bluuebunny/crossref_metadata_2025 huggingface dataset:
Patterns of genetic diversity and biogeographical history of the tropical wetland tree, Pterocarpus officinalis (Jacq.), in the Caribbean basin
Authors: Rivera‐Ocasio E. , Aide T. M. , McMillan W. O.
Published: March 2003

Studies examining intraspecific variation in plant species with widespread distributions and disjunct populations have mainly concentrated on temperate species. Here, we determined the genetic structure of a broadly distributed wetland tropical tree, Pterocarpus officinalis (Jacq.), from eight Neotropical populations using amplified length fragment polymorphisms (AFLP). AFLPs proved highly variable with almost half (48%) of the genetic variation at these loci occurring among individuals within populations. Nonetheless, there was a strong geographical pattern in the distribution of AFLP variation within P. officinalis. Caribbean and continental populations fell into two well‐defined genetic clusters supported by the presence of a number of unique AFLP bands. Within these two regions, there were also strong genetic differences among populations, caused mainly by frequency differences in AFLP bands, making it difficult to determine the evolutionary relationships among populations. In addition, our analysis of P. officinalis revealed striking differences in the levels of AFLP variation among the eight populations sampled. In general, Caribbean populations had lower genetic diversity than continental populations. Moreover, there was a clear loss in AFLP diversity with distance from the continent among Caribbean populations. The overall genetic pattern within P. officinalis suggests that past colonization history, coupled with genetic drift within local populations, rather than contemporary gene flow are the major forces shaping variation within this species.
Comparison of genetic diversity estimates within and among populations of maritime pine using chloroplast simple‐sequence repeat and amplified fragment length polymorphism data
Authors: Ribeiro M. M. , Mariette S. , Vendramin G. G. , Szmidt A. E. , Plomion C. , Kremer A.
Published: March 2003

We compared the genetic variation of Pinus pinaster populations using amplified fragment length polymorphism (AFLP) and chloroplast simple‐sequence repeat (cpSSR) loci. Populations’ levels of diversity within groups were found to be similar with AFLPs, but not with cpSSRs. The high interlocus variance associated with the AFLP loci could account for the lack of differences in the former. Although AFLPs revealed much lower genetic diversity than cpSSRs, the levels of among‐population differentiation found with the two types of marker were similar, provided that loci showing fewer than four null‐homozygotes, in any population, were pruned from the AFLP data. Moreover, the French and Portuguese populations were clearly differentiated from each other, with both markers. The Mantel test showed that the genetic distance matrix calculated using the AFLP data was correlated with the matrix derived from the cpSSRs. Because of the concordance found between markers we conclude that gene flow was indeed the predominant force shaping nuclear and chloroplastic genetic variation of the populations within regions, at the geographical scale studied.
Spawning times, reproductive compatibilities and genetic structuring in theAcropora asperagroup: evidence for natural hybridization and semi‐permeable species boundaries in corals
Authors: Van Oppen Madeleine J. H. , Willis Bette L. , Van Rheede Teun , Miller David J.
Published: March 2003

Species boundaries among five sympatric coral species of the Indo‐PacificAcropora asperagroup were examined by a combination ofin vitrobreeding trials, comparisons of spawning times and DNA sequence analysis of ribosomal DNA internal transcribed spacer (rDNA ITS) and 5.8S regions. The breeding trials showed that reproductive compatibility exists between at least some colonies of all the species pairs tested, suggesting a large potential for natural hybridization and introgression. TheAcroporaITS regions exhibited extremely high levels of variability (up to ∼62% for ITS1, ∼11% for 5.8S and ∼43% for ITS2), but most of the variation was shared among four of the five species,A. millepora,A. papillare,A. pulchraandA. spathulata,consistent with extensive introgression. Phylogenetic analyses did not resolve these four species as distinct clusters across a wide biogeographic region stretching from the southern Great Barrier Reef to Papua New Guinea. However, most colonies of the fifth species,A. aspera, constituted a distinct clade in phylogenetic analyses. This is consistent with our observations of a semi‐permeable temporal barrier involving differences in spawning times between this and the other four species. Although the majority of colonies of all five species generally spawned within 90 min of each other, in two out of four years, gametes were absent prior to mass spawning episodes from at least someA. asperacolonies. Hence, our data suggest that transient reproductive barriers may be the result of year‐to‐year variation in the date of spawning and that this difference in spawning time contributes to the genetic structure detected amongAcroporaspecies in this group. Occasional leakage through the reproductive barrier was confirmed by the observation ofA. aspera ×A. pulchraF1hybrids, identified based on additivity of ITS sequences.
Offspring sexual dimorphism and sex‐allocation in relation to parental age and paternal ornamentation in the barn swallow
Authors: Saino N. , Ambrosini R. , Martinelli R. , Calza S. , Møller A. P. , Pilastro A.
Published: March 2003

