bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonrandom mating in pikas Ochotona princeps: evidence for inbreeding between individuals of intermediate relatedness
Authors: Peacock M. M. , Smith A. T.
Published: November 2003

The extent and effect of inbreeding in natural populations remain largely undetermined. Pikas Ochotona princeps have been considered a likely candidate for close inbreeding in natural populations due to observations of frequent juvenile philopatry (colonization of natal home range or neighbouring home range) and high levels of spatial overlap and social tolerance between neighbouring individuals of the opposite sex. A 4‐year investigation of inbreeding in pikas, however, has revealed that dispersal and mating patterns are uncoupled in this species, i.e. explained by different hypotheses. DNA fingerprinting analysis revealed that band‐sharing scores between mated pairs, identified via parentage analysis, were not commensurate with band‐sharing among known first‐order relatives, but were similar to scores for a small sample of known second‐order relatives (i.e. half‐siblings, grandparent‐grandchild pairs). Band‐sharing scores between mated pairs were then compared with those between potential mated pairs within the population to assess whether mating was random or nonrandom with respect to genetic similarity. The results of Monte Carlo randomization tests show that pikas mated with individuals with intermediate genetic similarity in greater proportion than would be expected by chance. These data suggest mate choice in pikas may be based upon intermediate levels of relatedness.
Phylogenetic characterization of ineffective Frankia in Alnus glutinosa (L.) Gaertn. nodules from wetland soil inoculants
Authors: Wolters D. J. , Van Dijk C. , Zoetendal E. G. , Akkermans A. D. L.
Published: November 2003

Ineffective Frankia endophytes were retrieved from various wet soils by using Alnus glutinosa clones as trapping plants. No pure cultures could be isolated from these ineffective nodules. Therefore, the phylogenetic position of these endophytes was determined by sequence analysis of cloned PCR products of bacterial 16S rDNA, derived from nodules. The results showed that all nodule endophytes belong to a hitherto undescribed cluster of the Frankia phylogenetic tree. The position of these uncultured ineffective Frankia nodule endophytes is different from that of the ineffective Frankia isolates derived from A. glutinosa nodules, even when originating from the same geographical location. This suggests a bias in current isolation techniques.
Genetic isolation of populations of the gammaridean amphipod, Corophium volutator, in the Bay of Fundy, Canada
Authors: Wilson A. B. , Boates J. S. , Snyder M.
Published: November 2003

Selection experiments suggest that evolutionary modifications in amphipod demography can respond to local environmental changes and that local races of amphipods may be common. We tested this hypothesis in mudflat populations of Corophium volutator in the Bay of Fundy, Canada. Due to the unique topography of the Bay of Fundy, distinctive environmental conditions are prevalent in different branches of the Bay, while the impact of shorebird predation has also been shown to vary between populations. Several hypotheses have been suggested to explain ecological evidence which indicates that Corophium volutator (Pallas), a common amphipod crustacean, exhibits extensive life history variation in Bay of Fundy populations. We used RAPD‐PCR techniques to examine populations of C. volutator in an investigation of genetic isolation in marine environments. Our data suggest that variation in selection pressures have played a significant role in the genetic divergence of populations of C. volutator in the Bay of Fundy.
An evaluation of introgression of Atlantic coast striped bass mitochondrial DNA in a Gulf of Mexico population using formalin‐preserved museum collections
Authors: Wirgin I. , Maceda L. , Stabile J. , Mesing C.
Published: November 2003

Striped bass Morone saxatilis populations in drainages along the Gulf of Mexico coast (Gulf) were depleted in the 1950s and 1960s, probably because of anthropogenic influences. It is believed that only the Apalachicola‐Chattahoochee‐Flint (A‐C‐F) river system continually supported a naturally reproducing population of Gulf lineage. Striped bass juveniles of Atlantic coast (Atlantic) ancestry were introduced to restore population abundances in the A‐C‐F from the late 1960s to the mid 1970s and in many other Gulf rivers from the 1960s to the present. We previously identified mtDNA polymorphisms that were unique to ≊ 60% of striped bass from the A‐C‐F and which confirmed the continued successful natural reproduction of striped bass of Gulf maternal ancestry within the system. However, the genetic relatedness of the extant A‐C‐F population to ‘pure’ Gulf striped bass was not addressed. In this study, we determined the frequency of a diagnostic mtDNA XbaI polymorphism in samples of ‘pure’ Gulf striped bass that were collected from the A‐C‐F prior to the introduction of Atlantic fish, that were obtained from museum collections, and that were originally preserved in formalin. PCR primers were developed that allowed for amplification of a 191‐bp mtDNA fragment that contained the diagnostic XbaI restriction site. Using RFLP and direct sequence analyses of the PCR amplicons, we found no significant differences in mtDNA XbaI genotype frequencies between the archived samples and extant A‐C‐F samples collected over a 15‐year period. This indicates that significant maternally mediated introgression of Atlantic mtDNA genomes into the A‐C‐F gene pool has not occurred. Additionally, we found no evidence of the unique Gulf mtDNA genotype in striped bass from extant populations in Texas, Louisiana and the Mississippi River. These results highlight the importance of the A‐C‐F as a repository of striped bass to restore extirpated Gulf populations and the potential use of museum collections in retrospective population studies.
Spatially dependent genetic diversity within and between colonies of wild raspberry Rubus idaeus detected using RAPD markers
Authors: Graham J. , Squire G. R. , Marshall B. , Harrison R. E.
Published: November 2003

