bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinguishing between the extracellular DNases of Vibrio cholerae and development of a transformation system
Authors: Focareta T. , Manning P. A.
Published: October 2006

Vibrio cholerae is known to secrete DNase(s) into the extracellular environment. These proteins have been thought to be responsible for the difficulties in transforming this organism. In this work we demonstrate that the dns and xds genes differ and that their products are solely responsible for the extracellular DNase activity. By site‐directed mutagenesis, strains have been constructed which are mutant in one or both genes. These strains have been assessed for their ability to be transformed with plasmid DNA and for their virulence in the infant mouse cholera model. DNase‐deficient mutants can be readily transformed and the product of dns appears to be the more significant barrier. No effect on virulence was observed with the mutants.
The cell envelope of the hyperthermophilic archaebacterium Pyrobaculum organotrophum consists of two regularly arrayed protein layers: three‐dimensional structure of the outer layer
Authors: Phipps B. M. , Huber R. , Baumeister W.
Published: October 2006

The cell envelope of the hyperthermophilic sulphur‐reducing archaebacterium Pyrobaculum organotrophum H10 was found to be composed of two distinct hexagonally arranged crystalline protein arrays. Electron microscopic analysis of freeze‐etched cells and isolated envelopes in conjunction with image processing showed that the inner layer (lattice centre‐to‐centre spacing 27.9 nm) is essentially identical to the protein array of Pyrobaculum islandicum GE03, a complex, rigid structure implicated in the maintenance of cell shape. The outer layer has clear p6 symmetry and a lattice spacing of 20.6 nm. Its three‐dimensional structure was reconstructed from a negative stain tilt series of an intact double‐layered envelope using Fourier filtration to separate the desired information from the other lattices present. The outer layer is a unique, porous network of block‐like dimers disposed around six‐fold axes, and exhibits minimal asymmetry between its inner and outer faces. It appears Xo be rather loosely associated with the outer surface of the inner layer. In most H10 envelopes, the inner layer is orientated with one base vector exactly perpendicular to the long axis of the cell, so that the cylindrical portion is composed of a series of parallel cell‐girdling hoops of hexameric morphological units. All the other known Pyrobaculum strains were found to have a GEO3‐type envelope structure, consisting of a single rigid protein array and a fibrous capsule. Although H10 does not possess a capsule, fibrils appear to be sandwiched between the two protein layers.
Molecular cloning, partial purification, and characterization of a haemin‐binding lipoprotein from Haemophilus influenzae type b
Authors: Hanson M. S. , Hansen E. J.
Published: October 2006

A library of genomic DNA fragments from Haemophilus influenzae type b (Hib) DL42 was constructed in plasmid pBR322, transformed into Escherichia coli strain RR1, and screened for recombinant clones with haemin‐binding activity by plating onto haemin‐containing agar. Expression of haemin‐binding activity by clones correlated with the expression of a protein with an apparent molecular weight of 51000 (51K) that was also recognized by anti‐Hib strain DL42 serum in immunoblots. One recombinant clone, designated pHM2, with the smallest DNA insert (3.62kb) was characterized further. Ethanol inhibition of expression of pHM2 in minicells revealed that the 51K protein was the result of a processing event involving a larger precursor. E. coli RR1(pHM2) adsorbed haemin in liquid suspensions as well as from solid media. Subcloning of a 2.6 kb fragment of pHM2 into a shuttle vector permitted the construction of a recombinant Hib clone, DL42(pHM1002), which overexpressed the 51K haemin‐binding protein. This 51K protein appears to be peripherally associated with the inner, and possibly outer, membranes of Hib. Affinity chromatography on haemin‐agarose was utilized to purify the haemin‐binding protein from both E. coli RR1(pHM2) and Hib DL42(pHM1002) to near homogeneity. The use of the antibiotic globomycin in a minicell expression system and radioimmunoprecipitation analysis of Hib proteins intrinsically radiolabelled with [3H]‐palmitate indicated that the 51K haemin‐binding protein is a lipoprotein.
Pilin expression and processing in pilus mutants of Neisseria gonorrhoeae: critical role of Gly‐1 in assembly
Authors: Koomey M. , Bergstrom S. , Blake M. , Swanson J.
Published: October 2006

Spontaneous mutants of Neisseria gonorrheae failing to express pili or having diminished levels of piliation were studied with regard to pilin expression. All mutants displayed altered pilin processing detectable as the release of soluble, truncated pilin molecules (S‐pilin). Of particular interest was the finding, in one mutant, that substitution of serine for glycine at position ‐1 of propilin, a highly conserved residue among N‐metPhe and related pilins, abolished pilus expression but not S‐pilin release. The degree of S‐pilin processing and the levels of membrane‐associated pilin varied among the different classes of mutants, suggesting that each was blocked at a distinct step of pilus biogenesis. The data support a model in which increased S‐pilin processing is a result of a decreased rate of pilus polymerization.
Genetic analysis of superoxide dismutase, the 23 kilodalton antigen of Mycobacterium tuberculosis
Authors: Zhang Y. , Lathigra R. , Garbe T. , Catty D. , Young D.
Published: October 2006