We analysed the morphology of nestling barn swallows (Hirundo rustica) in relation to their sex, and laying and hatching order. In addition, we studied sex‐allocation in relation to parentage, parental age and expression of a secondary sexual character of fathers. Molecular sexing was conducted using the sex chromosome‐linked avian CHD1 gene. Sex of the offspring was not associated with laying or hatching order. None of nine morphological, serological and immunological variables varied in relation to offspring sex. Sexual dimorphism did not vary in relation to parental age and expression of a paternal secondary sexual character. The proportion of sons declined with brood size. Individual males and females had a similar proportion of sons during consecutive breeding years. The proportion of sons of individual females declined with age, but increased with the expression of a secondary sexual character of their current mate. The generalized lack of variation in sexual dimorphism among nestlings may suggest that barn swallows do not differentially invest in sons vs. daughters. Alternatively, male offspring may require different parental effort compared to their female siblings in order to attain the same morphological state. The lack of variation in offspring sexual dimorphism with paternal ornamentation suggests no adjustment of overall parental effort in relation to reproductive value of the two sexes. However, male‐biased sex ratio among offspring of highly ornamented males may represent an adaptive sex‐allocation strategy because the expression of male ornaments is heritable and highly ornamented males are at a sexual selection advantage.
Microsatellite variation in the reintroduced Pennsylvania elk herd
Authors: Lenney Williams Christen , Serfass Thomas L. , Cogan Rawland , Rhodes Olin E.
Published: March 2003

Relocation programs have restored elk (Cervus elaphus) to portions of its vast historical range. We examine the consequences of these relocation programs by assessing variation at 10 microsatellite loci in three elk herds, a source herd (Yellowstone National Park), a large herd reintroduced from Yellowstone (Custer State Park) and a bottlenecked herd reintroduced from both Yellowstone and Custer (the Pennsylvania herd). Observed single locus heterozygosities ranged from 0.000 to 0.739. Multi‐locus heterozygosities ranged from 0.222 to 0.589. Although significant differences were detected among all three herds, the Yellowstone National Park and Custer State Park herds possessed similar levels of variation and heterozygosity, and the genetic distance between these two herds was small. The Pennsylvania herd, on the other hand, experienced a 61.5% decrease in heterozygosity relative to its source herds, possessed no unique and few rare alleles, and the genetic distances between the Pennsylvania herd and its sources were large. Simulations were performed to identify bottleneck scenarios in agreement with levels of variation in the Pennsylvania herd. Our data confirm that the rate of population growth post‐relocation may have important genetic consequences and indicate that theoretical predictions regarding the maintenance of genetic variation during relocation events must be viewed with caution when small numbers of a polygynous species are released.
Population differentiation and nuclear gene flow in the Dominican anole (Anolis oculatus)
Authors: Stenson Andrew G. , Malhotra Anita , Thorpe Roger S.
Published: March 2003

Allele frequency data from nuclear microsatellite loci were used to investigate patterns of nuclear gene flow and population structure in the morphologically variable Dominican anole (Anolis oculatus). All six loci used proved to be highly polymorphic, with an average of 18.8 alleles per locus. Test for Hardy–Weinberg equilibrium revealed small numbers of heterozygote deficiencies at single loci in single populations and consistent patterns of increasingly significant heterozygote deficiency in global tests across populations and loci. No significant relationship between FST and patristic distances estimated from mitochondrial DNA sequences was detected and estimates of FIS were significantly higher in females than in males, indicating that gene flow may be sex‐biased and mediated mainly by male migration. A highly significant correlation between linearized FST and loge (geographical distance) indicates that geographical proximity is a significant factor in the genetic structure of A. oculatus populations. However, levels of gene flow between morphologically differentiated parapatric populations are frequently seen to be relatively high. This supports the hypothesis of natural selection being the driving force behind the development and maintenance of morphological variation and shows that adaptive differentiation may be maintained despite the homogenizing influence of gene flow. Generally, the morphologically variable populations of A. oculatus are seen to be poor candidates for in situ speciation, but an exceptional case on the west coast of Dominica indicates that isolation resulting from vicariant events may lead to rapid differentiation at both mitochondrial and nuclear loci. This provides a possible mechanism for anole speciation on other Caribbean islands.
Major histocompatibility complex variation in red wolves: evidence for common ancestry with coyotes and balancing selection
Authors: Hedrick P. W. , Lee R. N. , Garrigan D.
Published: March 2003

We examined variation at a class II major histocompatibility complex (MHC) gene (DRB1) in the captive red wolf population and samples of coyotes from Texas and North Carolina. We found 4 alleles in the 48 red wolves, 8 alleles in the 10 coyotes from Texas and 15 alleles in the 29 coyotes from North Carolina. Two of the four alleles found in red wolves, Caru‐2 and Caru‐4, were found in both the Texas and North Carolina coyote samples. Allele Caru‐1, previously found in gray wolves, was also found in the North Carolina sample. The most frequent red wolf allele, Caru‐3, was not found in any of the coyote samples. However, an allele found in both the Texas and North Carolina coyote samples is only one nucleotide (one amino acid) different from this red wolf allele. Overall, it appears from examination of this MHC gene that red wolves are more closely related to coyotes than to gray wolves. There were a number of different types of evidence supporting the action of balancing selection in red wolves. Namely, there was: (i) an excess of heterozygotes compared with expectations; (ii) a higher rate of nonsynonymous than synonymous substitution for the functionally important antigen‐binding site positions; (iii) an eight times higher average heterozygosity of individual amino acids at the positions identified as part of the antigen‐binding site than those not associated with it; (iv) the amino acid divergence of four red wolf alleles was greater than that expected from a simulation of genetic drift; and (v) the distribution of alleles, and the distributions of amino acids at many positions were more even than expected from neutrality. Examination of the level and pattern of linkage disequilibria between pairs of sites suggest that the heterozygosity, substitution and frequencies at individual amino acids are not highly dependent upon each other.
Wolbachiainfections and superinfections in cytoplasmically incompatible populations of the European cherry fruit flyRhagoletis cerasi(Diptera, Tephritidae)
Authors: Riegler Markus , Stauffer Christian
Published: March 2003