In the Tayside region of Scotland, red raspberry Rubus idaeus exists both as extensive plantations of clonally propagated cultivars, and as wild populations that inhabit both the cultivated areas and uncultivated uplands to the north. In order to explore the genetic diversity of the wild populations and their degree of similarity to the plantation clones, individual plants in wild populations were sampled from four distinct sites along a 25‐km transect northwards from the area of cultivation. The genetic material of each of 45 individuals and the commercial cv. Glen Clova were examined using six RAPD primers giving a total of 63 variable bands. Some identical banding patterns were observed, suggesting vegetative growth up to 20 m, but populations consisted mainly of genetically distinct individuals. Similarity matrices based on the marker bands showed, without exception, that plants were more similar within a site than between sites. None of the populations was closely related to the cv. Glen Clova. Although the most northerly site had the largest proportion of rare bands, there was no general trend between within‐site diversity, or similarity to the cultivar, and position on the north‐south line. All four sites had unique bands and bands not displayed by the cultivar. However, the genetic diversity of a site appeared to increase as the extent of the sampled area increased, implying that the genetic variation was spatially dependent. For example, maximum and minimum similarities were 100 and 80%, respectively, at interplant distances of 2 m; 100 and 60% at distances of 20 m; 85 and 55% at 200 m and 70 and 40% at 20 km.
Matriarchal genetic population structure of North American beluga whales Delphinapterus leucas (Cetacea: Monodontidae)
Authors: Brown Gladden J. G. , Ferguson M. M. , Clayton J. W.
Published: November 2003

The North American beluga whale Delphinapterus leucas population has been divided into a number of putative geographical stocks based upon migration routes and areas of summer concentration. Nucleotide sequences of the mitochondrial DNA (mtDNA) control region were used to assess whether these geographical stocks are genetically distinct. Beluga whale samples from 25 sites were collected primarily from aboriginal subsistence hunts across North America from 1984 to 1994. Thirty‐nine mtDNA haplotypes were identified in 628 beluga samples. No differences were found in the distribution of haplotypes between male and female beluga whales at any sampling site. These haplotypes segregated into two distinct assemblages in both a haplotype network and a neighbour‐joining tree. The haplotype assemblages had a geographically disjunct distribution that suggests postglacial recolonization of the North American Arctic from two different refugia.

An analysis of molecular variance based on haplotype relationships and frequency indicated genetic heterogeneity among beluga whale summering groups (P≤ 0.001). Sequence divergence estimates between sampling sites also indicated geographical differentiation, particularly between samples taken at east Hudson Bay or St Lawrence River and the western or central Arctic. The results of this study show a high degree of philopatry to specific summering areas by this highly mobile animal.
Social structure of the mound‐building mouse Mus spicilegus revealed by genetic analysis with microsatellites
Authors: Garza J. C. , Dallas J. , Duryadi D. , Gerasimov S. , Croset H. , Boursot P.
Published: November 2003

The Mound‐building mouse Mus spicilegus possesses a unique behaviour amongst mice. It constructs large earthen mounds and associated nesting chambers which serve to store food for immature individuals during the winter nesting period. We have used genetic analysis of four autosomal and four X‐linked microsatellite loci to determine relationships between individuals inhabiting 40 mounds in Bulgaria. We show that, in almost all cases, individuals in a mound are the product of multiple parentage. We estimate the minimum number of males and female parents contributing offspring to each mound and demonstrate that at least two male and two female parents contribute offspring to a minimum of seven mounds. Analyses of relatedness coefficients and allele sharing values demonstrate that parents of different sibships within mounds are more related than if they had been chosen at random from the population and suggest that it is the female parents that contribute this excess relatedness. These results suggest that the mechanism by which individuals congregate to build mounds is kin‐based and that the evolution of mound building and communal nesting in M. spicilegus is due in part to kin selection. This study represents a novel approach to the study of mammalian behavioural ecology. We have used a genetic dataset to construct an outline of social structure in the absence of behavioural data. These inferences can now be used to direct further work on this species.
Genetic structure of populations of the threatened eastern massasauga rattlesnake, Sistrurus c. catenatus: evidence from microsatellite DNA markers
Authors: Gibbs H. Lisle , Prior Kent A. , Weatherhead Patrick J. , Johnson Glenn
Published: November 2003

Throughout its distribution in North America, the threatened eastern massasauga rattlesnake (Sistrurus c. catenatus) persists in a series of habitat‐isolated disjunct populations of varying size. Here, we use six microsatellite DNA loci to generate information on the degree of genetic differentiation between, and the levels of inbreeding within populations to understand how evolutionary processes operate in these populations and aid the development of conservation plans for this species. Samples were collected from 199 individuals from five populations in Ontario, New York and Ohio. Our results show that all sampled populations: (i) differ significantly in allele frequencies even though some populations are < 50 km apart, and may contain genetically distinct subpopulations < 2 km apart; (ii) have an average of 23% of alleles that are population specific; and (iii) have significant FIS values (mean overall FIS= 0.194) probably due to a combination of Wahlund effects resulting from fine‐scale genetic differentiation within populations and the presence of null alleles. Our results imply that massasauga populations may be genetically structured on an extremely fine scale even within continuous populations, possibly due to limited dispersal. Additional information is needed to determine if dispersal and mating behaviour within populations can account for this structure and whether the observed differentiation is due to random processes such as drift or to local adaptation. From a conservation perspective, our results imply that these massasauga populations should be managed as demographically independent units and that each has high conservation value in terms of containing unique genetic variation.
Developing microsatellites when they are rare: trinucleotide repeat loci in the northern mockingbird Mimus polyglottos
Authors: Hughes C. R. , Moralez Deloach D.
Published: November 2003

