bluuebunny/crossref_metadata_2025_split huggingface dataset:
Suppression of rpsL phenotypes by tuf mutations reveals a unique relationship between translation elongation and growth rate
Authors: Tubulekas Ioannis , Hughes Diarmaid 
Published: October 2006

SummaryWe have found a simple relationship between bacterial growth rate and the translation elongation rate. Thus, for a set of defined ribosomal protein S12 mutations which reduce the efficiency of the ternary complex ribosome interaction (and restrict the frequency of translational errors) there is a linear relationship between growth rate and translation elongation rate. When these mutants are combined with defined EF‐Tu mutants (which increase the probability of translational errors) both the elongation rate and growth rate reductions are reversed. The reductions and reversals are described by a unique linear relationship. We interpret this to mean that these two types of mutation exert opposing effects on the same molecular interaction. We suggest that this interaction is in the initial selection of the aminoacyl‐tRNA on the ribosome. The slope of the relationship between translation elongation rate and growth rate, defined in per cent of the wild‐type rates, is close to 1. Interestingly, the reversal of the elongation and growth phenotypes is Incomplete, suggesting that the ribosomal mutants have an additional defect which is not compensated for by the ternary complex interaction. Our results show that the efficiency of the ternary complex ribosome interaction limits the translation elongation rate, which in turn correlates with changes in exponential growth rate.
Q pili enhance the attachment of Moraxella bovis to bovine corneas in vitro
Authors: Ruehl W. W. , Marrs C. , Beard M. K. , Shokooki V. , Hinojoza J. R. , Banks S. , Bieber D. , Mattick J. S. 
Published: October 2006

Summary Moraxella bovis, the causative agent of infectious bovine keratoconjunctivttis, exhibits several virulence factors, including pili, haemotysin, leukotoxin, and proteases. The pili are filamentous appendages which mediate bacterial adherence. Prior studies have shown that Q‐piliated M. bovis Epp63 are more infectious and more pathogenic than l‐piliated and nonpiliated isogenic variants, suggesting that Q pili perse or traits associated with Q‐pilin expression, promote the early association of Q‐pillated bacteria with bovine corneal tissue. In order to better evaluate the role of Q pili in M. bovis attachment, several M. bovis strains and a recombinant P. aeruginosa strain which elaborates M. bovis Q pili but not P. aeruginosa PAK pili, were evaluated using an in vitro corneal attachment assay. For each strain tested, piliated organisms attached better than non‐piliated bacteria. M. bovis Epp63 Q‐piIiated bacteria adhered better than either the l‐piliated or non‐piliated isogenic variants. Finally, recombinant P. aeruginosa organisms elaborating M. bovis Q pili adhered better than the parent P. aeruginosa strain which did not produce M. bovis pili. These results indicate that the presence of pili, especially Q pili, enhances the attachment of bacteria to bovine cornea In vitro.
The origin of greater‐than‐unit‐length plasmids generated during bacterial conjugation
Authors: Erickson Matthew J. , Meyer Richard J. 
Published: October 2006

SummaryIn Gram‐negative bacteria, the general mechanism of conjugal plasmid transfer, which is probably similar for many different groups of plasmids, involves the transfer of a single plasmid DNA strand with 5′ to 3′ polarity. Transfer is initiated by nicking of the duplex DNA at a particular site, i.e. the origin of transfer (oriT). We constructed plasmids containing two directly repeated copies of oriT, derived from the broad‐host‐range plasmid R1162 and flanking the lac operator. The number of lacO copies in the plasmid after transfer could be determined from the colour of transconjugant colonies on medium containing X‐Gal. When the oriTs were mutated to prevent initiation and termination of transfer at the same oriTs, almost all of the transconjugant cells contained greater‐than‐unit‐length plasmids with two copies of lacO and three copies of oriT. We show that these molecules were generated by an intramolecular, conjugation dependent mechanism unlikely to depend solely on a pre‐existing population of circular or linear multimers in donor cells. We propose that the greater‐than‐unit‐length molecules were instead generated by a rolling‐circle mechanism of DNA replication.
Signalling of Trichomonas vaginalis for amoeboid transformation and adhesin synthesis follows cytoadherence
Authors: Arroyo Rossana , González‐Robles Arturo , Martínez‐Palomo Adolfo , Alderete J. F. 
Published: October 2006

