bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Five 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

Although 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

The 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

Folate‐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

The 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

Most 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

The 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

The 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

De‐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

A 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

Bacteriophage 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.
Isolation and characterization of the aspartokinase and aspartate semialdehyde dehydrogenase operon from mycobacteria
Authors: Cirillo Jeffrey D. , Weisbrod Torin R. , Pascopella Lisa , Bloom Barry R. , Jacobs William R.
Published: October 2006

Diaminopimelic acid (DAP) is a major component of the peptidoglycan layer of the mycobacterial cell wall. The mycobacterial cell wall has been implicated as a potential virulence factor and is highly immunogenic. The pathway for biosynthesis of DAP may serve as a target in the design of antimycobacterial agents and construction of in vivo selection systems. Despite its significance, this biosynthetic pathway is poorly understood in mycobacteria. In order to develop a better understanding of mycobacterial DAP biosynthesis, the aspartate semialdehyde dehydrogenase (asd) genes of Mycobacterium smegmatis, bacille Calmette‐Guerin (BCG), Mycobacterium avium, Mycobacterium leprae, and Mycobacterium tuberculosis were isolated. The M. smegmatis asd gene was isolated by complementation in Escherichia coli. This gene was then used to isolate the asd genes from other mycobacteria. The asd‐complemening fragments from BCG and M. smegmatis were sequenced. An open reading frame upstream of the mycobacterial asd gene was identified as the mycobacterial aspartokinase gene (ask). Primer extension analysis revealed that the only transcriptional start in this region is found 5’of the ask gene. This observation indicates that the mycobacterial ask and asd genes are in an operon.
mRNAs in the methanogenic archaeon Methanococcus vannielii: numbers, half‐lives and processing
Authors: Hennigan Aidan N. , Reeve John N.
Published: October 2006

Cells from the early exponential growth phase of cultures of the methanogenic archaeon Methanococcus vannielii have been shown to contain c. 180 transcripts of the mcrBDCGA (mcr) operon, c. 100 transcripts of the MvaL1,L10,L12 (Mva) operon, c. 8 transcripts of the argG gene and c. 1 transcript of the secY gene. These values decreased to c. 50 mcr transcripts, c. 30 Mva transcripts, c. 3 argG transcripts and <1 secY transcript per cell as the cultures entered the stationary phase of growth. Addition of bromoethanesulphonate (BES) or removal of H2 inhibited growth and RNA synthesis in vivo and, at 37°C in the presence of BES, the half‐lives of the mcr, Mva, argG and secY transcripts were found to be 15 min, 30 min, 57 min and 7 min, respectively. Addition of puromycin, pseudomonic acid or virginiamycin also inhibited growth but did not inhibit transcription. In the presence of puromycin the half‐lives of the mcr and Mva transcripts increased c. 4.6‐fold and c. 3.5‐fold, respectively, and there was a net accumulation of the Mva transcript. Addition of pseudomonic acid or virginiamycin also increased the half‐life of the Mva transcript and also resulted in the accumulation of a second, shorter Mva transcript but did not increase the half‐life of the mcr transcript. Transcription of the mcr operon was not stimulated by partial inhibition of methanogenesis.
Regulation of nitrogen metabolism is altered in a glnB mutant strain of Rhizobium leguminosarum
Authors: Amar M. , Patriarca E. J. , Manco G. , Bernard P. , Riccio A. , Lamberti A. , Defez R. , Laccarino M.
Published: October 2006

We isolated a Rhizobium leguminosarum mutant strain altered in the glnB gene. This event, which has never been described in the Rhizobiaceae, is rare in comparison to mutants isolated in the contiguous gene, glnA. The glnB mutation removes the glnBA promoter but in vivo does not prevent glnA expression from its own promoter, which is not nitrogen regulated. The glnB mutant strain does not grow on nitrate as a sole nitrogen source and it is Nod+, Fix+. Two –24/–12 promoters, for the glnll and glnBA genes, are constitutively expressed in the glnB mutant, while two –35/–10‐like promoters for glnA and ntrBC are unaffected. We propose that the glnB gene product, the P11 protein, plays a negative role in the ability of NtrC to activate transcription from its target promoters and a positive role in the mechanism of nitrate utilization.
Isolation and characterization of IS 31831, a transposable element from Corynebacterium glutamicum
Authors: Vertès Alain A. , Inui Masayuki , Kobayashi Miki , Kurusu Yasurou , Yukawa Hideaki
Published: October 2006