The great value of microsatellite loci to population studies spurs their development. However, finding loci is difficult when microsatellites are uncommon in the genome. Because trinucleotide‐repeat loci are rare in northern mockingbirds Mimus polyglottos, we sought a new method for developing a suite of loci. Here we show that a bacteriophage cloning vector, Lambda Zap Express (Stratagene, La Jolla, CA, USA) has several features which make it suitable for this purpose. Using this vector, we made a library of 150 000 size‐selected clones and screened with an AAT10 probe; 97 positives were identified. From these, 12 pairs of PCR primers were developed, nine of which amplify polymorphic loci. Certain combinations of these primer pairs enable simultaneous amplification of up to three loci.
Mitochondrial DNA sequence variation and phylogeography among Salmo trutta L. (Greek brown trout) populations
Authors: Apostolidis A. P. , Triantaphyllidis C. , Kouvatsi A. , Economidis P. S.
Published: November 2003

To investigate the phylogenetic relationships and geographical structure among brown trout S. trutta L. populations from the South Adriatic‐Ionian and Aegean sea basins, mitochondrial DNA nucleotide sequence comparisons were used. A 310‐base‐pair (bp) segment of the control region (D‐loop), and an additional 280‐bp segment of the cytochrome b gene were sequenced from representatives of 13 brown trout populations. Phylogenetic analyses, conducted after combining the data presented with published data from other Eurasian brown trout, revealed four major phylogenetic groups, three of which were found widely distributed within the southern Balkan region. The phylogeographical patterns revealed by mtDNA represent one of the few cases where phylogenetic discontinuity in a gene tree exists without obvious geographical localization within a species' range and has most likely resulted from the differentiation of the major mtDNA clades during Messinian or early Pleistocene times. Finally, the genetic relationships among the populations suggested by mtDNA were generally not in accordance with either allozyme or morphological data.
Effects of systematic variation of the minimal Escherichia coli met consensus operator site: in vivo and in vitro met repressor binding
Authors: Wild Catherine M. , McNally Teresa , Phillips Simon E. V. , Stockley Peter G.
Published: November 2003

We have produced a set of sequence variants based upon the idealized, minimal Escherichia coli met operator in which each position within the basic recognition unit, the 8bp met box (dAGACGTCT), has been changed to all other possible sequences containing single symmetrical base substitutions. The effects of these sequence variations have been assayed in vivo by monitoring the production of β‐galactosidase from a standard promoter regulated by the operator variants, and in vitro by gel‐retardation assay. The two sets of data are consistent and correlate well with expectations based on the three‐dimensional structure of the holorepressor bound to a minimal idealized operator and the results of in vitro evolution experiments. Comparison with two natural operators, metA and metC, suggests that in vivo, with non‐consensus operators, the repressor binds to at least four consecutive met boxes.
Identification of σs‐dependent genes associated with the stationary‐phase acid‐resistance phenotype of Shigella flexneri
Authors: Waterman Scott R. , Small P. L. C.
Published: November 2003

Shigella flexneri grown to stationary phase has the ability to survive for several hours at pH 2.5. This acid resistance, which may contribute to the low infective dose associated with shigellosis, is dependent upon the expression of the stationary‐phase‐specific sigma factor σs. Using random TnphoA and TnlacZ mutagenesis we isolated five acid‐sensitive mutants of S. flexneri, which had lost their ability to survive at pH 2.5 for 2 h in vitro. Each transposon insertion with flanking S. flexneri DNA was cloned and sequenced. Database searches indicated that two TnlacZ mutants had an insertion within the hdeA gene, which is the first gene in the hdeAB operon. Acid resistance was restored in one of these mutants by a plasmid carrying the entire hdeAB operon. Further sequence analysis from the remaining TnlacZ and two TnphoA mutants demonstrated that they all had insertions within a previously unidentified open reading frame (ORF), which is directly downstream from the gadB gene. This putative ORF encodes a protein that has homology to a number of inner membrane amino acid antiporters. A 1.8 kb polymerase chain reaction (PCR) product containing this gene was cloned, which was able to restore acid resistance in each mutant. These fusions were induced during entry into late exponential phase and were positively regulated by RpoS. We confirmed that the expression of the acid‐resistance phenotype in acidified minimal media was dependent upon the supplementation of glutamic acid and that this glutamate‐dependent system was RpoS regulated. Southern hybridization revealed that both the gadC and hdeAB loci are absent in Salmonella. An rpoS deletion mutant of S. flexneri was also constructed to confirm the important role played by this gene in acid resistance. This rpoS − derivative was extremely acid sensitive. Two‐dimensional gel electrophoresis of this mutant revealed that it no longer expressed 27 proteins in late log phase that were present in its isogenic parent. These data indicate that the expression of acid resistance in S. flexneri may be multifactorial and involve proteins located at different subcellular locations.
A cyclophilin‐like peptidyl‐prolyl cis/trans isomerase from Legionella pneumophila – characterization, molecular cloning and overexpression
Authors: Schmidt Bettina , Tradler Thomas , Rahfeld Jens‐U. , Ludwig Birgit , Jain Bunty , Mann Karlheinz , Rücknagel K. Peter , Janowski Bernhard , Schierhorn Angelika , Küllertz Gerhard , Hacker Jörg , Fischer Gunter
Published: November 2003