SummaryThe cytoadherence of Trichomonas vaginalis, the sexually transmitted flagellated protozoan, to vaginal epithelial cells (VECs) is the key to infection. Electron microscopy revealed that in vitro‐grown parasites having typical globular shape transformed rapidly after contact with VECs into thin, flat, amoeboid cells, maximizing the area of adhesion to the surface of VECs. Amoebic trichomonads formed filopodia and pseudopodia, which interdigitated at distinct sites on the plasma membrane of target cells. In contrast, the amoeboid transformation did not occur for T. vaginalis interacting with He La cells, the previously used in vitro host model cell. Initial parasitism of VECs by a single organism was followed by establishment of a monolayer of trichomonads on the host cell. Finally, parasites adhering to either VECs or HeLa cells were induced to synthesize greater amounts of the four previously described adhesins. Therefore, distinct signals after contact with either epithelial cell type leads to the morphological transformation and/or induction of adhesin synthesis by T. vaginalis.
Regulation of transcription of genes required for fatty acid transport and unsaturated fatty acid biosynthesis in Escherichiacoli by FadR
Authors: DiRusso Concetta O. , Metzger Amy K. , Heimert Tamra L. 
Published: October 2006

SummaryFatty acid biosynthesis and fatty acid degradation in Escherichiacoliare co‐ordinately regulated at the level of transcription by the product of the fadRgene, FadR. In the present work we investigate FadR interaction with the fabA and fadL promoters. The FadR‐responsive operator within fabA, OA, was localized to a region −47 to −31 base pairs relative to the start of transcription using DNase I protection studies. The promoter and untranslated leader within fadL had two binding sites for FadR, OL1 at −25 to −9 and OL2 at −1 to +16 relative to the start of transcription. The binding affinity of FadR for OA and OL1 or OL2 was lower than that for the single site within fadB (OB) as measured using protein‐DNA gel retention assays. Overall, these experiments demonstrated that the affinity of FadR binding for DNA containing the fadB, fadL and fabA promoters was OB > OL1, OL2 > OA. We could not distinguish separate binding affinities for OL1 or OL2. We demonstrated repression of fadL transcription and activation of fabA transcription in vitro using run‐off transcription assays containing purified FadR and RNA polymerase.
Bafilomycin A1 inhibits Helicobacter pylori‐induced vacuolization of HeLa cells
Authors: Papini E. , Bugnoli M. , De Bernard M. , Figura N. , Rappuoli R. , Montecucco C. 
Published: October 2006

SummaryBafilomycin A1, a specific inhibitor of the vacuolar‐type H+‐ATPase, responsible for acidification of intra‐cellular compartments, prevents the vacuolization of Hela cells induced by H. pylori, with an inhibitory concentration giving 50% of maximal (ID50) of 4 nM. Bafilomycin A1 is also very efficient in restoring vacuolated cells to a normal appearance. The vacuolating activity of Helicobacter pylori is not inhibited by a series of specific inhibitors of vacuolar H+‐ATPases. These findings indicate that a transmembrane pH gradient is needed for the formation and growth of vacuoles caused by the bacterium and that this pH gradient is due to the activity of a vacuolar ATPase proton pump of HeLa cells.
Isolation and characterization of a topA mutant of Shigella flexneri
Authors: Bhriain Niamh Ni , Dorman Charles J. 
Published: October 2006

