bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intermolecular complementation of the kinase activity of CheA
Authors: Swanson Ronald V. , Bourret Robert B. , Simon Melvjn I. 
Published: October 2006

SummaryCheA is a dimeric autophosphorylating protein kinase that plays a critical role in the signal transduction network controlling chemotaxis In Escherichia coli. The autophosphorylation reaction was analysed using mutant proteins defective in kinase and regulatory functions. Proteins in which the site of autophosphorylation was mutated (CheA48HQ) or missing (CheAs) were found to phosphorylate the kinase‐defective mutant, CheA470GK. The kinetics of this reaction support the hypothesis that autophosphorylation is the result of trans‐phosphorylation within a dimer. The carboxy‐terminal portion of CheA was previously shown to be dispensable for autophosphorylation, but required for regulation in response to environmental signals transmitted through a transducer and CheW. Mixing of CheA48HQ or CheA470GK with a truncated protein lacking this regulatory domain demonstrated that regulated autophosphoryltion requires the presence of both carboxy‐terminal portions in a CheA dimer. These results indicate that the dimeric form of CheA plays an integral role in signal transduction in bacterial chemotaxis.
Molecular cloning and characterization of 13 out genes from Erwinia carotovora subspecies carotovora: genes encoding members of a general secretion pathway (GSP) widespread in Gram‐negative bacteria
Authors: Reeves P.J. , Whitcombe D. , Wharam S. , Gibson M. , Allison G. , Bunce N. , Barallon R. , Douglas P. , Mulholland V. , Stevens S. , Walker D. , Salmond G. P. C. 
Published: October 2006

SummaryThe chemical mutagen ethylmethanesulphonate (EMS) has been used to generate mutants of Erwinia carotovora subspecies carotovora which are defective in the secretion of pectinases (Pel) and cellulases (Cel) but unaltered for protease (Prt) secretion. Such mutants, called Out− still synthesize Pel and Cel but these enzymes accumulate within the periplasm. Cosmid clones carrying wild‐type E. carotovra ssp. carotovora DNA, identified by their ability to restore the Out+ phenotype when transferred to some Out− mutants, were classified into six complementation groups using cosmids and cosmid derivatives. Analysis of the nucleotide sequence of a 12.7 kb DNA fragment, encompassing complementing cosmid inserts, revealed a coding capacity for 13 potential open reading frames (ORFs), and these were designated outC‐outO. Some of the out gene products were visualized using a T7 gene 10 expression system. The predicted Out proteins are highly similar to components of extracellular enzyme secretion systems from a diverse range of eubacteria including Erwinia chrysanthemi, Klebsiella oxytoca, Aeromonas hydrophila, Pseudomonas aeruginosa and Xanthomonas campestris. Lower levels of similarity exist between Ecc Out proteins and components of macromolecular trafficking systems from Bacillus subtilis, Haemophilus influenzae, Agrobacterium tumefaciens, Yersinia pestis and a protein involved in the morphogenesis of filamentous bacteriophages such as M13.
Identification of a two‐component regulatory system controlling methanol dehydrogenase synthesis in Paracoccus denitrificans
Authors: Harms Nellie , Reijnders Willem N.M. , Anazawa Hideharu , van der Palen Carol J. N. M. , van Spanning Rob J. M. , Oltmann L. Fred , Stouthamer Adriaan H. 
Published: October 2006

