bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of amber mutations in bacteriophage Mu transposase: a functional analysis of the protein
Authors: Desmet L. , Faelen M. , Gama M.‐J. , Ferhat A. , Toussaint A.
Published: October 2006

We have characterized a series of amber mutations in the A gene of bacteriophage Mu encoding the phage transposase. We tested different activities of these mutant proteins either in a sup0 strain or in different sup bacteria. In conjunction with the results described in the accompanying paper by Bétermier et al. (1989) we find that the C‐terminus of the protein is not absolutely essential for global transposase function, but is essential for phage growth. Specific binding to Mu ends is defined by a more central domain. Our results also reinforce the previous findings (Bétermier et al., 1987) that more than one protein may be specified by the A gene.
Cloning a genomic region required for a high‐affinity iron‐uptake system in Rhizobium meliloti 1021
Authors: Gill P. R. , Neilands J. B.
Published: October 2006

A collection of transposon‐induced mutants of Rhizobium meliloti 1021 defective in siderophore‐mediated iron assimilation were obtained and classified as biosynthetic, transport or regulatory. Several of the mutations were cloned and the adjacent sequences were used to acquire complementing DNA from the wild type. A single genomic region of about 35kb complemented all of the mutants deficient in production of the siderophore.
The transition state transcription regulator AbrB of Bacillus subtilis is autoregulated during vegetative growth
Authors: Strauch M. A. , Perego M. , Burbulys D. , Hoch J. A.
Published: October 2006

The DNA‐binding AbrB protein of Bacillus subtilis is an ambiactive transcriptional regulator of genes expressed during the transition state between vegetative growth and the onset of stationary phase and sporulation. Studies on the transcriptional control of AbrB synthesis using abrB‐lacZ fusions indicated that the abrB gene was autoregulated. This was consistent with the observation that purified AbrB protein bound specifically to the promoter region of its own gene in DNase I protection experiments. The structural gene mutation abrB4 abolished the autoregulatlon and purified AbrB4 protein did not have the promoter binding properties associated with the wild‐type protein. Both AbrB and AbrB4 proteins were shown to be hexamers of 10500 Dalton subunits and subunit exchange occurred between the proteins in vitro. However, the presence of only one or two mutant subunits dramaticaly altered the DNA‐binding ability of the multimeric protein. The results support a model in which autoregulation of the abrB gene is an important factor in preventing sporulation‐associated genes from being expressed during vegetative growth.
Expression of the recE gene during induction of the SOS response in Bacillus subtilis recombination‐deficient strains
Authors: Gassel M. , Alonso J. C.
Published: October 2006

A transcriptional fusion of the recE gene to a reporter gene has been constructed. Expression of recE was found to be induced upon damage to DNA with either mitomycin C or nalidixic acid. This specific transcriptional induction is blocked by a recE mutation. Mutations affecting the recB, recF and recL gene products markedly reduced induction. However, de‐repression of recE seems to be independent of the ATP‐dependent DNase activity of the exonuclease V enzyme (also called AddAB enzyme).
Membrane topology of penicillin‐binding protein 3 of Escherichia coli
Authors: Bowler L. D. , Spratt B. G.
Published: October 2006

The β‐lactamase fusion vector, pJBS633, has been used to analyse the organization of penicillin‐binding protein 3 (PBP3) in the cytoplasmic membrane of Escherichia coli. The fusion junctions in 84 in‐frame fusions of the coding region of mature TEM β‐lactamase to random positions within the PBP3 gene were determined. Fusions of β‐lactamase to 61 different positions in PBP3 were obtained. Fusions to positions within the first 31 residues of PBP3 resulted in enzymatically active fusion proteins which could not protect single cells of E. coli from killing by ampicillin, indicating that the β‐lactamase moieties of these fusion proteins were not translocated to the peri‐plasm. However, all fusions that contained: ≥36 residues of PBP3 provided single ceils of E coli with substantial levels of resistance to ampicillin, indicating that the β‐lactamase moieties of these fusion proteins were translocated to the periplasm. PBP3 therefore appeared to have a simple membrane topology with residues 36 to the carboxy‐terminus exposed on the periplasmic side of the cytoplasmic membrane. This topology was confirmed by showing that PBP3 was protected from proteolytic digestion at the cytoplasmic side of the inner membrane but was completely digested by proteolytic attack from the periplasmic side. PBP3 was only inserted in the cytoplasmic membrane at its amino terminus since replacement of its putative lipoprotein signal peptide with a normal signal peptide resulted in a water‐soluble, periplasmic form of the enzyme. The periplasmic form of PBP3 retained its penicillin‐binding activity and appeared to be truly water‐soluble since it fractionated, in the absence of detergents, with the expected molecular weight on Sephadex G‐100 and was not retarded by hydrophobic interaction chromatography on Phenyl‐Superose.
The Bradyrhizobium japonicum fixBCX operon: identification of fixX and of a 5’mRNA region affecting the level of the fixSCX transcript
Authors: Gubler M. , Zürcher T. , Hennecke H.
Published: October 2006