The gene encoding a 23 kilodalton protein antigen has been cloned from Mycobacterium tuberculosis by screening of a recombinant DNA library with monoclonal antibodies. The product of the gene has been identified as the superoxide dismutase (SOD) of M. tuberculosis on the basis of sequence comparison and by expression of the recombinant protein in a functionally active form. The derived amino acid sequence of M. tuberculosis SOD reveals a close similarity to manganese‐containing SODs from other organisms, in spite of the fact that previous studies using the purified enzyme have identified iron as the preferred metal ion ligand. SOD is present in the extracellular fluid of logarithmic‐phase cultures of M. tuberculosis, but the structural gene is not preceded by a signal peptide sequence. Insertion of the M. tuberculosis SOD gene into a novel shuttle vector demonstrated the presence of a promoter which functions efficiently in mycobacteria but is ineffective in Escherichia coli
Sequence and expression analysis of a cAMP‐responsive gene regulated during late development of Dictyostelium discoideum
Authors: Ramji D. P. , Donovan J. T. J. , Hames B.D.
Published: October 2006

The 7E gene is expressed late in normal development of Dictyostelium discoideum after pseudoplasmodium formation. After disaggregation of the developing cells, transcription of this gene depends entirely on exogenous 3′5′ cyclic AMP (cAMP). The 5′ flanking region of the 7E gene contains two TATA box‐oligo (dT) promoter motifs but analysis of 7E gene expression by primer extension shows only a single primary transcript with transcription initiating immediately after the most proximal promoter motif during development or in disaggregated cells in the presence of exogenous cAMP. Four C‐rich sequence lie within 350 bp upstream of the cap site, analogus to the upstream elements implicated in the cAMP regulation of several other Dictyostelium genes expressed in development.
Two global regulators repress the anaerobic expression of MnSOD in Escherichia coli: Fur (ferric uptake regulation) and Arc (aerobic respiration control)
Authors: Tardat B. , Touati D.
Published: October 2006

The expression of sodA, the Escherichia coli gene encoding manganese superoxide dismutase (MnSOD) is induced by aerobiosis and superoxide generators such as paraquat. Analysis of variants expressing sodA in the absence of oxygen has revealed that mutations in genes for two global regulatory systems, Fur (ferric uptake regulation) and Arc (aerobic respiration control), are simultaneously required for the expression of sodA in anaerobiosis. The Fur protein still represses sodA in an iron‐dependent fashion in aerobiosis. Moreover, all mutants remain inducible by paraquat, indicating that the positive control of SoxR, which mediates the response to superoxide in E. coli, is still operative. Thus, in addition to the response to the superoxide‐mediated oxidative stress which depends on SoxR, two global controls regulate MnSOD expression: ArcA couples MnSOD expression to respiration, and Fur couples it to the intracellular concentration of iron.
Roles of DNA adenine methylation in controlling replication of the REPI replicon of plasmid pColV‐K30
Authors: Gammie A. E. , Crosa J. H.
Published: October 2006

DNA adenine methylation controls DNA replication of plasmids containing the prototypic REPI replicon by affecting protein recognition and by altering the helical stability of the origin. Denaturing gradient gel electrophoresis shows that adenine methylated origin DNA is more easily melted than unmethylated. However, because an added DNA adenine methylation (dam) site at the origin, whether in or out of phase with other helically aligned dam sites, actually prevents replication, we conclude that destabilization of the helix is not the exclusive function of adenine methyfation in REPI replication. We find that the conformation and degree of methylation at the origin, features which are important for protein recognition, are essential for replication. In fact, RepI, a protein required for replication initiation at REPI replicons, contains a region homologous with a domain in proteins which specifically recognize and bind 5′‐GATC‐3′. We propose that the dam sites in the origin play a dual role: one is destabilization of the helix, and the other is protein recognition.
Physical and genetic map of the Methanococcus voltae chromosome
Authors: Sitzmann J. , Klein A.
Published: October 2006

A physical map of the Methanococcus voltae chromosome was constructed on the basis of restriction mapping and cross‐hybridization experiments, employing total and partial digests obtained with rarely cutting restriction enzymes. On the basis of the sum of the fragment sizes of digests with seven enzymes the chromosome length was calculated to be approximately 1900kb. The derived map is circular. Hybridization of gene probes to mapped restriction fragments has led to a genetic map of genes for structural RNAs as well as proteins, including enzymes involved in the methanogenic pathway.
Control of maltase synthesis in yeast
Authors: Needleman R.
Published: October 2006

Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4 and MAL6. Each MAL locus is complex consisting of at least three genes: a trans‐acting activator, a maltose permease, and maltase. All the MAL loci show homology to each other both at the sequence level as determined by Southern transfer analysis and at the functional level as determined by complementation. We describe the organization of the MAL loci in yeast and the basic features of their regulation. The analysis of MAL has contributed to our understanding of the evolution of multigenic families. The global integration of carbohydrate metabolism, and gene regulation.
Molecular cloning, characterization and analysis of the regulation of the AR02 gene, encoding chorismate synthase, of Saccharomyces cerevisiae
Authors: Jones D. G. L. , Reusser U. , Braus G. H.
Published: October 2006

We describe here the cloning, characterization and analysis of the regulation of the ARO2 gene of Saccharomyces cerevisiae, the first reported study of a eukaryotic gene encoding chorismate synthase (E.C. 4.6.1.4). The gene contains an ORF of 1128 bp, encoding a protein with a calculated molecular mass of 40.8 kDa. ARO2 is regulated under the ‘general control system’ for amino acid biosynthesis by the transcriptional activator GCN4 which binds in vitro at three sites within the AR02 promoter.