A transposable element from a coryneform bacterium, Corynebacterium glutamicum ATCC 31831 was isolated and characterized. The element IS 31831 is a 1453 bp insertion sequence with 24 bp imperfect terminal inverted repeats. It contains one open reading frame highly homologous at the amino acid level to the transposase of IS 1096 from Mycobacterium smeg‐matis. Both IS 31831 and IS 1096 exhibit several common characteristics suggesting that they constitute a new family of insertion sequences. IS 31831 was isolated by taking advantage of the sucrose sensitivity of coryneform bacteria conferred by expression of the Bacillus subtilis sacB gene. An Escherichia coli/ Corynebacterium shuttle vector useful for the isolation of transposable elements from the coryneform group of bacteria was constructed.
The cellulose‐binding domain of endoglucanase A (CenA) from Cellulomonas fimi: evidence for the involvement of tryptophan residues in binding
Authors: Din Neena , Forsythe Ian J. , Burtnick Leslie D. , Gilkes Neil R. , Miller Robert C. , Warren R. Antony J. , Kilburn Douglas G.
Published: October 2006

Cellulomonas fimi endo‐β‐1, 4‐glucanase A (CenA) contains a discrete N‐terminal cellulose‐binding domain (CBDcenA)‐ Related CBDs occur In at least 16 bacterial glycanases and are characterized by four highly conserved Trp residues, two of which correspond to W14 and W68 of CBDcenA‐ The adsorption of CBDcenA to Crystalline cellulose was compared with that of two Trp mutants (W14A and W68A). The affinities of the mutant CBDs for cellulose were reduced by approximately 50‐ and 30‐fold, respectively, relative to the wild type. Physical measurements indicated that the mutant CBDs fold normally. Fluorescence data indicated that W14 and W68 were exposed on the CBD, consistent with their participation in binding to cellobiosyl residues on the cellulose surface.
An ABC‐transporter from Streptomyces longisporoflavus confers resistance to the polyether‐ionophore antibiotic tetronasin
Authors: Linton Kenneth J. , Cooper Howard N. , Hunter Lain S. , Leadlay Peter F.
Published: October 2006

Streptomyces longisporoflavus produces the poly‐ketide‐polyether antibiotic, tetronasin, which acts as an ionophore and depolarizes the membrane of bacteria sensitive to the drug. A genomic library of S. longisporoflavus DN A was cloned in Streptomyces Uvldans and screened to identify tetronasin‐resistance determinants. The inclusion of 0.2 M NaCl in the growth medium with tetronasin markedly improved the sensitivity of the screen. Two different resistance determinants, designated tnrB (ptetR51) and tnrA (ptetR11) respectively, were identified. The determinant tnrB (ptetR51) but not tnrA (ptetR11), also conferred resistance to tetronasin when cloned into Streptomyces albus. The tnrB determinant was further localized, by subcloning, to a 2.8 kb Kpnl fragment. DNA sequence analysis of this insert revealed one incomplete and two complete open reading frames (ORFs 1, 2 and 3). The deduced sequence of the gene product of ORF2 (TnrB2) revealed significant similarity to the ATP‐binding domains of the ABC (ATP binding cassette) superfamily of transport‐related proteins. The adjacent gene, ORF3, is translationally coupled to ORF2 and would encode a hydrophobic protein (TnrB3) with six transmembrane helices which probably constitutes the integral membrane component of the transporter. The mechanism of tetronasin resistance mediated by tnrB is probably an ATP‐dependent efflux system.
Entamoeba histolytica: electron‐dense granule secretion, collagenase activity and virulence are altered in the cytoskeleton mutant BG‐3
Authors: de Jesús Serrano José , de la Garza Mireya , Moreno Miguel Angel , Tovar Rosalinda , León Gloria , Tsutsumi Victor , de Lourdes Muñoz Maria
Published: October 2006

