bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structure and expression of the alternative sigma factor, RpoN, in Rhodobacter capsulatus; physiological relevance of an autoactivated nifU2‐rpoN superoperon
Authors: Cullen Paul J. , Foster‐Hartnett Dawn , Gabbert Karen K. , Kranz Robert G. 
Published: October 2006

SummaryThe alternative sigma factor, RpoN (σ54) is responsible for recruiting core RNA polymerase to the promoters of genes required for diverse physiological functions In a variety of eubacterial species. The RpoN protein In Rhodobacter capsulatus is a putative sigma factor specific for nitrogen fixation (nif) genes. Insertional mutagenesis was used to define regions important for the function of the R. capsulatus RpoN protein. Insertions of four amino acids in the predicted helix‐turn‐helix or in the highly conserved C‐terminal eight amino acid residues (previously termed the RpoN box), and an in‐frame deletion of the glutamine‐rich M‐terminus completely inactivated the R. capsulatus RpoN protein. Two separate insertions in the second hydrophobic heptad repeat, a putative leucine zipper, resulted in a partially functional RpoN protein. Eight other linkers in the rpoN open reading frame (ORF) resulted in a completeiy or partially functional RpoN protein. The rpoN gene in R capsulatus is downstream from the nifHDKU2 genes, in a nifU2‐rpoN operon. Results of genetic experiments on the nifU2‐rpoN locus show that the rpoN gene is organized in a nifU2‐rpoN superoperon. A primary promoter directly upstream of the rpoN ORF is responsible for the initial expression of rpoN. Deletion analysis and insertional mutagenesis were used to define the primary promoter to 50 bp, between 37 and 87 nucleotides upstream of the predicted rpoN translational start site. This primary promoter is expressed constitutively with respect to nitrogen, and it is necessary and sufficient for growth under nitrogen‐limiting conditions typically used in the laboratory. A secondary promoter upstream of nifU2 is autoactivated by RpoN and NifA to increase the expression of rpoN, which ultimately results in higher expression of RpoN dependent genes. Moreover. rpoN expression from this secondary promoter is physiologically beneficial under certain stressful conditions, such as nitrogen‐limiting environments that contain high salt (>50mM NaCl) or low iron (<400nM FeS04).
Molecular analysis of the Erwinia chrysanthemi region containing the kdgA and zwf genes
Authors: Hugouvieux‐Cotte‐Pattat Nicole , Robert‐Baudouy Janine 
Published: October 2006

SummaryThe pathways of pectin and galacturonate catabolism In Erwinia chrysantheml converge to form a common intermediate, 2‐keto‐3‐deoxygluconate, which is phosphorylated to form 2‐keto‐3‐deoxy‐6‐phospho‐giuconate (KDGP) and then cleaved by the aldolase encoded by the kdgA gene. We cloned the kdgA gene of the E. chrysanthemi strain 3937 by complementing an Escherichia coli kdgA mutation, using an RP4‐derivative plasm id. Restriction mapping of the kdgA region and isolation of kdgA‐lac fusions allowed the more precise localization of the kdgA gene and determination of its transcriptional direction. The nucleotide sequence of the kdgA region indicated that the kdgA reading frame is 639 bases long, corresponding to a protein of 213 amino acids with a molecular mass of 22187 Da. Comparison of the deduced primary amino acid sequences of the E. chrysanthemi KDGP‐aldolase to the E. coli, Zymomonas mobilis and Pseudomonas putida enzymes showed that they are highly conserved. The E. chrysanthemi kdgA structural gene begins 153 bases downstream of an open reading frame that has a high homology with the zwf E. coli gene encoding giucose‐6‐phosphate dehydrogenase. The zwf gene is also linked to eda (kdgA) in E. coli and P. putida but genetic organization is different. Regulation of zwf and kdgA expression in E. chrysanthemi was analysed using lacZ fusions. The expression of zwf is independent of the growth rate, but is repressed in the presence of glucose. Induction of kdgA by pectin‐degradation products is mediated in vivo by the negative regulatory gene kdgR, which also controls all the steps of pectin degradation. However, the KdgR repressor is unable to bind in vitro to the 5′‐untranslated end of the kdgA gene.
Evidence that residues −15 to −46 of the haemolysin secretion signal are involved in early steps in secretion, leading to recognition of the translocator
Authors: Kenny Brendan , Chervaux Christian , Holland I. Barry 
Published: October 2006