The AR02 gene product is highly similar to its Escherichia coli counterpart, with a 47% identity distributed over the entire length of the peptide. We therefore suggest that the S. cerevisiae chorismate synthase, in contrast to the Neurospora crassa enzyme, but like other chorismate synthases, is a monofunctional peptide, solely possessing chorismate synthase activity.
Characterization of kdgR, a gene of Erwinia chrysanthemi that regulates pectin degradation
Authors: Reverchon S. , Nasser W. , Robert‐Baudouy J.
Published: October 2006

Erwinia chrysanthemi is a phytopathogenic enterobacterium able to degrade the pectic fraction of plant cell walls. The kdgR negative regulatory gene controls all the genes involved in pectin catabolism, including the pel genes encoding pectate lyases. The E. chrysanthemi kdgR regulatory gene was subcloned in Escherichia coli where it was shown to be functional, since it repressed the expression of a pelE::uidA fusion. The nucleotide sequence of kdgR contained an open reading frame of 918bp preceded by classical transcriptional initiation signals. KdgR shows similarity to two other regulatory proteins, namely GylR, encoding an activator protein of the glycerol operon in Streptomyces coelicolor, and IclR, encoding a repressor of the acetate operon in Salmonella typhimurium and in Escherichia coli. Previously, comparison of regulatory regions of several genes controlled by kdgR revealed the existence of a conserved region which was proposed as a KdgR‐binding site. The 25 bp oligonucleotide AAAAAAGAAACATTG‐TTTCATTTGT corresponding to this consensus was substituted to the lac operator, at the beginning of transcription of the lacZ gene. This construct functioned as an operator for binding of the KdgR protein in vivo.
Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated
Authors: Mengaud J. , Dramsi S. , Gouin E. , Vazquez‐Boland J. A. , Milon G. , Cossart P.
Published: October 2006

Evidence for ptelotropic activation of virulence genes in Listeria monocytogenes is presented. A complementation study of a spontaneous prfA‐deletion mutant and analysis of cassette and transposon insertion mutants showed that the gene prfA activates the transcription of four independent genes which code for a phosphatidyl‐inositol‐specific phospholipase C (gene plcA), listeriolysin O (gene hlyA), a metallo‐protease (gene prtA) and a lecithinase (gene prtC). Transcription of prfA is not constitutive. During the growth phase, two peaks of prfA transcript accumulation were observed: the first was during exponential growth, and the second was at the beginning of the stationary phase. In addition, two prf4‐specific transcripts of 2.2 kb and 1 kb are detected. Early in exponential growth, prfA is co‐transcribed with plcA which lies upstream prfA, giving rise to the 2.2 kb plcA‐prfA transcript. In late‐exponential growth and at the beginning of the stationary phase, prfA transcripts of 1 kb are predominantly detected. Our results demonstrate that since prfA controls plcA transcription, it also regulates its own synthesis.
Genetic mapping of starch‐ and Lambda‐receptor sites in maltoporin: identification of substitutions causing direct and indirect effects on binding sites by cysteine mutagenesis
Authors: Francis G. , Brennan L. , Stretton S. , Ferenci T.
Published: October 2006

Cysteine mutagenesis was used to test the proximity of 16 residues to protein‐ligand interaction sites in maltoporin (LamB protein). LamB protein with additional cysteines was incorporated into the outer membrane of Escherichia coli except with a Ser‐30→ Cys substitution. Phage Lambda and starch binding was assayed before and after incubation of mutants with six thiol‐specific reagents. Four categories of mutation were recognized on the basis of phenotype and modification for each of the Lambda‐ and starch binding sites. The thiol modification experiments helped to clarify whether the phenotype of a mutation was due to a substitution at the binding site or an indirect perturbation of the structure. This study suggests that the cysteine mutagenesis/thiol modification approach may be usefully applied to the operational mapping of surface‐accessible binding sites or epitopes.
Glucose repression in the yeast Saccharomyces cerevisiae
Authors: Trumbly R. J.
Published: October 2006

Understanding the mechanism of glucose repression in yeast has proved to be a difficult and challenging problem. A multitude of genes in different pathways are repressed by glucose at the level of transcription. The SUC2 gene, which encodes invertase, is an excellent reporter gene for glucose repression, since its expression is controlled exclusively by this pathway. Genetic analysis has identified numerous regulatory mutations which can either prevent derepression of SUC2 or render its expression insensitive to glucose repression. These mutations allow us to sketch the outlines of a pathway for general glucose repression, which has several key elements: hexo‐kinase PII, encoded by HXK2, which seems to play a role in the sensing of glucose levels; the protein kinase encoded by SNF1, whose activity is required for derepression of many glucose‐repressible genes; and the MIG1 repressor protein, which binds to the upstream regions of SUC2 and other glucose‐repressible genes. Repression by MIG1 requires the activity of the CYC8 and TUP1 proteins. Glucose repression of other sets of genes seems to be controlled by the general glucose repression pathway acting in concert with other mechanisms. In the cases of the GAL genes and possibly CYC1, regulation is mediated by a cascade in which the general pathway represses expression of a positive transcriptional activator.
Isolation and analysis of IS6120, a new insertion sequence from Mycobacterium smegmatis
Authors: Guilhot C. , Gicquel B. , Davies J. , Martin C.
Published: October 2006

