bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular population genetic analysis of the enn subdivision of group A streptococcal emm‐like genes: horizontal gene transfer and restricted variation among enn genes
Authors: Whatmore Adrian M. , Kapur Vivek , Musser James M. , Kehoe Michael A. 
Published: October 2006

SummaryThe group A streptococcal emm‐like genes, which encode the cell‐surface M and M‐like proteins, are divided into distinct mrp, emm and enn subdivisions and are clustered together in a region of the chromosome called the vir regulon. In order to understand the mechanisms involved in the evolution of emm‐like genes, a 180bp fragment of the 5 variable region of the enn gene was characterized in 31 strains for which emm sequences and multilocus enzyme electrophoretic profiles have been previously determined. The results demonstrate that nucleotide polymorphisms at the enn locus are generated predominantly by point mutations and short deletions or insertions, and that variation among enn and emm genes has arisen by similar mechanisms. However, diversity at the enn locus is restricted in comparison to the emm locus. Moreover, there is strong evidence for intragenic recombination at the enn locus and the pattern of distribution of emm and enn alleles among strains suggests that these genes may be independently acquired by horizontal transfer and recombination from distinct donor strains, thereby generating a mosaic structure for the vir regulon. The results add to a growing body of evidence that horizontal gene transfer has played a major role in the evolution of Streptococcus pyogenes vir regulons.
Protein kinase‐dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram‐positive bacteria
Authors: Deutscher Josef , Küster Elke , Bergstedt Uta , Charrier Véronique , Hillen Wolfgang 
Published: October 2006

SummaryCcpA, the repressor/activator mediating carbon catabolite repression and glucose activation in many Gram‐positive bacteria, has been purified from Bacillus megaterium after fusing it to a His tag. CcpA‐his immobilized on a Ni‐NTA resin specifically interacted with HPr phosphorylated at seryl residue 46. HPr, a phosphocarrier protein of the phosphoenolpyruvate: glycose phosphotransferase system (PTS), can be phosphorylated at two different sites: (i) at His‐15 in a PEP‐dependent reaction catalysed by enzyme I of the PTS; and (ii) at Ser‐46 in an ATP‐dependent reaction catalysed by a metabolite‐activated protein kinase. Neither unphosphorylated HPr nor HPr phosphorylated at His‐15 nor the doubly phosphorylated HPr bound to CcpA. The interaction with seryl‐phosphorylated HPr required the presence of fructose 1,6‐bisphosphate. These findings suggest that carbon catabolite repression in Gram‐positive bacteria is a protein kinase‐triggered mechanism. Glycolytic intermediates, stimulating the corresponding protein kinase and the P‐ser‐HPr/CcpA complex formation, provide a link between glycolytic activity and carbon catabolite repression. The sensitivity of this complex formation to phosphorylation of HPr at His‐15 also suggests a link between carbon catabolite repression and PTS transport activity.
L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria
Authors: Sarkis Gary J. , Jacobs William R. , Hatfulll Graham F. 
Published: October 2006

SummaryRecombinant bacteriophages provide efficient delivery systems for introducing reporter genes into specific bacterial hosts. We have constructed mycobacteriophage L5 recombinants carrying the firefly luciferase gene inserted into the tRNA region of the phage genome. Infection of Mycobacterium smegmatis by these phages results in expression of the luciferase gene and light emission. Fortuitously, the luciferase gene is expressed continuously in lysogens surviving infection. Synthesis of luciferase from a mycobacterial promoter created by cloning enables the detection of extremely small numbers of M. smegmatis cells. These reporter phages can be used to discriminate between drug‐sensitive and drug‐resistant strains of M. smegmatis, and may provide tools for the rapid identification and classification of antimycobacterial agents.
Mutations in the chemotactic response regulator, CheY, that confer resistance to the phosphatase activity of CheZ
Authors: Sanna M. Germana , Swanson Ronald V. , Bourret Robert B. , Simon Melvin I. 
Published: October 2006

SummaryCheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway. The concentration of phospho‐CheY is thought to determine the swimming behaviour of the cell: high levels of phospho‐CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phos‐phorylated form of the protein allow counter‐ciockwise rotation of the flagella and smooth swimming. The phosphorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphorylation of phospho‐CheY. The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho‐CheY and by the CheZ protein, which accelerates dephosphorylation. We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY proteins. Two point mutations were identified which were 50‐fold and 5‐fold less sensitive to the activity of CheZ than was the wild‐type protein. Nonetheless, the phosphorylation and autodephos‐phorylation rates of these mutants, CheY23ND and CheY26KE, were observed to be identical to those of wild‐type CheY in the absence of CheZ. These are the first examples of CheY mutations that reduce sensitivity to the phosphatase activity of CheZ without being altered in terms of their intrinsic phosphorylation and autodephospborylation rates, interestingly, the residues Asn‐23 and Lys‐26 are located on a face of CheY far from the phosphorylation site (Asp‐57), distinct from the previously described site of inter‐action with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch.
The hrp gene locus of Pseudomonas solanacearum, which controls the production of a type III secretion system, encodes eight proteins related to components of the bacterial flagellar biogenesis complex
Authors: van Gijsegem Frédérique , Gough Clare , Zischek Claudine , Niqueux Eric , Arlat Matthieu , Genin Stéphane , Barberis Patrick , German Sylvie , Castello Philippe , Boucher Christian 
Published: October 2006

SummaryFive transcription units of the Pseudomonas solanacearum hrp gene cluster are required for the secretion of the HR‐inducing PopA1 protein. The nucleotide sequences of two of these, units 1 and 3, have been reported. Here, we present the nucleotide sequence of the three other transcription units, units 2, 4 and 7, which are together predicted to code for 15 hrp genes. This brings the total number of Hrp proteins encoded by these five transcription units to 20, including HrpB, the positive regulatory protein, and HpaP, which is apparently not required for plant interactions., Among the 18 other proteins, eight belong to protein families regrouping proteins involved in type III secretion pathways in animal and plant bacterial pathogens and in flagellum biogenesis, while two are related solely to proteins involved in secretion systems. For the various proteins found to be related to P. solanacearum Hrp proteins, those in plant‐pathogenic bacteria include proteins encoded by hrp genes. For Hrp‐related proteins of animal pathogens, those encoded by the spa and mxi genes of Shigella flexneri and of Salmonella typhimurium and by the ysc genes of Yersinia are involved in type III secretion pathways. Proteins involved in flagellum biogenesis, which are related to Hrp proteins of P. solanacearum, include proteins encoded by fli and fli genes of S. typhimurium, Bacillus subtils and Escherichia coli and by mop genes of Erwinia carotovora. P. solanacearum Hrp proteins were also found to be related to proteins of Rhizobium fredii involved in nodulation specificity.
Repression of lipopolysaccharide biosynthesis in Escherichia coli by an antisense RNA of Acetobacter methanolicus phage Acm1
Authors: Mamat U. , Rietschel E. Th. , Schmidt G. 
Published: October 2006

SummaryLysogenic Acetobacter methanolicus strains carrying the prophage Acm1 were found to be unable to synthesize both the capsutar polysaccharide (CPS) and the O‐specific side‐chain of lipopolysaccharide (LPS) and to represent rough variants of the host bacterium. A 262 bp DNA fragment of phage Acm1, obviously required for interference with LPS biosynthesis, was cloned and expressed in Escherichia coli Independently of the O‐type, transformation of various E. coli strains with the recombinant DNA resulted in a suppression of biosynthesis of the O‐specific chains. The DNA fragment of phage Acm1 contained three very short open reading frames of 21, 24, and 36 bp. However, attempts to express phage‐encoded peptides were not successful. Instead, the Acm1‐derived DNA fragment was shown to code for the synthesis of a trans‐acting RNA molecule of 97 nucleotides, designated lbi (LPS biosynthesis‐interfering) RNA. This RNA contains sequence complementarity to E. coli target RNA sequences and appears to have the ability to form intracellularly RNA hybrid duplexes with mRNA. The data presented in this study support the hypothesis that the phenotypic effect of conversion to rough‐type LPS is accompanied by the expression of an antisense RNA of phage Acm1.
Molecular genetics of a chromosomal locus involved in copper tolerance in Escherichia coli K‐12
Authors: Fong Sheik‐Tao , Camakaris James , Lee Barry T. O. 
Published: October 2006