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires’disease). A major virulence factor, the Mip protein (FK506‐binding protein, FKBP25mem), belongs to the enzyme family of peptidyl‐prolyl cis/trans isomerases (PPIases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPiase at a level of enzyme activity comparable to that of FKBP25mem. The N‐terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPIases. The Icy gene (Legionella cycophn) was cloned and sequenced. It encodes a putative 164‐amino‐acid protein with a molecular mass of 17 968 Da called L. pneumophila cyclophilin 18 (L. p. Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coll with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19‐amino‐acid polypeptide extension including a 6‐histi‐dine tag and an enterokinase cleavage site exhibits
Ligand‐induced conformational change in the ferrichrome–iron receptor of Escherichia coli K‐12
Authors: Moeck Gregory S. , Tawa Paul , Xiang Hui , Ismail Ashraf A. , Turnbull Joanne L. , Coulton James W.
Published: November 2003

Ferrichrome–iron is actively transported across the outer membrane of Escherichia coli by the TonB‐dependent receptor FhuA. To obtain FhuA in a form suitable for secondary‐structure analyses, a hexahistidine tag was inserted into a surface‐located site and the recombinant protein was purified by metal chelate chromatography. Functional studies indicated that the presence of the hexahistidine tag did not interfere with FhuA localization or with ligand‐binding activity. Ferrichrome protected lysine 67 but not lysine 5 of purified recombinant FhuA from trypsinolysis. Results from trypsin digestion were interpreted as a conformational change in FhuA which had occurred upon ferrichrome binding, thereby preventing access of trypsin to lysine 67. Circular dichroism and Fourier transform infrared spectroscopy revealed a predominance of ‐sheet structure for the purified protein. In the presence of ferrichrome, FhuA exhibited a secondary structure and a thermostability which were similar to FhuA without ligand. The addition of ferrichrome to purified FhuA reduced the ability of certain anti‐FhuA monoclonal antibodies to bind to the receptor. All antibodies which could in this manner discriminate between FhuA and FhuA bound to ferrichrome had their determinants within a loop which is toward the N‐terminus and which is exposed to the periplasm. These data indicate that the binding of ferrichrome induces a structural change that is propogated across the outer membrane and results in an altered conformation of a periplasmically exposed loop of FhuA. It is proposed that by such an alteration of FhuA conformation, TonB is triggered to energize the active transport of the bound ligand across the outer membrane.
Analysis of bacterial carbapenem antibiotic production genes reveals a novel β‐lactam biosynthesis pathway
Authors: McGowan S. J. , Sebaihia M. , Porter L. E. , Stewart G. S. A. B. , Williams P. , Bycroft B. W. , Salmond G. P. C.
Published: November 2003

Carbapenems are β‐lactam antibiotics which have an increasing utility in chemotherapy, particularly for nosocomial, multidrug‐resistant infections. Strain GS101 of the bacterial phytopathogen, Erwinia carotovora, makes the simple β‐lactam antibiotic, 1‐carbapen‐2‐em‐3‐carboxylic acid. We have mapped and sequenced the Erwinia genes encoding carbapenem production and have cloned these genes into Escherichia coli where we have reconstituted, for the first time, functional expression of the β‐lactam in a heterologous host. The carbapenem synthesis gene products are unrelated to enzymes involved in the synthesis of the so‐called sulphur‐containing β‐lactams, namely penicillins, cephamycins and cephalosporins. However, two of the carbapenem biosynthesis genes, carA and carC, encode proteins which show significant homology with proteins encoded by the Streptomycesclavuligerus gene cluster responsible for the production of the β‐lactamase inhibitor, clavulanic acid. These homologies, and some similarities in genetic organization between the clusters, suggest an evolutionary relatedness between some of the genes encoding production of the antibiotic and the β‐lactamase inhibitor. Our observations are consistent with the evolution of a second major biosynthetic route to the production of β‐lactam‐ring‐containing antibiotics.
Site‐directed mutagenesis of the katG gene of Mycobacterium tuberculosis: effects on catalase–peroxidase activities and isoniazid resistance
Authors: Rouse David A. , DeVito Joseph A. , Li Zhongming , Byer Heather , Morris Sheldon L.
Published: November 2003

Recent studies examining the molecular mechanisms of isoniazid (INH) resistance in Mycobacterium tuberculosis have demonstrated that a significant percentage of drug‐resistant strains are mutated in the katG gene which encodes a catalase–peroxidase, and the majority of these alterations are missense mutations which result in the substitution of a single amino acid. In previous reports, residues which may be critical for enzymatic activity and the drug‐resistant phenotype have been identified by evaluating INH‐resistant clinical isolates and in vitro mutants. In this study, site‐directed mutagenesis techniques were utilized to alter the wild‐type katG gene from M. tuberculosis at 13 of these codons. The effects of these mutations were determined using complementation assays in katG‐defective, INH‐resistant strains of Mycobacterium smegmatis and Mycobacterium bovis BCG. This mutational analysis revealed that point mutations in the katG gene at nine of the 13 codons can cause drug resistance, and that enzymatic activity and resistance to INH are inversely related. In addition, mutations in the mycobacterial catalase–peroxidase which reduce catalase activity also decrease peroxidase activity.
Highly conserved toxicity of Saccharomyces cerevisiae Rap1p
Authors: Chambers Alistair
Published: November 2003