Insertion sequence IS6120 from Mycobacterium smegmatis was identified by its ability to transpose into different sites in the lambda repressor gene, d857, carried on an Escherichia coli/mycobacteria shuttle plasmid. IS6120 is a novel 1.5 kb insertion sequence, which has 24‐bp imperfect terminal inverted repeats and generates 9‐bp duplications of the target DNA following insertion. IS6120 is present in at least three copies in M. smegmatis but was not found in other species, including Mycobacterium tuberculosis. Nucleotide sequence analysis revealed that IS6120 contains two open reading frames, one of which encodes a putative transposase with similarities to those found in IS256 from Staphylococcus aureus, IST2 from Thiobacillus ferrooxidans, and ISRm3 from Rhizobium meliloti. The fact that IS6120 does not recognize a consensus target sequence for insertion and has no homologous sequences in the other strains studied makes IS6120 useful for transposon mutagenesis in mycobacteria.
Outer membranes of Gram‐negative bacteria are permeable to steroid probes
Authors: Plésiat Patrick , Nikaido Hiroshi
Published: October 2006

The permeability of bacterial outer membranes was assayed by coupling the influx of highly hydrophobic probes, 3‐oxosteroids, with their subsequent oxidation catalysed by 3‐oxosteroid δ1‐dehydrogenase, expressed from a gene cloned from Pseudomonas testosteroni. In Salmonella typhimurium producing wild‐type lipopolysaccharide, the permeability coefficients for uncharged steroids were 0.45 to 1 × 10‐5 cm S‐1 and the diffusion appeared to occur mainly through the lipid bilayer domains of the outer membrane. These rates are one or two magnitudes lower than that expected for their diffusion through the usual biological membranes. The permeation rates were markedly increased (up to 100 times) when the lipopolysaccharide leaflet was perturbed either by adding deacylpolymyxin or by introducing mutations leading to the production of deep rough lipopolysaccharides. An amphiphilic, negatively charged probe, testosterone hemisuccinate, penetrated much more slowly than the uncharged steroids. Study of various Gram‐negative species revealed that P. testosteroni, Pseudomonas acidovorans, and Acinetobacter calcoaceticus showed higher outer membrane permeability to steroid probes and higher susceptibility to hydrophobic agents such as fusidic acid, novobiocin and crystal violet relative to S. typhimurium and Escherichia coli.
Characterization of the Escherichia coli codBA operon encoding cytosine permease and cytosine deaminase
Authors: Danielsen S. , Kilstrup M. , Barilla K. , Jochimsen B. , Neuhard J.
Published: October 2006

The nucleotide sequence of a 3.1 kb segment carrying the cytosjne deaminase gene (codA) from Esctierichia coli was determined. The sequence revealed the presence of two open reading frames, the first (codB) specifying a highly hydrophobic polypeptide and the second specifying cytosine deaminase. A two‐codon overlap between the two reading frames indicates that they constitute an operon. Transcription of the operon was found to be regulated by exogenous purines. Polypeptides specified by each of the two reading frames were expressed in minicells, and the codB gene product was found to be highly enriched in the membrane fraction. Uptake experiments showed that the CodB protein is required for cytosine transport into the cell and that the intracellular accumulation of cytosine correlated with the codB gene dose. A topological model for the cytosine permease in the cytoplasmic membrane is proposed.
Nucleotide sequence analysis and serologic characterization of the Mycobacterium intracellulare homologue of the Mycobacterium tuberculosis 19 kDa antigen
Authors: Nair Jaygopal , Rouse David A. , Morris Sheldon L.
Published: October 2006

Disseminated Mycobacterium avium/Mycobacterium intracellulare complex (MAC) disease is a frequent complication in patients with the acquired immune deficiency syndrome (AIDS). In this report, we present the nucleotide sequence of the M. intracellulare MI22 gene. Computer sequence comparisons reveal that the MI22 gene, which encodes a serologically active protein, has 78% DNA sequence identity and 77% protein sequence identity with the seroreactive 19 kDa Mycobacterium tuberculosis lipoprotein antigen. Southern blot hybridizations indicate that an MI22 gene probe binds similar—‐sized restriction fragments in M. tuberculosis and M. intracellulare genomic DNA. In addition, immunoblot analyses demonstrate that MI22 is recognized by sera from tuberculosis patients. These data further support the existence of 19 kDa MAC and M. tuberculosis protein homologues. Phase partitioning experiments and the presence of a consensus lipid modification site in the deduced MI22 protein sequence strongly suggest that MI22 is also a lipoprotein. Comparative analyses of these mycobacterial antigenic homologues may provide the basis for the design of species‐specific diagnostic reagents.
Identification of individual amino acids required for secretion within the haemolysin (HlyA) C‐terminal targeting region
Authors: Kenny Brendan , Taylor Sally , Holland I. Barry
Published: October 2006

The release of haemolysin from Escherichia coli involves direct secretion across both the inner and outer membranes. Secretion of HlyA is dependent upon a specific membrane export complex composed of HlyB, ‐D and possibly TolC. HlyA is targeted to the medium via the membrane translocation complex, by a novel C‐terminal secretion signal. Previous studies involving deletion and fusion analyses have given contradictory results for the minimal length (20–60 residues) of this HlyA signal region and little is known of the nature of the specific residues and structural features required for function.

