bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple paternity and mating patterns in the American alligator, Alligator mississippiensis
Authors: Davis Lisa M. , Glenn Travis C. , Elsey Ruth M. , Dessauer Herbert C. , Sawyer Roger H.
Published: March 2003

Eggs were sampled from 22 wild American alligator nests from the Rockefeller Wildlife Refuge in south‐west Louisiana, along with the females guarding the nests. Three nests were sampled in 1995 and 19 were sampled in 1997. Females and offspring from all clutches were genotyped using five polymorphic microsatellite loci and the three nests from 1995 were also genotyped using one allozyme locus. Genotypes of the hatchlings were consistent with the guarding females being the mothers of their respective clutches. Multiple paternity was found in seven of the 22 clutches with one being fathered by three males, and the remaining six clutches having genotypes consistent with two males per clutch. Paternal contributions of multiply sired clutches were skewed. Some males sired hatchlings of more than one of the 22 clutches either as one of two sires of a multiple paternity clutch, as the sole sire of two different clutches, or as the sole sire of one clutch and one of two sires of a multiply sired clutch. There was no significant difference between females that had multiple paternity clutches and those that had singly sired clutches with respect to female total length (P = 0.844) and clutch size (P = 0.861). Also, there was no significant correlation between genetic relatedness of nesting females and pairwise nest distances (r2 = 0.003, F1,208 = 0.623, P = 0.431), indicating that females in this sample that nested close to one another were no more related than any two nesting females chosen at random. Eleven mutations were detected among hatchlings at the five loci over the 22 clutches. Most of these mutations (eight of 11) occurred at Amiµ‐17, the only compound microsatellite locus of the five used in this study, corresponding to a mutation rate of 1.7 × 10−3. Finally, most of the mutations (82%) were homoplasious, i.e., mutating to an allelic state already present in this Louisiana population.
Noninvasive paternity assignment in Gombe chimpanzees
Authors: Constable Julie L. , Ashley Mary V. , Goodall Jane , Pusey Anne E.
Published: March 2003

The relative success of chimpanzee male mating strategies, the role of male dominance rank and the success of inbreeding avoidance behaviour can only be assessed when paternities are known. We report the probable paternities of 14 chimpanzees included in a long‐term behavioural study of chimpanzees (Pan troglodytes schweinfurthii) at Gombe National Park, Tanzania. DNA samples were collected noninvasively from shed hair and faeces and genotyped using 13–16 microsatellite loci characterized in humans. All 14 offspring could be assigned to fathers within the community. While there is a positive relationship between male rank and reproductive success, we demonstrate that a range of male mating strategies (possessiveness, opportunistic mating and consortships) can lead to paternity across all male ranks. Several adult females were at risk of breeding with close male relatives. Most successfully avoided close inbreeding but in one case a high‐ranking male in the community mated with his mother and produced an offspring. In contrast to recent data on chimpanzees (P. t. verus) from the Taï forest, Côte d’Ivoire, no evidence of extra‐group paternity was observed in our study. Reanalysis of Taï data using a likelihood approach casts doubt on the occurrence of extra‐group paternity in that community as well.
High levels of extra‐pair paternity in an isolated, low‐density, island population of tree swallows (Tachycineta bicolor)
Authors: Conrad K. F. , Johnston P. V. , Crossman C. , Kempenaers B. , Robertson R. J. , Wheelwright N. T. , Boag P. T.
Published: March 2003

Molecular genetic studies have suggested that apparently nonbreeding males (‘floaters’) may account for a significant proportion of extra‐pair paternity (EPP) in avian populations. Attempts to determine the influence of breeding density on EPP are therefore confounded by the presence of a subpopulation of floaters whose numbers are difficult to estimate. To study EPP in a tree swallow (Tachycineta bicolor) population with few floaters, we chose a nestbox grid on an island with an excess of available breeding sites and very few floaters. We obtained DNA samples from 13 complete families and performed DNA profiling on them using four microsatellite loci. For comparison, we also obtained a sample of 58 extra‐pair young (EPY) from a mainland population typed at five microsatellite loci. Paternity assignments among resident males in both populations were made using the microsatellite profiles and a likelihood‐based statistical method. Of the 67 island nestlings typed, we found 37 (55%) nestlings from 11 (85%) different nests that were EPY. The proportion of nestlings that were EPY and the proportion of broods containing EPY did not differ significantly between island and mainland populations studied previously. There was no significant difference between island and mainland populations in the proportion of extra‐pair paternities assigned among neighbouring resident males. Male breeding density does not appear to affect the ability of female tree swallows to obtain extra‐pair fertilizations, at least over the range of densities studied so far. The rate of EPP has remained remarkably consistent over many years, studies and populations implying an important role of active female choice in determining EPP.
Mating patterns, relatedness and the basis of natal philopatry in the brown long‐eared bat,Plecotus auritus
Authors: Burland T. M. , Barratt E. M. , Nichols R. A. , Racey P. A.
Published: March 2003