Budding yeast (Saccharomyces cerevisiae) Rap1p has been expressed in fission yeast (Schizosaccharo‐myces pombe) under the control of the regulatable fructose bisphosphatase (fbp) promoter. When the fbp promoter was derepressed, cells containing the complete RAP1 gene failed to show any significant growth, suggesting that Rap1p is toxic. A derivative of Rap1p that has a temperature‐sensitive mutation in the DNA‐binding domain was not toxic in cells grown at 37°C, a temperature at which DNA binding by rap1pts is severely inhibited. Removal of a short region downstream of the DNA‐binding domain, including a region previously shown to be essential for Rap1p toxicity in budding yeast, also abolished the toxic effect. The toxic effect of Rap1p has therefore been conserved between two distantly related yeasts. In budding yeast, overexpression of Rap1p also caused changes to the lengths of the telomeric repeats. No effects on telomeres were detected in fission yeast.
Quantitative analysis of the metabolism of soluble cytoplasmic peptidoglycan precursors of glycopeptide‐resistant enterococci
Authors: Arthur Michel , Depardieu Florence , Reynolds Peter , Courvalin Patrice
Published: November 2003

Transposon Tn1546 from Enterococcus faecium BM4147 mediates high‐level resistance to the glycopeptide antibiotics vancomycin and teicoplanin. Tn1546 encodes a dehydrogenase (VanH) and a ligase (VanA) that synthesize D‐alanyl‐D‐lactate (D‐Ala‐D‐Lac), a DD‐dipeptidase (VanX) that hydrolyses D‐Ala‐D‐Ala and a two‐component regulatory system (VanR–VanS) that controls transcription of the vanHAX operon. Strains of Enterococcus faecalis harbouring various copy numbers of the vanRSHAX cluster were tested to determine if there was a correlation between the levels of resistance to glycopeptides, the levels of expression of the corresponding resistance genes and the relative proportions of the different cytoplasmic peptidoglycan precursors. Increased transcription of the vanHAX operon was associated with increased incorporation of D‐Ala‐D‐Lac into peptidoglycan precursors to the detriment of D‐Ala‐D‐Ala, and with a gradual increase in the vancomycin‐resistance levels. More complete elimination of D‐Ala‐D‐Ala‐containing precursors was required for teicoplanin resistance. The VanY and VanZ proteins also encoded by Tn1546 were not effectors of the regulation of the vanHAX operon but contributed to vancomycin and teicoplanin resistance, respectively. Differences at the regulatory level accounted for phenotypic diversity in acquired glycopeptide resistance by production of D‐lac‐ending precursors.
Purification and characterization of the DNA‐binding protein DnrI, a transcriptional factor of daunorubicin biosynthesis in Streptomyces peucetius
Authors: Tang Li , Grimm A. , Zhang Ying‐Xin , Hutchinson C. Richard
Published: November 2003

The DnrI protein, essential for the biosynthesis of daunorubicin in Streptomyces peucetius, was purified almost to homogeneity from dnrI expression strains of Escherichia coli and S. peucetius through several steps of chromatography. The proteins purified from both organisms had identical chromatographic and electrophoretic behaviour. Purified His‐tagged or native DnrI was used to conduct DNA‐binding assays by gel mobility‐shift analysis, and the results showed no significant difference in the DNA‐binding activity of native or His‐tagged proteins. DnrI binds specifically to DNA segments containing the intergenic regions separating the putative dnrG–dpsABCD and dpsEF operons, and the dnrC gene and dnrDKPSQ operon. DNase I footprinting assays indicated that the DNA‐binding sites for DnrI extended from upstream of the −10 to −35 regions of the dnrG or dpsE promoters to include about 65 bp of the dnrG–dpsE intergenic region and about 80 bp of the dnrC–dnrD intergenic region. Both binding sites contain imperfect inverted repeat sequences of 6–10 bp with a 5′‐TCGAG‐3′ consensus sequence that was present in 4 out of 10 other promoter regions in the cluster of daunorubicin biosynthesis genes.
Introducing mutations into a chromosomal rRNA gene using a genetically modified eubacterial host with a single rRNA operon
Authors: Sander Peter , Prammananan Therdsak , Bo¨ttger Erik C.
Published: November 2003

Gene‐inactivation techniques were employed to construct a eubacterial organism harbouring a single functional rRNA operon. This mutant of Mycobacterium smegmatis permits replacement of the single remaining rRNA operon with a homologous fragment from a vector‐borne gene. By homologous recombination with the chromosome a plasmid‐borne rDNA segment with resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, resulting in a homogeneous population of mutated ribosomes in the cell. As a first result we demonstrate that the single allelic knock‐out strain allows for isolation of rRNA mutants with a drug‐resistant phenotype, circumventing the problem of recessivity which prohibits the isolation of such mutants in organisms with multiple rRNA operons. Subsequently, by allelic exchange experiments, it was demonstrated that the rRNA mutation found indeed confers drug resistance in vivo. This system provides intriguing potential for the study of the structure and function of ribosomal RNAs.
Isolation and characterization of Pseudomonas aeruginosa genes inducible by respiratory mucus derived from cystic fibrosis patients
Authors: Wang Jingyi , Lory Stephen , Ramphal Reuben , Jin Shouguang
Published: November 2003