In this study we have analysed, quantitatively, the effect upon secretion of many point mutations introduced into the HlyA C‐terminus. The results indicate the presence of a minimal secretion signal domain whose proximal boundary extends to at least residue –46 and which contains at least four Individual residues essential for maximal secretion levels. We propose that such residues act co‐operatively, forming multiple contact points with the translocator proteins, with the ‘best fit’ promoting maximal levels of secretion.
Identification and characterization of a novel insertion sequence, IS1106, downstream of the porA gene in B15 Neisseria meningitidis
Authors: Knight A. I. , Ni H. , Cartwright K. A. V. , McFadden J. J.
Published: October 2006

Examination of Neisseria meningitidis strains associated with endemic meningococcal disease demonstrated differences in the number of copies of a repetitive sequence. Characterization of a copy of this repetitive sequence present in B15 strains has revealed the presence of a novel insertion sequence (S11106) located within a complex repetitive region downstream of the gene for the major surface antigen (porA). IS1106 has a length of 1137 bp and is flanked by 36bp inverted repeats. Two open reading frames (0RF1 and 0RF2) are present in opposite strands in codon–codon register with 0RF2 entirely located within ORF1. The predicted protein from 0RF1 demonstrates homology with the 5A protein of IS5 (Kroger and Hobom, 1962). Strains from two independent outbreaks of B15 meningococcal disease in the UK were found to contain the same genomic deletion removing a copy of IS1106 downstream of the porA gene.
Amino acid sequences of several Bacillus subtilis proteins modified by apparent guanylylation
Authors: Mitchell Clayton , Morris Paul W. , Vary James C.
Published: October 2006

Bacillus subtilis cell extracts, prepared at different times during growth, contained several proteins that were apparently guanylylated in vitro with [α‐32P]‐GTP. Four of the proteins were partially purified and the N‐terminal amino acid sequences (13 to 20 residues) were determined. One sequence had 84% identity to Bacillus stearothermophlius triosephosphate isomerase, two were 100% identical to the predicted sequences of the B. subtilis ptsI and ptsH genes while no identity was found for the fourth sequence. This apparent guanylylation occurred with proteins involved in glucose metabolism, although the significance is unknown.
The role of the pro‐sequence in the processing and secretion of the thermolysin‐like neutral protease from Bacillus cereus
Authors: Wetmore Diana R. , Wong Sui‐Lam , Roche Rodney S.
Published: October 2006

The Bacillus cereus cnp gene coding for the thermolysin‐like neutral protease (TNP) has been cloned, sequenced, and expressed in Bacillus subtilis. The protease is first produced as a pre‐pro‐protein (Mr= 61000); the pro‐peptide is approximately two‐thirds of the size of the mature protein. The pro‐sequence has been compared with those of six other TNPs, and significant homologies have been found. Additionally, the TNP pro‐sequences are shown to be homologous to the pro‐sequence of Pseudomonas aeruginosa elastase. A mutant has been constructed from cnp, in which 23 amino acids upstream from the pro‐protein processing site have been deleted. This region has no homologous analogue in any of the other TNP pro‐sequences. The detection results in a delay of six to eight hours in detection of active protease in the growth medium, as well as a 75% decrease in maximum protease production. N‐terminal analysis of the mutant mature protein demonstrates that the processing site is unaltered by the pro‐sequence deletion. The deletion must, therefore, modulate the kinetics of processing and/or secretion of the pro‐protein.
Characterization of transformation‐deficient mutants of Acinetobacter calcoaceticus
Authors: Palmen Ronald , Vosman Ben , Kok Ruben , van der Zee J. Rob , Hellingwerf Klaas J.
Published: October 2006

Three Acinetobacter calcoaceticus transformation‐deficient mutants, obtained by insertional mutagenesis with the nptII gene, have been characterized physiologically. One mutant (AAC211) was found to be completely transformation deficient, while two others, AAC213 and AAC214, were severely impaired in transformation efficiency (100–1000 times lower than the wild type). The latter applied to both chromosomal as well as plasmid DNA. Analysis of the chromosomal DNA fragments flanking the nptll gene in the mutants showed that mutants AAC213 and AAC214 had an insertion of the nptll gene in the same chromosomal region, but that they were the result of two independent mutational events, whereas the insertion in mutant AAC211 was at a different position. None of the three mutants showed phenotypic or genotypic characteristics typical of a RecA‐deficient strain.
NtcA, a global nitrogen regulator from the cyanobacterium Synechococcus that belongs to the Crp family of bacterial regulators
Authors: Vega‐Palas M. A. , Flores E. , Herrero A.
Published: October 2006

The gene ntcA is required for full expression of proteins subject to ammonium repression in the cyanobacterium Synechococcus. A 3.1 kb DNA fragment able to complement an ntcA mutant was digested with exonuclease III, and deleted fragments of different sizes were tested for complementation of that mutant, allowing the localization of its mutation within a BamHI‐HindIII genomic fragment of c. 0.4 kb. Insertion of a chloramphenicol‐resistance‐encoding gene cassette into both the BamHI and the HindiIII sites of wild‐type Synechococcus resulted in a pleiotropic, nitrogen‐assimilation‐minus phenotype, corroborating the presence of the ntcA gene in that genomic region. Sequencing of DNA in this region showed the presence of an open reading frame that included both the BamHI and the HindIII sites. The ntcA gene product, NtcA, is a protein of 24817 Da which belongs to a family of bacterial transcriptional activators that, among others, includes Crp and Fnr from Escherichia coli. Of special biological significance, it appears, is the presence of a conserved helix‐turn‐helix motif in the sequence close to the C‐terminal end of all the proteins in the family. The gene ntcA is proposed to encode a transcriptional activator of genes subject to nitrogen control in Synechococcus.
Identification and characterization of narQ, a second nitrate sensor for nitrate‐dependent gene regulation in Escherichia coli
Authors: Chiang Robin C. , Cavicchioli Ricardo , Gunsalus Robert P.
Published: October 2006