HM‐1:IMSS, a pathogenic strain of Entamoeba histolytica, and its mutant BG‐3, Identified by resistance to cytochalasin D, were tested for their capacity to: (i) secrete electron‐dense granules; (ii) adhere and digest native type I collagen gels; and (iii) produce liver abscesses in new‐born hamsters. The results demonstrate that the mutant has low adherence to collagen, low electron‐dense granule secretion and collagenolytic activity, and low capacity to produce liver lesions in vivo, compared with the parental strain HM1:IMSS.
Nodulation protein NodL of Rhizobium leguminosarum O‐acetylates lipo‐oligosaccharides, chitin fragments and N‐acetylglucosamine in vitro
Authors: Bloemberg Guido V. , Thomas‐Oates Jane E. , Lugtenberg Ben J. J. , Spaink Herman P.
Published: October 2006

Upon induction of their nodulation genes, the root nodule‐inducing Rhizobium bacteria produce lipo‐oligosaccharide signal molecules. All lipo‐oligosaccharides identified from Rhizobium leguminosarum bv. viciae carry an O‐acetyl group at the C‐6 position of the non‐reducing terminal sugar, the presence of which is important for biological activity and host specificity. Previously we showed that a functional nodL gene product is required for the presence of this O‐acetyl moiety. The production of polyclonal antibodies against isolated NodL protein, using a NodL‐ overproducing Escherichia coli strain is described. These antibodies were used (i) to elucidate the subcellular localization of the NodL protein, which appeared to be present in the cytosol, and (ii) for the purification of native NodL protein from E. coli. Here we provide biochemical proof that purified NodL protein has transacetylating activity in vitro with acetyl‐CoA as the acetyl donor. NodL protein appeared to be able to acetylate various substrates, such as lipo‐oligosaccharides, chitin fragments and N‐acetylglucosamine. For chitinpentaose as the substrate we have shown, using mass spectrometry and NMR spectroscopy, that NodL protein substitutes one O‐acetyl group at the C‐6 position of the non‐reducing terminal sugar.
Cdc7 protein kinase for DNA metabolism comes of age
Authors: Sclafani Robert A. , Jackson Aimee L.
Published: October 2006

The Cdc7 protein kinase is the product of an essential cell cycle gene, and Is involved in three aspects of DNA metabolism: mitotic DNA replication, meiotic DNA recombination, and replication‐dependent DNA repair. The mechanism by which Cdc7 regulates each of its cellular functions is an issue of considerable interest. Recently, much of the research regarding the regulation of cell cycle progression has focused on the regulatory action of cyclins on their catalytic counterparts. We propose that the function of Cdc7 in ceil cycle progression is mediated in a similar manner, in that Dbf4, a protein whose transcript level is known to fluctuate in the cell cycle, is essential for Cdc7 kinase activity. The periodic association of Dbf4 with Cdc7 may account for the regulation of Cdc7 kinase function and progression through the cell cycle.
Cloning and characterization of the RNase P RNA genes from tow porcine mycoplasmas
Authors: Svärd Staffan G. , Mattsson Jens G. , Johansson Karl‐Erik , Kirsebom Leif A.
Published: October 2006

We report the cloning of the RNase P RNA genes from the primary aetiological agent of porcine pneumonia, Mycoplasma hyopneumoniae, and the closely related commensal, Mycoplasma flocculare. The monocistronic genes each have promoters with AT‐rich ‐35 regions and Rho‐independent‐like transcription terminators which are retained in the RNase P RNA. Both of these RNase P RNA variants are shown to be catalytically active in vitro in spite of a low overall GC content (30%). Our results suggest a new example of a stable mini‐helix in the conserved core of the mycoplasmal RNase P RNAs. Deletion of the corresponding structural element in Escherichia coli RNase P RNA (M1 RNA) generated an RNase P RNA with an impaired substrate interaction. Displacement of this structural element with the mycoplasmal mini‐helix resulted in an enzyme with a phenotype similar to that of wild‐type M1 RNA. in addition, this structural element is important for lead ion‐induced cleavage at specific sites in M1 RNA.
The DnaK homologue of the marine Vibrio sp. strain S14 binds to the unprocessed form of a carbon starvation‐specific periplasmic protein
Authors: Holmquist Louise , Nelson David R. , Kjelleberg Staffan
Published: October 2006