Wolbachiais an obligately intracellular, maternally inherited bacterium which has been detected in many arthropods.Wolbachiainfections disperse in host populations by mechanisms such as cytoplasmic incompatibility (CI). CI leads to embryonic mortality which occurs when infected males mate with uninfected females or females with a differentWolbachiastrain. Populations of the European cherry fruit flyRhagoletis cerasi(Diptera, Tephritidae) were found to be infected by two differentWolbachiastrains,wCer1 andwCer2. Superinfections with both strains occurred throughout southern and central Europe and infections withwCer1 were found in northern, western and eastern Europe. Strong unidirectional CI between European populations ofR. cerasiwere first reported in the 1970s. From the conformity in the recent geographical distribution of theWolbachiainfections and the CI expression patterns found 25 years ago it was deduced thatwCer2 potentially causes CI inR. cerasi. The comparison of the geographical distributions indicated thatwCer1 + 2 must have spread intowCer1‐infected populations in some areas. In other regions, a spread ofwCer1 + 2 was probably prevented by dispersal barriers. There, a sharp transition from infected to superinfected populations suggested regional isolation betweenwCer1 andwCer1 + 2‐infected populations.
Slow mitochondrial DNA sequence evolution in the Anthozoa (Cnidaria)
Authors: Shearer T. L. , van Oppen M. J. H. , Romano S. L. , Wörheide G.
Published: March 2003

Mitochondrial genes have been used extensively in population genetic and phylogeographical analyses, in part due to a high rate of nucleotide substitution in animal mitochondrial DNA (mtDNA). Nucleotide sequences of anthozoan mitochondrial genes, however, are virtually invariant among conspecifics, even at third codon positions of protein‐coding sequences. Hence, mtDNA markers are of limited use for population‐level studies in these organisms. Mitochondrial gene sequence divergence among anthozoan species is also low relative to that exhibited in other animals, although higher level relationships can be resolved with these markers. Substitution rates in anthozoan nuclear genes are much higher than in mitochondrial genes, whereas nuclear genes in other metazoans usually evolve more slowly than, or similar to, mitochondrial genes. Although several mechanisms accounting for a slow rate of sequence evolution have been proposed, there is not yet a definitive explanation for this observation. Slow evolution and unique characteristics may be common in primitive metazoans, suggesting that patterns of mtDNA evolution in these organisms differ from that in other animal systems.
Similar estimates of population genetic composition and sex ratio derived from carcasses and faeces of Eurasian otter Lutra lutra
Authors: Dallas John F. , Coxon Karen E. , Sykes Tim , Chanin Paul R. F. , Marshall Freda , Carss David N. , Bacon Philip J. , Piertney Stuart B. , Racey Paul A.
Published: March 2003

Collecting faeces is viewed as a potentially efficient way to sample elusive animals. Nonetheless, any biases in estimates of population composition associated with such sampling remain uncharacterized. The goal of this study was to compare estimates of genetic composition and sex ratio derived from Eurasian otter Lutra lutra spraints (faeces) with estimates derived from carcasses. Twenty per cent of 426 wild‐collected spraints from SW England yielded composite genotypes for 7–9 microsatellites and the SRY gene. The expected number of incorrect spraint genotypes was negligible, given the proportions of allele dropout and false allele detection estimated using paired blood and spraint samples of three captive otters. Fifty‐two different spraint genotypes were detected and compared with genotypes of 70 otter carcasses from the same area. Carcass and spraint genotypes did not differ significantly in mean number of alleles, mean unbiased heterozygosity or sex ratio, although statistical power to detect all but large differences in sex ratio was low. The genetic compositions of carcass and spraint genotypes were very similar according to confidence intervals of θ and two methods for assigning composite genotypes to groups. A distinct group of approximately 11 carcass and spraint genotypes was detected using the latter methods. The results suggest that spraints can yield unbiased estimates of population genetic composition and sex ratio.
American marten (Martes americana) in the Pacific Northwest: population differentiation across a landscape fragmented in time and space
Authors: Small Maureen P. , Stone Karen D. , Cook Joseph A.
Published: March 2003