In response to nitrate availability, Escherichia coli regulates the synthesis of a number of enzymes involved in anaerobic respiration and fermentation. When nitrate is present, nitrate reductase (narGHJI) gene expression is induced, while expression of the DMSO/TMAO reductase (dmsABC, fumarate reductase (frdABCD)and fermentation related genes are repressed. The narL and narX gene products are required for this nitrate‐dependent control, and apparently function as members of a two‐component regulatory system. NarX is a presumed sensor‐transmitter for nitrate and possibly molybdenum detection. The presumed response‐regulator, NarL, when activated by NarX then binds at the regulatory DNA sites of genes to modulate their expression. In this study a third nitrate regulatory gene, narQ, was identified that also participates in nitrate‐dependent gene regulation. Strains defective in either narQ or narX alone exhibited no nitrate‐dependent phenotype whereas mutants defective in both narQ and narX were fully inactive for nitrate‐dependent repression or activation. In all conditions tested, this regulation required a functional narL gene product. These findings suggest that the narX and narQ products have complementary sensor‐transmitter functions for nitrate detection, and can work independently to activate NarL, for eliciting nitrate‐dependent regulation of anaerobic electron transport and fermentation functions. The narQ gene was cloned, sequenced, and compared with the narX gene. Both gene products are similar in size, hydrophobicity, and sequence, and contain a highly conserved histidine residue common to sensor–transmitter proteins.
NADH formation by Na+‐coupled reversed electron transfer in Klebsiella pneumoniae
Authors: Dan Pfenninger‐Li Xiao , Dimroth Peter
Published: October 2006

Citrate is fermented by Klebsiella pneumoniae to 2 acetate, 0.5 formate and 1.2 CO2. The formation of <1 formate and >1 CO2 per citrate can be accounted for by the oxidation of formate to CO2 in order to provide reducing equivalents for the assimilation of citrate into cell carbon. A membrane‐bound electron transport chain is apparently involved in NADH synthesis by these cells. The electrons from formate oxidation to CO2 are used to reduce ubiquinone to ubiquinol by membrane‐bound formate dehydrogenase and ubiquinol further delivers its electrons to NAD+, if this endergonic reaction is powered by δμTNa+. The endogenous NADH level of K. pneumoniae cells thus increased in the presence of formate in response to a δpNa+ >‐100 mV. NADH formation was completely abolished in the presence of oxygen or after addition of hydroxyquinoline‐N‐oxide, a specific inhibitor of the Na+‐translocating NADH:ubiqurnone oxidoreductase. The increase of endogenous NADH was dependent on the δpNa+ applied to the cells. Inverted membrane vesicles of K. pneumoniae catalysed the reduction of NAD+ to NADH with formate as electron donor after application of delta;μTNa+ of about 120 mV consisting of δpNa+ of 60 mV and ΔΨ of the same magnitude. Neither the δpNa+ nor the ΔΨ of this size alone was sufficient to drive the endergonic reaction. Strictly anaerobic conditions were required for NADH formation and hydroxyquinoline‐N‐oxide completely inactivated the reaction. It is suggested that NADH formation by reversed δμTNa+‐coupled electron transfer in these cells is an essential requirement for the synthesis of cell material from citrate.
IS231 D, E and F, three new insertion sequences in Bacillus thuringiensis: extension of the IS231 family
Authors: Rezsöhazy René , Hallet Bernard , Delcour Jean
Published: October 2006

IS231 constitutes a family of insertion sequences widespread among Bacillus thuringiensis subspecies. Three new IS231 variants have been isolated from B. thuringiensis subspecies finitimus (IS231 D and E) and israelensis (IS231 F). Like the previously described IS231 A, B and C, these 1.7 kb elements display single open reading frames encoding 477/478‐amino‐acid proteins which share between 72% and 88% identity with those of the other members of the family. Sequence comparisons also reveal that all the iso‐IS231 terminal inverted repeats are strongly conserved 20bp sequences. A region susceptible to forming a stable hairpin structure is found just upstream of the open reading frame. Nucleotide substitutions occurring on one strand of the hairpin stems are compensated for by complementary changes at facing positions, giving credence to the hypothesis that this secondary structure plays a role in the regulation of transposition. Examination of IS231 D, E and F flanking sequences reveals that IS231 F is bordered by a 12bp direct repeat. No direct repeats were found flanking IS231 D or IS231 E.
Mycobacteria contain two groEL genes: the second Mycobacterium leprae groEL gene is arranged in an operon with groES
Authors: de Wit Rinke Tobias F. , Bekelie Siraj , Osland Arve , Miko Tivadar L. , Hermans Peter W. M. , van Soolingen Dick , Drijfhout Jan‐Wouter , Schöningh Ron , Janson Anneke A. M. , Thole Jelle E. R.
Published: October 2006

