bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of a functional neisserial fbp gene in Escherichia coli
Authors: Berish Sally A. , Chen Cheng‐Yen , Mietzner Timothy A. , Morse Stephen A. 
Published: October 2006

SummaryThe ability to acquire iron from a human host is a major determinant in the pathogenesis of Neisseria gonorrhoeae and Neisseria meningitidis. Pathogenic Neisseria spp. do not synthesize siderophores and instead express a receptor‐mediated, high‐affinity iron acquisition system in the iron‐restricted environment of its host. A ferric‐iron‐binding protein (Fbp) of Neisseria spp. is also iron‐regulated and may play a central role in this novel iron‐uptake system. To define the physical properties of Fbp further, we used polymerase chain reaction to synthesize DNA fragments containing the fbp structural gene with and without the sequence encoding the Fbp leader peptide. These fragments were ligated into pUC13 to create in‐frame fusions with the alpha peptide of lacZ. The expression of Fbp was under the control of the lacZ promoter. Both fusion clones produced Fbp in large amounts, facilitating the purification of quantities of Fbp sufficient for elucidating the biochemical, immunologic, and functional properties of this protein.
Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102 kb of chromosomal DNA which is flanked by a repetitive element
Authors: Fetherston Jacqueline D. , Schuetze Paul , Perry Robert D. 
Published: October 2006

SummaryThe pigmentation. (Pgm+) phenotype of Yersinia pestis encompasses a variety of different physiological traits, all of which are missing in Pgm‐ mutants. We have previously shown that loss of the Pgm+ phenotype is accompanied by the spontaneous deletion of at least 45 kb of chromosomal DNA, referred to as the pgm locus. Using chromosomal walking, we have now mapped the full extent of the pgm locus in Y. pestis strain KIM6+. Our results indicate that the locus spans 102 kb of DNA which is absent in the spontaneous Pgm‐ mutant, KIM6. Yersinia pseudo‐tuberculosis PB1/0 contains sequences homologous to the entire pgm locus while only part of this region hybridized to Yersinia enterocolitica WA‐LOX DNA. Restriction enzyme mapping and hybridization studies revealed the presence of a repetitive element at both ends of the pgm locus and in multiple copies elsewhere in the Y. pestis genome. This element may be responsible for generating the deletion.
Frameshifting in the expression of the Escherichia coli trpR gene
Authors: Benhar Itai , Miller Chaya , Engelberg‐Kulka Hanna 
Published: October 2006

SummaryThe trpR gene of Escherichia coli carries an open reading frame that encodes the trp repressor, 108 amino acids long. Here we show that translation of an additional (+1) reading frame of trpR occurs both in vivo and in vitro. This results in the synthesis of a stable +1 frame polypeptide. Using site‐specific mutagenesis, immunological techniques and amino acid sequencing we have found that the N‐terminus of the +1 frame product and that of the known 0 frame product are identical but that their C‐termini differ. Our results are discussed in relation to the role of natural frameshifting as a regulatory mechanism of gene expression in general, and with respect to tryptophan biosynthesis in particular.
The glucose kinase gene of Streptomyces coelicolor A3(2): its nucleotide sequence, transcriptional analysis and role in glucose repression
Authors: Angell S. , Schwarz E. , Bibb M. J. 
Published: October 2006

SummaryMutants (glk) of Streptomyces coelicolor A3(2) that are resistant to the non‐utilizable glucose analogue 2‐deoxyglucose are deficient in glucose kinase activity, defective in glucose repression, and usually unable to utilize glucose. A 2.9 kb Bcll fragment, previously shown to restore a wild‐type phenotype to a glk deletion mutant that lacks the entire segment, contains two complete open reading frames that would encode proteins of 20.1 kDa (ORF2) and 33.1 kDa (ORF3). ORF3 is transcribed from its own promoter, and also from a promoter that initiates transcription upstream of ORF2. A derivative of the temperate phage πC31 containing ORF3 alone restored a wild‐type phenotype when used to lysogenize the deletion mutant. The product of ORF3 is homologous to members of a family of repressor proteins encoded by xylR In Bacillus subtilis and Lactobacilius pentosus, and by nagC in Escherichia coli. Although this might suggest that ORF3 encodes a positive activator for glucose kinase, rather than the enzyme itself, ORF3 restored the ability to metabolize glucose to an E. coli glk mutant, and activity gels of cell extracts of E. coli containing ORF3 cloned in the pT7‐7 expression vector demonstrated that the ORF3 product has glucose kinase activity.
Separation of two lipopolysaccharide populations with different contents of O‐antigen factor 122 in Salmonella enterica serovar Typhimurium
Authors: Helander Ilkka M. , Moran Anthony P. , Mäkela P. Helena 
Published: October 2006

