bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

SummaryDiaminopimelic 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

SummaryCells 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

SummaryWe 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

SummaryA 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

Summary 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

Summary 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

SummaryHM‐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

SummaryUpon 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

SummaryThe 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

SummaryWe 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

SummaryThe 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

SummaryWithin 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

SummaryThe 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

Summary 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

SummaryThe 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

SummaryThe 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

SummaryBacteriophage 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.