The Bradyrhizobfum japonicum fixX gene was identified and shown to be essential for symbiotic and free‐living, microaerobic nitrogen fixation. The fixX gene encodes a ferredoxin‐like protein which may be Involved in a redox process (electron transport?) essential for nitrogenase activity. This gene was localized downstream of fixC and its expression was dependent on the fixB promoter, providing evidence for the existence of a fixBCX operon. Mutagenesis and sequence analysis of the unusually long, 709 bp leader region between the fixB promoter and the fixB structural gene did not reveal the presence of a nif or fix gene that was absolutely essential for nitrogen fixation. However, a short open reading frame (ORF) within this region encoding a polypeptide of 35 amino acids (ORF35) was shown to be efficiently translated. Chromosomal deletion of a 400bp DNA fragment covering ORF35 resulted in a three‐fold reduction of the fixSCX mRNA level, which in turn also reduced the nitrogen fixation activity of this mutant. This suggests a possible post‐transcriptional control mechanism for the expression of the fixBCX operon involving the stabilization of fixBCX mRNA by ribosomes actively translating ORF35.
Cloning of a surface protein antigen gene from serotype c Streptococcus mutans
Authors: Okahashi N. , Sasakawa C. , Yoshikawa M. , Hamada S. , Koga T.
Published: October 2006

The structural gene for a 190kD protein antigen (PAc) of Streptococcus mutans MT8148 (serotype c) was cloned into the plasmid vector pUC118. SDS‐polyacrylamide gel electrophoresis and Western immunoblotting showed that the Escherichia coli harbouring the chimaeric plasmid produced multiple polypeptides of 190‐210kD. Immunodiffusion analysis revealed that the cloned PAc had the same specific determinants as S. mutans PAc. The cloned pac gene was mapped, and its transcriptional orientation was determined by characterizing deletion mutants of the chimaeric plasmid. Southern blot analysis with the cloned gene sequence as a probe revealed the presence of a homologous sequence in DNAs from sero‐types e and f S. mutans. PAc‐defective mutants were constructed by inserting an erythromycin‐resistance gene into the pac gene. The cell‐surface hydrophobicity of the mutants was lower than that of the parent strain.
Mutations produced by DNA polymerase III holoenzyme of Escherichia coli after in vitro synthesis in the absence of single‐strand binding protein
Authors: Carraway M. , Rewinski C. , Marinus M. G.
Published: October 2006

Single‐stranded plasmid DNA, containing the mnt gene, was replicated in vitro with DNA polymerase III holoenzyme. Escherichia colimutH bacteria, defective in mismatch repair, were transformed with the products of in vitro synthesis. Mutations in mnt were readily identified and 33 out of 65 isolates were single base changes including transition, transversion and frameshift mutations. The remaining 32 isolates were deletions of apparently random length and substitutions (deletion/insertions). The intergenic deletions as well as the transition and frameshift mutations were identical to those previously isolated from mismatch repair‐defective cells in vivo.
Geographically distinct isolates of Mycobacterium leprae exhibit no genotypic diversity by restriction fragment‐length polymorphism analysis
Authors: Williams D. L. , Gillis T. P. , Portaels F.
Published: October 2006