American marten (Martes americana) have a close association with mature temperate forests, a habitat that expanded throughout the Pacific Northwest as glaciers receded at the end of the Pleistocene. Similar to several other forest‐associated mammals in North America (e.g. black bear), genetic analysis of the marten shows a deep phylogeographical subdivision that reflects populations with distinctive evolutionary histories. Using a suite of 14 microsatellite markers, we explored the genetic structure of marten populations in two reciprocally monophyletic clades in the Pacific Northwest identified previously as M. caurina and M. americana by mitochondrial haplotypes and morphology. Microsatellite phylogeographical patterns were congruent with mitochondrial analyses. These independent data sets shed light upon hybridization patterns, population structure and evolutionary histories. Hybridization between M. caurina and M. americana individuals was documented in two regions of sympatry (Kuiu Island in southeastern Alaska and southern Montana). Northern insular populations of M. caurina exhibited higher differentiation and lower variability relative to northern populations of M. americana. Greater divergence among M. caurina populations may reflect longer isolation and persistence in coastal forest habitat that was fragmented by rising sea level in the early Holocene. Lower differentiation among northern M. americana populations and close relationship to other continental M. americana populations may reflect more recent expansion into the Pacific Northwest and/or continued gene flow among populations. Differentiation among M. caurina populations was attributed to habitat fragmentation (i.e. rising sea level), as opposed to isolation‐by‐distance; oceanic straits pose significant barriers to gene flow among M. caurina populations and between populations of M. caurina and M. americana.
Nuclear DNA identification of migrating bull trout captured at the Puget Sound Energy diversion dam on the White River, Washington State
Authors: Baker J. D. , Moran P. , Ladley R.
Published: March 2003

Bull trout (Salvelinus confluentus) is a char listed as threatened under the United States Endangered Species Act throughout its range in the coterminous United States. Substantial morphological similarities between bull trout and Dolly Varden (S. malma) make field identification difficult. This has resulted in an incomplete understanding of their distribution and abundance in Washington State where these two species occur sympatrically. We used three diagnostic nuclear loci to determine the species of char collected at a trap on the White River in southern Puget Sound (Washington State, USA). Each of the 104 samples revealed the expected bull trout genotype at all three loci. This work presents three principle results: (i) the presence of a migratory bull trout population in southern Puget Sound; (ii) no evidence of migratory Dolly Varden over 3 years; and (iii) no evidence of hybridization was detected. These results also demonstrate how molecular markers can provide information essential to the conservation and management of these species.
Control of the expression of the manXYZ operon in Escherichia coli: Mlc is a negative regulator of the mannose PTS
Authors: Plumbridge Jacqueline
Published: March 2003

The manXYZ operon of Escherichia coli encodes a sugar transporter of the phosphoenol pyruvate (PEP)‐dependent phosphotransferase system, which is capable of transporting many sugars, including glucose, mannose and the aminosugars, glucosamine and N‐acetylglucosamine. Transcription of manX is strongly dependent on cyclic AMP (cAMP)/cAMP receptor protein (CAP). A cAMP/CAP binding site is located at −40.5, and activation by cAMP/CAP is shown to be typical of a class II promoter. The 5′ end of a transcript, potentially encoding two proteins, is expressed divergently from the manXYZ operon. Previously, two binding sites for the NagC repressor were detected upstream of manX, but a mutation in nagC has very little effect on manX expression. However, a mutation in the mlc gene, encoding a homologue of nagC, results in a threefold derepression of manX expression, suggesting that this protein is a more important regulator of manX expression than NagC. The Mlc protein binds to the NagC operators, binding preferentially to the promoter‐proximal operator. Plasmids overproducing either the NagC protein or the Mlc protein repress the expression of manX, but the effect of the Mlc protein is stronger. The mlc gene is shown to be allelic with the previously characterized dgsA mutation affecting the mannose phosphoenolpyruvate‐dependent phosphotransferase system (PTS).
MvaL1 autoregulates the synthesis of the three ribosomal proteins encoded on the MvaL1 operon of the archaeon Methanococcus vannielii by inhibiting its own translation before or at the formation of the first peptide bond
Authors: Mayer Christine , Köhrer Caroline , Gröbner Peter , Piendl Wolfgang
Published: March 2003

The control of ribosomal protein synthesis has been investigated extensively in Eukarya and Bacteria. In Archaea, only the regulation of the MvaL1 operon (encoding ribosomal proteins MvaL1, MvaL10 and MvaL12) of Methanococcus vannielii has been studied in some detail. As in Escherichia coli, regulation takes place at the level of translation. MvaL1, the homologue of the regulatory protein L1 encoded by the L11 operon of E. coli, was shown to be an autoregulator of the MvaL1 operon. The regulatory MvaL1 binding site on the mRNA is located about 30 nucleotides downstream of the ATG start codon, a sequence that is not in direct contact with the initiating ribosome. Here, we demonstrate that autoregulation of MvaL1 occurs at or before the formation of the first peptide bond of MvaL1. Specific interaction of purified MvaL1 with both 23S RNA and its own mRNA is confirmed by filter binding studies. In vivo expression experiments reveal that translation of the distal MvaL10 and MvaL12 cistrons is coupled to that of the MvaL1 cistron. A mRNA secondary structure resembling a canonical L10 binding site and preliminary in vitro regulation experiments had suggested a co‐regulatory function of MvaL10, the homologue of the regulatory protein L10 of the β‐operon of E. coli. However, we show that MvaL10 does not have a regulatory function.
Histoplasma acquisition of calcium and expression of CBP1 during intracellular parasitism
Authors: West Batanghari Janet , Deepe George S. , Di Cera Enrico , Goldman William E.
Published: March 2003