SummaryUpstream of the moxFJGIR genes of Paracoccus denitrificans a regulatory region involved in methanol oxidation was identified. The nucleotide sequence of this region was determined and revealed three genes, moxZ, moxY and moxX, which are transcribed opposite to moxF and which encode proteins of 16.4, 48.2 and 24.5kDa, respectively. Computer alignment analysis revealed that the gene products of moxyand moxX have homology with the protein histidine kinases and the response regulators, respectively, forming the two‐component regulatory systems. No significant homology of the moxZ gene product with any known protein, sequenced thus far, was found. The MoxZ, MoxY and MoxX proteins were identified in Escherichia coli in a heterologous expression system. Mutants with an insertion of a kanamycin‐resistance marker in moxZ, moxY and moxX were isolated. These mutant strains were unable to grow on methanol while growth on methylamine was not affected. In the moxZ mutant both subunits of methanol dehydrogenase and cytochrome c5511 were not synthesized, methanol dehydrogenase activity was absent, and hardly any expression of a moxZ‐lacZ transcriptional fusion was found. Complementation of the mutation was observed after addition of the three genes moxZ, Y and X, in trans. This indicates that the two‐component regulatory system is involved in activation of the moxF promoter. A mutant with an unmarked deletion in moxZ was isolated. This mutant showed reduced growth on methanol relative to the wild type. Expression of the moxF‐lacZ transcriptional fusion gene and methanol dehydrogenase activity in this strain were also lower than those found in the wild type. Therefore, besides the two proteins of the two‐component regulatory pair, a third protein, MoxZ, appears to be involved in regulation of methanol dehydrogenase synthesis.
Molecular analysis of a microaerobically induced operon required for hydrogenase synthesis in Rhizobium leguminosarum biovar viciae
Authors: Rey Luis , Murillo Jesus , Hernando Yolanda , Hidalgo Elena , Cabrera Ezequiel , Imperial Juan , Ruiz‐Argüeso Tomás 
Published: October 2006

SummaryThe nucleotide sequence (6138 bp) of a microaerobically inducible region (hupV/VI) from the Rhizobium leguminosarum bv. viciae hydrogenase gene cluster has been determined. Six genes, arranged as a single operon, were identified, and designated hypA, B, F, C, D and E based on the sequence similarities of all of them, except hypF, to genes from the hydrogenase pleiotropic operon (hyp) from Escherichia coli. The gene products from hypBFCDE were identified by in vivo expression analysis in E. coli, and their molecular sizes were consistent with those predicted from the nucleotide sequence. Transposon Tn5 insertions into hypB, hypF, hypD and hypE resulted in R. leguminosarum mutants that lacked any hydrogenase activity in symbiosis with peas, but still were able to synthesize the polypeptide for the hydrogenase large subunit. The gene products HypA, HypB, HypF and HypD contained CX2C motifs characteristic of metal‐binding proteins. In addition, HypB bore a long histidine‐rich stretch of amino acids near the N‐terminus, suggesting a possible role in nickel binding for this protein. The gene product HypF, which was translationally coupled to HypB, presented two cysteine motifs (CX2CX81CX2C) with a capacity to form zinc finger‐like structures in the N‐terminal third of the protein. A role in nickel metabolism in relation to hydrogenase synthesis is postulated for proteins HypB and HypF.
Expression and analysis of two gyrB genes from the novobiocin producer, Streptomyces sphaeroides
Authors: Thiara Amrik S. , Cundliffe Eric 
Published: October 2006

SummaryA novobiocin producer, Streptomyces sphaeroides, contains two genes, designated gyrBs and gyrBR, that encode novobiocin‐sensitive and ‐resistant DNA gyrase B proteins, respectively. The cloning of gyrBR was reported earlier; here, we describe the cloning of gyrBS. Both genes have been sequenced (the deduced products of gyrBs and gyrBR have Mr values of 87.6K and 86.5 K, respectively) and their transcripts have been mapped. Downstream of gyrBS, and co‐transcribed with it, is the sole gyrA gene (encoding DNA gyrase A protein). By constructing hybrid gyrBS genes, using fragments of gyrBS and gyrBR, a specific portion of the N‐terminal domain of the gyrase B protein (corresponding to amino acid residues 134–256 of Escherichia coli gyrase B) has been implicated in the binding of novobiocin.
Mechanism of initiation of transcription by Escherichia coli RNA polymerase on supercoiled template
Authors: Mishra Rakesh K. , Chatterji Dipankar 
Published: October 2006

SummaryDNA supercoiling is known to influence the pattern of gene expression in prokaryotes. Thus the mechanism of transcription initiation and the topological state of the template are intimately related. Using in vitro reconstituted transcription assays, composed of purified RNA polymerase and promoters in their natural topological state, we have conducted a detailed study of transcription initiation from T7 early promoters including the following steps: the formation of ternary complexes, acquisition of rifampicin resistance, release of sigma factor and the capacity for RNA chain elongation in complexes. We determined the order of these events and the length of the transcripts when each step occurred during initiation of transcription on supercoiled templates. The length of the transcripts varied in a promoter‐specific manner. Analysis of abortive products formed during the initiation showed that stronger promoters go to the elongation mode at transcript lengths shorter than that required for weaker promoters.
Trichomonas vaginalis and early evolving DNA and protein sequences of the CDC2/28 protein kinase family
Authors: Riley Donald E. , Campbell Lee Ann , Puolakkainen Mirja , Krieger John N. 
Published: October 2006