The Escherichia coli DnaK homoiogue in Vibrio sp. strain S14 was shown to possess chaperone function for translocation during carbon starvation. This was demonstrated by using the method of co‐immunoprecipitation. DnaK co‐precipitated with the carbon starvation‐specific periplasmic space protein Csp5 three hours after the onset of carbon starvation. Pulse‐chasing of the protein with radiolabelled methlonine followed by the addition of an excess of unlabelled methionine demonstrated that the Csp5 protein was translocated across the inner membrane. Only the cytoplasmic unprocessed precursor form of Csp5 co‐precipitated with DnaK. The non‐covalent binding between the two proteins was found to be ATP‐dependent, as the addition of ATP released the interaction between DnaK and the precursor form of Csp5, as was shown both on silver‐stained SDS‐poly‐acrylamide gels and by Western blot analysis. We suggest that DnaK maintains the carbon starvatlon‐Inducible protein Csp5 in a translocation‐competent form In the cytoplasm.
Contribution of genes from the capsule gene complex (cps) to lipooligosaccharide biosynthesis and serum resistance in Neisseria meningitidis
Authors: Hammerschmidt Sven , Birkholz Carola , Zähringer Ulrich , Robertson Brian D. , van Putten Jos , Ebeling Olaf , Frosch Matthias
Published: October 2006

Within the capsule gene complex (cps) of Neisseria meningitidis B a 5.5 kb DNA fragment encodes proteins with strong homologies to enzymes of the lipopolysaccharide biosynthetic pathway of Salmonella typhimurium and Escherichia coli, GalE, RfbB, RfbC and RfbD. A meningococcal galE mutant expressed a truncated lipooligosaccharide (LOS), which terminated at the glucose residue between inner and outer core, and a second gaiE gene present outside the cps cluster was found to be transcriptionally and functionally inactive and, thus, unable to complement this defect. Because of the defect in the outer core, the LOS of the galE‐defective meningococcal mutant was not sialylated. In contrast, carbohydrate analysis of the LOS of an rfb‐defective meningococcal mutant revealed no difference from the LOS of the wild‐type strain, suggesting that the rfb genes are inactive. This was supported by Northern blot analysis, which showed that expression of the rfb gene products was transcriptionally regulated. The inability of the meningococcal galE mutant, which cannot sialylate the LOS, allowed us to investigate the significance of LOS sialylation in relation to the presence of the polysialic acid capsule. Sialylated LOS, but not the polysialic acid capsule, is necessary to confer complete serum resistance on the meningococcus by inhibition of the alternative complement pathway.
Open complex formation by DnaA initiation protein at the Escherichia coli chromosomal origin requires the 13‐mers precisely spaced relative to the 9‐mers
Authors: Hsu Julia , Bramhill David , Thompson Chris M.
Published: October 2006

The 245 bp chromosomal origin, oriC, of Escherichia coli contains two iterated motifs. Three 13‐mers tandemly repeated at one end of the origin and four 9‐mers in a nearby segment of oriC are highly conserved in enteric bacteria, as is the distance separating these two sequence clusters. Mutant origins were constructed with altered spacing of the 9‐mers relative to the 13‐mers. Loss or addition of even a single base drastically reduced replication, both in vivo and in vitro. Spacing mutant origins bound effectively to DnaA protein but failed to support efficient open complex formation. These results suggest that interaction with the 9‐mers positions at least one subunit of DnaA to recognize directly the nearest 13‐mer for DNA melting.
DNA supercoiling and thermal regulation of unsaturated fatty acid synthesis in Bacillus subtilis
Authors: Grau Roberto , Gardiol Daniela , Glikin Gerardo Claudio , de Mendoza Diego
Published: October 2006

Bacillus subtilis growing at 37° C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37°C is transferred to 20°C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20° C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20°C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37°C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.
Anaerobic activation of arcA transcription in Escherichia coli: roles of Fnr and ArcA
Authors: Compan Inès , Touati Danlèle
Published: October 2006

