bluuebunny/crossref_metadata_2025_split huggingface dataset:
Properties of Acinetobacter calcoaceticus recA and its contribution to intracellular gene conversion
Authors: Gregg‐Jolly Leslie A. , Ornston L. Nicholas 
Published: October 2006

SummaryThe Acinetobacter calcoaceticus pcaJ and catJ genes, nearly identical in DNA sequence, differ in transcriptional control and are separated by more than 20 kb of chromosomal DNA. The pcaJ3125 mutation is repaired frequently in organisms containing the wild‐type catJ gene. This high‐frequency repair is eliminated in strains iacking the catJ gene, which suggests that recombination between the homologous catJ and pcaJ genes contributes to the high‐frequency repair of the pcaJ3125 mutation. We report here that the high‐frequency repair also requires a functional recA gene. The A. calcoaceticus recA gene was cloned in Escherichia coli by complementation of a recA mutation in the host strain. The nucleotide sequence of a 1506bp DNA fragment containing A. calcoaceticus recA was determined. The amino acid sequences of RecA from E. coli and A. calcoaceticus shared 71% identity. The DNA sequences differed in that a consensus binding site for binding of LexA repressor, represented upstream from recA in E. coli, is not evident in the corresponding region of the A. calcoaceticus DNA sequence. A Tn5 insertion was introduced into the A. calcoaceticus recA gene. Selection for Tn5‐encoded kanamycin resistance allowed the inactivated recA gene to be recombined by natural transformation into the A. calcoaceticus chromosome. Strains that had acquired the mutant gene were sensitive to both MMS and u.v. light, were deficient in natural transformation, and failed to carry out catJ‐dependent high‐frequency repair of the pcaJ3125 mutation.
Mosaic pbpX genes of major clones of penicillin‐resistant Streptococcus pneumoniae have evolved from pbpX genes of a penicillin‐sensitive Streptococcus oralis
Authors: Sibold Claus , Henrichsen Jørgen , König Andrea , Martin Christiane , Chalkley Lynda , Hakenbeck Regine 
Published: October 2006

SummaryPenicillin‐resistant clinical isolates of Streptococcus pneumoniae contain mosaic penicillin‐binding protein (PBP) genes that encode PBPs with decreased affinity for β‐lactam antibiotics. The mosaic blocks are believed to be the result of gene transfer of homologous PBP genes from related penicillin‐resistant species. We have now identified a gene homologous to the pneumococcal PBP2x gene (pbpX) in a penicillin‐sensitive Streptococcus oralis isolate M3 from South Africa that diverged by almost 20% from pbpX of penicillin‐sensitive pneumococci, and a central sequence block of a mosaic pbpX gene of Streptococcus mitis strain NCTC 10712. In contrast, it differed by only 2‐4% of the 1 to 1.5 kb mosaic block in pbpX genes of three genetically unrelated penicillin‐resistant S. pneumoniae isolates, two of them representing clones of serotype 6B and 23F, which are prevalent in Spain and are also already found in other countries. With low concentrations of cefotaxime, transformants of the sensitive S. pneumoniae R6 strain could be selected containing pbpX genes from either S. mitis NCTC 10712 or S. oralis M3, demonstrating that genetic exchange can already occur between β‐lactam‐sensitive species. These data are in agreement with the assumption that PBPs as penicillin‐resistance determinants have evolved by the accumulation of point mutations in genes of sensitive commensal species.
Prokaryotic HU and eukaryotic HMG1: a kinked relationship
Authors: Bianchi Marco E. 
Published: October 2006

SummaryHU and IHF proteins have long been considered the prokaryotic analogues of eukaryotic histones. Their ability to bend DNA, however, is distinctly similar to that of eukaryotic HMG‐box proteins, a recently identified family of chromatin components and transcription factors. In some conditions, HU and HMG1‐like proteins can even be swapped, both in vitro and in vivo. In spite of this, HU/IHF and HMG‐box proteins are not evolutionarily related, and represent two independent solutions for the same biochemical problem.
Altered pH lysine signalling mutants of cadC, a gene encoding a membrane‐bound transcriptional activator of the Escherichia coli cadBA operon
Authors: Dell Cheryl L. , Neely Melody N. , Olson Eric R. 
Published: October 2006