Differentiation of microorganisms for taxonomic purposes is based primarily on phenotypic characteristics, which are the direct or cumulative result of gene expression. Since expression of phenotypic characteristics usually relies on in vitro growth of a microorganism, non‐cultivable organisms, such as Mycobacterium leprae, present major problems for the identification of potential variants based on phenotypic similarities or differences between individual isolates. We have employed the use of restriction fragment‐length polymorphism (RFLP) analysis of chromosomal DNA of M. leprae isolates, including human isolates from geographically distinct regions of the world and isolates from a Sooty Mangabey monkey and an armadillo, to assess the relatedness among these isolates. Restriction endonuclease (Eco RI, Bst EII, Pst I, and Pvu I) digests of chromosomal DNA were analysed using DNA probes encoding all or part of the 12kD, 18kD, 28kD, 65kD and 70kD proteins of M. leprae as well as a probe containing an M. leprae‐specific sequence repeated up to 20 times in the M. leprae chromosome. Comparison of the resulting autoradiographs showed that the RFLP patterns were all identical, indicating that these isolates contained no polymorphism with respect to the restriction endonuclease sites analysed. In addition, RFLP patterns of two separate human M. leprae isolates remained unchanged after three cycles of experimental infection in the armadillo model. These results indicated that the M. leprae isolates tested in this study were indistinguishable at the genotypic level, strongly suggesting homogeneity among members of this species.
Functional importance of the Escherichia coli ribosomal RNA leader box A sequence for post‐transcriptional events
Authors: Theißen G. , Behrens S. E. , Wagner R.
Published: October 2006

To shed more light on the controversial findings concerning the functional participation of the highly conserved nut‐like leader box A sequence element in ribosomal RNA transcription antitermination we have carried out a mutational study. We have substituted the box A and combined this mutation with several deletions comprising the rRNA leader elements box B, box C and the tL region. The mutations are located within the genuine rrnB operon cloned on multicopy plasmids. We determined the effects of the mutations on cell growth, rRNA accumulation and ribosomal subunit stoichiometry. Cells transformed with the mutated plasmids were affected in their growth rate, and showed a surprising deficiency of the promoter‐proximal 16S compared to the 23S RNA, indicative of a post‐transcriptional degradation event. Accordingly, we could demonstrate a reduced amount of free 305 relative to 50S ribosomal subunits in exponentially growing cells. Similar stoichiometric aberrations in the ribosome pool were detected in conditionally Nus factor‐defective strains. The results show that the leader box A sequence within rRNA operons has important post‐transcriptional functions for 16S RNA stability and ribosomal subunit stoichiometry. A model is proposed, describing the biogenesis and quality control of ribosomes based on rRNA leader and Nus‐factor interactions. It is compatible with the previously observed effects of box A in antitermination.
Spore colour in Streptomyces coelicolor A3(2) involves the developmentally regulated synthesis of a compound biosynthetically related to polyketide antibiotics
Authors: Davis N. K. , Chater K. F.
Published: October 2006

Streptomyces coelicolor produces spores whose development of a grey colour requires the activity of the whiE locus. The cloned whiE locus was identified after mobilization into a whiE mutant of a library of S. coelicolor DNA inserted into a transmissible plasmid vector. The whiE region of the cloned DNA was localized both by subcloning and by mutagenesis of the cloned DNA with the Streptomyces transposon Tn4560. Nucleotide sequencing of this region revealed seven open reading frames, of which six show homo‐logy at the level of deduced gene products with genes involved in the synthesis of polyketide antibiotics. A previously described S. coelicolor DNA segment encoding biosynthesis of a brown pigment (Horinouchi and Beppu, 1985) corresponds to the cloned whiE DNA. It is proposed that whiE is normally expressed only in the aerial hyphae, where the biosynthetic product is responsible for spore colour.
Transcriptional analysis of the glnB‐glnA region of Rhizobium leguminosarum biovar viciae
Authors: Chiurazzi M. , Laccarino M.
Published: October 2006

We report that the glnB and gln A genes of Rhizobium leguminosarum biovar viciae are preceded by promoters located upstream of each gene. We find the presence of a glnB–glnA and a glnA mRNA whose intracellular concentration changes two‐ to three‐fold when R leguminosarum is grown on different nitrogen sources. Primer extension analysis shows unique transcriptional initiation sites upstream of glnB and glnA. The glnB promoter is rpoN(ntrA)‐dependent, while the glnA promoter does not contain a typical consensus sequence for previously described promoters.