SummaryThe cutA locus, presumably involved in copper tolerance in Escherichia coli, was characterized by a mutation leading to copper sensitivity. Copper‐accumulation measurements with radioactive 64Cu6+ showed increased uptake by cutA copper‐sensitive mutant cells, and reduced uptake when the cutA mutation was complemented in trans. The locus was mapped using complementation of the cutA mutant to partial copper tolerance with wild‐type chromosomal fragments. The 3.2 kb DNA region involved in cutA was sequenced and analysed, revealing three significant open reading frames, none of which had been previously published. The products of all three open reading frames were identified, when synthesized with the T7 phage promoter expression system, as polypeptides of about 50kDa, 24kDa, and 13kDa, consistent with the sizes predicted from the DNA sequences. The 50kDa and 24kDa polypeptides were found in the bacterial inner membrane, and the 13kDa polypeptide with the cytoplasmic fraction. In addition to being required for copper tolerance, cutA affects tolerance levels to zinC., nickel, cobalt and cadmium salts. Transcriptional fusions of cutA with the lux operon showed induction by copper, zinc, nickel, cobalt and, to a lesser extent, cadmium, manganese and silver salts.
The biogenesis of c‐type cytochromes in Escherichia coli requires a membrane‐bound protein, DipZ, with a protein disulphide isomerase‐like domain
Authors: Crooke Helen , Cole Jeff 
Published: October 2006

SummaryA mutant of Escherichia coli K‐12, JCB606, which lacks all five c‐type cytochromes synthesized during anaerobic growth in the presence of nitrite or tri‐methylamine‐N‐oxide (TMAO), was totally defective in Nrf activity and also partially defective in TMAO reductase activity. The mutation in strain JCB606 was shown to affect expression of the tor operon, which contributes almost equally with the products of the dms operon to the rate of TMAO reduction by bacteria during anaerobic growth in the presence of TMAO. The mutation in strain JCB606, dipZ, was mapped by P1 transduction close to the mel operon at co‐ordinate 4425 on the E. coli chromosome, the gene order being nrf–fdhF–mel–dipZ–ampC. Recombinant plasmids that restored Nrf activity to test‐tube cultures of the mutant were isolated from a cosmid library. A 2.7 kb EcoRV–Smal fragment (co‐ordinates 4443 to 4446 kb on the physical map of the E. coli chromosome) was found potentially to encode three genes arranged in at least two operons. The second gene, dipZ, was sufficient to complement the JCB606 mutation. The translated DNA sequence predicts that DipZ is a 53kDa integral membrane protein with a 37kDa N‐terminal domain including at least six membrane‐spanning helices and a 16kDa carboxy‐terminal hydrophilic domain which includes a protein disulphide isomerase‐like motif. It is suggested that DipZ is essential for maintaining cytochrome c apoproteins in the correct conformations for the covalent attachment of haem groups to the appropriate pairs of cysteine residues.
Treponema pallidum in gel microdroplets: a novel strategy for investigation of treponemal molecular architecture
Authors: Cox David L. , Akins Darrin R. , Porcella Stephen F. , Norgard Michael V. , Radolf Justin D. 
Published: October 2006