The brown long‐eared bat,Plecotus auritus, is unusual among temperate zone bats in that summer maternity colonies are composed of adult males and females, with both sexes displaying natal philopatry and long‐term association with a colony. Here, we describe the use of microsatellite analysis to investigate colony relatedness and mating patterns, with the aim of identifying the evolutionary determinants of social organization inP. auritus. Mean colony relatedness was found to be low (R = 0.033 ± 0.002), with pairwise estimates ofRwithin colonies ranging from −0.4 to 0.9. The proportion of young fathered by males in their own colony was investigated using a Bayesian approach, incorporating parameters detailing the number of untyped individuals. This analysis revealed that most offspring were fathered by males originating from a different colony to their own. In addition, we determined that the number of paternal half‐sibs among cohorts of young was low, inferring little or no skew in male reproductive success. The results of this study suggest that kin selection cannot account for colony stability and natal philopatry inP. auritus, which may instead be explained by advantages accrued through the use of familiar and successful roost sites, and through long‐term associations with conspecifics. Moreover, because the underlying causes of male natal dispersal in mammals, such as risk of inbreeding or competition for mates, appear to be avoided via extra‐colony copulation and low male reproductive skew, bothP. auritusmales and females are able to benefit from long‐term association with the natal colony.
Phylogeography of the African buffalo based on mitochondrial and Y‐chromosomal loci: Pleistocene origin and population expansion of the Cape buffalo subspecies
Authors: Van Hooft W. F. , Groen A. F. , Prins H. H. T.
Published: March 2003

Population genetics and phylogeography of the African buffalo (Syncerus caffer) are inferred from genetic diversity at mitochondrial D‐loop hypervariable region I sequences and a Y‐chromosomal microsatellite. Three buffalo subspecies from different parts of Africa are included. Nucleotide diversity of the subspecies Cape buffalo at hypervariable region I is high, with little differentiation between populations. A mutation rate of 13–18% substitutions/million years is estimated for hypervariable region I. The nucleotide diversity indicates an estimated female effective population size of 17 000–32 000 individuals. Both mitochondrial and Y‐chromosomal diversity are considerably higher in buffalo from central and southwestern Africa than in Cape buffalo, for which several explanations are hypothesized. There are several indications that there was a late middle to late Pleistocene population expansion in Cape buffalo. This also seems to be the period in which Cape buffalo evolved as a separate subspecies, according to the net sequence divergence with the other subspecies. These two observations are in agreement with the hypothesis of a rapid evolution of Cape buffalo based on fossil data. Additionally, there appears to have been a population expansion from eastern to southern Africa, which may be related to vegetation changes. However, as alternative explanations are also possible, further analyses with autosomal loci are needed.
Extensive exchange of fungal cultivars between sympatric species of fungus‐growing ants
Authors: Green A. M. , Mueller U. G. , Adams R. M. M.
Published: March 2003

Fungal cultivars of fungus‐growing ants (Attini, Formicidae) are carried by dispersing queens from parent to offspring nest. This vertical cultivar transmission between generations is thought to result in long‐term ant‐fungus coevolution and selection for beneficial cultivar traits that maximize harvests and thus colony productivity. In contrast to this traditional view of vertical cultivar transmission, frequent horizontal cultivar transmission between ant species is implicated by a phylogenetic analysis of 72 cultivars propagated by two fungus‐growing ant species coexisting sympatrically in central Panama. The two ant species are specialized on the same group of closely related cultivars, but in six of 12 cultivar clades identifiable within this group, cultivars from both ant species were united in the same clade. Five of these ‘mixed’ clades were supported by bootstrap values of about 90% or higher. In one instance, colonies from the two ant species cultivated the same, genetically identical, cultivar clone. These phylogenetic patterns indicate that: (i) cultivar exchanges between the two ant species occur routinely throughout ecological time; and that (ii) coevolutionary processes between ants and their fungi are more diffuse than previously assumed. Because the two ant species are specialized on a narrow group of closely related cultivars that they regularly exchange among each other, but not with other sympatric ant species, cultivar exchanges are constrained, most likely, by ant preferences for their own cultivar group or by stringent selection against transitions of ant lineages to distantly related cultivars.
Population genetic subdivision in the New Zealand greenshell mussel (Perna canaliculus) inferred from single‐strand conformation polymorphism analysis of mitochondrial DNA
Authors: Apte S. , Gardner J. P. A.
Published: March 2003

Single‐strand conformation polymorphism (SSCP) analysis of the NADH IV region of the mitochondrial DNA (mtDNA) molecule in greenshell mussels (Perna canaliculus) indicated strong population genetic structuring in this endemic New Zealand species. A northern and a southern group were differentiated by frequency shifts in common haplotypes and by the occurrence of a unique southern haplotype at ≈ 20% frequency. This split occurred south of Cook Strait (the body of water between the North and the South Island) at ≈ 42° S latitude. Northern populations were less genetically diverse than southern populations and mussels from the west coast of the South Island were most distinct from northern mussels. We hypothesize that the unique haplotype VIII originated in the lower South Island, and that its spread northwards was obstructed by the opening of Cook Strait ≈ 15 000–16 000 years ago and the subsequent establishment of present‐day surface water circulation patterns in Greater Cook Strait. We suggest that present‐day strong tidal flows and turbulent mixing of water masses in Cook Strait, and intense up‐welling on the east and west coasts in this region, represent a barrier to gene flow between mussels located in the North Island and northern South Island vs. mussels in most of the South Island and Stewart Island.
Field experiments on the distributions of eggs of different phosphoglucomutase (PGM) genotypes in the yellow dung fly Scathophaga stercoraria (L.)
Authors: Ward Paul I. , Vonwil Judith , Scholte Ernst‐Jan , Knop Eva
Published: March 2003