In Klebsiella pneumoniae the glnB promoter requires active ntrC and ntrA genes and a DNA fragment containing 53 nucleotides upstream of the transcription initiation site shows full promoter activity, thus indicating that no NtrC binding sites are necessary for this activation in the glnB upstream region.
Direction of DNA entry in competent cells of Bacillus subtilis
Authors: Vagner V. , Claverys J.‐P. , Ehrlich S. D. , Méjean V.
Published: October 2006

Direction of DNA entry in Bacillus subtilis competent cells was studied using molecules in which only one of the two strands was radioactively labelled. The label was either distributed homogeneously or was localized in a small region of the strand, in the centre or at one of the ends. Regardless of the distribution and the position of the label, similar amounts of radioactivity were taken up by the cells exposed to the labelled molecules. This suggests that DNA enters B. subtilis either by two different uptake systems having opposite polarities, or by a single non‐polar system.
The yeast phosphatase system
Authors: Vogel K. , Hinnen A.
Published: October 2006

Yeast cells produce a set of enzymes which are involved in the metabolism of phosphate, and include acid and alkaline phosphatases as well as permeases. Most of these enzymes are synthesized in response to the presence or absence of inorganic phosphate. In the past few years a considerable amount of genetic and molecular evidence has accumulated and a rather precise overall picture emerges which describes the mechanism of phosphate control at the level of gene activation. This mini‐review summarizes these data. The main focus lies on the regulatory features associated with the control of transcription of PH05, a gene coding for most of the regulated acid phosphatase activity produced by yeast cells.
TonB and the Gram‐negative dilemma
Authors: Postle K.
Published: October 2006

TonB protein serves as an energy transducer to couple cytoplasmic membrane energy to high‐affinity active transport of iron siderophores and vitamin B12 across the outer membranes of Gram‐negative bacteria. The biochemical mechanism of the energy transduction remains to be determined, but important details are already known. TonB is targeted to and anchored in the cytoplasmic membrane by a single membrane‐spanning domain and spans the periplasm to physically interact with outer‐membrane receptors of the transport ligands. TonB‐dependent energy transduction is modulated by ExbB protein, which stabilizes TonB, and possibly by several other proteins including ExbC, ExbD, and TolQ. TonB has a relatively short functional half‐life that is accelerated when rates of active transport across the outer membrane are increased. A model that incorporates this information, as well as some tempered speculation, is presented.
Vitamin B12 transport in Escherichia coli: energy coupling between membranes
Authors: Kadner R. J.
Published: October 2006

Cells of Escherichia coli possess high‐affinity active transport systems of vitamin B12 and iron‐siderophore complexes. Specific outer‐membrane proteins carry out the energy‐dependent transport across the outer membrane, in conjunction with the TonB coupling protein. Mutagenesis experiments have identified a conserved region near the amino‐terminus of the outer‐membrane transporters that is necessary for energy‐coupled transport. The ability of extragenic suppressor mutations in tonB to correct the transport defect indicates that TonB couples the proton‐motive force to the outer‐membrane proteins by direct contact.
Efflux‐mediated antiseptic resistance gene qacA from Staphylococcus aureus: common ancestry with tetracycline‐ and sugar‐transport proteins
Authors: Rouch D. A. , Cram D. S. , Di Berardino D. , Littlejohn T. G. , Skurray R. A.
Published: October 2006

Resistance to intercalating dyes (ethidium, acriflavine) and other organic cations, such as quaternary ammonium‐type antiseptic compounds, mediated by the Staphylococcus aureus ptasmid pSK1 is specified by an energy‐dependent export mechanism encoded by the qacA gene. From nucleotide sequence analysis, qacA is predicted to encode a protein of Mr 55017 containing 514 amino acids. The gene is likely to initiate with a CUG codon, and a 36bp palindrome immediately preceding qacA, along with an upstream reading frame with homology to the TetR repressors, may be components of a regulatory circuit. The putative polypeptide specified by qacA has properties typical of a cytoplasmic membrane protein, and is indicated to be a member of a transport protein family that includes proteins reponsible for export‐mediated resistance to tetracycline and methylenomycin, and uptake of sugars and quinate. The analysis suggests that N‐ and C‐terminal regions of these proteins are involved in energy coupling (proton translocation) and substrate transport, respectively. The last common ancestor of the qacA and related tet (tetracycline resistance) lineages is inferred to have been repressor controlled, as occurs for modern tet determinants from Gram‐negative, but not those from Gram‐positive, bacteria.
Isolated DNA repeat region from fcrA76, the Fc‐binding protein gene from an M‐type 76 strain of group A streptococci, encodes a protein with Fc‐binding activity
Authors: Heath D. G. , Boyle M. D. P. , Cleary P. P.
Published: October 2006