The ArcA and Fnr regulators of Escherichia coli, both of which are activated in anaerobic conditions, negatively regulate the sodA gene (coding for manganese superoxide dismutase), but Fnr has no effect on anaerobic sodA expression in a δarcAδfnr background (Compan and Touati, 1993). We show here that the sdh gene (coding for succinate dehydrogenase) is also negatively regulated by Fnr, but again Fnr exerts no control in a δarcA background. One interpretation of these results is that Fnr activates arcA transcription. Using arcA–lac transcriptional and translational fusions, we show that arcA expression increases (about fourfold) in anaerobiosis and that both Fnr and ArcA are required for full expression, in a δfnr background, there is no autoactivation, suggesting that ArcA enhances activation by Fnr. Transcript and sequence analyses reveal that the arcA upstream regulatory region lies within a 530 bp non‐coding DNA fragment, which contains five putative promoter sequences and a putative Fnr‐binding site, identification of the transcription start sites indicates that transcription occurs in aerobiosis from three constitutive upstream promoters (Pe, Pd, Pc). In anaerobiosis an additional completely Fnr‐dependent transcript starting at Pa, is present; expression from Pa is reduced in the absence of ArcA, and Fnr activation at Pa blocks the weak anaerobic‐dependent expression from Pb Fnr activation of arcA transcription may play an important role in the co‐ordination of expression of genes associated with aerobic and anaerobic metabolism during environmental changes.
The growth phase‐dependent synthesis of cyclopropane fatty acids in Escherichia coli is the result of an RpoS(KatF)‐dependent promoter plus enzyme instability
Authors: Wang Ai‐Yu , Cronan John E.
Published: October 2006

The formation of cyclopropane fatty acids (CFAs) in Escherichia coli is a post‐synthetic modification of the phospholipid bilayer that occurs predominantly as cultures enter the stationary phase of growth. The mechanism of this growth phase‐dependent regulation of CFA synthesis was unclear, since log‐phase and stationary‐phase cultures had been reported to contain similar levels of the enzyme catalysing the reaction (CFA synthase). We report that the timing of CFA synthesis can be explained by two unusual features. First, the gene encoding CFA synthase (cfa) was found to be transcribed from two promoters and the 5′ ends of both transcripts were mapped by primer extension. One of the promoters was active only during the log‐to‐stationary phase transition and depended on the putative sigma factor encoded by the rpoS(katF) gene whereas the other promoter had a standard σ70 promoter consensus sequence and was expressed throughout the growth curve. Second, CFA synthase activity was shown to be unstable in vivo and a Cfa fusion protein was found to have a half life of <5min. The combination of these factors meant that, although CFA synthase was synthesized throughout the growth curve, a large increase in activity occurred during the log‐to‐stationary phase transition. As stationary phase progressed, the Increased CFA synthase activity rapidly declined to the basal level. This transient increase In CFA synthase activity coupled with the cessation of net phospholipid synthesis in stationary phase provides an explanation for the unusual time course of CFA synthesis.
Phosphorylation of elongation factor G and ribosomal protein S6 in bacteriophage T7‐infected Escherichia coli
Authors: Robertson Erle S. , Aggison Lee A. , Nicholson Allen W.
Published: October 2006

Bacteriophage T7 expresses a serine/threonine‐specific protein kinase activity during Infection of Its host, Escherichia coli. The protein kinase (gpO.7 PK), encoded by the T7 early gene 0.7, enhances phage reproduction under sub‐optimal growth conditions. It was previously shown that ribosomal protein S1 and translation initiation factors IF1, IF2, and IF3 are phosphoryiated in T7‐infected cells, and it was suggested that phosphorylation of these proteins may serve to stimulate translation of the phage late mRNAs. Using high‐resolution two‐dimensional gel electrophoresis and specific immunoprecipitation, we show that elongation factor G and ribosomal protein S6 are phosphorylated following T7 infection. The gel electro‐phoretic data moreover indicate that elongation factor P is phosphorylated in T7‐infected cells. T7 early and late mRNAs are processed by ribonuclease III, whose activity is stimulated through phosphorylation by gp0.7 PK. Specific overexpression and phosphorylation was used to locate the RNase III polypeptide in the standard two‐dimensional gel pattern, and to confirm that serine is the phosphate‐accepting amino acid. The two‐dimensional gels show that the in vivo expression of gp0.7 PK results in the phosphorylation of over 90 proteins, which Is a significantly higher number than previous estimates. The protein kinase activities of the T7‐related phages T3 and BA14 produce essentially the same pattern of phosphorylated proteins as that of T7. Finally, several experimental variables are analysed which influence the production and pattern of phosphorylated proteins in both uninfected and T7‐rnfected cells.
Cloning and characterization of ppiB, a Bacillus subtilis gene which encodes a cyclosporine A‐sensitive peptidyl‐prolyl cis‐trans isomerase
Authors: Herrler Michael , Bang Holger , Marahiel Mohamed A.
Published: October 2006