Female yellow dung flies can, in the laboratory, influence the probability that stored sperm from different males are used to fertilize eggs. This matches offspring phosphoglucomutase genotypes to the environmental conditions in which the larvae will grow, increasing larval growth success. We conducted field experiments in which dung topology or shading conditions were controlled. The proportions of the five common phosphoglucomutase alleles in eggs laid in north‐facing slopes or in shaded conditions was related to their electrophoretic mobility. We suggest that females lay eggs of different genotypes, by appropriately choosing their fathers, in different places.
Mitochondrial DNA phylogeography and population history ofMelademadiving beetles on the Atlantic Islands and in the Mediterranean basin (Coleoptera, Dytiscidae)
Authors: Ribera I. , Bilton D. T. , Vogler A. P.
Published: March 2003

The phylogeny and population history ofMelademadiving beetles (Coleoptera, Dytiscidae) were examined using mitochondrial DNA sequence from 16S ribosomal RNA and cytochrome oxidase I genes in 51 individuals from 22 populations of the three extant species (M. imbricataendemic to the western Canary Islands,M. lanioendemic to Madeira andM. coriaceawidespread in the Western Mediterranean and on the western Canaries), using a combination of phylogenetic and nested clade analyses. Four main lineages are observed withinMeladema, representing the three recognized species plus Corsican populations ofM. coriacea. Phylogenetic analyses demonstrate the sister relationship of the two Atlantic Island taxa, and suggest the possible paraphyly ofM. coriacea. A molecular clock approach reveals that speciation within the genus occurred in the Early Pleistocene, indicating that the Atlantic Island endemics are not Tertiary relict taxa as had been proposed previously. Our results point to past population bottlenecks in all four lineages, with recent (Late‐Middle Pleistocene) range expansion in non‐CorsicanM. coriaceaandM. imbricata. Within the Canary Islands,M. imbricataseems to have independently colonized La Gomera and La Palma from Tenerife (although a colonization of La Palma from La Gomera cannot be discarded), andM. coriaceahas independently colonized Tenerife and Gran Canaria from separate mainland lineages. In the Mediterranean basin this species apparently colonized Corsica on a single occasion, relatively early in its evolutionary history (Early Pleistocene), and has colonized Mallorca recently on multiple occasions. On the only island whereM. coriaceaandM. imbricataare broadly sympatric (Tenerife), we report evidence of bidirectional hybridization between the two species.
Condition‐dependent sex allocation in a lek‐breeding wader, the ruff (Philomachus pugnax)
Authors: Thuman Katherine A. , Widemo Fredrik , Griffith Simon C.
Published: March 2003

Sex allocation theory predicts that females should bias the production of offspring towards the sex that will maximize maternal fitness. Here we demonstrate evidence for nonrandom sex allocation by female ruffs (Philomachus pugnax), at both the individual and population level in relation to female condition. At the population level, female condition varies significantly across 3 years and is mirrored by population sex ratio, such that in years when females are in poor condition the population offspring sex ratio is female‐biased, while in years when females are in better condition there was little or no bias. In the year when females were in overall poor condition, females in better condition produced more daughters. The same relationship is also revealed by comparing the sex ratios of individual females breeding in two consecutive years in different condition. As the condition of an individual female improves (across years) she tends to produce more female offspring. Although we have shown that, as in other birds, female condition is an important determinant of sex allocation, our results also suggest that such nonrandom allocation does not occur in every year, being particularly strong in a year when females, on average, are in poorer condition. We suggest that our results are consistent with the idea that skewing the sex ratio is likely to carry a cost to females and that it is adaptive only when the fitness differential between sons and daughters is sufficient to outweigh probable costs.
The recent evolutionary origin of the phenotypically novel amphipodHyalella montezumaoffers an ecological explanation for morphological stasis in a closely allied species complex
Authors: Witt Jonathan D. S. , Blinn Dean W. , Hebert Paul D. N.
Published: March 2003

Numerous molecular studies have identified morphologically cryptic, freshwater invertebrate species, but have not suggested possible mechanisms for their phenotypic stasis. The amphipod crustacean genusHyalellacontains numerous morphologically cryptic species in theH. aztecacomplex, as well as a small number of morphologically very divergent, narrowly endemic taxa. One such taxon,Hyalella montezuma, is the sole planktonic filter‐feeder within the North American amphipod fauna, and is known only from Montezuma Well, a fishless travertine spring mound in Arizona, USA. In this study, we conduct a phylogenetic analysis of mtDNA sequence data using likelihood, Bayesian and cladistic approaches to determine both the relationship ofH. montezumato theH. ‘azteca’ species complex, and to ascertain if its morphological and ecological differentiation have been comparatively recent. The results show thatH. montezumahas a very close phylogenetic affiliation with one lineage in theH. aztecacomplex, indicating that its origin has been recent. We present evidence suggesting that fish predation is an important ecological factor, which constrains morphological and ecological diversification within the genusHyalella, and that Montezuma Well has provided a relaxation on this constraint.
Extinction‐colonization dynamics structure genetic variation of spotted sunfish (Lepomis punctatus) in the Florida Everglades
Authors: Mcelroy T. C. , Kandl K. L. , Garcia J. , Trexler J. C.
Published: March 2003