SummaryWe previously identified three well‐dispersed mutations, E978‐K, F989‐L and D1009‐R within the haemolysin A signal region, located at positions –46, –35 and –15, with respect to the C‐terminus, respectively. Each mutation reduces the efficiency of secretion two‐ to threefold leaving 30–45% of the wild‐type activity. We have constructed by in vitro manipulations double mutants of HlyA carrying all combinations of these mutations and a triple mutant carrying all three mutations. The effects on secretion were determined and the results, including residual levels of secretion with the triple mutant of only 0.6%, compared with the wild type, indicated that these residues may interact to form a single function in the wild‐type signal. To test this further, we developed a secretion competition assay in order to classify signal mutations. We demonstrated that a CIZ‐HlyA fusion protein, containing the C‐terminal 81 kDa of HlyA fused to virtually the whole LacZ protein, strongly inhibits the secretion of the wild‐type HlyA co‐expressed In the same cell. The properties of the fusion indicate that it blocks the translocator. The three mutations singly and in combinations were recombined in vitro into the 3′‐end of the hybrid gene. In every case, the presence of a mutation in the secretion signal of the hybrid protein alleviated the inhibition of secretion of the co‐expressed HiyA. All the mutations are therefore essentially recessive and we propose that they all affect an early function, probably recognition of the translocator, rather than a subsequent step involved in translocation or final release of the toxin to the medium. This would indicate that residues involved in recognition for steps
The relative contributions of transcription and translation to plasmid DNA supercoiling in Salmonella typhimurium
Authors: Spirito Flavia , Figueroa‐Bossi Nara , Bossl Lionello 
Published: October 2006

SummaryMutations affecting DNA topoisomerase I (topA) in Saimonella typhimurium were isolated and graded on the basis of their ability to reverse the effects of gyrB mutations on his operon expression. Different topA and gyrB alleles (in otherwise isogenic strains) were used to gather insights into the transcription‐dependent variability of plasmid DNA‐linking deficit in growing bacteria. This study showed that modulation of DNA supercoiling by transcription results from the action of two components: one is highly dependent on the coupling of translation to RNA‐chain elongation; and the other is unrelated to protein synthesis and entirely dependent on promoter determinants. The former greatly predominates In DNA topoisomerase I mutants (topA and topA gyrB) while the latter is the sole contributor to plasmid DNA‐linking deficit in wild‐type cells. Altogether, these data suggest that whereas translation acts by enhancing the formation of twin supercoiled domains during elongation, the promoter‐dependent effects bear no relation to the twin‐supercoiled‐domain model and are latter explained by a mechanism which responds to the binding/unwinding of template DNA by RNA polymerase.
Characterization of a Pseudomonas aeruginosa gene cluster involved in pilus biosynthesis and twitching motility: sequence similarity to the chemotaxis proteins of enterics and the gliding bacterium Myxococcus xanthus
Authors: Darzins Aldis 
Published: October 2006

SummaryThe type 4 pill of Pseudomonas aeruginosa are important cell‐associated virulence factors that play a crucial role in mediating (i) bacterial adherence to, and colonization of, mucosal surfaces, (ii) a novel mode of fiagetia‐independent surface translocation known as‘twitching motility, and (iii) the initial stages of the infection process for a number of bacteriophages. A new set of loci involved in pilus biogenesis and twitching motility was identified based on the ability of DNA sequences downstream of the pilG gene to complement the non‐piliated (pil) strain, PAO6609. Sequence analysis of a 3.2 kb region directly downstream of pilG revealed the presence of three genes, which have been designated pilH, pill, and pilJ. The predicted translation product of the pilH gene (13 272 Da), like PilG, exhibits significant amino acid identity with the enteric single‐domain response regulator CheY. The putative Pili protein (19933 Da) is 28% identical to the FrzA protein, a CheW homologue of the gliding bacterium Myxococcus xanthus, and the PMJ protein (72 523 Da) is 26% identical to the enteric methyl‐accepting chemotaxis protein (MCP) Tsr. Mutants containing insertions in pill and pilJ were severely impaired in their ability to produce pili and did not translocate across solid surfaces. The pilH mutant remained capable of pilus production and twitching motility, but displayed an altered motility pattern characterized by the presence of many doughnut‐shaped swirls. Each of these pil mutants, however, produced zones that were at least as large as the parent in flagellar‐mediated swarm assays. The sequence similarities between the putative pilG, H, I and J gene products and several established chemotaxis proteins, therefore, lend strong support to the hypothesis that these proteins are part of a signal‐transduction network that controls P. aeruginosa pilus biosynthesis and twitching motility.
Immunogenicity and evolutionary variability of epitopes within IgA1 protease from serogroup A Neisseria meningitidis
Authors: Morelli Giovanna , del Valle Jesus , Lammel Claudia J. , Pohlner Johannes , Müller Kerstin , Blake Milan , Brooks Geo. F. , Meyer Thomas F. , Koumaré Brehima , Brieske Norbert , Achtman Mark 
Published: October 2006