A highly adapted parasite of macrophages, the yeast phase of Histoplasma capsulatum, survives and proliferates within phagolysosomes, while the mycelial phase exists only as a saprophyte in the soil. We have shown previously that these two phases of Histoplasma differ in their calcium requirements for growth and in the production of a released calcium‐binding protein (CBP). Cloning and sequencing the CBP1 gene revealed two introns, a putative signal peptide and potential calcium‐binding sites. We also evaluated CBP1 expression by reverse transcription–polymerase chain reaction (RT–PCR) of yeasts grown in broth culture and within two host cell types, a macrophage‐like cell line and respiratory epithelial cells. H. capsulatum yeasts expressed CBP1 in all of these settings. Splenocytes from mice immunized with H. capsulatum yeasts responded to purified CBP in proliferation assays, providing evidence for the production of CBP during the infection of mammalian hosts. In addition, after H. capsulatum yeasts were subjected to a calcium‐free shock, exogenously added CBP allowed yeasts to incorporate more calcium than yeasts incubated without added CBP. These results suggest that CBP may function to provide yeasts with calcium when they are in a low‐calcium environment, such as the phagolysosomal compartment within macrophages.
Activation of the toluene‐responsive regulator XylR causes a transcriptional switch between σ54 and σ70 promoters at the divergent Pr/Ps region of the TOL plasmid
Authors: Bertoni Giovanni , Marqués Silvia , De Lorenzo Víctor
Published: March 2003

The mechanism by which XylR, the toluene‐responsive activator of the σ54‐dependent Pu and Ps promoters of the Pseudomonas TOL plasmid pWW0, downregulates its own σ70 promoter Pr has been examined. An in vitro transcription system was developed in order to reproduce the repression of Pr observed in cells of P. putida (pWW0) both in the presence and in the absence of the XylR inducer, benzyl alcohol. DNA templates bearing the two σ70‐RNA polymerase (RNAP) binding sites of Pr, which overlap the upstream activating sequences (UAS) for XylR in the divergent σ54 promoter Ps, were transcribed in the presence of a constitutively active XylR variant deleted of its N‐terminal domain (XylRΔA). The addition of ATP, known to trigger multimerization of the regulator at the UAS, enhanced the repression of Pr by XylR. Furthermore, we observed activation of the divergent σ54 promoter Ps during Pr downregulation by XylRΔA. These results support the notion that activation of XylR by aromatic inducers in vivo triggers a transcriptional switch between Pr and PsSuch a switch is apparently caused by the ATP‐dependent multimerization and strong DNA binding of the protein required for activation of the σ54 promoter. This device could reset the level of XylR expression during activation of the σ54Pu and Ps promoters of the TOL plasmid.
Differential interaction of the transcription factor PrfA and the PrfA‐activating factor (Paf) of Listeria monocytogenes with target sequences
Authors: Dickneite Carmen , Böckmann Regine , Spory Andrea , Goebel Werner , Sokolovic Zeljka
Published: March 2003

The interaction of the purified PrfA transcription factor with the regulatory sequences located upstream of the PrfA‐dependent listeriolysin (hly) and internalin (inlA) genes was studied in the presence and in the absence of Paf (PrfA‐activating factor)‐containing extracts. It is shown that PrfA protein is able to bind, independently of additional factors, to a 109 bp DNA fragment including the entire hly promoter sequence with the anticipated PrfA binding site (‘PrfA‐box’). PrfA alone, but not in combination with Paf, can also bind to a shorter target sequence of 28 bp comprising essentially the PrfA‐box of the hly promoter. The addition of a Paf‐containing extract does not lead to significant protein binding to these two hly target sequences in the absence of PrfA but converts the complex (CIII) consisting of PrfA and the 109 bp hly DNA fragment to a slower migrating PrfA–Paf–DNA complex (CI). Incubation of cell‐free extracts of wild‐type Listeria monocytogenes with the 109 bp DNA fragment leads to the formation of CI. The addition of polyclonal PrfA antibodies causes a supershift of CIII. Purified PrfA and PrfA–Paf also bind to a DNA fragment containing the PrfA‐dependent promoter P2 of inlA, albeit at a lower rate when compared with the corresponding hly sequence. In contrast to the hly target DNA, the inlA promoter sequence efficiently binds Paf alone, and this Paf binding reduces that of PrfA and PrfA–Paf to the inlA target DNA. DNase I footprint experiments show that purified PrfA protects sequences of dyad symmetry previously proposed as PrfA binding sites in the hly and in the inlA promoter regions.
ClpX and ClpP are essential for the efficient acquisition of genes specifying type IA and IB restriction systems
Authors: Makovets Svetlana , Titheradge Annette J. B. , Murray Noreen E.
Published: March 2003

Efficient acquisition of genes that encode a restriction and modification (R–M) system with specificities different from any already present in the recipient bacterium requires the sequential production of the new modification enzyme followed by the restriction activity in order that the chromosome of the recipient bacterium is protected against attack by the restriction endonuclease. We show that ClpX and ClpP, the components of ClpXP protease, are necessary for the efficient transmission of the genes encoding EcoKI and EcoAI, representatives of two families of type I R–M systems, thus implicating ClpXP in the modulation of restriction activity. Loss of ClpX imposed a bigger barrier than loss of ClpP, consistent with a dual role for ClpX, possibly as a chaperone and as a component of the ClpXP protease. Transmission of genes specifying EcoKI was more dependent on ClpX and ClpP than transmission of the genes for EcoAI. Sensitivity to absence of the protease was also influenced by the mode of gene transfer; conjugative transfer and transformation were more dependent on ClpXP than transduction. In the absence of either ClpX or ClpP transfer of the EcoKI genes by P1‐mediated transduction was impaired, transfer of the EcoAI genes was not.
Sites internal to the coding regions of phoA and pstS bind PhoP and are required for full promoter activity
Authors: Liu Wei , Qi Ying , Hulett F. Marion
Published: March 2003

