IHF bends the DNA to bring the distal UP sequence in closer proximity to the promoter core sequences to allow the docking of the αCTD of RNA polymerase. Furthermore, IHF may also participate in protein–protein interactions with the αCTD. In support of this model, we found that alanine substitutions in αCTD at positions 265, 268, 270 and 275 reduced PL promoter activity. Mutations in the IHF DNA binding site, as well as IHF mutant proteins exhibiting a decreased ability to bend the DNA, were both defective in stimulating the PL promoter. In addition, some of the mutated IHF residues are clustered at a protein surface that interacts with the UP DNA sequence. These residues may also participate in protein–protein interactions with the αCTD.
Conversion to bidirectional replication after unidirectional initiation from R1 plasmid origin integrated at oriC in Escherichia coli
Authors: Maisnier‐Patin Sophie , Dasgupta Santanu , Krabbe Margareta , Nordström Kurt
Published: March 2003

The cell division phenotypes of Escherichia coli with its chromosome replication driven by oriR (from plasmid R1) were examined by fluorescence microscopy and flow cytometry. Chromosome replication patterns in these strains were followed by marker frequency analyses. In one of the strains, the unidirectional oriR was integrated so that the replication fork moved clockwise from the oriC region, and bacterial growth and division were similar to those of the wild‐type parent. The bacteria were able to convert the unidirectional initiation from oriR into bidirectional replication. The site for conversion of uni‐ to bidirectional replication seemed to be localized and could be mapped genetically within 6 min to the immediate right of the minimal oriC. Replication starting in the counterclockwise direction from the R1 replicon integrated at the same site in the opposite orientation could not be described as either bi‐ or unidirectional, as no single predominant origin could be discerned from the more or less flat marker frequency pattern. These strains also showed extensive filamentation, irregular nucleoid distribution and the presence of anucleate cells, indicative of segregation and division defects. Comparison among intR1 derivatives differing in the position of the integrated oriR relative to the chromosome origin suggested that the oriC sequence itself was dispensable for the conversion to bidirectionality. However, passage of the replication fork over the 6 min region to the right of oriC seemed important for the bidirectional replication pattern and normal cell division phenotype.