SummaryFive murine epitopes were defined and mapped within IgA1 protease produced by Neisseria meningitidis. Epitopes 1 and 2 were present in IgA1 protease from all strains, and from Neisseria gonorrhoeae. Epitopes 3 through to 5 varied between subgroups of serogroup A meningococci. but have remained constant over decades within the subgroups, except for epitope 4, which changed between 1983 and 1987 during the spread of subgroup III meningococci from Asia to Africa. Binding of monoclonal antibodies to epitopes 1, 4 and 5 neutralized enzymatic function. Human sera containing antibodies to lgA1 protease as a result of natural infection inhibited binding of monoclonal antibodies to epitope 4 but not to the other epitopes.
Glycogen in Bacillus subtilis: molecular characterization of an operon encoding enzymes involved in glycogen biosynthesis and degradation
Authors: Kiel J. A. K. W. , Boels J. M. , Beldman G. , Venema G. 
Published: October 2006

SummaryAlthough it has never been reported that Bacillus subtilis is capable of accumulating glycogen, we have isolated a region from the chromosome of B. subtilis containing a glycogen operon. The operon is located directly downstream from trnB, which maps at 275 on the B. subtilis chromosome, it encodes five poly‐peptides with extensive similarity to enzymes involved in glycogen and starch metabolism in both prokaryotes and eukaryotes. The operon is presumably expressed by an EσE‐controlled promoter, which was previously identified downstream from trnB. We have observed glycogen biosynthesis in B. subtilis exclusively on media containing carbon sources that allow efficient sporulation. Sporulation‐independent synthesis of glycogen occurred after integration of an EσA controlled promoter upstream of the operon.
Identification of a DNA‐contacting surface in the transcription factor sigma‐54
Authors: Cannon W. , Claverie‐Martin F. , Austin S. , Buck M. 
Published: October 2006

SummaryThe transcription factor sigma‐54 (σ54) is a sequence‐specific DNA‐binding protein that directs RNA polymer a se to a particular class of promoter. The interaction of σ54 with promoter DNA has been analysed by protein‐DNA crosslinking and enzymatic and chemical proteolysis. Direct physical evidence for a DNA‐contacting surface within the carboxy‐terminal one‐third of the protein has been obtained. This region of σ54 is likely to be close to the surface of the protein, and contacts DNA when either o54 or the σ54‐holoenzyme bind specifically to promoter DNA. The amino‐terminal region of σ54 appears to be highly susceptible to proteolysis, and its integrity influences the accessibility towards proteolysis of a second region of σ54, which includes the DNA‐contacting surface. Thus the amino‐terminal region of σ54 may have a role in influencing its DNA‐binding properties, the major determinants of which appear to reside in the carboxy‐terminal one‐third of the protein.
Folate responsiveness during growth and development of Dictyostelium: separate but related pathways control chemotaxis and gene regulation
Authors: Blusch Jürgen H. , Nellen Wolfgang 
Published: October 2006