SummaryThe human sexually transmittted parasite Trichomonas vaginalis is a representative of one of the three earliest evolving eukaryotic lineages. We investigated whether T. vaginalis has DNA sequences and peptides related to cell division control molecules universal among yeasts and higher eukaryotes. A T. vaginalis ceil division control (CDC2/28) homologue was amplified by the polymerase chain reaction and sequenced. The absolute similarity with other CDC2/28 genes was 47%, with conservative replacement similarity of 67%. Western blots demonstrated a single T. vaginalis peptide reactive with antiserum to the PSTAIRE peptide, an expressed component of CDC2/28 genes in higher eukaryotes. Although eukaryotic, T. vaginalis has properties similar to those of bacteria and is the earlist evolving eukaryote reported to possess CDC2/28 DNA and peptide homologues. These observations suggest that the molecular origins of cell division control in eukaroytes preceded mitochondria, 28S ribosomes and regulated glycolysis.
Transformation with katG restores isoniazid‐sensitivity in Mycobacterium tuberculosis isolates resistant to a range of drug concentrations
Authors: Zhang Ying , Garbe Thomas , Young Douglas 
Published: October 2006

SummaryIsoniazid‐resistant isolates of Mycobacterium tuberculosis were transformed with a plasmid vector carrying the functional catalase‐peroxidase (katG) gene. Expression of katG restored full drug susceptibility in isolates initially resistant to concentrations ranging from 3.2 to >50μgml−1. Transformation with the corresponding katG gene from Escherichia coli resulted in low‐level expression of catalase and peroxidase activities and conferred partial isoniazid sensitivity.
Charged amino‐terminal amino acids affect the lethal capacity of Lambda lysis proteins S107 and S105
Authors: Steiner Martin , Bläsi Udo 
Published: October 2006

SummaryThe lysis inhibitor protein S107 and the lysis effector protein S105 start at Met codons 1 and 3 of the Lambda S gene, respectively. The antagonistic action of both proteins precisely schedules lysis by formation of a non‐specific lesion in the inner membrane through which the Lambda‐encoded murein transglycosylase can pass. Here, we show that the main difference between lysis—effector and lysis—inhibitor is the degree by which an energized membrane inhibits either protein from hole formation. To dissect the structural parameters responsible for intrinsic inhibition of both proteins, charged amino acids were replaced proximal to the first putative membrane‐spanning region in both S proteins. Our results show that the distribution of amino‐terminal charged amino acids as well as the total amino‐terminal net charge of S107 and S105 influence their lethal potential. The data are interpreted in terms of a model in which the electrostatic status of the amino‐terminus of both S107 and S105 is an important feature affecting their conf or mat ional change required for formation of the S‐dependent hole.
Regulation of the synthesis of unsaturated fatty acids by growth temperature in Bacillus subtilis
Authors: Grau Roberto , de Mendoza Diego 
Published: October 2006

Summary Bacillus subtilis synthesizes, almost exclusively, saturated fatty acids, when grown at 37° C. When cultures were transferred from 37° C to 20° C, a chloramphenicol‐ and rifampicin‐sensitive synthesis of a C‐16 unsaturated fatty acid was observed. Synthesis of this compound reached a plateau after 5 h at 20° C, reaching levels of 20% of the total fatty acid content. [14C]‐labelled fatty acids attached as thioesters to acyl‐carriers compounds, such as coenzyme A (CoA) or acyl‐carrier protein (ACP) synthesized de novo by glycerol‐requiring auxotrophs deprived of glycerol to arrest phospholipid synthesis, could not be desaturated at 20° C. Desaturation of these fatty acids was readily observed when glycerol was restored to the cultures allowing resumption of transfer of acyl‐moieties from acyl‐thioesters to phospholipid. It was also observed that depletion of the pools of CoA and ACP by starvation of pantothenate auxotrophs had no effect on the observed synthesis of unsaturated fatty acid at 20° C. The overall results indicate that synthesis of unsaturated fatty acids in B. subtilis is a cold‐inducible process and that phospholipids are obligate intermediates in this fatty acid desaturation pathway.
Nucleotide sequence of a 13.9 kb segment of the 90 kb virulence plasmid of Salmonella typhimurium: the presence of fimbriai biosynthetic genes
Authors: Friedrich Mary Jane , Kinsey Nicole E. , Vila Jordi , Kadner Robert J. 
Published: October 2006