SummaryLipopolysaccharide (LPS) extraction from smooth‐type Salmonella enterica sv. Typhimurium was carried out with the modified phenol/chloroform/ petroleum ether method (volume ratio 5: 5:8). In this procedure, LPS was precipitated from 90% phenol sequentially with water and acetone to yield LPS‐H2O (minute amounts) and LPS‐Ac (major amounts), respectively. Chemical analyses of the LPS fractions revealed that in the O antigen of LPS‐H2O position C4 of the D‐galactose was extensively glucosylated, corresponding to the O‐antigen factor 122. In LPS‐Ac, this glucosylation was negligible. Inspection of the LPS fractions by sodium dodecyl sulphate polyacryl‐amide gel electrophoresis and silver staining suggested that the glucosylation in LPS‐H2O was present only in LPS species with a chain length higher than six repeating units.
Cloning of mycobacterial histidine synthesis genes by complementation of a Mycobacterium smegmatis auxotroph
Authors: Hinshelwood S. , Stoker N. G. 
Published: October 2006

SummaryHistidine‐requiring auxotrophs of Mycobacterium smegmatis were isolated following N‐methyl‐N′‐nitro‐ N‐nitrosoguanidine treatment. One of these mutants, his 5, was transformed with an M. smegmatis shuttle cosmid library, and complementing clones were isolated at a frequency of approximately 1%. A 2.3 kb fragment was subcloned and sequenced, and found to contain the start of an operon including the hisD gene and part of the hisC gene. No hisG gene was detected upstream of hisD, suggesting that the regulation of histidine biosynthesis in mycobacteria may differ from that of Escherichia coli. The strategy used here will allow the molecular genetics of complex mycobacterial‐specific biosynthetic pathways involved in the virulence of pathogenic species to be studied.
Mechanisms of the cytopathic action of actin‐ADP‐ribosylating toxins
Authors: Aktories K. , Wegner A. 
Published: October 2006

Summary Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, and Clostridium spiroforme toxin ADP‐ribosylate actin monomers. Toxin‐induced ADP‐ribosylation disturbs the cellular equilibrium between monomeric and polymeric actin and traps monomeric actin in its unpolymerized form, thereby depolymerizing actin filaments and destroying the microfilament network. Furthermore, the toxins ADP‐ribosylate gelsolin actin complexes. These modifications may contribute to the cytopathic action of the toxins.
cis‐Unsaturated fatty acids specifically inhibit a signal‐transducing protein kinase required for initiation of sporulation in Bacillus subtilis
Authors: Strauch Mark A. , de Mendoza Diego , Hoch James A. 
Published: October 2006

SummaryThe initiation of sporutation in Bacillus subtilis is controlled by the Spo0A transcription factor which is activated by phosphorylation through a phosphorelay mechanism that is dependent upon the activity of one or more protein kinases. The enzymatic activity of one of these protein kinases, KinA, was found to be inhibited in vitro by certain fatty acids. The most potent inhibitors have at least one unsaturated double bond in the cis configuration and a chain length of 16–20 carbon atoms. Homologous isomers with a trans double bond are not inhibitory. Saturated straight‐ or branched‐chain fatty acids are either much weaker inhibitors or have no effect. The inhibitors prevent autophosphorylation of KinA and are non‐competitive with ATP. B. subtilis phospholipids were found to contain at least one as yet unidentified type of fatty acid that, when present in an unesterified form, inhibited KinA. The results suggest that the concentration of a specific unsaturated fatty acid may act as a signal linking the initiation of sporulation to the status of membrane synthesis and septation or some other specific membrane‐associated activity.
Cloning and characterization of a gene encoding flagellin of Listeria monocytogenes
Authors: Dons L. , Rasmussen O.F. , Olsen J.E. 
Published: October 2006