SummaryFolate‐controtled gene expression and chemotaxis have been examined in Dictyostelium wild‐type and mutant strains. We show that regulation of the discoidin genes is sensitive to foiate in growing ceiis as weli as in suspension development. The signal is transferred via the N10‐methylfoiate‐sensitive folate receptor sites, which also appear to confer the chemotactic response. The strain HG5145 has previously been isolated as a mutant that does not display chemotactic movement towards folate. Nevertheless, these cells are fully functional in foiate‐mediated downregulation of discoidin I expression. The strain ga 93 has been isolated as an overproducer mutant of the cyclic nucleotide phosphodiesterase inhibitor. Simultaneously, these cells fail to downregulate discoidin I in response to folate but are fully functional in folate chemotaxis. Therefore we conclude that the pathways for chemotaxis and for gene regulation diverge downstream of a common receptor type.
Identification and molecular characterization of a major ring‐forming surface protein from the gastric pathogen Helicobacter mustelae
Authors: O'Toole Paul W. , Austin John W. , Trust Trevor J. 
Published: October 2006

SummaryThe spiral microaerophilic bacterium Helicobacter mustelae is linked to gastritis and gastric ulcers in ferrets. Electron microscopy of H. mustelae showed the presence of a laterally extensive array of 8.5‐nm‐diameter rings on the cell surface, which was shown to be composed of a 150 kDa protein. This protein was purified, and the sequence of 10 amino‐terminal residues was determined. Polyclonal antibody against the purified 150 kDa protein labelled the ring structures on the homologous strain by means of immuno‐gold. Cross‐reactive proteins were identified in three H. mustelae strains, but not in Helicobacter pylori or Helicobacter felis. The hsr gene encoding this protein was cloned, and the protein expressed in Escherichia coli independently of vector promoters. The 1519‐codon nucleotide sequence of the gene was determined, and comparison with the chemically derived protein sequence indicated a 47‐residue leader pep‐tide, and a mature protein with a molecular weight of 152 300. Thus the cell surface of H. mustelae differs markedly from other members of the genus Helicobacter in being covered by an array of 8.5 nm rings composed of a 150 kDa protein.
Horizontal gene transfer in the evolution of group A streptococcal emm‐like genes: gene mosaics and variation in Vir regulons
Authors: Whatmore Adrian M. , Kehoe Michael A. 
Published: October 2006

SummaryMost M type 5 group A streptococcal strains were found to contain a single emm‐like gene between virR and scpA (the Vir reguion), but two distinct emm‐like genes were identified in the Vir regulon of the MS strain NCTC8193. The complete sequences of both of these genes were determined. One, called emm5.8193, was shown to be a minor variant of the previously described emm5 gene from strain Manfredo. The second, designated enn5.8193, expresses an IgG‐binding protein when cloned in Escherichia coli. A comparison of enn5.8193 with emm‐like gene sequences from other strains indicated that it has a mosaic structure, consisting of distinct segments originating from emm‐like genes in different OF+ and OF−strains. These data provide the first clear evidence that the horizontal transfer of emm‐like sequences between distinct strains contributes to the evolution of group A streptococcal emm‐like genes and Vir regulons.
The Clostridium perfringens Tet P determinant comprises two overlapping genes: tetA(P), which mediates active tetracycline efflux, and tetB(P), which is related to the ribosomal protection family of tetracycline‐resistance determinants
Authors: Sloan Joan , McMurry Laura M. , Lyras Dena , Levy Stuart B. , Rood Julian I. 
Published: October 2006

SummaryThe complete nucleotide sequence and mechanism of action of the tetracycline‐resistance determinant Tet P, from Clostridium perfringens has been determined. Analysis of the 4.4 kb of sequence data revealed the presence of two open reading frames, designated as tetA(P) and tetB(P), The tetA(P) gene appears to encode a 420 amino acid protein (molecular weight 46079) with twelve transmembrane domains. This gene was shown to be responsible for the active efflux of tetracycline from resistant ceils. Although there was some amino acid sequence similarity between the putative TetA(P) protein and other tetracycline efflux proteins, analysis suggested that TetA(P) represented a different type of efflux protein. The tetB(P) gene would encode a putative 652 amino acid protein (molecular weight 72639) with significant sequence similarity to Tet(M)‐like cytoplasmic proteins that specify a ribosomal‐protection tetracycline‐resistance mechanism. In both C. perfringens and Escherichia coli. tetB(P) encoded low‐level resistance to tetracycline and minocycline whereas tetA(P) only conferred tetracycline resistance. The tetA(P) and tetB(P) genes appeared to be linked in an operon, which represented a novel genetic arrangement for tetracycline‐resistance determinants. It is proposed that tetB(P) evolved from the conjugative transfer into C. perfringens of a fer (M)‐like gene from another bacterium.
Bacillus anthracis pXO1 virulence plasmid encodes a type 1 DNA topoisomerase
Authors: Fouet Agnès , Sirard Jean‐Claude , Mock Michèle 
Published: October 2006