The population genetics of aquatic animals in the Florida Everglades may be strongly influenced by extinction and colonization dynamics. We combined analyses of allozyme and microsatellite loci to test the hypothesis that two levels of population structure are present for spotted sunfish (Pisces: Centrarchidae: Lepomis punctatus) inhabiting the Everglades. We hypothesized that annual cycles of marsh dry‐down increase local‐scale genetic variation through a process of local extinction and colonization; we hypothesized that barriers to gene flow by levee/canal systems create a second, regional level of genetic variation. In 1996 and 1997, we sampled spotted sunfish from 11 Everglades sites that were distributed in three regions separated by levees. We documented patterns of genetic variation at 7 polymorphic allozyme loci and 5 polymorphic microsatellite loci. Most genetic variation was present among local populations, according to both types of genetic markers. Furthermore, samples from marsh sites were heterogeneous, while those from canals were not. These data supported our hypothesis that dry‐down events and local population dynamics in the marsh have a significant effect on population genetic structure of spotted sunfish. We found no support for our hypothesis that water‐management structures superimpose a second level of genetic structure on this species, possibly because canals obscure historical structure by facilitating gene flow or because the complete canal system has been in place for fewer than 20 generations of this species. Our data suggests a continent‐island (canal‐marsh) structure of populations with high gene flow among regions and recurrent mixing in marshes from canal and creek habitats.
The role of glyoxalase I in the detoxification of methylglyoxal and in the activation of the KefB K+ efflux system in Escherichia coli
Authors: MacLean M. J. , Ness L. S. , Ferguson G. P. , Booth I. R.
Published: March 2003

The glyoxalase I gene (gloA) of Escherichia coli has been cloned and used to create a null mutant. Cells overexpressing glyoxalase I exhibit enhanced tolerance of methylglyoxal (MG) and exhibit elevated rates of detoxification, although the increase is not stoichiometric with the change in enzyme activity. Potassium efflux via KefB is also enhanced in the overexpressing strain. Analysis of the physiology of the mutant has revealed that growth and viability are quite normal, unless the cell is challenged with MG either added exogenously or synthesized by the cells. The mutant strain has a low rate of detoxification of MG, and cells rapidly lose viability when exposed to this electrophile. Activation of KefB and KefC is diminished in the absence of functional glyoxalase I. These data suggest that the glutathione‐dependent glyoxalase I is the dominant detoxification pathway for MG in E. coli and that the product of glyoxalase I activity, S‐lactoylglutathione, is the activator of KefB and KefC.
Regulation of RssB‐dependent proteolysis in Escherichia coli: a role for acetyl phosphate in a response regulator‐controlled process
Authors: Bouché Sandrine , Klauck Eberhard , Fischer Daniela , Lucassen Magnus , Jung Kirsten , Hengge‐Aronis Regine
Published: March 2003

σS (RpoS) is a highly unstable global regulatory protein in Escherichia coli, whose degradation is inhibited by various stress signals, such as carbon starvation, high osmolarity and heat shock. As a consequence, these stresses result in the induction of σS‐regulated stress‐protective proteins. The two‐component‐type response regulator, RssB, is essential for the rapid proteolysis of σS and is probably involved in the transduction of some of these stress signals. Acetyl phosphate can be used as a phosphodonor for the phosphorylation of various response regulators in vitro and, in the absence of the cognate sensor kinases, acetyl phosphate can also modulate the activities of several response regulators in vivo. Here, we demonstrate increased in vivo half‐lives of σS and the RpoS742::LacZ hybrid protein (also a substrate for RssB‐dependent proteolysis) in acetyl phosphate‐free (pta–ackA) deletion mutants, even though no sensor kinase was eliminated. The in vivo data indicate that acetyl phosphate acts through the response regulator, RssB. In vitro, efficient phosphotransfer from radiolabelled acetyl phosphate to the Asp‐58 residue of RssB (the expected site of phosphorylation in the RssB receiver domain) was observed. Via such phosphorylation, acetyl phosphate may thus modulate RssB activity even in an otherwise wild‐type background. While acetyl phosphate is not essential for the transduction of specific environmental stress signals, it could play the role of a modulator of RssB‐dependent proteolysis that responds to the metabolic status of the cells reflected in the highly variable cellular acetyl phosphate concentration.
The bacteriophage T4 AsiA protein: a molecular switch for sigma 70‐dependent promoters
Authors: Colland Frédéric , Orsini Gilbert , Brody Edward N. , Buc Henri , Kolb Annie
Published: March 2003

The AsiA protein, encoded by bacteriophage T4, inhibits Eσ70‐dependent transcription at bacterial and early‐phage promoters. We demonstrate that the inhibitory action of AsiA involves interference with the recognition of the −35 consensus promoter sequence by host RNA polymerase. In vitro experiments were performed with a C‐terminally labelled sigma factor that is competent for functional holoenzyme reconstitution. By protease and hydroxyl radical protein footprinting, we show that AsiA binds region 4.2 of σ70, which recognizes the −35 sequence. Direct interference with the recognition of the promoter at this locus is supported by two parallel experiments. The stationary‐phase sigma factor containing holoenzyme, which can initiate transcription at promoters devoid of a −35 region, is insensitive to AsiA inhibition. The recognition of a galP1 promoter by Eσ70 is not affected by the presence of AsiA. Therefore, we conclude that AsiA inhibits transcription from Escherichia coli and T4 early promoters by counteracting the recognition of region 4.2 of σ70 with the −35 hexamer.
nodD2 of Rhizobium sp. NGR234 is involved in the repression of the nodABC operon
Authors: Fellay R. , Hanin M. , Montorzi G. , Frey J. , Freiberg C. , Golinowski W. , Staehelin C. , Broughton W.J. , Jabbouri S.
Published: March 2003