SummaryThe gene, flaA, encoding the flagellin protein of Listeria monocytogenes (strain 12067) has been isolated from an expression library in Escherichia coli using a flagellin‐specific monoclonal antibody. DNA sequence analysis of a positive clone revealed the presence of an open reading frame of 287 amino acid residues with a calculated molecular mass of 30.4 kDa. Comparison of this sequence with flagellins from other bacteria showed a significant degree of homology in both the N‐ and C‐terminal parts of the protein. The flagellin mRNA was determined to be 1 kb in size, which is the expected size for a monocistronic mRNA, and the temperature‐dependent expression of flagellin was found to be regulated at the transcriptional level. Southern blot analysis, using the flagellin gene as probe, indicated that L. monocytogenes can be divided into two groups. These groups correspond to the flagellar antigens AB and ABC, respectively, as well as to the two types of L. monocytogenes based on the DNA sequence of the listeriolysin gene.
Gene disruption identifies a 290 kDa cell‐surface polypeptide conferring hydrophobicity and coaggregation properties in Streptococcus gordonii
Authors: McNab R. , Jenkinson H. F. 
Published: October 2006

SummaryThe C‐terminal coding region of the gene (denoted csh A) encoding a high‐molecular‐mass (290 kDa) cell‐surface polypeptide in the oral bacterium Streptococcus gordonii was cloned and sequenced. Insertion of ermAM into the S. gordonii chromosome at the 3′ end of the coding region of cshA led to the production of isogenic mutants that secreted a truncated form (260 kDa) of the CshA polypeptide into the growth medium. Mutants had reduced cell‐surface hydrophobicity and were impaired in their ability to coaggregate with oral actinomyces. The results identify a carboxyl terminus‐anchored cell‐surface protein determinant of hydrophobicity and coaggregation in S. gordonii
Determinants of an unusually stable mRNA in the bacterium Myxococcus xanthus
Authors: Romeo Joseph M. , Zusman David R. 
Published: October 2006

Summary Myxococcus xanthus is a Gram‐negative bacterium which has a complex life cycle that includes development (fruiting body formation). The gene for myxo‐bacterial haemagglutinin, mbhA, is developmentally regulated and highly expressed. In this report we show that the mbhA mRNA is exceptionally stable for a prokaryotic organism, exhibiting a chemical half life (t1/2) of 150 min at 18 h of development. The mbhA mRNA was not stable in negetatively growing cells nor was it stable when expressed in Escherichia coli. We have used site–directed mutagenesis of the mbhA gene to analyse some of the determinants which mediate the stability of the mbhA transcript. Sequences within the 3′‐untranslated region (3′‐UTR) were found to be crucial for mRNA stability. This region of mRNA can potentially form an extremely stable stem‐loop structure immediately adjacent to the translational stop codon. A deletion within this region caused a 10‐fold increase in the decay rate of the transcript. Furthermore, conditions which were associated with reduced mbhA translation or mutations that caused premature termination of translation drastically reduced mRNA stability even in the presence of the wild type 3′‐UTR. These results suggest that a significant aspect of mbhA mRNA stability involves a synergistic interaction of the translational machinery with sequence elements within the 3′‐UTR.
Identification of functional regions of the positively acting regulatory gene amdR from Aspergillus nidulans
Authors: Parsons Linda M. , Davis Meryl A. , Hynes Michael J. 
Published: October 2006

SummaryThe amdR (intA) regulatory gene of Aspergillus nidulans encodes a 765‐amino‐acid polypeptide which determines the omega‐amino acid induction of at least five structural genes. The AmdR polypeptide contains a potential Zn(ll)2Cys6 DNA‐binding motif which has been shown to be present in the N‐terminal region of a large number of fungal activator proteins. In vitro mutagenesis of the fourth cysteine of this motif abolishes AmdR function as shown by loss of complementation of an amdR− mutation and by the AmdR− phenotype of a mutant gene replacement strain. Studies using constructs in which the proposed AmdR DNA‐binding motif is replaced with that from another activator, FacB, shows that induction is independent of DNA‐binding specificity and that sequences in the C‐terminal region of AmdR are activation domains. Sequencing of several amdR mutant alleles which affect activation and/or induction, together with studies of deletion constructs indicate that changes in the conformation of the protein determines its activity and that this is modulated by inducers.
The 5′ to 3′ exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae
Authors: Díaz Asunción , Lacks Sanford A. , López Paloma 
Published: October 2006