Bacillus subtilis PhoP and PhoR, a pair of two‐component regulatory proteins, regulate the phosphate starvation response. Here, we used two other pho regulon promoters, the phoA and pstS promoters, to examine the mechanism of PhoP‐specific activation of its target promoters. Both gel shift and DNase I footprinting assays indicate that PhoP bound to the two promoters. Unphosphorylated PhoP bound only to the multiple TTAACA‐like sequences upstream of these two promoters, while phosphorylated PhoP extended the binding region in both the 5′ and the 3′ direction and, additionally, protected sequences internal to the coding region of these two genes. The PhoP binding sites in the coding region were necessary for full induction from either promoter during phosphate starvation. Deletion of these sites eliminated approximately 75% and 45% of the induced promoter activity of the phoA and pstS promoters respectively. In vitro transcription assays using the phoA promoters with various 3′ ends confirmed the requirement of the PhoP∼P binding to the coding region for full promoter activity. The multiple TTAACA‐like sequences in the phoA and pstS promoters were essential for promoter activity, and deletion of one or more of these sequences in either promoter eliminated the promoter activity. Two pairs of TTAACA‐like sequences were required for efficient PhoP binding and were suggested to be one B. subtilis Pho box. Based on our data, we have proposed a model for activation of the phoA and the pstS promoter by PhoP.
Structure of the has operon promoter and regulation of hyaluronic acid capsule expression in group A Streptococcus
Authors: Albertí Sebastián , Ashbaugh Cameron D. , Wessels Michael R.
Published: March 2003

Group A streptococcal strains vary widely in the amount of hyaluronic acid capsule they produce, although the has operon, which encodes the enzymes required for hyaluronic acid synthesis, is highly conserved. The three genes making up the has operon are transcribed from a single promoter located upstream of the first gene in the operon, hasA. To investigate transcriptional regulation of capsule synthesis, we studied the structure and function of the has operon promoter sequences from two strains of group A Streptococcus: a highly encapsulated M‐type 18 strain and a poorly encapsulated M‐type 3 strain. Transcriptional fusions of the has operon promoter to a promoterless chloramphenicol acetyltransferase gene were constructed in a temperature‐sensitive shuttle vector. The influence of promoter structure on has operon transcription was reflected by chloramphenicol acetyl transferase activity in cell lysates of Escherichia coli harbouring the recombinant plasmids and in group A Streptococcus after integration of the promoter fusions into the streptococcal chromosome. Fusions including as few as 12 nucleotides upstream from the −35 site of the has promoter exhibited full activity, indicating that sequences further upstream do not affect has gene transcription. A transcriptional fusion of the has promoter from the highly encapsulated M‐type 18 strain was threefold more active than a similar construct from the poorly encapsulated M‐type 3 strain. Analysis of the promoter sequences for the two strains revealed differences in three nucleotides in the −35, −10 spacer region of the promoter and in four nucleotides in the + 2 to + 8 positions relative to the start site of hasA transcription. To determine the relative importance of the two groups of nucleotide substitutions, chimeric promoter sequences were constructed in which either of the two clusters of variant nucleotides from the M18 has promoter was substituted for the corresponding positions in the M3 has promoter. Analysis of these chimeric promoter fusions showed that sequence changes in both regions influenced promoter strength. These results define the limits of cis‐acting chromosomal sequences that influence transcription of the has operon and indicate that the fine structure of the promoter is an important determinant of capsule gene expression in group A Streptococcus.
Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen‐insensitive NADPH nitroreductase
Authors: Goodwin Avery , Kersulyte Dangeruta , Sisson Gary , Veldhuyzen van Zanten Sander J. O. , Berg Douglas E. , Hoffman Paul S.
Published: March 2003