Transcriptional regulators of the lysR family largely control the expression of bacterial symbiotic genes. Rhizobium sp. NGR234 contains at least four members of this family: two resemble nodD, while two others are more closely related to syrM. Part of the extremely broad host range of NGR234 can be attributed to nodD1, although the second gene shares a high degree of DNA sequence homology with nodD2 of R. fredii USDA191. A nodD2 mutant of NGR234 was constructed by insertional mutagenesis. This mutant (NGRΩnodD2) was deficient in nitrogen fixation on Vigna unguiculata and induced pseudonodules on Tephrosia vogelii. Several other host plants were tested, but no correlation could be drawn between the phenotype and nodule morphology. Moreover, nodD2 has a negative effect on the production of Nod factors: mutation of this gene results in a fivefold increase in Nod factor production. Surprisingly, while the structure of Nod factors from free‐living cultures of NGRΩnodD2 remained unchanged, those from V. unguiculata nodules induced by the same strain are non‐fucosylated and have a lower degree of oligomerization. In other words, developmental regulation of Nod factor production is also abolished in this mutant. Competitive RNA hybridizations, gene fusions and mobility shift assays confirmed that nodD2 downregulates expression of the nodABC operon.
EspE, a novel secreted protein of attaching and effacing bacteria, is directly translocated into infected host cells, where it appears as a tyrosine‐phosphorylated 90 kDa protein
Authors: Deibel Christina , Krämer Sylvia , Chakraborty Trinad , Ebel Frank
Published: March 2003

Shiga toxin‐producing Escherichia coli (STEC), enteropathogenic E. coli (EPEC) and some strains of Hafnia alvei are capable of inducing attaching and effacing (A/E) lesions, characterized by tight apposition of the bacteria to the eukaryotic membrane and formation of actin‐based pedestals. In this study, we report on the identification of EspE, a novel secreted 80 kDa protein of A/E bacteria. During infection, EspE is delivered into the cytoplasm of the infected host cell, where it is detected as a higher‐molecular‐weight form of 90 kDa. We present evidence that translocated EspE becomes tyrosine phosphorylated and that this modified form of EspE may be identical to Hp90, the putative receptor of EPEC intimin. Bacteria of the classic enterohaemorrhagic E. coli (EHEC) serotype O157:H7 fail to induce a tyrosine phosphorylation of EspE and differ in this respect from other A/E bacteria. Translocated EspE, whether tyrosine phosphorylated or not, becomes incorporated into the bacteria‐induced cytoskeletal structures, where it normally colocalizes with filamentous actin. EPEC are also able to induce ‘pseudopods’, elongated pedestals that have recently been implicated in a novel kind of actin‐based motility. EspE is enriched at the tip of these structures, suggesting its involvement in the process of actin dynamics, which is triggered during the attaching and effacing process.
Initiation factors of protein biosynthesis in bacteria and their structural relationship to elongation and termination factors
Authors: Brock Stephan , Szkaradkiewicz Karol , Sprinzl Mathias
Published: March 2003

Initiation of protein biosynthesis in bacteria requires three initiation factors: initiation factor 1, initiation factor 2 and initiation factor 3. The mechanism by which initiation factors form the 70S initiation complex with initiator fMet‐tRNAfMet interacting with the initiation codon in the ribosomal P site and the second mRNA codon exposed in the A site is not yet understood. Here, we present a model for the function of initiation factors 1 and 2 that is based on the analysis of sequence homologies, biochemical evidence and the present knowledge of the three‐dimensional structures of translation factors and ribosomes. The model predicts that initiation factors 1 and 2 interact with the ribosomal A site mimicking the structure of the elongation factor G. We present data that extend the mimicry hypothesis to initiation factors 1 and 2, originally postulated for the aminoacyl‐tRNA·elongation factor Tu·GTP ternary complex, elongation factor G and release factors.
Evidence that trypanothione reductase is an essential enzyme in Leishmania by targeted replacement of the tryA gene locus
Authors: Tovar Jorge , Wilkinson Shane , Mottram Jeremy C. , Fairlamb Alan H.
Published: March 2003