SummaryControversy exists regarding the constituents and antigenic properties of the Treponema pallidum outer membrane; a major point of contention concerns the cellular location(s) of the spirochaete's lipoprotein immunogens. To address these issues and circumvent problems associated with prior efforts to localize treponemal surface antigens, we developed a novel strategy for investigating T. pallidum molecular architecture. Virulent treponemes were encapsulated in porous agarose beads (gel microdroplets) and then probed in the presence or absence of Triton X‐100. Intact., encapsulated treponemes were not labelled by monospecific antisera directed against four major T. pallidum lipoproteins or a candidate T. pailidum outer membrane protein (TpN50) with C‐terminal sequence homology to Escherichia coli OmpA or by human or rabbit syphilitic serum. Each of these immunologic reagents, however, labelled encapsulated treponemes co‐incubated with detergent. In contrast, antibodies generated against isolated T, pal‐lidum outer membranes labelled intact organisms and the pattern of fluorescence was consistent with the distribution of rare outer membrane proteins visualized by freeze‐fracture electron microscopy. In addition to providing strong evidence that the protein portions of treponemal lipoproteins are located within the periplasmic space, these studies have extended our understanding of the topographical relationships among T. pallidum cell envelope constituents. They also demonstrate the feasibility of generating antibodies against rare outer membrane proteins and detecting them on the surfaces of virulent treponemes.
Low‐molecular‐weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterized by a periplasmic N‐terminal domain and a missing C‐terminal domain
Authors: Becker Anke , Niehaus Karsten , Pühler Alfred 
Published: October 2006

SummaryThe membrane topology of the Rhizobium meliloti 2011 ExoP protein involved in polymerization and export of succinoglycan was analysed by translational fusions of lacZ and phoA reporter genes to the exoP gene. Based on this analysis, the ExoP protein could be divided into an N‐terminal domain mainly located in the periplasmic space and a C‐terminal domain located in the cytoplasm. Whereas the C‐terminal domain of ExoP is characterized by a potential nucleotide‐binding motif, the N‐terminal ExoP domain contains the sequence motif‘PX2pX4SPKX11GXMXG1′, which is also present in proteins involved in the determination of O‐antigen chain length. R. meliloti strains carrying mutated exoP* genes, exclusively encoding the N‐terminal ExoP domain, produced a reduced amount of succinoglycan. This reduction could be suppressed by a mutation in the regulatory gene exoR. The ratio of low‐molecular‐weight to high‐molecular‐weight succinoglycan was significantly increased in the exoP* mutant strain. In the exoP*lexoR mutant strain only low‐molecular‐weight succinoglycan could be detected. Based on sequence homologies and similar hydropathic profiles, the N‐terminal domain of ExoP was proposed to be a member of a protein family thought to be involved in polysaccharide chain‐length determination.
Activation of the transcriptional regulator XylR of Pseudomonas putida by release of repression between functional domains
Authors: Fernández Silvia , de Lorenzo Víctor , Pérez‐Martin José 
Published: October 2006

SummaryIn the presence of toluene, xylenes and other structural analogues, the regulatory protein XylR, of the family of transcriptional regulators which act in concert with the σ54 factor, activate the promoter Pu of the TOL (toluene degradation) plasmid pWW0 of Pseudomonas putida. Amino acid changes Val‐219‐Asp and Ala‐220‐Pro, introducing a proline kink at the hinge region between the N‐terminal A domain and the central portion of XylR, resulted in a semi‐constitutive phenotype which mimicked the activating effect of aromatic inducers. This phenotype was further exacerbated by inserting extra amino acid residues within the same inter‐domain region. A truncated XylR protein devoid of the signal‐receiving, amino‐terminal portion of the protein stimulated the cognate promoter Pu at high levels independently of inducer addition, both in Escherichia coli and in Pseudomonas putida. Replacement of the amino‐terminal domain by a heterologous peptide derived from the MS2 virus polymerase resulted in a hybrid protein still able to bind DNA to the same extent in vivo as XylR, but unable to stimulate transcription. These data indicate that a key event in the activation of XyIR by toluene/xylenes is the release of the repression caused by the A domain of the protein on surfaces located at the central domain of the regulator.
The genetic basis of colony opacity in Streptococcus pneumoniae: evidence for the effect of box elements on the frequency of phenotypic variation
Authors: Saluja Sunil K. , Weiser Jeffrey N. 
Published: October 2006