SummaryThe Escherichia coli CadC protein is required for activation of cadBA transcription under conditions of low external pH and exogenous lysine. cadBA encodes proteins involved in the decarboxylation of lysine to cadaverine, and cadaverine excretion. Sequence analysis suggested that CadC contains a single transmembrane segment separating a DNA‐binding domain in the amino terminus from a periplasmic domain. Western analysis of subcellular fractions demonstrated that CadC is expressed and concentrated in the cytoplasmic membrane in cells grown either at an inducing pH (pH5.8) or at a non‐inducing pH (pH7.6.). Eight cadC mutants were isolated based on their ability to confer expression of a cadA‐lacZ fusion independent of low external pH or exogenous lysine. Five of these mutants expressed the cadA‐lacZ fusion at both pH 5.8 and pH 7.6, but retained the requirement for the lysine signal while the other three mutants displayed pH independence in the presence of lysine, and lysine independence at pH 5.8 but not at pH 7.6. These results support a model in which CadC is a membrane‐bound transcriptional activator of the cadBA operon capable of sensing (directly or indirectly) signals generated outside the cytoplasmic membrane as a consequence of acidic pH and lysine.
Information essential for cell‐cycle‐dependent secretion of the 591‐residue Caulobacter hook protein is confined to a 21‐amino‐acid sequence near the N‐terminus
Authors: Kornacker M. G. , Newton A. 
Published: October 2006

SummaryRecent findings suggest that axial flagellar proteins and virulence proteins of Gram‐negative bacteria are exported from the cytoplasm via conserved trans‐location systems. To identify residues essential for secretion of flagellar axial proteins we examined the 591‐residue Caulobacter crescentus flagellar hook protein. Western blot assays of the culture media of strains producing mutant hook proteins show that only residues 38–58 are essential for its secretion to the cell surface. We discuss the observation that this unprocessed 21‐residue sequence is not conserved in other axial proteins and does not correspond to the SGL‐, ANN LAN‐ and heptad repeat motifs that are located Just upstream of the essential secretion information in the hook protein and are conserved near the N‐termini of other axial proteins. These motifs, for which an essential role in export or assembly has been proposed, are required for motility. However, we also demonstrate that hook protein can only be secreted when the flagellar basal body is present in the cell envelope. The cell‐cycle regulation of hook protein secretion confirms the specificity of the assay used in these studies and suggests that the basal body itself may serve as a secretion channel for the hook protein.
Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans
Authors: Hube B. , Monod M. , Schofield D. A. , Brown A. J. P. , Gow N. A. R. 
Published: October 2006

SummaryThe opportunistic fungal pathogen Candida albicans produces secretory aspartyl proteinases, which are believed to be virulence factors in infection. We have studied the in vitro expression of seven known members of the SAP gene family in a range of strains and serotypes by Northern analysis. SAP1 and SAP3 were regulated during phenotypic switching between the white and opaque forms of the organism. The SAP2 mRNA, which was the dominant transcript in the yeast form, was found to be autoinduced by peptide products of Sap2 activity and to be repressed by amino acids. The expression of the closely related SAP4‐SAP6 genes was observed only at neutral pH during serum‐induced yeast to hyphal transition. No SAP7 mRNA was detected under any of the conditions or in any of the strains tested. Our data suggest that the various members of the SAP gene family may have distinct roles in the colonization and invasion of the host.
Proteolytic cleavage and cell wall anchoring at the LPXTG motif of surface proteins in Gram‐positive bacteria
Authors: Navarre William Wiley , Schneewind Olaf 
Published: October 2006

SummaryMany surface proteins are thought to be anchored to the cell wall of Gram‐positive bacteria via their C‐terminus. Cell wall anchoring requires a specific sorting signal, normally located at the predicted C‐terminus of surface proteins. Here we show that when placed into the middle of a polypeptide chain, the sorting signal causes the specific cleavage of the precursor as well as the cell wall anchoring of its N‐ terminal fragment, while the C‐terminal fragment remains within the cytoplasm. N‐terminal sequencing of the C‐terminal cleavage fragment suggests that the cleavage site is located between threonine (T) and glycine (G) of the LPXTG motif, the signature sequence of cell wall sorting signals. All surface proteins harbouring an LPXTG sequence motif may therefore be cleaved and anchored by a universal mechanism. We also propose a novel hypothesis for the cell wall linkage of surface proteins in Gram‐positive bacteria.
Escherichia coli tyrT gene transcription is sensitive to DNA supercoiling in its native chromosomal context: effect of DNA topoisomerase IV overexpression on tyrT promoter function
Authors: Free Andrew , Dorman Charles J. 
Published: October 2006