Sequencing of N‐terminal and internal peptide fragments of the purified 17kDa Bacillus subtilis peptldyl‐prolyl cls‐trans isomerase (PPlase) revealed sequence identity to conserved regions of a number of eukaryotic and prokaryotic cyclophilins. Using two oligonucleotide primers corresponding to the N‐terminus and a highly conserved internal amino acid sequence, polymerase chain reactions (PCR) with B. subtilis genomic DNA were carried out. The resultant PCR fragment of 335 bp was cloned, sequenced and subsequently used as a probe for screening a λZap II gene library of B. subtilis. Two overlapping positive clones of 5 and 7 kb containing the B. subtilis PPlase gene (ppiB), which is 432 bp in length and encodes a protein of 144 amino acid residues, were identified and two distinct transcriptional initiation sites at the 5′ end of ppiB were mapped. The entire region (35 kb) between spoVA and serA was recently sequenced in B. subtilis, and an open reading frame (ORF) that encodes a putative peptidyl‐prolyl cis‐trans isomerase at about 210° on the B. subtilis genetic map was located. This putative PPlase is identical to PPiB. We have overexpressed the ppiB gene in Escherichia coli, purified the encoded protein to apparent homology and shown that it exhibits PPlase activity. In addition, the recombinant PPiB shows a significant inhibition of PPlase activity by cyclosporin A (CsA) at a level comparable to that observed for the B. subtilis enzyme. Interestingly the B. subtilis PPlase shows about 40% identity to eukaryotic PPlases and less similarity to those of Gram‐negative bacteria (27–32% identity). Like other interruption mutants of yeast and Neurospora, which iack a functionai cyclophilin gene, a B. subtiiis mutant containing ppiBv.cat, a caf‐interrupted copy of ppiB in the chromosome, is viable.
Interaction of the Chlamydia trachomatis histone H1‐like protein (Hc1) with DNA and RNA causes repression of transcription and translation in vitro
Authors: Pedersen Lotte Bang , Birkelund Svend , Christiansen Gunna
Published: October 2006

The 18 kDa histone H1‐like protein from Chlamydia trachomatis (Hc1) is a DNA–binding protein thought to be involved in condensation of the chlamydial chromosome during late stages in the chlamydial life cycle. Expression of Hc1 in Escherichia coli results in an overall relaxation of DNA and severely affects DNA, RNA and protein synthesis. We have analysed the interaction of Hc1 with single‐stranded DNA and RNA by Southwestern and Northwestern blotting. Furthermore, we show that purified, recombinant Hc1 dramatically affects transcription and translation in vitro at physiologically relevant concentrations. These results were found to coincide with the formation of condensed Hc1–DNA and Hc1–RNA complexes as revealed by agarose gel electrophoresis and electron microscopy. The implications of these results for possible functions of Hc1 in vivo are discussed.
The Pseudomonas fluorescens lipase has a C‐terminal secretion signal and is secreted by a three‐component bacterial ABC‐exporter system
Authors: Duong Frank , Soscia Chantal , Lazdunski Andrée , Murgier Maryse
Published: October 2006