Summary Streptococcus pneumoniae undergoes spontaneous phase variation in colony morphology. Differences in colony opacity have previously been shown to correlate with differences in the ability of organisms to colonize the mucosal surface of the nasopharynx in an animal model. The genetic basis of opacity variation was identified in transformation experiments. A DNA library, from a strain that varies at high frequency, was screened to identify a single clone capable of transforming a transparent recipient strain which varies at low frequency to an opaque phenotype. Analysis of this opacity locus revealed two genes, glpD and glpF, with similarity to genes required for glycerol metabolism in other bacteria. Following the pneumococcal glpF, repetitive intergenic elements, boxes A and C, were identified. These stem‐loop‐forming elements were not present in the same locus of the recipient strain. Although not required for phase variation in colony opacity, the box element was necessary for expression of phase variation at high frequency. Introduction of the box elements during transformation affected colony morphology, possibly by altering expression of a putative regulatory gene downstream from the box element. Mutagenesis within this region confirmed the contribution of the putative regulatory gene to the expression of colony opacity. Growth characteristics of strains generated in this study provide additional evidence for an association of differences in cell wall autolysis and variation in colony opacity.
Genetic studies reveal that myristoylCoA:protein N‐myristoyltransferase is an essential enzyme in Candida albicans
Authors: Weinberg Robin A. , McWherter Charles A. , Freeman Sandra K. , Wood David C. , Gordon Jeffrey I. , Lee Stephen C. 
Published: October 2006

SummaryMyristoylCoA:protein N‐myristoyltransferase (Nmt) catalyses the co‐transiational, covalent attachment of myristate (C14:0) to the amino‐terminal glycine residue of a number of eukaryotic proteins involved in cellular growth and signal transduction. The NMT1 gene is essential for vegetative growth of Saccharomyces cerevisiae. Studies were carried out to determine if Nmt is also essential for vegetative growth of the pathogenic fungus Candida albicans. A strain of C. albicans was constructed in which one copy of NMT was partially deleted and disrupted. A Gly‐447 — Asp mutation was Introduced into the second NMT allele. This mutation produced marked reductions in catalytic efficiency at 24 and 37° C, as judged by in vitro kinetic studies of the wild‐type and mutant enzymes which had been expressed in, and purified from, Escherichia coli. The growth characteristics of isogenic NMT/NMT, NMT/Δnmt, and nmtΔ/nmtG447D C. albicans strains were assessed under a variety of conditions. Only the nmtδ/nmtG447D strain required myristate for growth. This was true at both 24 and 37°C. Palmitate could not substitute for myristate. Incubation of nmtΔ/nmtG447D cells at 37° C in the absence of myristate resulted in cell death as observed by the inability to form colonies on media supplemented with 500 μM myristate. Studies in an immunosuppressed‐mouse model of C. albicans infection revealed that the NMT/Δnmt strain produced 100% lethality within 7 d after intravenous administration while the isogenic nmtΔ/nmtG447G strain produced no deaths even after 21 d. These observations establish that Nmt is essential for vegetative growth of C. albicans and suggest that Inhibitors of this acyltransferase may be therapeutically useful fungicidal agents.
Purification and characterization of LasD: a second staphylolytic proteinase produced by Pseudomonas aeruginosa
Authors: Park Sukjoon , Galloway D. R. 
Published: October 2006

SummaryWe have previously described studies of a 22 kDa active fragment of the LasA proteinase. In follow‐up studies of LasA, we have discovered the separate existence of a 23 kDa proteinase which shares many of the enzymatic properties of LasA, including the ability to lyse heat‐killed staphylococoi. However, this apparent serine proteinase, which we designate LasD, is distinct from the 22 kDa active LasA protein for the following reasons: (i) the N‐terminal sequence of LasD shares no homology with LasA or the LasA precursor sequence; (ii) Pseudomonas aeruginosa LasA mutant strains AD1825 and FRD2128 do not produce LasA yet produce LasD; and (iii) specific antibodies to each proteinase do not show any cross‐reactivity. LasD appears to be produced as a 30 kDa protein, which is possibly cleaved to produce a 23 kDa active fragment. The purified LasD fragment (23 kDa) shows strong staphylolytic activity only at higher pH conditions, while LasA exhibits staphylolytic activity over a broad pH range, in addition to their ability to cleave at internal diglycine sites, both the LasD and LasA endoproteinases efficiently cleave β‐casein.