SummaryThe virulence plasmid pXO1 is responsible for toxin production in Bacillus anthracis. A DNA fragment from pXO1 was isolated and was shown, by sequence analysis, to contain part of a type 1 DNA topoisomerase gene. Attempts to clone the entire wild‐type gene, designated topX, in Escherichia coli, were unsuccessful. In order to obtain the complete gene, it was first insertionally inactivated and then cloned in the mutated form. The deduced amino acid sequence of Topo X1 shows similarities to that of the two E. coli type 1 DNA topoisomerases. The N‐terminal two‐thirds of the putative B. anthracis protein exhibits strongest sequence similarity to topoisomerase III, whereas the C‐terminal portion contains cysteine residues that could form three zinc‐binding domains, as they do in topoisomerase I. The suggested active‐site tyrosine is conserved in all three proteins. The regulation of expression from the topX promoter is modified by addition of a gyrase inhibiting antibiotic. The Topo X1 protein is likely to be involved in the stability of pXO1.
Increased substitution of phosphate groups in lipopolysaccharides and lipid A of the polymyxin‐resistant pmrA mutants of Salmonella typhimurium: a 31P‐NMR study
Authors: Helander Ilkka M. , Kilpeläinen Ilkka , Vaara Martti 
Published: October 2006

SummaryDe‐O‐acylated lipopolysaccharides (LPS) of three polymyxin‐resistant Salmonella typhimurium pmrA mutants and their parent strains were analysed by 31P‐NMR (nuclear magnetic resonance) in order to assess, in relation to polymyxin resistance, the types and degree of substitution of phosphates of the LPS and lipid A. in the pmrA mutant LPS phosphate diesters predominated over phosphate monoesters, whereas the latter were more abundant in the parent wild‐type LPS. The increase in the proportion of phosphate diesters was traced to both the core oligosaccharide and the lipld A part. In the latter, the ester‐linked phosphate at position 4’was to a large extent (79–88%) substituted with 4‐amino‐4‐deoxy‐l‐arabinose, whereas in the wild‐type LPS the 4′‐phosphate was mainly present as monoester. In each LPS, regardless of the pmrA mutation, the glycosidically linked phosphate of lipid A was largely unsubstituted.
Trachoma and LGV biovars of Chlamydia trachomatis share the same glycosaminoglycan‐dependent mechanism for infection of eukaryotic cells
Authors: Chen Joseph C.‐R. , Stephens Richard S. 
Published: October 2006

SummaryA sulphated glycosaminoglycan‐dependent mechanism of microbial infection for mammailan cells was characterized for the Chlamydia trachomatis trachoma and lymphogranuloma venereum (LGV) biovars. We demonstrated that the trachoma and LGV biovars compete for the same receptor(s) on host cells and that their infectivity was inhibited by heparin or heparan sulphate. Using a specific heparan suiphate lyase (heparitinase) to treat organisms, the Infectivity of both biovars was abolished. Furthermore, exogenous heparan sulphate rescued chlamydial infectivity following treatment with heparitinase and the restored infectivity was neutralized by an anti‐heparan sulphate monoclonal antibody. These data suggest that heparan sulphate‐like‐mediated Interactions between C. trachomatis and eukaryotic cells are essential for infectivity.
Genetic selection for mutations that impair the co‐operative binding of lambda repressor
Authors: Benson Nicholas , Adams Curtis , Youderian Philip 
Published: October 2006

SummaryBacteriophage λ repressor binds co‐operatively to adjacent pairs of DNA target sites. A novel combination of positive genetic selections, involving two different operon fusions derived from P22 challenge phages, was used to isolate mutant λ repressors that have lost the ability to bind co‐operatively to tandem sites yet retain the ability to bind a strong, single site. These cb (co‐operative binding) mutations result in 10 different amino acid changes, which define eight residues in the carboxyl‐terminus of repressor. Because challenge phage derivatives may be applied to study essentially any specific protein‐DNA interaction, analogous combinations of genetic selections may be used to explore the ways that a variety of proteins interact to assemble regulatory complexes.