In contrast to other bacterial species, mycobacteria were thus far considered to contain groEL and groES genes that are present on separate loci on their chromosomes, Here, by screening a Mycobacterium leprae lambda gt11 expression library with serum from an Ethiopian lepromatous leprosy patient, two DNA clones were isolated that contain a groEL gene arranged in an operon with a groES gene. The complete DNA sequence of this groESL operon was determined. The predicted amino acid sequences of the GroES and GroEL proteins encoded by this operon are 85–90% and 59–61% homologous to the sequences from previously characterized mycobacterial GroES and GroEL proteins. Southern blotting analyses with M. leprae groES‐ and groEL‐specific probes demonstrate that similar groESL homologous DNA is present in the genomes of other mycobacteria, including Mycobacterium tuberculosis. This strongly suggests that mycobacteria contain a groESL operon in addition to a separately arranged second groEL gene. Using five T‐cell clones from two leprosy patients as probes, expression of the M. leprae GroES protein in Escherichia coli after heat shock was demonstrated. Four of these clones recognized the same M. leprae‐specific GroES‐derived peptide in a DR2‐restricted fashion. No expression of the groEL gene from this operon was detected in E. coli alter heat shock, as tested with a panel of T‐cell clones and monoclonal antibodies reactive to previously described GroEL proteins of mycobacteria.
Interaction between maltose‐binding protein and the membrane‐associated maltose transporter complex in Escherichia coli
Authors: Dean David A. , Hor Lien I. , Shuman Howard A. , Nikaido Hiroshi
Published: October 2006

Active transport of maltose in Escherichia coli requires the presence of both maltose‐binding protein (MBP) in the periplasm and a complex of MalF, MalG, and MalK proteins (FGK2) located in the cytoplasmic membrane. Earlier, mutants in malF or MalG were isolated that are able to grow on maltose in the complete absence of MBP. When the wild‐type malE+ allele, coding for MBP, was introduced into these MBP‐independent mutants, they frequently lost their ability to grow on maltose. Furthermore, starting from these Mal‐ strains, Mal+ secondary mutants that contained suppressor mutations in malE were isolated. In this study, we examined the interaction of wild‐type and mutant MBPs with wild‐type and mutant FGK2 complexes by using right‐side‐out membrane vesicles. The vesicles from a MBP‐independent mutant (malG511) transported maltose in the absence of MBP, with Km and Vmax values similar to those found in intact cells. However, addition of wild‐type MBP to these mutant vesicles produced unexpected responses. Although male+ malG511 cells could not utilize maltose, wild‐type MBP at low concentrations stimulated the maltose uptake by malG511 vesicles. At higher concentrations of the wild‐type MBP and maltose, however, maltose transport into malG511 vesicles became severely inhibited. This behaviour of the vesicles was also reflected in the phenotype of male+ malG511 cells, which were found to be capable of transporting maltose from a low external concentration (1 μM), but apparently not from millimolar concentrations present in maltose minimal medium. We found that the mutant FGK2 complex, containing MalG511, had a much higher apparent affinity towards the wild‐type MBP than did the wild‐type FGK2 complex. We propose that the wild‐type FGK2 complex exists in at least two conformations, active and inactive, and that the binding of the liganded MBP converts the latter into the former. The mutant complex presumably exists predominantly in the active form that has a higher affinity toward liganded MBP, and the Inhibition of the mutant complex by an excess of maltose and wild‐type MBP may be explained as a form of inhibition by excess substrate.
Chromosomal and genetic analysis of the electrophoretic karyotype of Trichoderma reesei: mapping of the cellulase and xylanase genes
Authors: Carter Gina L. , Allison Dan , Rey Michael W. , Dunn‐Coleman Nigel S.
Published: October 2006

An electrophoretic karyotype has been established for Trichoderma reesei strain QM6a, and several of its derivatives, by pulsed‐field gel electrophoresis. All strains examined appear to have seven chromosomes with a total genome size of approximately 33 megabases (Mb). The sizes of the chromosomal bands in strain QM6a are approximately 6.2, 6.0, 5.1, 4.2 (doublet), 3.6 and 3.2 Mb. Genes encoding the cellulase complex and xylanases of T. reesei have been mapped, as have several other genes.
Decreasing transcription elongation rate in Escherichia Coli exposed to amino acid starvation
Authors: Vogel Ulla , Sørensen Michael , Pedersen Steen , Jensen Kaj Frank , Kilstrup Mogens
Published: October 2006

The time required for transcription of the lacZ gene in Escherichia coli was determined during exponential growth and under conditions, when the bacterium was exposed to partial isoleucine starvation. To do this, RNA was extracted from the cells at 10 s intervals following induction and quantified by Northern hybridization with probes complementary to either the beginning or the end of the lacZ mRNA. The time lag between inducer addition and the appearance of a hybridization signal at the ‘late’ probe represents the transit time for RNA polymerase on the lacZ gene, and this parameter and the known length of the transcribed sequence were used to calculate the lacZ mRNA chain growth‐rate. The transcription elongation rate was c. 43 nucleotides s‐1 during exponential growth and decreased abruptly to c. 20 nucleotides s‐1 in a relA+ strain after the onset of isoleucine starvation, when massive concentrations of guanosine tetraphosphate (ppGpp) accumulated in the cells. The starvation condition did not affect initiation of transcription at the lec‐promoter, but a substantial fraction of the initiated lacZ mRNA chains was never completed. For the rel+ strain the polarity was moderate, since c. 25% of the initiated lacZ mRNA’ chains were continued into full‐length mRNAs, but for the relA strain the polarity was so strong that no completed lacZ mRNA could be detected. The protein chain elongation rates decreased from 13 amino acids (aa) s‐1 in the unperturbed growth phase to approximately 6 aa s‐1, when the cells starved for isoleucine. In combination, these results suggest that ppGpp plays a major role in maintaining the coupling between transcription and translation during the downshift by inhibiting mRNA chain elongation. The implications of this result for the control of stable RNA synthesis during the stringent response are discussed.
Disulphide bridge formation in the periplasm of Escherichia coli: β‐lactamase::human lgG3 hinge fusions as a model system
Authors: De Sutter K. , Remaut E. , Fiers W.
Published: October 2006