Pseudomonas aeruginosa, an opportunistic human pathogen, is a major causative agent of mortality and morbidity in immunocompromised individuals and those with cystic fibrosis (CF). In CF patients, the secretion of abnormally high amounts of mucus into the airways contributes to their susceptibility to infection by P. aeruginosa. To identify virulence genes of P. aeruginosa that are important in infection of CF patients, an in vivo selection system (IVET) was used to identify promoters that are specifically inducible by respiratory mucus derived from CF patients. Three genetic loci that are highly inducible by the mucus were identified. One of them is a well‐characterized virulence gene (fptA), encoding the receptor for pyochelin, which is a P. aeruginosa iron siderophore. Induction of the fptA gene by mucus is suppressed by the addition of exogenous iron, demonstrating that the mucus is an iron chelator and generates an iron‐deficient environment in CF lungs. Therefore, as a part of the host‐defence mechanism, the mucus could also be responsible for induction of iron‐regulated virulence factors of bacterial pathogens. The second locus, np20, encodes a peptide that shares sequence homology to a number of transcriptional regulators. An identical locus was previously identified to be inducible in vivo during infection of mice and was shown to be important in bacterial virulence in a neutropenic‐mouse infection model. The third locus, designated migA (mucus inducible gene), was sequenced and found to encode a 299‐amino‐acid peptide which is homologous to glycosyltransferases of other bacteria, and is involved in the biosynthesis of lipopolysaccharides or exopolysaccharides. Inducibilities of the np20 and migA genes are not affected by iron and the exact nature of the inducing signals in the mucus is not known. The possible implications of the migA inducibility by respiratory mucus is discussed in relation to the P. aeruginosa infection in CF.
Saccharomyces cerevisiae chorismate synthase has a flavin reductase activity
Authors: Henstrand John M. , Schaller Andreas , Braun Martin , Amrhein Nikolaus , Schmid Ju¨rg
Published: November 2003

Chorismate synthase (CS) catalyses the conversion of 5‐enolpyruvylshikimate 3‐phosphate (EPSP) to form chorismate, which is the last common intermediate in the synthesis of the three aromatic amino acids phenylalanine, tyrosine and tryptophan. Despite the overall redox‐neutral reaction, catalysis has an absolute requirement for reduced flavin. In the fungus Neurospora crassa, a flavin reductase (FR) activity able to generate reduced flavin mononucleotide in the presence of NADPH is an intrinsic feature of a bifunctional CS. In all bacterial and plant species investigated to date, purified CSs lack an FR activity and are correspondingly 8–10 kDa smaller than the N. crassa CS (on the basis of SDS–PAGE). The cloning of N. crassa CS and subsequent characterization of the purified heterologously expressed enzyme indicates that, surprisingly, the FR probably resides within a region conserved amongst both mono‐ and bifunctional CSs and is not related to non‐homologous sequences which contribute to the larger molecular mass of the N. crassa CS. This information directed this work towards the smaller Saccharomyces cerevisiae CS, the sequence of which was known, although the protein has not been extensively characterized biochemically. Here the characterization of the S. cerevisiae CS is reported in more detail and it is shown that the protein is also bifunctional. With this knowledge, S. cerevisiae could be used as a genetic system for studying the physiological consequences of bifunctionality. The phylogenetic relationship amongst known CSs is discussed.
Identification and cellular localization of the actin‐binding protein ABP‐120 from Entamoeba histolytica
Authors: Vargas Miguel , Sansonetti Philippe , Guillén Nancy
Published: November 2003

Several actin‐binding proteins participate in the morphological changes that occur during amoeboid movement. The gene encoding one of these proteins, the gelation factor ABP‐120, was identified and characterized from trophozoites of Entamoeba histolytica. The sequence contains 2574 nucleotides, with an open reading frame of 858 amino acids, giving a protein of 93 kDa belonging to the spectrin family. The N‐terminal domain of ABP‐120 from E. histolytica revealed a consensus site for actin binding homologous to the actin‐binding sites of ABP‐120 of Dictyostelium discoideum, α‐actinin and spectrin. Analysis of the central domain revealed the presence of four repeats of a 73‐amino‐acid motif constituting 31% of the protein. In addition, a stretch of 105 amino acids was highly divergent when compared with the C‐terminal domain of D. discoideum ABP‐120. This sequence showed short motifs that are homologous to microtubule‐binding domains. We found that ABP‐120 from E. histolytica binds to F‐actin. In addition, upon motility of the parasite, this protein localized in the pseudopod and the uroid region, implying a role for ABP‐120 in movement and capping of surface receptors in E. histolytica.
RecA relieves negative autoregulation of rdgA, which specifies a component of the RecA–Rdg regulatory circuit controlling pectin lyase production in Erwiniacarotovora ssp. carotovora
Authors: Liu Yang , Wang Xiaohong , Mukherjee Asita , Chatterjee Arun K.
Published: November 2003