Both Pseudomonas aeruginosa and Pseudomonas fluorescens secrete a lipase into the extracellular medium. Unlike the lipase of P. aeruginosa, the lipase produced by P. fluorescens does not contain any N‐terminal signal sequence. We show that the P. fluorescens lipase is secreted through the signal peptide‐independent pathway of the alkaline protease that we previously identified in P. aeruginosa. Secretion of this protease (AprA) is dependent on the presence of three genes located adjacent to the aprA gene, aprD, aprE and aprF. The three secretion functions permit an efficient secretion of P. fluorescens lipase. Inactivation of one of them (AprE) prevented this secretion. In Escherichia coli, the three proteins AprD, AprE, AprF are necessary and sufficient for efficient secretion of lipase to the extracellular medium. The secretion signal is located within the C‐terminal part of the lipase sequence and can promote efficient secretion of a passenger protein. Thus the P. fluorescens lipase secretion system belongs to the group of the three‐component bacterial ABC‐exporter systems.
pecS: a locus controlling pectinase, cellulase and blue pigment production in Erwinia chrysanthemi
Authors: Reverchon Sylvie , Nasser William , Robert‐Baudouy Janine
Published: October 2006

Erwinia chrysanthemi mutants (designated as pecS) displaying derepressed pectate lyase and cellulose synthesis were isolated. In addition, the pecS mutation is responsible for production of an extracellular insoluble blue pigment whose synthesis is cryptic in the wild‐type 3937 strain. Transduction analysis indicates that the phenotype is due to a single mutation located near the xyl marker on the strain 3937 chromosome. This mutation was complemented by an R‐prime plasmid carrying the xyl and argG genes of E. chrysanthemi, suggesting that the pecS product acts in trans to modulate pectinase, cellulase and blue pigment production. Insertion mutagenesis of the cloned region and recombination of the corresponding mutations in the bacterial chromosome by marker exchange revealed the existence of two divergently transcribed genes, pecS and pecM, that are both involved in the pectate lyase and cellulase regulation. The nucleotide sequences of pecS and pecM were determined. The pecS gene encodes a 166 amino acid polypeptide that shows similarity to the MprA regulatory protein of Escherichia coli whereas the pecM gene encodes a 297 amino acid polypeptide that was shown to be an integral membrane protein. The possible functions of the PecS and PecM proteins derived from the mutant phenotype and sequence analysis are discussed in terms of signal transduction and transcription regulation.
Cloning of the nupC gene of Escherichia coli encoding a nucleoside transport system, and identification of an adjacent Insertion element, IS 186
Authors: Craig Jane E. , Zhang Yianbiao , Gallagher Maurice P.
Published: October 2006

Escherichia coli is known to contain more than one active transport system for nucleoside uptake. In the present study we report the sequence of a gene encoding a second nucleoside transport system, nupC (in addition to nupG.) An open reading frame (ORF) of 1200bp was identified that codes for a hydrophobic polypeptide of 43 560 Da and an NupC fusion protein was shown to be membrane associated. The native NupC protein is also identified, following over‐expression. NupC exhibits short regions of homology to several membrane‐associated proteins, including LacY and Cyd. Analysis of the nupC promoter region revealed the presence of at least two putative CRP‐binding sites, centred at–40bp and–89bp, which probably flank a CytR‐binding site. In addition, an adjacent IS186 element was identified and found to reside within a putative terminator structure, downstream from the nupC ORF. This arrangement is shown to reflect the previously established gene order on the E. coli chromosome.
TMAO anaerobic respiration in Escherichia coli: involvement of the tor operon
Authors: Méjean Vincent , Lobbi‐Nivol Chantal , Lepelletier Michèle , Giordano Gérard , Chippaux Marc , Pascal Marie‐Claire
Published: October 2006