SummaryWe have investigated the in vivo DNA supercoiling sensitivity of the Escherichia coli tRNAtyr1 gene (tyrT) promoter in its normal chromosomal location. Here, the native tyrT promoter is found to be exquisitely sensitive to mutations and to drugs which alter the level of DNA supercoiling. We show that the response of the tyrT promoter to supercoiling is qualitatively similar to that of a known supercoiling‐sensitive tRNA gene promoter, hisR. Specifically, treatments which increase in vivo DNA supercoiling levels enhance transcription of these tRNA genes. Particularly striking is the strong enhancement of expression from both promoters by a transposon insertion mutation in the topA gene encoding DNA topoisomerase i. This phenotypic effect can be complemented by providing active topoisomerase I in trans from a recombinant plasmid. Interestingly, it can also be complemented by overexpression of the genes encoding the subunits of DNA topoisomerase IV. We believe that this is the first demonstration that DNA topoisomerase IV can influence gene expression and it suggests that DNA topoisomerase I is partially redundant, at least in E. coli.
Repeated polyketide synthase modules involved in the biosynthesis of a heptaene macrolide by Streptomyces sp. FR‐008
Authors: Hu Zhihao , Bao Kai , Zhou Xiufen , Zhou Qi , Hopwood David A. , Kieser Tobias , Deng Zixln 
Published: October 2006

SummaryGenes for biosynthesis of a Streptomyces sp. FR‐008 heptaene macrolide antibiotic with antifungal and mosquito larvicidal activity were cloned in Escherichia coli using heterologous DNA probes. The cloned genes were implicated in heptaene biosynthiesis by gene replacement. The FR‐008 antibiotic contains a 38‐membered, poiyketide‐derived macrolide ring. Southern hybridization using probes encoding domains of the type i modular erythromycin polyketide synthase (PKS) showed that the Streptomyces sp. FR‐008 PKS gene cluster contains repeated sequences spanning c. 105 kb of contiguous DNA; assuming c. 5 kb for each PKS module, this is in striking agreement with the expectation for the 21‐step condensation process required for synthesis of the FR‐008 carbon chain. The methods developed for transformation and gene replacement in Streptomyces sp. FR‐008 make it possible to genetically manipulate polyene macrolide production, and may later lead to the biosynthesis of novel polyene macrolides.
Iron, DtxR, and the regulation of diphtheria toxin expression
Authors: Tao Xu , Schiering Nikolaus , Zeng Hui‐yan , Ringe Dagmar , Murphy John R. 
Published: October 2006

SummaryIn recent years considerable advances have been made in the understanding of the molecular basis of iron‐mediated regulation of diphtheria toxin expression. The tox gene has been shown to be regulated by the heavy metal ion‐activated regulatory element DtxR. In the presence of divalent heavy metal ions, DtxR becomes activated and binds to a 9 bp interrupted palindromic sequence. The consensus‐binding site has been determined by both the sequence analysis of DtxR‐responsive operators cloned from genomic libraries of Corynebacterium diphtheriae as well as by in vitro genetic methods using cyclic amplification of selected targets (CAST‐ing). it is now clear that DtxR functions as a global iron‐sensitive regulatory element in the control of gene expression in C. diphtheriae. In addition, the metal ion‐activation domain of DtxR is being characterized by both mutational analysis and determination of the X‐ray structure at 3.0 Å resolution.
Building bridges: disulphide bond formation in the cell
Authors: Bardwell James C. A. 
Published: October 2006

SummaryDisulphides are often vital for the folding and stability of proteins. Dedicated enzymatic systems have been discovered that catalyse the formation of disulphides in the periplasm of prokaryotes. These discoveries provide compelling evidence for the actual catalysis of protein foldingin vivo.Disulphide bond formation inEscherichia coliis catalysed by at least three ‘Dsb’ proteins; DsbA, ‐B and ‐C. The DsbA protein has an extremely reactive, oxidizing disulphide which it simply donates directly to other proteins. DsbB is required for the reoxidation of DsbA. DsbC is active in disulphide rearrangements and appears to work synergistically with DsbA. The relative rarity of disulphides in cytoplasmic proteins appears to be dependent upon a disulphide‐destruction machine. One pivotal cog in this machine is thioredoxin reductase.
The RTX haemolysins ApxI and ApxII are major virulence factors of the swine pathogenActinobacillus pleuropneumoniae:evidence from mutational analysis
Authors: Tascón Rubén I. , Vázquez‐Boland José A. , Gutiérrez‐Martín César B. , Rodríguez‐Barbosa Ignacio , Rodríguez‐Ferri ElZías F. 
Published: October 2006