Summary Shigella flexneri was shown to possess a homologue of the Escherichia coli K‐12 topA gene which encodes DNA topoisomerase I. The S. flexneri topA gene was replaced by a copy of the E. coli K‐12 topA gene which has been insertionally inactivated by transposon Tn10. The topoisomer distribution of reporter plasmids showed that the presence of this topA lesion in S. flexneri correlated with an increase in the level of negative DNA supercoiling in the mutant, indicating that topoisomerase I is required to relax DNA in S. flexneri as it is in E. coli and Salmonella typhimurium. The introduction of the topA mutation also resulted in repression of transcription of a thermally regulated invasion gene located on the 230 kb virulence plasmid. In addition, the topA mutant was hypersensitive to growth medium osmolarity, was unable to grow on MacConkey indicator plates and exhibited an increased doubling time under all growth conditions tested. All of these phenotypes were fully complemented in trans by a cloned copy of the E. coli topA gene carried on a recombinant plasmid. Unlike E. coli topA mutants which acquire compensatory mutations at a high frequency, such compensatory mutations were not detected in the S. flexneri topA::Tn 10 mutant.
Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte‐like Caco‐2 and mucus‐secreting HT29 cells in culture
Authors: Eveillard Matthieu , Fourel Valérie , Bare Marie‐Claude , Kernéis Sophie , Coconnier Marie‐Hélène , Karjalainen Tuomo , Bourlioux Pierre , Servin Alain L. 
Published: October 2006

SummaryExperiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco‐2 cells and the human mucus‐secreting HT29‐MTX cells. Scanning electron microscopy revealed that the bacteria interacted with well‐defined apical microvilli of differentiated Caco‐2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29‐MTX cells. The binding of C. difficile to Caco‐2 cells developed in parallel with the differentiation features of the Caco‐2 cells, suggesting that the protein(s) which constitute C. difficile‐binding sites are differentiation‐related brush border protein(s). To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C. difficile with adherence assays. It was shown that heating of C. difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte‐like Caco‐2 cells and the human mucus‐secreting HT29‐MTX cells. A labile surface‐associated component was involved in C. difficile adhesion since washes of C. difficile grown in the presence of blood without heat shock decreased adhesion. After heating, washes of C. difficile grown in the presence of blood did not modify adhesion. Analysis of surface‐associated proteins of C. difficile subjected to different culture conditions was con‐ducted. After growth of C. difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS‐extractable proteins with molecular masses of 12 and 27 kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared. Direct involvement of the 12 and 27 kDa surface‐associated proteins in the adhe‐sion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27kDa proteins. Indeed, adhesion to Caco‐2 cell monoiayers of C. difficiie strains grown in the presence of blood, without or with heat‐shock, was blocked. Taken together, our results suggest that C. difficiie may utilize blood components as adhesins to adhere to human intestinal cultured cells.
The TonB‐dependent ferrichrome receptor FcuA of Yersinia enterocolitica: evidence against a strict co‐evolution of receptor structure and substrate specificity
Authors: Koebnik Ralf , Hantke Klaus , Braun Volkmar 
Published: October 2006

SummaryA Yersinia enterocolitica receptor mutant was isolated which is impaired in ferrichrome uptake. The receptor‐encoding gene fcuA was cloned in Escherichia coli K‐12. A fcuA mutant of Y. enterocolitica could be complemented by the cloned DNA fragment. The FcuA‐encoding region was sequenced and an open reading frame encoding 758 amino acids including a signal sequence of 36 amino acids was found. FcuA shared 34.6% amino acid sequence homology with FatA, the anguibactin receptor of Vibrio anguillarum, but only 20.6% homology with FhuA, the ferrichrome receptor of E. coli Since the structure of anguibactin differs strongly from that of ferrichrome there seems to be no co‐evolution of receptor structure and substrate specificity.The ferrichrome receptors FcuA from Y. enterocolitica and FhuA from E. coli had slightly different substrate specificities. In contrast to FhuA from E. coli, FcuA from Y. enterocolitica was more stereoselective and failed to transport enantio ferrichrome. Three additional ferrichrome receptors were cloned from Pantoea aggiomerans (formerly Erwinia herbicola), Salmonella paratyphi B and Salmonella typhimurium. Their substrate specificity was similar but not identical.
DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics
Authors: Hatfult Graham F. , Sarkis Gary J. 
Published: October 2006

SummaryGenetic studies of Mycobacterium tuberculosis and other mycobacterial pathogens have suffered from the lack of a sophisticated genetic system. To address this issue we have developed a viral system through a detailed characterization of mycobacteriophage L5, a temperate phage that infects both fast‐ and slow‐growing mycobacteria. We describe here the complete DNA sequence of the L5 genome and initial characterization of L5 virion structure and gene expression. In addition to providing a genetic‘toolbox’for the mycobacteria we find that L5 offers a new paradigm for dsDNA phages, being phenotypically temperate but employing genetic strategies for phage growth usually associated with lytic bacteriophages.
Bacterial cholesterol oxidases are able to act as flavoprotein‐linked ketosteroid monooxygenases that catalyse the hydroxylation of cholesterol to 4‐cholesten‐6‐ol‐3‐one
Authors: Molnár I. , Hayashi N. , Choi K.‐P. , Yamamoto H. , Yamashita M. , Murooka Y. 
Published: October 2006