The DNA repeat region of fcrA76, the gene encoding a group A streptococcal Fc‐binding protein, was sub‐cloned in‐frame into an Escherichia coli plasmid expression vector. The expressed protein product displayed the same Fc‐binding properties as the full‐length Fc‐binding protein expressed from fcrA76. The affinity‐purified, full‐length Fc‐binding protein was found to compete with staphylococcal protein A or streptococcal protein G for binding to beads coated with human IgG. These results are consistent with earlier studies suggesting that the binding sites on human IgG for protein A, protein G and the type II Fc‐binding protein from group A streptococci are located at the interface of the CH2 and CH3 domains of the Fc region.
Activation of a cryptic crystal protein gene of Bacillus thuringiensis subspecies kurstaki by gene fusion and determination of the crystal protein insecticidal specificity
Authors: Dankocsik C. , Donovan W. P. , Jany C. S.
Published: October 2006

DNA hybridization with the insecticidal crystal protein gene cryIIA (formerly cryBI) of Bacillus thuringiensis supspecies kurstaki has shown that subspecies kurstaki contains a cryIIA‐related sequence in addition to the cryIIA gene (Donovan et al., 1988a). We have cloned the cryIIA‐related sequence and have determined that the sequence, which has been designated cryIIB, is 89% identical to the cryIIA gene. Recombinant B. thuringiensis cells harbouring the cloned cryIIB gene produced very little CryIIB protein. A high level of production of the CryIIB protein was achieved by fusing the regulatory region of the cryIIIA crystal protein gene to the cryIIB gene. The CryIIB protein was found to be highly toxic to Lymantria dispar, Heliothis virescens and Trichoplusia ni, and was not toxic to Aedes aegypti.
RNase E, an endoribonuclease, has a general role in the chemical decay of Escherichia coli mRNA: evidence that rne and ams are the same genetic locus
Authors: Mudd E. A. , Krisch H. M. , Higgins C. F.
Published: October 2006

Escherichia coli RNase E is known to process RNA precursors at specific sites. We show that this endoribonuclease has a general role in E. coli mRNA turnover and affects the stability of specific transcripts. The effect of the rne mutation on functional stability of mRNA was much less pronounced than that on chemical stability, although the expression of some genes was affected. The E. coli ams (altered mRNA stability) mutation was found to have phenotypes indistinguishable from those of the rne mutation, affecting both 9S RNA and T4 gene 32 mRNA processing. The rne and ams mutations were both complemented by the same 3.7kb fragment of E. coli DNA and are probably allelic. RNase E is the first endoribonuclease Identified as having a general role in the chemical decay of E. coli mRNA.
Identification of hypoxanthine and guanine as the co‐repressors for the purine regulon genes of Escherichia coli
Authors: Meng L. M. , Nygaard P.
Published: October 2006

Addition of purine compounds to the growth medium of Escherichia coli and Salmonella typhimurium causes repressed synthesis of the purine biosynthetic enzymes. The repression is mediated through a regulatory protein, PurR. To identify the co‐repressor(s) of PurR, two approaches were used: (i) mutations were introduced into purine salvage genes and the effects of different purines on pur gene expression were determined; (ii) purine compounds which dictate the binding of the PurR protein to its operator DNA were resolved by gel retardation. Both the in vivo and the in vitro data indicated that guanine and hypoxanthine are co‐repressors. The toxic purine analogues 6‐mercaptopurjne and 6‐thioguanine also activated the binding of PurR to its operator DNA.
Cloning and characterization of mepA, the structural gene of the penicillin‐insensitive murein endopeptidase from Escherichia coli
Authors: Keck W. , Van Leeuwen A. M. , Huber M. , Goodell E. Wm.
Published: October 2006