The production of pectin lyase (Pnl) in Erwiniacarotovora ssp. carotovora strain 71 is induced by DNA‐damaging agents such as mitomycin C (MC). This induction requires functions of recA, rdgA and rdgB genes. Based upon sequence homology, rdgA was predicted to encode a repressor and rdgB was presumed to specify a transcriptional activator. To elucidate the function of rdgA, the gene has been over‐expressed in Escherichia coli, and the 30 kDa product purified by ammonium‐sulphate precipitation, heparin–agarose chromatography and gel filtration. The results of gel mobility‐shift and DNase I protection assays revealed that purified RdgA specifically binds the rdgA operator sequence located between the −10 and −35 boxes. The expression of a rdgA–lacZ gene fusion in E. coli MC4100 is suppressed upon overproduction of RdgA from a Ptac–rdgA construct induced by isopropyl‐β‐d‐thiogalactopyranoside (IPTG). However, the suppression of rdgA–lacZ expression is relieved by MC in the RecA+E. coli strain MC4100, but not in its RecA− derivative, MC4160. An immunoblot analysis revealed RecA‐dependent in vivo cleavage of the 30 kDa RdgA protein upon MC treatment. These results demonstrate that the transcription of rdgA is autoregulated, and strongly support the idea that proteolytic activity of RecA* is responsible for the derepression of rdgA expression.
Comparative analysis of the virulence control systems of Bordetella pertussis and Bordetella bronchiseptica
Authors: Martínez de Tejada Guillermo , Miller Jeff F. , Cotter Peggy A.
Published: November 2003

Bordetella pertussis and Bordetella bronchiseptica contain nearly identical BvgAS signal‐transduction systems that mediate a biphasic transition between virulent (Bvg+) and avirulent (Bvg–) phases. In the Bvg+ phase, the two species express a similar set of adhesins and toxins, and in both organisms the transition to the Bvg– phase occurs in response to the same environmental signals (low temperature or the presence of nicotinic acid or sulphate anion). These two species differ, however, with regard to Bvg–‐phase phenotypes, host specificity, the severity and course of the diseases they cause, and also potentially in their routes of transmission. To investigate the contribution of the virulence‐control system to these phenotypic differences, we constructed a chimeric B. bronchiseptica strain containing bvgAS from B. pertussis and compared it with wild‐type B. bronchisepticain vitro and in vivo. The chimeric strain was indistinguishable from the wild type in its ability to express Bvg+‐ and Bvg–‐phase‐specific factors. However, although the chimeric strain responded to the same signals as the wild type, it differed dramatically in sensitivity to these signals; significantly more nicotinic acid or MgSO4 was required to modulate the chimeric strain compared with the wild‐type strain. Despite this difference in signal sensitivity, the chimeric strain was indistinguishable from the wild type in its ability to cause respiratory‐tract infections in rats, indicating that the bvgAS loci of B. pertussis and B. bronchiseptica are functionally interchangeable in vivo. By exchanging discrete fragments of bvgAS, we found that the periplasmic region of BvgS determines signal sensitivity.
The secretin‐specific, chaperone‐like protein of the general secretory pathway: separation of proteolytic protection and piloting functions
Authors: Hardie Kim R. , Seydel Anke , Guilvout Ingrid , Pugsley Anthony P.
Published: November 2003

The chaperone‐like protein of the main terminal branch of the general secretory pathway from Klebsiella oxytoca, the outer membrane lipoprotein PulS, protects the multimeric secretin PulD from degradation and promotes its correct localization to the outer membrane. To determine whether these are separable functions, or whether resistance to proteolysis results simply from correct localization of PulD, we replaced the lipoprotein‐type signal peptide of PulS by the signal peptide of periplasmic maltose‐binding protein. The resulting periplasmic PulS retained its ability to protect PulD, but not its ability to localize PulD to the outer membrane and to function in pullulanase secretion. Periplasmic PulS competed with wild‐type PulS to prevent pullulanase secretion, presumably again by causing mislocalization of PulD. A hybrid protein comprising the mature part of PulS fused to the C‐terminus of full‐length maltose‐binding protein (MalE–PulS) had similar properties to the periplasmic PulS protein. Moreover, MalE–PulS was shown to associate with PulD by amylose‐affinity chromatography. The MalE–PulS hybrid was rendered completely functional (i.e. it restored pullulanase secretion in a pulS mutant) by replacing its signal peptide with a lipoprotein‐type signal peptide. However, this fatty‐acylated hybrid protein was only functional if it also carried a lipoprotein sorting signal that targeted it to the outer membrane. Thus, the two functions of PulS are separate and fully dissociable. Incorrect localization, rather than proteolysis, of PulD in the absence of PulS was shown to be the factor that causes high‐level induction of the phage shock response. The Erwinia chrysanthemi PulS homologue, OutS, can substitute for PulS, and PulS can protect the secretin OutD from proteolysis in Escherichia coli, indicating the possible existence of a family of PulS‐like chaperone proteins.
The RpoN‐box motif of the RNA polymerase sigma factor σN plays a role in promoter recognition
Authors: Taylor Mark , Butler Robin , Chambers Sheila , Casimiro Matthew , Badii Farah , Merrick Mike
Published: November 2003