SummaryA new metabolite of cholesterol was found in reaction mixtures containing cholesterol or 4‐cholesten‐3‐one as a substrate and extra‐ or intracellular protein extracts from recombinant Streptomyces lividans and Escherichia coli strains carrying cloned DNA fragments of Streptomyces sp. SA‐COO, the producer of Streptomyces cholesterol oxidase. The new metabolite was identified as 4‐cholesten‐6‐ol‐3‐one based on comparisons of its high‐performance liquid chromatography, gas chromatography/mass spectrometry, infrared and proton‐nuclear magnetic resonance spectra with those of an authentic standard. Genetic analyses showed that the enzyme responsible for the production of 4‐cholesten‐6‐ol‐3‐one is cholesterol oxidase encoded by the choA gene. Commercially purified cholesterol oxidase (EC 1.1.3.6.) of a Streptomyces sp., as well as of Brevibacterium sterolicum and a Pseudomonas sp., and a highly purified recombinant Streptomyces cholesterol oxidase were also able to catalyse the 6‐hydroxylation reaction. Hydrogen peroxide accumulating in the reaction mixtures as a consequence of the 3β‐hydroxysteroid oxidase activity of the enzyme was shown to have no role in the formation of the 6‐hydroxylated derivative. We propose a possible scheme of a branched reaction pathway for the concurrent formation of 4‐cholesten‐3‐one and 4‐chotesten‐6‐ol‐3‐one by cholesterol oxidase, and the observed differences in the rate of formation of the 6‐hydroxy‐ketosteroid by the enzymes of different bacterial sources are also discussed.
Characterization of genes involved in molybdenum transport in Azotobacter vinelandii
Authors: Luque F. , Mitchenall L. A. , Chapman M. , Christine R. , Pau R. N. 
Published: October 2006

SummaryExpression of alternative nitrogenases in Azotobacter vinelandii is repressed by molybdenum. Two strains with Tn5 insertion mutations showed alternative nitrogenase‐dependent diazotrophic growth in the presence of Mo. The mutations were in a region which contained four open reading frames (ORFs 1–4). The genetic structure and predicted products of ORFs 2, 3 and 4 are typical of the membrane‐associated elements of the ATP‐binding cassette (ABC) superfamily of transport systems. The products of 0RF3 and 0RF4 are homologous with the products of the Escherichia coli genes chlD and the partially sequenced chlJ, respectively, both of which are implicated in molybdenum transport. ORF1, which is in the relative position of bacterial permease genes commonly specifying periplasmic binding proteins, encodes a 29 kDa protein with a novel primary structure. It lacks a potential signal sequence, and its C‐terminal half consists of a tandem repeat of a segment which is homologous with the Mr 7 kDa molybdenum‐pterin binding protein Mop from Clostridium pasteurianum. This suggests that a substituted pterin may be involved in the initial capture or early metabolism of molybdenum.
Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin
Authors: Henderson Douglas P. , Payne Shelley M. 
Published: October 2006

Summary Vibrio cholerae can utilize haemin or haemoglobin as its sole source of iron. Four haem utilization mutants of a classical strain of V. cholerae were isolated. These mutations were complemented with pHUT1, a cosmid clone isolated from a library of wild‐type CA401 DNA. Two independent Tn5 insertions into the cloned sequence disrupted function in all of the complemented mutants. Escherichia coli 1017 transformed with pHUT1 failed to utilize haemin as an iron source; a second plasmid containing a different cloned fragment of V. cholerae DNA (pHUT3) was required in addition to pHUT1 to reconstitute the system in E. coli. Minicell analysis and SDS‐PAGE of protein fractions indicate that pHUT10 (a subclone of p>HUT1) encodes a 26 kDa inner membrane protein, and pHUT3 encodes a 77 kDa outer membrane protein. Loss of either protein by Tn5 mutagenesis abolishes haem utilization in E. coli. An E. coli hemA mutant that cannot synthesize porphyrins was transformed with the recombinant plasmids to determine whether the plasmids encoded the ability to transport the porphyrin as well as the iron. The transformants grew aerobically in media containing haemin, whereas the parental strain was unable to grow under these conditions. This indicates that V. cholerae haem‐iron utilization genes allow transport of the entire haem moiety into the cell.
The two faces of Bacillus thuringiensis: insecticidal proteins and post‐exponential survival
Authors: Aronson Arthur I. 
Published: October 2006