We report the construction and the expression in Escherichia coli of three different fusion genes encoding the extended human lgG3 hinge region (Hi) fused in‐phase to the C‐terminal end of bacterial TEM1 β‐lactamase (Bla). In the first fusion gene blahi, the hinge sequence was directly coupled to the 3′ end of the β‐lactamase gene, whereas in the two other constructs, blal1hi and blal2hi, a linker encoding 14 and 10 amino acids, respectively, was inserted between the two subunits. After expression (24 h, 20° C) under control of the constitutive kanamycin phosphoribosyl transferase promoter, the fusion proteins, BlaHi, BlaL 1Hi and BlaL2Hi, respectively, were almost exclusively detected in the periplasmic fraction, and they conferred carbenicillin‐resistance to the cells. These results indicate that β‐lactamase can efficiently direct the export of proteins fused to its C‐ terminus, and moreover, at least some of the exported fusion proteins must carry the β‐lactamase moiety in a properly folded form. Analysis of their assembly, however, revealed that only a minor fraction was recovered as the expected F(ab′)2‐nke dimer. The presence in the periplasm of oxidized’monomers (with intrachain disulphide bonds) as well as of several high‐molecular‐mass proteins, probably resulting from the association between monomers and other cysteine‐rich proteins, strongly suggests that the conditions in the bacterial periplasm are insufficient to allow proper assembly of multimeric proteins with several interchain disulphide bonds.
(A)BC excinuclease: the Escherichia coli nucleotide excision repair enzyme
Authors: Lin Jing‐Jer , Sancar Aziz
Published: October 2006

Nucleotide excision repair is the major pathway for removing damage from DNA. (A)BC excinuclease is the nuclease activity which initiates nucleotide excision repair in Escherichia coli. in this review, we focus on current understanding of the structure‐function of the enzyme and the reaction mechanism of the repair pathway. In addition, recent biochemical studies on preferential repair of actively transcribed genes in E. coli are summarized.
Interrelated effects of DNA supercoiling, ppGpp, and low salt on melting within the Escherichia coli ribosomal RNA rrnB P1 promoter
Authors: Ohlsen Kari L. , Gralla Jay D.
Published: October 2006

The formation of complexes containing high levels of DNA melting at the ribosomal RNA rrnB P1 promoter in vitro is shown to be facilitated by DNA supercoiling or low salt. The effector nucleotide ppGpp is ineffective under these conditions. The loss of supercoils or addition of salt increases the effectiveness of ppGpp in inhibiting formation of these complexes. In vivo plasmid DNA supercoiling is shown to decrease during starvation protocols that also increase levels of ppGpp. The results suggest that ppGpp regulation may be affected by the state of DNA supercoiling in vivo.
Surface protein‐CAT reporter fusions demonstrate differential gene expression in the wr regulon of Streptococcus pyogenes
Authors: Podbielski A. , Peterson J. A. , Cleary P.
Published: October 2006

Streptococcus pyogenes expresses at least two virulence factors, the anti‐phagocytic M protein and an inhibitor of chemotaxis, the C5a peptidase (ScpA), under control of the virR locus. To facilitate studies of this regulatory unit, we constructed a new shuttle vector with a staphylococcal chloramphenicol acetyl transferase (CAT) reporter box which replicates in S. pyogenes. We cloned polymerase chain reaction (PCR)‐derived potential promoter regions of the virR, M protein (emm12), and ScpA (scpA) genes from an M type 12 5. pyogenes, strain CS24. Promoter activity was assessed by measurements of specific mRNAs, transacetylase activity, and minimum inhibitory concentrations (MICs) for chloramphenicol resistance. We demonstrated that VirR is a necessary but not always sufficient positive trans‐acting regulator of emm12 and scpA expression; however, virR is not autoregulated. A potential virR‐bindlng consensus sequence is postulated for emm12, scpA and other M‐like protein genes. Promoter activity of the structural genes was found to be dramatically influenced by growth conditions such as anaerobiosis. Levels of control, over and above the requirement for virR, are realized. The virR and scpA promoters were mapped for the first time using primer extension analysis. The observed mRNA start sites did not completely agree within the sequence predicted start sites. Data suggest that scpA could be subject to transcription attenuation.
A metalloprotease gene from Streptomyces coelicolor‘Müller’ and its transcriptional activator, a member of the LysR family
Authors: Dammann Tanja , Wohlleben Wolfgang
Published: October 2006

A metalloprotease gene (mprA) and its regulatory gene (mprR) from Streptomyces coelicolor‘Müller’ DSM3030 were isolated and their DNA sequences determined. The protease secreted by the heterologous host Streptomyces lividans was characterized biochemically as a metalloprotease with a M, of 20000, which is in good agreement with data derived from DNA sequence analysis. The mprA gene is transcribed divergently from mprR, the deduced protein of which displays homology to prokaryotic transcriptional regulators of the LysR family. The regulatory protein (MprR) was shown to bind to the intergenic region between mprH and mprA. It was found to activate transcription of mprA in 5. lividans and also in Escherichia coli.