SummaryThe 90 kb plasmid resident in Salmonella typhimurium confers increased virulence in mice by promoting the spread of infection after invasion of the intestinal epithelium. The nucleotide sequence of a 13.9 kb segment of this plasmid known to encode an outer membrane protein related in sequence to components of fimbrial biosynthesis in enteric bacteria was determined. This cloned segment between the repB and repC replicon regions programmed expression of abundant surface fimbriae in Escherichia coli and S. typhimurium cells. A 7 kb region contained seven open reading frames, the protein products of five of which were related in sequence to regulatory, structural, and assembly proteins of adherence fimbriae/pili, such as the P and K88 piM. These five genes and two adjacent ones which were not markedly related to proteins in the data bases comprise the pef (plasmid‐encoded fimbriae) locus. Transposon TnphoA insertions In four genes in the pef locus (pefA, pefC, orf5 and orf6) resulted in active PhoA fusions and blocked or reduced the surface presentation of fimbriae, indicating that the proteins encoded by these four genes are translocated at least across the cytoptasmic membrane and contribute to formation of the fimbrial structure. The differences in genetic organization and protein sequence relatedness from other fimbrial gene clusters suggest that the pef locus might encode a novel type of fimbria. Between the pef and the repB loci, there were five open reading frames, one of which (orfS) gave rise to active PhoA fusions but was not necessary for fimbriai expression. Two of the other proteins were homologous to transcription regulatory proteins and a third was the rck gene, which encodes an outer membrane protein that confers complement resistance to serum‐sensitive hosts.
Molecular genetics of phosphofructokinase in the yeast Kluyveromyces lactis
Authors: Heinisch Jürgeni , Kirchrath Lutz , Liesen Thomas , Vogelsang Kathrin , Hollenberg Cornelis P. 
Published: October 2006

SummaryWe have undertaken a study of phosphofructokinase (PFK; E.C. 2.7.1.11) in the yeast Kluyveromyces lactis. Like other eukaryotic PFKs, the K. lactis enzyme is activated by the allosteric effectors AMP and fructose‐2,6‐bisphosphate. PFK activity is induced in cells grown on glucose as compared to ethanol‐grown cells, in contrast to the constitutive expression of PFK in Saccharomyces cerevisiae. We show here that phosphofructokinase of the yeast K. lactis is composed of two non‐identical types of sub‐units, encoded by the genes KIPFK1 and KIPFK2. We have cloned and sequenced both genes. KIPFK1 and KIPFK2 encode the α‐ and the β‐PFK subunits with deduced molecular weights of 109.336 Da and 104.074Da, respectively. Sequence analysis indicates that the genes evolved from a double duplication event. Null mutants in either of the genes lack detectable PFK activity in vitro and the respective subunits cannot be detected on Western blots. In contrast to the situation in S. cerevisiae, Klpfk1 Klpfk2 double mutants retain the ability to grow on glucose. However. Klpfk2 mutants and the double mutants do not grow on glucose, when respiration is blocked. These data suggest that the pentose phosphate pathway and respiration play a substantial role in glucose utilization by K. lactis. The K. lactis PFK genes can be expressed independently in S. cerevisiae and each of them complements the glucose‐negative phenotype of pfk1 pfk2 double deletion mutants in this yeast. Expression of both K. lactis PFK genes simultaneously in S. cerevisiae pfk double deletion mutants complements for PFK activity. However, expression of a combination of PFK genes from K. lactis and S. cerevisiae does not lead to the production of a functional enzyme.
Streptomyces antibioticus contains at least three oleandomycin‐resistance determinants, one of which shows similarity with proteins of the ABC‐transporter superfamily
Authors: Ma Rodriguez Ana , Olano Carlos , Vilches Carmen , Méndez Carmen , Salas José A. 
Published: October 2006