SummaryThree different mutations were introduced in the polA gene of Streptococcus pneumoniae by chromosomal transformation. One mutant gene encodes a truncated protein that possesses 5′ to 3′ exonuclease but has lost polymerase activity. This mutation does not affect cell viability. Other mutated forms of polA that encode proteins with only polymerase activity or with no enzymatic activity could not substitute for the wild‐type polA gene in the chromosome unless the 5′ to 3′ exonuclease domain was encoded elsewhere in the chromosome. Thus, it appears that the 5′ to 3′ exonuclease activity of the DNA polymerase I is essential for cell viability in S. pneumoniae. Absence of the polymerase domain of DNA polymerase I slightly diminished the ability of S. pneumoniae to repair DNA lesions after ultraviolet irradiation. However, the polymerase domain of the pneumococcal DNA polymerase I gave almost complete complementation of the polA5 mutation in Escherichia coli with respect to resistance to ultraviolet irradiation.
Identification, genetic analysis and characterization of a sugar‐non‐specific nuclease from the cyanobacterium Anabaena sp. PCC 7120
Authors: Muro‐Pastor A. M. , Flores E. , Herrero A. , Wolk C. P. 
Published: October 2006

SummaryA nuclease that could be recovered from the supernatant of cultures, as well as from cell‐free extracts, of the cyanobacterium Anabaena sp. PCC 7120 was identified as a 29 kDa polypeptide by its ability to degrade DNA after electrophoresis in DNA‐containing SDS‐polyacrylamide gels. Some clones of a gene library of strain PCC 7120 established in Escherichia coli were found to produce the 29 kDa nuclease. The nucA gene encoding this nuclease was subcloned and sequenced. The deduced polypeptide, NucA, had a molecular weight of 29650, presented a presumptive signal peptide in its N‐terminal region and showed homology to the products of the nuc gene from Serratia marcescens and the NUC1 gene from Saccharomyces cerevisiae. The NucA protein from Anabaena itself, or from the cloned nucA gene expressed in E. coli, catalysed the degradation of both RNA and DNA, had the potential to act as an endonuclease, and functioned best in the presence of Mn2+ or Mg2+. An Anabaena nucA insertional mutant was generated which failed to produce the 29 kDa nuclease.
Direct visualization of plasmid DNA in bacterial cells
Authors: Eliasson Åsa , Bernander Rolf , Dasgupta Santanu , Nordström Kurt 
Published: October 2006

SummaryThe direct visualization of plasmid DNA inside Escherichia coli cells is demonstrated using phase‐fluorescence microscopy of DAPI (4′,6‐diamidino‐2‐phenylindole)‐stained bacteria. Small as well as large plasmids could be detected, both in minicells and in cells of larger size. For large plasmids, even single molecules appeared to be within the detection limit. The fluorescence generated from monomers of small plasmids was probably below this limit, and for these plasmids the observed signals may represent aggregates. The distribution of the fluorescence foci might reflect specific plasmid positioning during partition and/or replication.
Purification and functional characterization of the KdgR protein, a major repressor of pectinolysis genes of Erwinia chrysanthemi
Authors: Nasser W. , Reverchon S. , Robert‐Baudouy J. 
Published: October 2006