Metronidazole (Mtz) is a critical component of combination therapies that are used against Helicobacter pylori, the major cause of peptic ulcer disease. Many H. pylori strains are Mtz resistant (MtzR), however, and here we show that MtzR results from loss of oxygen‐insensitive NADPH nitroreductase activity. The underlying gene (called ‘rdxA’) was identified in several steps: transformation of Mtz‐susceptible (MtzS) H. pylori with cosmids from a MtzR strain, subcloning, polymerase chain reaction (PCR) and DNA sequencing. We also found that (i) E. coli (normally MtzR) was rendered MtzS by a functional H. pylori rdxA gene; (ii) introduction of rdxA on a shuttle vector plasmid into formerly MtzRH. pylori rendered it MtzS; and (iii) replacement of rdxA in MtzSH. pylori with an rdxA::camR null insertion allele resulted in a MtzR phenotype. The 630 bp rdxA genes of five pairs of H. pylori isolates from infections that were mixed (MtzR/MtzS), but uniform in overall genotype, were sequenced. In each case, the paired rdxA genes differed from one another by one to three base substitutions. Typical rdxA genes from unrelated isolates differ by ≈ 5% in DNA sequence. Therefore, the near identity of rdxA genes from paired MtzR and MtzS isolates implicates de novo mutation, rather than horizontal gene transfer in the development of MtzR. Horizontal gene transfer could readily be demonstrated under laboratory conditions with mutant rdxA alleles. RdxA is a homologue of the classical nitroreductases (CNRs) of the enteric bacteria, but differs in cysteine content (6 vs. 1 or 2 in CNRs) and isoelectric point (pI = 7.99 vs. 5.4–5.6), which might account for its reduction of low redox drugs such as Mtz. We suggest that many rdxA (MtzR) mutations may have been selected by prior use of Mtz against other infections. H. pylori itself is an early risk factor for gastric cancer; the possibility that its carcinogenic effects are exacerbated by Mtz use, which is frequent in many societies, or the reduction of nitroaromatic compounds to toxic, mutagenic and carcinogenic products, may be of significant concern in public health.
Antisense inhibition of expression of cysteine proteinases does not affect Entamoeba histolytica cytopathic or haemolytic activity but inhibits phagocytosis
Authors: Ankri Serge , Stolarsky Tamara , Mirelman David
Published: March 2003

Inhibition of most of the expression of the cysteine proteinases of Entamoeba histolytica strain HM‐1: IMSS was successfully performed by transcription of ehcp5 antisense RNA using the promoter of ehg34, which encodes a L21 ribosomal protein of E. histolytica. We have generated a stable transfectant in which the overall level of cysteine proteinase activity is strongly reduced (≈ 90%). This transfectant has a normal growth rate in Diamond's TYI‐S‐33 medium, a cytopathic and haemolytic activity similar to the control HM‐1:IMSS pEhAct‐Neo transfectant but with a significantly lower phagocytic activity.
Replication at the telomeres of the Streptomyces linear plasmid pSLA2
Authors: Qin Zhongjun , Cohen Stanley N.
Published: March 2003

The Streptomyces linear plasmid pSLA2 initiates DNA replication bidirectionally towards its telomeres from a site located near the centre of the molecule; at the telomeres, the recessed ends of lagging strands are filled in by non‐displacing DNA synthesis. Here, we report experiments that test three proposed mechanisms for lagging‐strand fill‐in. We present data inconsistent with recombinational or terminal hairpin models for the formation of full‐length duplex pSLA2 DNA. Instead, we find that deletions in short, distantly separated homologous palindromes in the leading‐strand 3′ overhang prevent propagation of linear pSLA2 DNA, implicating a mechanism of palindrome‐mediated leading‐strand fold‐back in telomere replication. We further show that circularized pSLA2 DNA molecules are opened in vivo precisely at the terminal nucleotides of telomeres, generating functional linear replicons containing native telomeres covalently bound to a protein at their 5′ DNA termini. Together, our results support a model in which pairing of multiple widely separated pSLA2 palindromes anchors the 3′ end of the leading‐strand overhang to a site near the overhang's base — providing a recognition site for terminal‐protein‐primed DNA synthesis and subsequent endonucleolytic processing. Thus, the replication of Streptomyces plasmid telomeres may have features in common with the mechanism proposed for telomere replication in autonomous parvoviruses.
Viral escape from antisense RNA
Authors: Bull J. J. , Jacobson A. , Badgett M. R. , Molineux I. J.
Published: March 2003

RNA coliphage SP was propagated for several generations on a host expressing an inhibitory antisense RNA complementary to bases 31–270 of the positive‐stranded genome. Phages evolved that escaped inhibition. Typically, these escape mutants contained 3–4 base substitutions, but different sequences were observed among different isolates. The mutations were located within three different types of structural features within the predicted secondary structure of SP genomic RNA: (i) hairpin loops; (ii) hairpin stems; and (iii) the 5′ region of the phage genome complementary to the antisense molecule. Computer modelling of the mutant genomic RNAs showed that all of the substitutions within hairpin stems improved the Watson–Crick pairing of the stem. No major structural rearrangements were predicted for any of the mutant genomes, and most substitutions in coding regions did not alter the amino acid sequence. Although the evolved phage populations were polymorphic for substitutions, many substitutions appeared independently in two selected lines. The creation of a new, perfect, antisense RNA against an escape mutant resulted in the inhibition of that mutant but not of other escape mutants nor of the ancestral, unevolved phage. Thus, at least in this system, a population of viruses that evolved to escape from a single antisense RNA would require a cocktail of several antisense RNAs for inhibition.
Domain structure and RNA annealing activity of the Escherichia coli regulatory protein StpA
Authors: Cusick Michael E. , Belfort Marlene
Published: March 2003