SummaryThree different DNA fragments of an oleandomycin producer, Streptomyces antibioticus, conferring oleandomycin resistance were cloned in plasmid pIJ702 and expressed in Streptomyces lividans and in Streptomyces albus. These oleandomycin resistance determinants were designated as oleA (pOR400), oleB (pOR501) and oleC (pOR800). oleA and oleC are closely linked in the chromosome as they were both obtained together in two cosmid clones that were isolated from a genomic library. Sequencing of the oleC resistance determinant revealed four complete open reading frames (ORFs) and the C‐terminal end of a fifth. The functions of orf1 and orf2 are unknown since they did not show significant similarity with other sequences in the data bases. The orf3 gene product has similarity with some proteins involved in iron and vitamin B12 uptake in bacteria. The orf4 gene product had a hydrophilic profile and showed important similarity with proteins containing typical ATP‐binding domains characteristic of the ABC‐transporter superfamily and involved in membrane transport and, particularly, with several genes conferring resistance to various macrolide antibiotics and anticancer drugs. The last gene, orf5, is translationally coupled to orf4 and codes for a hydrophobic polypeptide containing several trans‐membrane domains characteristic of integral membrane proteins. Subcloning and deletion experiments limited the resistance determinant to a 0.9kb Pst1‐Sph1 fragment and only orf4 is included in this fragment. These results suggest that resistance to oleandomycin conferred by oleC (orf4) is probably due to an efflux transport system of the ABC‐transporter superfamily.
Genetic analysis of the alginate biosynthetic gene cluster of Pseudomonas aeruginosa shows evidence of an operonic structure
Authors: Chitnis Chetan E. , Ohman Dennis E. 
Published: October 2006

SummaryStrains of Pseudomonas aeruginosa causing pulmonary infections in cystic fibrosis patients have an unusual mucoid phenotype because of production of the capsule‐like exopolysaccharide, alginate. Transcriptional activation of algD, the first gene of a large alginate biosynthetic gene cluster, is associated with conversion to the alginate‐producing (Alg+) phenotype. In this study, we examined the regulation of alginate genes immediately downstream of algD. Mutants of the Alg+ strain FRD1 were constructed by gene replacement with defined Tn501 (8.2 kb) insertions in the alginate biosynthetic gene cluster, resulting in an Alg− phenotype. The Alg+ phenotype of these mutants was restored by integration of narrow‐host‐range plasmids containing DNA fragments from P. aeruginosa that reconstructed a continuous alginate gene cluster. A broad‐host‐range plasmid containing the entire alginate gene cluster except for the terminal gene, algA was unable to complement an aIG::Tn501 mutant unless algA was transcribed from a second plasmid. This indicated that any Tn501 insertion in the cluster was polar on downstream alginate genes. Northern blot hybridization experiments also showed that a transposon insertion downstream of algD adversely affected algG and algA transcription. These results provided evidence that the alginate biosynthetic gene cluster has an operonic structure and is cotranscribed from the algD promoter.
Identification and characterization of the pupB gene encoding an inducible ferric‐pseudobactin receptor of Pseudomonas putida WCS358
Authors: Koster Margot , van de Vossenberg Jack , Leong John , Weisbeek Peter J. 
Published: October 2006

Summary Pseudomonas putida WCS358 can transport iron complexed to a wide variety of pseudobactins produced by other Pseudomonas strains. The pupB gene encoding an outer membrane ferric‐pseudobactin receptor was isolated from a genomic library of P. putida WCS358. The PupB receptor facilitated iron transport via two distinct heterologous siderophores, i.e. pseudobactin BN8 and pseudobactin BN7. The amino acid sequence deduced from the nucleotide sequence consisted of 804 amino acids (molecular weight 88369) of which the N‐terminal part was very similar to a prokaryotic leader peptide. The mature protein shared significant homology with the receptor for ferric‐pseudobactin 358 (PupA) and contained three regions common to TonB‐dependent receptor proteins of Eschenchia coli. Interestingly, PupB expression was only observed in cells cultured in iron‐deficient medium containing pseudobactin BN8 or pseudobactin BN7. This expression required a transcriptional unit, pupR, identified upstream of the structurai pupB gene. Transposon Tn5 insertion mutants defective in PupB production still exhibited uptake of iron via pseudobactin BN8, although with reduced efficiency. Apparently, an additional transport system for this ferric‐siderophore complex operates in this strain. In addition to pseudobactin BN8 also other heterologous siderophores were capable of inducing synthesis of specific high‐molecular‐weight outer membrane proteins in strain WCS358, which suggests the existence of multiple siderophore‐inducible iron transport systems in this strain.