The RNA polymerase sigma factor σN (σ54) is characterized by the presence, near the C‐terminal end of the protein, of a highly conserved sequence of 10 amino acids (ARRTVAKYRE) that has been termed the RpoN box. In order to examine the function of this motif, which is predicted to adopt an α‐helical stucture, we have isolated a number of mutations that alter residues within the box and examined the properties of the σN derivatives encoded by them. Certain mutations that alter charged and potentially exposed residues within the motif result in transcriptionally inactive proteins with impaired promoter recognition but no impairment in core RNA polymerase binding. We therefore suggest that the RpoN box could play a direct or indirect role in recognition of the −24, −12 promoter consensus that is characteristic of σN‐dependent genes.
Degradation by proteases Lon, Clp and HtrA, of Escherichia coli proteins aggregated in vivo by heat shock; HtrA protease action in vivo and in vitro
Authors: Laskowska Ewa , Kuczyńska‐Wiśnik Dorota , Skórko‐Glonek Joanna , Taylor Alina
Published: November 2003

Thermally aggregated, endogenous proteins of Escheri‐chia coli form a distinct fraction, denoted S, which is separable by sucrose‐density‐gradient centrifugation. It was shown earlier that DnaK, DnaJ, IbpA and IbpB heat‐shock proteins are associated with the S fraction. Comparison of the rise and decay of the S fraction in mutants defective for heat‐shock proteases Lon (La), Clp, HtrA (DegP, Do) and in wild‐type strains made studies of proteolysis and the function of the heat‐shock response possible in vivo. Different timing and the extent of action of particular proteases was revealed by the initial size and decay kinetics of the S fraction. The proteases Lon, Clp, and HtrA all participated in removal of the aggregated proteins. Mutation in the gene encoding ClpB caused the most prominent effect (47% stabilization of the S fraction). The correlation between the disappearance of the S fraction and proteolytic activity was supported by the result of the in vitro reaction. Approximately one third of the isolated S fraction was converted to trichloroacetic acid‐soluble products by the purified HtrA protease. Mg2+ ions stimulated the reaction, in contrast to the reaction of the HtrA protease with casein. The digestion of the aggregated proteins, unlike the digestion of casein, by HtrA protease in vitro was inhibited by added DnaJ, which might reflect protection of the aggregated proteins in vivo by DnaJ from excessive degradation. One might expect that such an activity of DnaJ would promote denatured protein renaturation versus proteolysis. Moreover, among the aggregated proteins that are discernible by electrophoresis, none could be identified as being more susceptible than any other to HtrA degradation. The separation pattern of these proteins before and after the in vitro digestion did not show a difference corresponding to the loss of about 30% of constituting proteins. This was interpreted as recognition by the HtrA protease of a state of protein denaturation rather than specific amino acid sequences in particular proteins. We conclude that the fraction consisting of proteins heat‐ aggregated in vivo (i.e. the S fraction) contains endogenous substrates for the heat‐shock proteases tested. Their use for in vitro reaction reveals information that is in some respects different from that obtained with exogenous substrates such as casein.
Different roles of CheY1 and CheY2 in the chemotaxis of Rhizobium meliloti
Authors: Sourjik Victor , Schmitt Rüdiger
Published: November 2003

Cells of Rhizobium meliloti swim by the unidirectional, clockwise rotation of their right‐handed helical flagella and respond to tactic stimuli by modulating the flagellar rotary speed. We have shown that wild‐type cells respond to the addition of proline, a strong chemoattractant, by a sustained increase in free‐swimming speed (chemokinesis). We have examined the role of two response regulators, CheY1 and CheY2, and of CheA autokinase in the chemotaxis and chemokinesis of R. meliloti by comparing wild‐type and mutant strains that carry deletions in the corresponding genes. Swarm tests, capillary assays, and computerized motion analysis revealed that (i) CheY2 alone mediates 60 to 70% of wild‐type taxis, whereas CheY1 alone mediates no taxis, but is needed for the full tactic response; (ii) CheY2 is the main response regulator directing chemokinesis and smooth swimming in response to attractant, whereas CheY1 contributes little to chemokinesis, but interferes with smooth swimming; (iii) in a CheY2‐overproducing strain, flagellar rotary speed increases upon addition and decreases upon removal of attractant; (iv) both CheY2 and CheY1 require phosphorylation by CheA for activity. We conclude that addition of attractant causes inhibition of CheA kinase and removal causes activation, and that consequent production of CheY1‐P and CheY2‐P acts to slow the flagellar motor. The action of the chief regulator, CheY2‐P, on flagellar rotation is modulated by CheY1, probably by competition for phosphate from CheA.
Acetylglutamate synthase from Neurospora crassa: structure and regulation of expression
Authors: Yu Yeon Gyu , Turner Gloria E. , Weiss Richard L.
Published: November 2003

A DNA clone which complemented an arg‐14 mutation of Neurospora crassa was isolated by sib selection from a cosmid library (pMOcosX). Southern and restriction‐fragment‐length polymorphism (RFLP) analysis confirmed that the cloned DNA contained the arg‐14 gene. The arg‐14 gene was identified as the structural gene for acetylglutamate synthase by immunodepletion of enzyme activity with antibodies prepared against an arg‐14 fusion protein and by the thermal instability of acetylglutamate synthase in a temperature‐sensitive arg‐14 mutant. The fungal acetylglutamate synthase has little sequence homology to its bacterial counterpart, unlike other arginine biosynthetic enzymes. Expression of the arg‐14 gene is regulated by cross‐pathway control similar to many amino acid biosynthetic genes. However, expression of acetylglutamate synthase occurs throughout the developmental growth cycle, unlike other arginine biosynthetic enzymes.