SummaryThe involvement of the RTX haemolysins (Apxl and ApxII) of the swine pathogenActinobacillus pleuropneumoniae in virulencewas investigated using haemolysin‐deficient mutants constructed by a mini‐Tn10mutagenesis procedure. Two types of haemolysin mutant with single insertions of the transposon were obtained from a serotype 1 strain producing both ApxI and ApxII. One presented a complete loss of haemolytic activity because of the absence of ApxI and ApxII production. The other displayed weaker haemolysis than the wild type and produced only ApxII. The chromosomal regions flanking mini‐Tn10were cloned and sequenced. In the non‐haemolytic mutant, the transposon had inserted inapxIB, a gene involved in the exportation of ApxI and ApxII toxins. The weakly haemolytic mutant resulted from the disruption of the structural gene for ApxI. Both mutations In theapxIoperon were associated with a significant loss of virulence for mice and pigs, demonstrating that haemolysins are involved inA. pleuropneumoniaepathogenicity. The non‐haemolytic mutant was apathogenic and the weakly haemolytic mutant retained some virulence for pigs, suggesting that both ApxI and ApxII are needed for full virulence.
Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of Campylobacter jejuni
Authors: Konkel Michael E. , Marconi Richard T. , Mead David J. , Cieplak Witold 
Published: October 2006

SummaryCampylobacter jejuni is a significant cause of bacterial enteritis in humans. Three of seven C. jejuni isolates examined were found to contain fragmented 23S rRNA. The occurrence of fragmented 23S rRNA correlated with the presence of an intervening sequence (IVS) within the 23S rRNA genes. The IVS is 157 nucleotides in length and replaces an eight nucleotide sequence in the 23S rRNA genes of C. jejuni isolates that contain intact 23S rRNA. The two ends of the IVS share 31 bases of complementarity that could form a stem‐loop structure. Fragmentation of the 23S ribosomal RNA results from the excision of the IVS from the transcribed RNA; the 3’ cleavage site maps within the putative stem‐loop formed by the IVS. Southern hybridization analysis revealed that the IVS is not present in the genomes of isolates that contain intact 23S rRNA, suggesting that the IVS is not derived from Campylobacter chromosomal sequences. The C. jejuni IVS is located at a position analogous to that of the IVSs found in both Salmonella and Yersinia spp.
Proximal and distal sites bind LuxR independently and activate expression of the Vibrio harveyi lux operon
Authors: Miyamoto Carol M. , Smith Eric E. , Swartzman Eiana , Cao Jie‐Gang , Graham Angus F. , Meighen Edward A. 
Published: October 2006

SummaryThe LuxR regulatory protein of Vibrio harveyi as well as the autoinducer molecule, N‐(3‐hydroxybutanoyl) homoserine lactone, are known to be required for expression of luminescence. Although LuxR has been implicated in the activation of the promoter of the lux operon of V. harveyi, and can bind to two distinct sites upstream of the transcription initiation start site, its mode of action is unknown, in the present experiments, mobility shift assays were used to demonstrate that LuxR bound to the distal and proximal sites in an independent rather than co‐operative interaction with a much tighter binding to the distal site. Deletion and mutation analyses of DNA upstream of the lux promoter followed by transconjugation Into V. harveyi in trans using the chloramphenicol acetyl‐transferase (cat) gene as a reporter demonstrated, however, that the proximal site for LuxR was absolutely critical for promoter activation while the distal LuxR site was only necessary for maximum activation. This result was confirmed by mutation of the proximal site which blocked activation of the lux promoter and binding of LuxR to this site, but did not prevent LuxR binding to the distal site.
Characterization of trimethoprim‐ and sulphisoxazole‐resistant Chlamydia trachomatis
Authors: Wang Ling Ling , Henson Elizabeth , McClarty Grant 
Published: October 2006