SummaryPost‐exponential Bacillus thuringiensis cells produce both an endospore and a variety of intracellular inclusions. The latter are comprised of protoxins, each being specific for the larvae of certain species from at least three orders of insects. Following ingestion of spores and inclusions, toxicity results in the spores gaining access to haemolymph, a source of nutrients suitable for germination and growth. Most B. thuringiensis subspecies contain multiple, plasmid‐encoded protoxin genes, often with several on the same plasmid. These genes have been manipulated in order to understand the basis of toxicity and specificity, information which is important to the use of these toxins as biological control agents. Some protoxin genes are in operons, and others are in close proximity, perhaps to enhance the chances of recombination, and some are on unstable plasmids. The arrangement of these genes is probably important for flexibility in the variety of protoxins packaged into inclusions by a particular subspecies and thus the capacity to adapt to changing populations of insects. Protoxins accumulate over a prolonged period during sporulation because of the sequential transcription from two promoters, each being dependent upon a specific sporulation sigma factor, the relative stability of the messenger RNA, and the synthesis of proteins which stabilize protoxins and perhaps facilitate inclusion assembly. During the post‐exponential phase, spore and inclusion formation must be balanced so as to ensure that both are available to contribute to the survival of these bacilli.
Electrophoretic karyotype and gene assignment to resolved chromosomes of Trichoderma spp.
Authors: Herrera‐Estrella A. , Goldman G. H. , Van Montagu M. , Geremia R. A. 
Published: October 2006

SummaryA molecular karyotype for three different Trichoderma species (T. harzianum, T. viride, and T. reesei) was determined by using two different systems: countour‐clamped electric‐field and rotating‐electrode electrophoresis. Six chromosomal DNA bands were observed in T. harzianum and T. reesei and five in T. viride. The sizes of these molecules were estimated by their mobility relative to the Schizosaccharomyces pombe chromosomes and ranged between 2.2 and 7.4 megabase pairs (mbp). The estimated genome sizes range from 31 to 39 mbp. A number of genes were located in the different chromosomes by means of Southern analysis. The implications of these findings are discussed.
The RpoS Sigma factor relieves H‐NS‐mediated transcriptional repression of csgA, the subunit gene of fibronectin‐binding curli in Escherichia coli
Authors: Olsén Arne , Arnqvist Anna , Hammar Måing;rten , Sukupolvi Soila , Normark Staffan 
Published: October 2006

SummaryCurli encoded by the curlin subunit gene, csgA, are fibronectin‐ and laminin‐binding fibres expressed by many natural Escherichia coli and E. coli K‐12 strains in response to low temperature, low osmolarity and stationary‐phase growth conditions. Curli expression is dependent on RpoS, a sigma factor that controls many stationary phase‐inducible genes. Many commonly used K‐12 strains carry an amber mutation in rpoS. Strains able to form Curli carry an amber suppressor whereas curli‐negative E. coli K‐12 strains, in general, are sup°. Introduction of supD, supE, or supF suppressors into sup0 strains resulted in expression of temperature‐regulated curli. In curli‐deficient, RpoS−E. coli K‐12 strains, csgA is transcriptionally activated by mutations in hns, which encodes the histone‐like protein H‐NS. Curli expression, fibronectin binding, and csgA transcription remain temperature‐ and osmoregulated in such double mutants. Our data suggest that RpoS+ strains, and hence curli‐proficient strains of E. coli K‐12, are relieved for the transcriptional repression mediated by the H‐NS protein upon accumulating RpoS as cells reach stationary phase.