SummaryThe phytopathogenicity of the enterobacterium Erwinia chrysanthemi chiefly results from its capacity to degrade pectin, which is the major component of plant cell walls. This degradation requires the product of 12 genes which constitute independent transcriptional units. All these genes, including kdgT which encodes the 2‐keto‐3‐deoxygluconate (KDG) transport system, are negatively regulated by the KdgR protein. The E. chrysanthemi KdgR gene was cloned into an expression vector and overexpressed in Escherichia coli. KdgR was then purified to homogeneity by two chromatographic steps as a dimer of approximately 62kDa. Using gel retardation assays, we demonstrated that this purified repressor binds to the 25 bp oligonucleotide (AAAAAAGAAACATTGTTTCATTTGT) present in the kdgT regulatory region. Dimethyl sulphate interference experiments revealed that the repressor interacts with four guanine bases and 10 adenine bases in the two strands of this KdgR box. KDG, an actual inducer of pectinolysis, releases the repressor from the operator complexes, whereas galacturonate, which is the precursor of the actual inducer, does not. These results suggest the existence of a specific interaction between KDG and KdgR protein. This study opens discussion on the relative affinity of the KdgR protein for the different operators of pectinolysis genes which are interpreted in terms of differential regulation.
Purification and initial characterization of AhrC: the regulator of arginine metabolism genes in Bacillus subtilis
Authors: Czaplewski L. G. , North A. K. , Smith M. C. M. , Baumberg S. , Stockley P. G. 
Published: October 2006

SummaryThe arginine‐dependent repressor‐activator from Bacillus subtilis, AhrC, has been overexpressed in Escherichia coli and purified to homogeneity. AhrC, expressed in E. coli, is able to repress a Bacillus promoter (argCp), which lies upstream of the argC gene. The purified protein is a hexamer with a subunit molecular mass of 16.7 kDa. Its ability to recognize DNA has been examined in vitro using argCp in both DNase I and hydroxyl radical protection assays. AhrC binds at two distinct sites within the argCp fragment. One site, argCO1, with the highest affinity for protein, is located within the 5′ promoter sequences, whilst the other, argCO2, is within the coding region of argC. The data are consistent with the binding of a single hexamer of AhrC to argCO1 via four of its subunits, possibly allowing the remaining two subunits to bind at argCO2in vivo forming a repression loop similar to those observed for the E coli Lac repressor.
Transcription activation at Class I CAP‐dependent promoters
Authors: Ebright Richard H. 
Published: October 2006

SummaryCatabolite gene activator protein (CAP)‐dependent promoters can be grouped into three classes, based on the requirement for transcription activation and the position of the DNA site for CAP. Class I CAP‐dependent promoters require only CAP for transcription activation and have the DNA site for CAP located upstream of the DNA site for RNA polymerase. Amino acids 156 to 162 of the promoter‐proximal subunit of CAP are essential for transcription activation at Class I CAP‐dependent promoters, but are not essential for DNA binding, and are not essential for DNA bending. In the structure of the CAP‐DNA complex, these amino acids are located in a surface loop and form a cluster on the surface of the CAP‐DNA complex. Amino acids 261, 265, and 270 of the α subunit of RNA polymerase are essential for response to transcription activation by CAP at Class I CAP‐dependent promoters. Several lines of evidence indicate that transcription activation at Class I CAP‐dependent promoters requires a direct protein‐protein contact between amino acids 156 to 162 of the promoter‐proximal subunit of CAP and a molecule of RNA polymerase bound adjacent to CAP on the same face of the DNA helix. It is a strong possibility that this direct protein–protein contact involves amino acids 261 and 265 of the α subunit of RNA polymerase.
Location and orientation of an activating region in the Escherichia coli transcription factor, FNR
Authors: Bell Andrew , Busby Steve 
Published: October 2006

SummaryWe have characterized a number of mutations in fnr that interfere with FNR‐dependent transcription activation at two promoters where the FNR‐binding site is centred around 41½ bp upstream from the transcription start site. The substituted residues in all but one of these FNR mutants are clustered around a presumed surface‐exposed beta‐turn containing G85 which, we suggest, forms an activating region that contacts RNA polymerase at these promoters. Using the‘oriented heterodimers’method described elsewhere, we show that this activating region on the promoter‐proximal subunit of the FNR dimer is sufficient to activate transcription initiation. In contrast, this region is not essential for activation of a third FNR‐dependent promoter where the FNR‐binding site is centred at 61 1/2 bp upstream from the transcription start site. However, a substitution at S73 interferes with FNR‐dependent activation at both this promoter and promoters in which the FNR site is located at 41 1/2 bp from the transcript start, suggesting that FNR may contain a second activating region.