SummaryTrimethoprim and sulphisoxazole were used as selective agents in culture to isolate, by a stepwise procedure, a series of Chlamydia trachomatis L2 populations resistant to the cytotoxic effects of the drugs. Two trimethoprim‐resistant populations. L2TriR‐60 and L2TriR‐100, and one sulphonamide‐resistant population. L2SulfR‐100, were characterized in more detail. In addition to being resistant to trimethoprim, L2TriR‐100 was cross‐resistant to metho‐trexate, sensitive to sulphisoxazole and displayed a ribonucleotide auxotrophy similar to that of its parental wild type, C. trachomatis L2. Surprisingly, L2TriR‐100 and L2SulfR‐100 appeared phenotypically identical. Both mutants were highly resistant to trimethoprim, sulphisoxazole, and methotrexate. In contrast to wild‐type C. trachomatis L2, these populations were sensitive to 5‐fluorouracil L2TriR‐100 and L2SulfR‐100 were incapable of taking pyrimidine ribonucleotides from the host cell and no longer synthesized thymidine nucleotides de novo. The pyrimidine requirement of these mutants was met by salvaging host‐cell uracil and thymidine, a property which can account for their drug‐resistance characteristics. L2TriR‐100 and L2Sulfn‐100 could also salvage adenine and guanine. Using L2TriR‐100 as a starting stock, a mutant population resistant to the cytotoxic effects of trimethoprim and 5‐fluorouracil (L2Tri/5‐FU) was selected. L2TFi/5‐FU was resistant to 5‐fluoro‐uracil because it had regained the capacity to take pyrimidine ribonucleotides from the host cell.
Analysis of the rnc locus of Coxiella burnetii
Authors: Zuber Mohammed , Hoover Timothy A. , Powell Bradford S. , Court Donald L. 
Published: October 2006

SummaryA 3.2 kb EcoRI genomic DNA fragment of Coxiella burnetii was isolated by virtue of Its ability to suppress mucoidy in Eschertchia coli. Nucleotide sequence analysis revealed the presence of the genes homologous to rnc, era and recO of E. coli. Suppression of capsule synthesis, measured by β‐galactosidase expression in Ion cps‐lac fusion strains of E. coli, is caused by gene‐dosage effects of the plasmid‐borne rnc genes of either C. burnetii or E. coli. The rnc gene of C. burnetii complemented rn– E. coli hosts for lambda plaque morphology and stimulation of lambda N gene expression. We also demonstrated heterologous complementation of an E coli strain defective for the expression of Era, an essential protein in E. coli, using the plasmid‐borne C. burnetii era. Under the control of the bacteriophage lambda PL promoter, this 3.2 kb EcoRI DNA fragment directed the synthesis in E. coli of three proteins with approximate molecular masses of 35,27 and 25 kDa. Antibodies against purified E. coli Era protein cross‐reacted with the 35 kDa protein of C. burnetii on Western blots.
Strain variation in the katG region of Mycobacterium tuberculosis
Authors: Zhang Ying , Young Douglas 
Published: October 2006

SummarySouthern blot analysis of chromosomal DNA from clinical isolates of Mycobacterium tuberculosis using cosmid DNA probes revealed extensive strain variation in the katG region of the genome. In addition to deletion of the katG gene itself in some isoniazid‐resistant strains, adjacent DNA fragments were missing or altered in a range of drug‐sensitive and drug‐resistant isolates. A species‐specific 2 kb Kpnl fragment located 10 kb upstream of katG in M. tuberculosis H37Rv hybridized to fragments of differing size in different clinical isolates and was characterized in detail. Sequence analysis of this fragment showed that it comprised three tandem copies of a novel 75 bp repeat element flanked by multiple copies of the previously described 10 bp major polymorphic tandem repeat of M. tuberculosis (MPTR). The copy number of the 75 bp repeat was found to vary between strains, allowing application of a poly‐merase chain reaction amplification strategy for strain differentiation. These results indicate that the katG region of the M. tuberculosis genome is highly variable and unstable. The presence of repetitive sequences may contribute to instability in this region of the genome.
The tail of a chaperonin: the C‐terminal region of Esctierichia coli GroEL protein
Authors: McLennan Neil F. , McAteer Sean , Masters Millicent 
Published: October 2006

SummaryThe active form of the KSP60 molecular chaperone of Escherichia coli, GroEL, is a pair of seven‐membered rings. We have used site‐directed mutagenesis to construct forms of the 547‐amino‐acid monomer truncated at the C‐terminus. We show here that forms that are 520 amino acids long or longer are close to being fully functional. Removing one further amino acid, however, results in a protein, GroEL519, which retains little function. This truncated form is metabolically stable but is not recovered from the cell in particle form. When synthesized at high levels, it prevents the normal assembly of GroEL547 present in the same cell. When synthesized at low levels, it can be included, probably at low molar ratios, in particles formed by assembly‐competent forms of GroEL. This can be seen as partial complementation of the temperature‐sensitive mutant groEL44. We conclude that amino acid 520 is cruical for particle assembly. GroEL516 has in vivo properties similar to those of GroEL519, but the still shorter form, GroEL504, appears to be inactive.