The Escherichia coli regulatory protein StpA bears striking similarity to the chromatin‐associated protein H‐NS. These two proteins have many structural, functional and mechanistic parallels. Although H‐NS is more abundant in the cell, both proteins act as transcriptional regulators, both bind to curved DNA and both restrain DNA supercoils. However, StpA is better able to promote RNA annealing and trans‐splicing in vitro. In this study, phylogenetic analyses and experiments to examine the protease sensitivity of StpA and H‐NS suggest a similar structure for the two proteins. Both proteins consist of two structured domains separated by an exposed protease‐sensitive linker. The N‐terminal (StpA‐NterL) and C‐terminal (StpA‐CterL) domains of StpA, as well as the full‐length StpA and H‐NS proteins, were cloned, overproduced in E. coli and purified to homogeneity. StpA‐CterL, but not StpA‐NterL, promotes strand annealing of complementary RNA oligonucleotides and in vitro trans‐splicing of a model group I intron. Both StpA and StpA‐CterL exhibited stronger RNA‐modulating activity than H‐NS. Phylogenetic analyses showed that the N‐terminal and C‐terminal domains can exist autonomously. The phylogenetic and experimental data are compatible with a two‐domain model for StpA and H‐NS, with independently functioning modules joined by a non‐conserved linker, and with the observed RNA‐related activities residing entirely within the C‐terminal domain.
On the birth and fate of bacterial division sites
Authors: Bouché Jean‐Pierre , Pichoff Sébastien
Published: March 2003

Thanks to genetics, to the study of protein–protein interactions and to direct viewing of subcellular structures by the use of immunofluorescence and green fluorescent protein (GFP) fusions, the organization of the constriction apparatus of walled bacteria is gradually coming to light. The tubulin‐like protein FtsZ assembles as a ring around the site of constriction and operates as an organizer and activator of septum‐shaping proteins. Much less is known about the factors specifying the location of FtsZ rings. Circumstantial evidence favours the presence at future ring positions of fixed elements, the potential division sites (PDS), before FtsZ assembles. FtsZ polymerization is initiated from a point on a PDS, the nucleation site, still to be identified, and proceeds bidirectionally around the cell. We hypothesize that new PDS are specified in a manner that depends on the functioning of an active chromosome partition apparatus. This view is supported by the fact that formation of mid‐cell PDS requires initiation of DNA replication, and by recent studies supporting the existence of a specialized partition apparatus in a variety of microorganisms. Although PDS may be specified directly by the partition apparatus, indirect localization linked to compartmentalized gene expression during chromosome segregation is also possible. Once created, PDS are used in a regulated manner, and several mechanisms normally operate to direct constriction to selected PDS at the correct time. One, dedicated to the permanent suppression of polar PDS, rests on the minicell suppression system and involves a protein that is able to discriminate between polar and non‐polar sites. Another is involved in asymmetric site selection at the early stages of sporulation in Bacillus subtilis. Finally, a mechanism observed only in certain multinucleated cells appears to favour division at non‐polar PDS related to the most ancient replication/DNA segregation events.
Discovery of critical Tol A‐binding residues in the bactericidal toxin colicin N: a biophysical approach
Authors: Raggett Elaine M. , Bainbridge Graeme , Evans Lucy J. A. , Cooper Alan , Lakey Jeremy H.
Published: March 2003

Colicins translocate across the Escherichia coli outer membrane and periplasm by interacting with several receptors. After first binding to outer membrane surface receptors via their central region, they interact with TolA or TonB proteins via their N‐terminal regions. Finally, the toxic C‐terminal region is inserted into or across the cytoplasmic membrane. We have measured the binding of colicin N to TolA by isothermal titration microcalorimetry (ITC) and tryptophan fluorescence. The isolated N‐terminal domain exhibits a higher affinity for TolA (Kd = 1 μM) than does the whole colicin (18 μM), and similar behaviour has been observed when the N‐terminal domain of the g3p protein of the bacteriophage fd, which also binds TolA, is examined in isolation and in situ. This may indicate a similar mechanism in which a cryptic TolA binding site is revealed after primary receptor binding. The isolated colicin N N‐terminal domain appears to be unstructured in circular dichroism and fluorescence studies. We have used mutagenesis and ITC to characterize the TolA binding site and have shown it to be of a different sequence and much further from the N‐terminus than previously thought.
Sequence‐specific binding by Aspergillus nidulans AflR, a C6 zinc cluster protein regulating mycotoxin biosynthesis
Authors: Fernandes Mary , Keller Nancy P. , Adams Thomas H.
Published: March 2003

The Aspergillus nidulans aflR gene is found within a 60 kb gene cluster that includes ≈24 other genes that putatively function in the production of the aflatoxin‐related mycotoxin sterigmatocystin. Previous work showed that AflR is a C6 zinc binuclear cluster protein that is conserved across Aspergillus spp. and functions as a pathway‐specific transcription factor in activating expression of other cluster genes. In this report, we demonstrate that A. nidulans AflR (AnAflR) is a 45 kDa protein that binds to the palindromic sequence 5′‐TCG(N5)CGA‐3′ found in the promoter regions of several aflatoxin and sterigmatocystin cluster genes (stc genes). The in vivo relevance of this AnAflR binding site was assessed by examining the contribution of the three TCG(N5)CGA elements in the 1.1 kb promoter region of stcU using gene fusions with the bacterial uidA gene encoding β‐glucuronidase (GUS). By mutating one, two or all three of the AnAflR‐binding elements and examining GUS activity in wild‐type aflR or ΔaflR A. nidulans strains, we found that stc gene activation required both AnAflR and at least one TCG(N5)CGA AflR binding site.