Trypanothione reductase (TR), a flavoprotein oxidoreductase central to the unique thiol‐redox system that operates in trypanosomatid protozoa, has been proposed as a potential target for the chemotherapy of trypanosomatid infections. In this study, targeted gene replacement was used to obtain evidence that TR is an essential cellular component and that its physiological function is crucial for parasite survival. Precise replacement of the Leishmania donovani tryA gene encoding TR was only possible upon simultaneous expression of the tryA coding region from an episome; in its absence, attempted removal of the last tryA allele invariably led to the generation of an extra copy of tryA, seemingly as a result of selective chromosomal polysomy. Partial replacement mutants were drastically affected in their ability to survive inside cytokine‐activated macrophages in a murine model of Leishmania infection. As no compensatory mechanism for the partial loss of TR activity was observed in these mutants and as it was not possible to obtain viable Leishmania devoid of TR catalytic activity, specific inhibitors of this enzyme are likely to be useful anti‐leishmanial agents for chemotherapeutic use.
Regulation of Pseudomonas aeruginosa hemF and hemN by the dual action of the redox response regulators Anr and Dnr
Authors: Rompf Alexandra , Hungerer Christoph , Hoffmann Tamara , Lindenmeyer Maja , Römling Ute , Groß Ulrich , Doss Manfred O. , Arai Hiroyuki , Igarashi Yasuo , Jahn Dieter
Published: March 2003

The oxidative decarboxylation of coproporphyrinogen III catalysed by an oxygen‐dependent oxidase (HemF) and an oxygen‐independent dehydrogenase (HemN) is one of the key regulatory points of haem biosynthesis in Pseudomonas aeruginosa. To investigate the oxygen‐dependent regulation of hemF and hemN, the corresponding genes were cloned from the P. aeruginosa chromosome. Recognition sequences for the Fnr‐type transcriptional regulator Anr were detected −44.5 bp from the 5′ end of the hemF mRNA transcript and at an optimal distance of −41.5 bp with respect to the transcriptional start of hemN. An approximately 10‐fold anaerobic induction of hemN gene expression was mediated by the dual action of Anr and a second Fnr‐type regulator, Dnr. Regulation by both proteins required the Anr recognition sequence. Surprisingly, aerobic expression of hemN was dependent only on Anr. An anr mutant did not contain detectable amounts of hemN mRNA and accumulated coproporphyrin III both aerobically and anaerobically, indicating the importance of HemN for aerobic and anaerobic haem formation. Mutation of hemN and hemF did not abolish aerobic or anaerobic growth, indicating the existence of an additional HemN‐type enzyme, which was termed HemZ. Expression of hemF was induced approximately 20‐fold during anaerobic growth and, as was found for hemN, both Anr and Dnr were required for anaerobic induction. Paradoxically, oxygen is necessary for HemF catalysis, suggesting the existence of an additional physiological function for the P. aeruginosa HemF protein.
A five‐nucleotide sequence protects DNA from exonucleolytic degradation by AddAB, the RecBCD analogue of Bacillus subtilis
Authors: Chédin Frédéric , Noirot Philippe , Biaudet Véronique , Ehrlich S. Dusko
Published: March 2003

Homologous recombination in Bacillus subtilis requires the product of the addA and addB genes, the AddAB enzyme. This enzyme, which is both a helicase and a powerful nuclease, is thought to be the counterpart of the Escherichia coli RecBCD enzyme. From this analogy, it is expected that the nuclease activity of AddAB can be downregulated by a specific DNA sequence, which would correspond to the chi site in E. coli. Using protection of linear double‐stranded DNA as a criterion, we identified the five‐nucleotide sequence 5′‐AGCGG‐3′, or its complement 5′‐CCGCT‐3′, as being sufficient for AddAB nuclease attenuation. We have shown further that this attenuation occurs only if the sequence is properly oriented with respect to the translocating AddAB enzyme. Finally, inspection of the complete B. subtilis genome revealed that this five‐nucleotide sequence is over‐represented and is, in a majority of cases, co‐oriented with DNA replication. Based on these observations, we propose that 5′‐AGCGG‐3′, or its complement, is the B. subtilis analogue of the E. coli chi sequence.
Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages
Authors: Hensel Michael , Shea Jacqueline E. , Waterman Scott R. , Mundy Rosanna , Nikolaus Thomas , Banks Geoff , Vazquez‐Torres Andrés , Gleeson Colin , Fang Ferric C. , Holden David W.
Published: March 2003

The type III secretion system of Salmonella pathogenicity island 2 (SPI‐2) is required for systemic infection of this pathogen in mice. Cloning and sequencing of a central region of SPI‐2 revealed the presence of genes encoding putative chaperones and effector proteins of the secretion system. The predicted products of the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA, EspD and EspB, which are secreted by the type III secretion system encoded by the locus of enterocyte effacement of enteropathogenic Escherichia coli. The transcriptional activity of an sseA::luc fusion gene was shown to be dependent on ssrA, which is required for the expression of genes encoding components of the secretion system apparatus. Strains carrying non‐polar mutations in sseA, sseB or sseC were severely attenuated in virulence, strains carrying mutations in sseF or sseG were weakly attenuated, and a strain with a mutation in sseE had no detectable virulence defect. These phenotypes were reflected in the ability of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA, sseB or sseC failed to accumulate, whereas the growth rates of strains carrying mutations in sseE, sseF or sseG were only modestly reduced. These data suggest that, in vivo, one of the functions of the SPI‐2 secretion system is to enable intracellular bacterial proliferation.
Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function
Authors: Papavinasasundaram K. G. , Colston M. Joseph , Davis Elaine O.
Published: March 2003