The putative structural gene mepA of the penicillin‐insensitive murein endopeptidase from Escherichia coli was cloned and sequenced. N‐terminal sequence determination with the isolated endopeptidase protein showed that this enzyme is coded by the mepA gene and that it is synthesized initially with an N‐terminal signal peptide. No significant sequence homoiogy with the other (penicillin‐sensitive) murein endopeptidase (dacB) or any other protein was found. The precise chromosomal mapping position of mepA relative to two other genes, aroC and fabB, was shown to be 50.4 min. E. coli strains carrying multicopy plasmids with the mepA gene produced 5‐6‐fold more endopeptidase and secreted it into the periplasm, where it appeared to function normally in vivo since the release of ceil wall peptides into the medium increased in parallel. The transformed cells were, however, not unusually sensitive to penicillin and their murein had a normal degree of cross‐bridges.
Molecular analysis of the Haemophilus influenzae type b pilin gene
Authors: Langermann S. , Wright A.
Published: October 2006

A Haemophilus influenzae DNA library was prepared in the vector lambda EMBL3, and recombinant phage were screened for the pilin gene (pil) using a synthetic oligonucleotide. Southern blot analysis of the positive clones revealed a 2.5kb Pstl/Pvul fragment that hybridized with the oligonucleotide probe. This fragment was subcloned into pBR322 and sequenced. The nucleotide sequence disclosed an open reading frame of 653 bases. The deduced amino acid sequence corresponded with the known amino acid sequence of the purified pilin protein. Primer extension analysis using total RNA from piliated H. influenzae cells delineated a start site for the gene, ‐10 and ‐35 promoter regions, and a ribosome‐binding site. No transcripts were seen with the RNA derived from a non‐piliated strain. Southern blots of DNA from a number of H. influenzae strains revealed homology with the pil structural gene. DNA from a non‐piliated strain of H. influenzae also hybridized with the pil probe. Transcriptional and translational studies were performed in Escherichia coli with plasmids containing: (i) the pil gene on the 2.5kb Pstl/Pvul fragment, (ii) the pil gene fused to the phoA gene, and (iii) the pil gene present on a 12.2 kb insert containing extensive H. influenzae DNA flanking the pil gene. The results suggest that the H. influenzae pil gene is expressed in Escherichia coli, but from a promoter other than the one used in H. influenzae.
The regulation of transcription of the gerA spore germination operon of Bacillus subtilis
Authors: Feavers I. M. , Foulkes J. , Setlow B. , Sun D. , Nicholson W. , Setlow P. , Moir A.
Published: October 2006

The gerA operon of Bacillus subtilis 168 comprises three genes concerned with the triggering of spore germination by l‐alanine and its analogues. The expression of this operon has been characterized using chromosomal lacZ fusions to the gerA promoter. The gerA promoter is switched on 2.5–3 hours after the initiation of sporulation, in parallel with glucose dehydrogenase. A high proportion of the gerA‐driven β‐galactosidase detected in sporulating cells is found in the mature spore; the gerA promoter is therefore active in the forespore compartment of the sporulating cell. The gerA promoter is not expressed in spo0, spoII or spoIIIA, B, E and G mutant backgrounds, but is expressed in spoIIICand D and in spoIV and V mutants. The in vivo transcriptional startpoint of the operon has been mapped by primer extension experiments; sequences upstream from this startpoint show significant homology with recognition sequences for RNA polymerase containing σG(EσG). The gerA operon was transcribed in vitro by EσG with a startpoint identical to that used in vivo, and expression of the gerA operon was rapidly induced in vegetative cells by induction of σG synthesis. These data indicate that the gerA operon is an additional member of the σG regulon, which includes a number of genes expressed in parallel only in the forespore compartment of sporulating B. subtilis cells.
Isolation of a secY homologue from Bacillus subtilis: evidence for a common protein export pathway in eubacteria
Authors: Suh J.‐W. , Boylan S. A. , Thomas S. M. , Dolan K. M. , Oliver D. B. , Price C. W.
Published: October 2006

Genetic and biochemical studies have shown that the product of the Escherichia coli secY gene is an integral membrane protein with a central role in protein secretion. We found the Bacillus subtilis secY homologue within the spc‐alpha ribosomal protein operon at the same position occupied by E. coli secY. B. subtilis secY coded for a hypothetical product 41% identical to E. coli SecY, a protein thought to contain 10 membrane‐spanning segments and 11 hydrophilic regions, six of which are exposed to the cytoplasm and five to the periplasm. We predicted similar segments in B. subtilis SecY, and the primary sequences of the second and third cytoplasmic regions and the first, second, fourth, fifth, seventh, and tenth membrane segments were particularly conserved, sharing greater than 50% identity with E. coli SecY. We propose that the conserved cytoplasmic regions interact with similar cytoplasmic secretion factors in both organisms and that the conserved membrane‐spanning segments actively participate in protein export. Our results suggest that despite the evolutionary differences reflected in cell wall architecture, Gram‐negative and Gram‐positive bacteria possess a similar protein export apparatus.
The role of transformation in the variability of the Neisseria gonorrhoeae cell surface
Authors: Scocca J. J.
Published: October 2006