The trimethylamine N‐oxide (TMAO) respiratory system is subject to a strict positive control by the substrate. This property was exploited in the performance of miniMu replicon‐mediated in vivo cloning of the promoter region of gene(s) positively regulated by TMAO. This region, located at 22 min on the chromosome, was shown to control the expression of a transcription unit composed of three open reading frames, designated torC, torA and torD, respectively. The presence of five putative c‐type haem‐binding sites within the TorC sequence, as well as the specific biochemical characterization, indicated that torC encodes a 43 300 Da c‐type cytochrome. The second open reading frame, torA, was identified as the structural gene for TMAO reductase. A comparison of the predicted amino‐terminal sequence of the torA gene product to that of the purified TMAO reductase indicated cleavage of a 39 amino acid signal peptide, which is in agreement with the periplasmic location of the enzyme. The predicted TorA protein contains the five molybdenum cofactor‐binding motifs found in other molybdoproteins and displays extensive sequence homology with BisC and DmsA proteins. As expected, insertions in torA led to the loss of TMAO reductase. The 22 500 Da polypeptide encoded by the third open reading frame does not share any similarity with proteins listed in data banks.
Antigenic drift of non‐encapsulated Haemophilus influenzae major outer membrane protein P2 in patients with chronic bronchitis is caused by point mutations
Authors: Duim Birgitta , van Alphen Loek , Eijk Paul , Jansen Henk M. , Dankert Jacob
Published: October 2006

The sequence of the gene encoding major outer membrane protein (MOMP) P2 of antigenic variants of non‐encapsulated Haemophilus influenzae isolated from persistently infected chronic bronchitis patients was analysed. Antigenic drift was shown to result from single base changes in the P2 gene, all generating amino acid changes in the surface‐exposed loops of MOMP P2, predominantly in loop 6. Similar single base changes were observed in H. influenzae persistently present in a subcutaneous cage implanted in rabbits, as well as in a spontaneous H. influenzae mutant that had survived MOMP P2 specific monoclonal‐antibody‐dependent bactericidal killing in vitro. We hypothesize that accumulation of point mutations under the selection pressure of immunity is a mechanism of antigenic drift of a surface‐exposed protein during persistent H. influenzae infection
DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans
Authors: Minton Kenneth W.
Published: October 2006

Deinococcus radiodurans and other members of the same genus share extraordinary resistance to the lethal and mutagenic effects of ionizing and u.v. radiation and to many other agents that damage DNA. While it is known that this resistance is due to exceedingly efficient DNA repair, the molecular mechanisms responsible remain poorly understood. Following very high exposures to u.v. irradiation (e.g. 500 Jm−2, which is non‐lethal to D. radiodurans), this organism carries out extremely efficient excision repair accomplished by two separate nucleotide excision repair pathways acting simultaneously. One pathway requires the uvrA gene and appears similar to the UvrABC excinuclease pathway defined in Escherichia coli. The other excision repair pathway is specific for u.v. dimeric photoproducts, but is not mediated by a pyrimidine dimer DNA glycosylase. Instead, it is initiated by a second bona fide endonuclease that may recognize both pyrimidine dimers and pyrimidine‐(6–4)pyrimidones. After high doses of ionizing‐radiation (e.g. 1.5Mrad), D. radiodurans can mend >100 double‐strand breaks (dsb) per chromosome without lethality or mutagenesis. Both dsb mending and survival are recA‐dependent, indicating that efficient dsb mending proceeds via homologous recombination. D. radiodurans contains multiple chromosomes per cell, and it is proposed that dsb mending requires extensive recombination amongst these chromosomes, a novel phenomenon in bacteria. Thus, D. radiodurans may serve as an easily accessible model system for the double‐strand‐break‐initiated interchromosomal recombination that occurs in eukaryotic cells during mitosis and meiosis.
Rhizobium meliloti DctD, a σ54‐dependent transcriptional activator, may be negatively controlled by a subdomain in the C‐terminal end of its two‐component receiver module
Authors: Gu Baohua , Lee Joon H. , Hoover Timothy R. , Scholl Dean , Nixon B. Tracy
Published: October 2006

Rhizobium meliloti DctD is believed to have three functional domains: an N‐terminal, two‐component receiver domain; and like other σ54‐dependent activators, C‐terminal and central domains for DNA binding and transcription activation. We have characterized a progressive series of M‐terminal deletions of R meliloti DctD. The N‐terminal domain was not needed for binding the dctA upstream activation sequence. Only 25% of the C‐terminal end of the receiver domain was needed to significantly inhibit the central domain, and proteins lacking up to 60% of the N‐terminal end of the receiver domain were‘inducible’in R. meliloti cells. We hypothesize that the W‐terminal two‐thirds of the DctD receiver domain augments and controls an adjacent subdomain for inhibiting the central domain.