A recA deletion mutant of Mycobacterium smegmatis has been isolated by homologous recombination using a sacB counterselection strategy. Deletion of the recA gene from the chromosome was demonstrated by Southern hybridizations and by polymerase chain reaction (PCR). Western analysis using anti‐RecA antibodies confirmed that the RecA protein was not made by the mutant strain. The recA deletion strain exhibited enhanced sensitivity to UV irradiation and failed to undergo homologous recombination. The results obtained from the recombination assays suggest that in wild‐type M. smegmatis the majority of colonies arise from single cross‐over homologous recombination events with only a very minor contribution from random integrations. The deficiencies in UV survival and recombination were complemented by introduction of the cloned M. smegmatis recA gene. Overexpression of RecA was found to be toxic in the absence of recX, which is found downstream of and co‐transcribed with recA and is thus also affected by the deletion of recA. The M. smegmatis recA deletion strain was also complemented by the M. tuberculosis recA gene with or without its intein; most importantly, the frequency of double cross‐over homologous recombination events was identical regardless of whether the M. tuberculosis recA gene contained or lacked the intein. Thus, the low frequency of homologous recombination observed in M. tuberculosis is not due to the presence of an intein‐coding sequence in its recA gene per se.
Role of the Myb‐like protein Bas1p in Saccharomyces cerevisiae: a proteome analysis
Authors: Denis Valérie , Boucherie Hélian , Monribot Christelle , Daignan‐Fornier Bertrand
Published: March 2003

The effect of extracellular adenine and the role of the transcriptional activator Bas1p on expression of the yeast genome was assessed by two‐dimensional (2D) analysis of the yeast proteome. These data combined with LacZ fusions and northern blot analysis allow us to show that synthesis of enzymes for all 10 steps involved in purine de novo synthesis is repressed in the presence of adenine and requires BAS1 and BAS2 for optimal expression. We also show that expression of ADE12 and ADE13, the two genes required for synthesis of AMP from inosine 5′monophosphate (IMP), is co‐regulated with the de novo pathway genes. The same combined approach, used to study histidine biosynthesis gene expression, showed that HIS1 and HIS4 expression is co‐regulated with purine biosynthesis genes whereas HIS2, HIS3, HIS5 and HIS6 expression is not. This work, together with previously published data, gives the first comprehensive overview of the regulation of purine and histidine pathways in a eukaryotic organism. Finally, the expression of two pyrimidine biosynthesis genes URA1 and URA3 was found to be severely affected by bas1 and bas2 mutations in the absence of adenine, establishing a regulatory link between the two nucleotide biosynthesis pathways.
Role of fimbriae‐mediated adherence for neutrophil migration across Escherichia coli‐infected epithelial cell layers
Authors: Godaly G. , Frendéus B. , Proudfoot A. , Svensson M. , Klemm P. , Svanborg C.
Published: March 2003

This study examined the role of P and type 1 fimbriae for neutrophil migration across Escherichia coli‐infected uroepithelial cell layers in vitro and for neutrophil recruitment to the urinary tract in vivo. Recombinant E. coli K‐12 strains differing in P or type 1 fimbrial expression were used to infect confluent epithelial layers on the underside of transwell inserts. Neutrophils were added to the upper well, and their passage across the epithelial cell layers was quantified. Infection with the P‐ and type 1‐fimbriated recombinant E. coli strains stimulated neutrophil migration to the same extent as a fully virulent clinical E. coli isolate, but the isogenic non‐fimbriated vector control strains had no stimulatory effect. The enhancement of neutrophil migration was adhesion dependent; it was inhibited by soluble receptor analogues blocking the binding of P fimbriae to the globoseries of glycosphingolipids or of type 1 fimbriae to mannosylated glycoprotein receptors. P‐ and type 1‐fimbriated E. coli triggered higher interleukin (IL) 8 secretion and expression of functional IL‐8 receptors than non‐fimbriated controls, and the increase in neutrophil migration across infected cell layers was inhibited by anti‐IL‐8 antibodies. In a mouse infection model, P‐ or type 1‐fimbriated E. coli stimulated higher chemokine (MIP‐2) and neutrophil responses than the non‐fimbriated vector controls. The results demonstrated that transformation with the pap or fim DNA sequences is sufficient to convert an E. coli K‐12 strain to a host response inducer, and that fimbriation enhances neutrophil recruitment in vitro and in vivo. Epithelial chemokine production provides a molecular link between the fimbriated bacteria that adhere to epithelial cells and tissue inflammation.
Cellular function of elastase in Pseudomonas aeruginosa: role in the cleavage of nucleoside diphosphate kinase and in alginate synthesis
Authors: Kamath Shilpa , Kapatral Vinayak , Chakrabarty A. M.
Published: March 2003

Elastase is a major virulence factor in Pseudomonas aeruginosa that is believed to cause extensive tissue damage during infection in the human host. Elastase is secreted in non‐mucoid P. aeruginosa. It is known that secretion of most virulence factors such as elastase, lipase, exotoxin A, etc., in P. aeruginosa is greatly reduced in alginate‐secreting mucoid cells isolated from the lungs of cystic fibrosis (CF) patients. We have previously reported that in mucoid P. aeruginosaan intracellular protease cleaves the 16 kDa form of nucleoside diphosphate kinase (Ndk) to a truncated 12 kDa form. This smaller form is membrane associated and has been observed to form complexes with specific proteins to predominantly generate GTP, an important molecule in alginate synthesis. The main aim of this study was to purify and characterize this protease. The protease was purified by hydrophobic interaction chromatography of the crude extract of mucoid P. aeruginosa 8821, a CF isolate. Further analysis using a gelatin containing SDS–polyacrylamide gel detected the presence of a 103 kDa protease, which when boiled, migrated as a 33 kDa protein on a SDS–polyacrylamide gel. The first 10 amino acids from the N‐terminus of the 33 kDa protease showed 100% identity to the mature form of elastase. An elastase‐negative lasB ::Cm knock‐out mutant in the mucoid 8821 background was constructed, and it showed a non‐mucoid phenotype. This mutant showed the presence of only the 16 kDa form of Ndk both in the cytoplasm and membrane fractions. We present evidence for the retention of active elastase in the periplasm of mucoid P. aeruginosa and its role in the generation of the 12 kDa form of Ndk. Finally, we demonstrate that elastase, when overproduced in both mucoid and non‐mucoid cells, stimulates alginate synthesis. This suggests that the genetic rearrangements that trigger mucoidy in P. aeruginosa also allow retention of elastase in the periplasm in an active oligomeric form that facilitates cleavage of 16 kDa Ndk to its 12 kDa form for the generation of GTP, required for alginate synthesis.
The macrolide–ketolide antibiotic binding site is formed by structures in domains II and V of 23S ribosomal RNA
Authors: Hansen Lykke Haastrup , Mauvais Pascale , Douthwaite Stephen
Published: March 2003

The macrolide antibiotic erythromycin interacts with bacterial 23S ribosomal RNA (rRNA) making contacts that are limited to hairpin 35 in domain II of the rRNA and to the peptidyl transferase loop in domain V. These two regions are probably folded close together in the 23S rRNA tertiary structure and form a binding pocket for macrolides and other drug types. Erythromycin has been derivatized by replacing the L‐cladinose moiety at position 3 by a keto group (forming the ketolide antibiotics) and by an alkyl‐aryl extension at positions 11/12 of the lactone ring. All the drugs footprint identically within the peptidyl transferase loop, giving protection against chemical modification at A2058, A2059 and G2505, and enhancing the accessibility of A2062. However, the ketolide derivatives bind to ribosomes with widely varying affinities compared with erythromycin. This variation correlates with differences in the hairpin 35 footprints. Erythromycin enhances the modification at position A752. Removal of cladinose lowers drug binding 70‐fold, with concomitant loss of the A752 footprint. However, the 11/12 extension strengthens binding 10‐fold, and position A752 becomes protected. These findings indicate how drug derivatization can improve the inhibition of bacteria that have macrolide resistance conferred by changes in the peptidyl transferase loop.
A new level in the Vibrio cholerae ToxR virulence cascade: AphA is required for transcriptional activation of the tcpPH operon
Authors: Skorupski Karen , Taylor Ronald K.
Published: March 2003

The expression of the ToxR virulence regulon is dependent upon the regulatory proteins ToxR/ToxS, TcpP/TcpH and ToxT. We describe here a previously unidentified gene in Vibrio cholerae, aphA (activator of tcpP and tcpH expression), which is required for the transcription of the tcpPH operon. Under conditions normally optimal for virulence gene expression, an in frame aphA deletion decreased the expression of a cholera toxin promoter fusion (ctx–lacZ ) and prevented the production of the toxin co‐regulated pilus (TCP). Plasmids producing ToxT or TcpP/H, but not ToxR, restored ctx–lacZ expression and TCP production in the ΔaphA strain, suggesting that the mutation interferes with toxT expression by influencing the transcription of tcpPH. Indeed, the expression of a chromosomal tcpP–lacZ fusion was reduced in the ΔaphA mutant and increased in both V. cholerae and Escherichia coli by introducing aphA expressed from an inducible promoter. These results support a model in which AphA functions at a previously unknown step in the ToxR virulence cascade to activate the transcription of tcpPH. TcpP/TcpH, together with ToxR/ToxS, then activate the expression of toxTresulting ultimately in the production of virulence factors such as cholera toxin and TCP.
The alarmone (p)ppGpp mediates physiological‐responsive control at the σ54‐dependent Po promoter
Authors: Sze Chun Chau , Shingler Victoria
Published: March 2003

Transcription from the Pseudomonas‐derived σ54‐dependent Po promoter of the dmp operon is mediated by the aromatic‐responsive regulator DmpR. However, physiological control is superimposed on this regulatory system causing silencing of the DmpR‐mediated transcriptional response in rich media until the transition between exponential and stationary phase is reached. Here, the positive role of the nutritional alarmone (p)ppGpp in DmpR regulation of the Po promoter has been identified and investigated in vivo. Overproduction of (p)ppGpp in a Pseudomonas reporter system was found to allow an immediate transcriptional response under normally non‐permissive conditions. Conversely (p)ppGpp‐deficient Escherichia coli strains were found to be severely defective in DmpR‐mediated transcription, demonstrating the requirement for this metabolic signal. A subset of mutations in the β, β′ and σ70 subunits of RNA polymerase, which confer prototrophy on ppGpp0E. coli, was also found to restore specific DmpR‐mediated transcription from Po, suggesting that the metabolic signal is mediated directly through the σ54‐RNA polymerase. These data provide a direct mechanistic link between the physiological status of the cell and expression from σ54 promoters.