This review discusses the genetic basis for surface changes in Neisseria gonorrhoeae and the role of specific transformation reactions in producing them. Variation in the structure of pilin, the subunit of gonococcal pili, occurs by transformation‐mediated recombination of DNA segments in storage loci with the expression locus. These pilin loci have low recombination potential since their sequences contain only short uninterrupted identical sequences. The DNA within storage or silent loci are also relatively deficient in the short defined sequences which target DNA for efficient uptake and thus have relatively low affinity for the DNA transport system. Consequently, pilin‐encoding DNA segments constitute relatively poor substrates for the general transformation system of gonococci. These considerations suggest the existence of locus‐specific factors which increase the efficiency of genetic exchange between pilin loci. I raise the speculative hypothesis that one function of transformation‐mediated DNA entry is to provide a regulatory stimulus signalling the death of neighbouring gonococci. This regulatory shift might lead to production of factors which accelerate genetic reshuffling of pilin loci either by transformation per se using external DNA as donor, or via a recombinational process which utilizes internally derived DNA segments as donors. A signalling function for transforming DNA also clarifies several general properties of specific transformation reactions.
Characterization of the repertoire of hypervariable regions in the Protein II (opa) gene family of Neisseria gonorrhoeae
Authors: Connelly T. D. , Shaffer D. , Cannon J. G.
Published: October 2006

P.11 outer membrane proteins of Neisseria gonorrhoeae are encoded by a family of closely related genes. Although the genes are highly conserved, major differences in sequence among them occur in two short regions, designated hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of the hypervariable regions in the P.11 genes of strain FA1090. The FA1090 chromosome contained at least eleven P.11 loci, having six different versions each of HV1 and HV2 among them. Southern blotting with HV‐specific oligonucleotides showed that each version was present in one to three copies, and that there were nine unique combinations of HV1 and HV2 in the P.M genes. Although each of the versions of HV1 or HV2 had a unique DNA sequence, there were some similarities among them, particularly when certain ones were compared. Restriction fragments containing only the HV regions were cloned into an expression vector to demonstrate that the epitopes recognized by a set of monoclonal antibodies specific for different FA1090 P.11 proteins were completely encoded by either HV1 or HV2.
Identification of regB, a gene required for optimal exotoxin A yields in Pseudomonas aeruginosa
Authors: Wick M. J. , Frank D. W. , Storey D. G. , Iglewski B. H.
Published: October 2006

The yield of exotoxin A from Pseudomonas aeruginosa has been shown to be strain‐dependent. Exotoxin A production requires the presence of the positive regulatory gene, regA. We cloned the regA genetic locus from the prototypical P. aeruginosa strain PAO1 and examined its ability to influence exotoxin A yields compared to the same region cloned from the hyper‐toxin‐producing strain, PA103. The P. aeruginosa regA mutant strain, PA103‐29, containing the PAO1 regA locus in trans produced approximately five to seven times less extracellular exotoxin A than PA103‐29 containing the regA locus cloned from the hyper‐toxigenic strain, PA103. Nucleotide sequence analysis of the PAO1 regA locus revealed several differences, the most striking of which was the absence of a second open reading frame that was present in the analogous PA103 DNA. In addition, an amino acid substitution was found at position 144 of RegA (Thr in PAO1 and Ala in PA103). Recombinant molecules were constructed to test the contribution of each of these changes in nucleotide sequence on extracellular exotoxin A yields. The amino acid substitution in the PAO1 RegA protein was found not to affect overall exotoxin A yields. In contrast, the presence of the second open reading frame immediately downstream of the PA103 regA gene was found to influence extracellular exotoxin A yields. This open reading frame encodes a gene which we call regB. Nucleotide sequence analysis indicates that regB is 228 nucleotides in length and encodes a protein of 7527 Daltons. Our data suggest that regB is required for optimal exotoxin A production and its absence in strain PAO1 partially accounts for the difference in yield of extracellular exotoxin A between P. aeruginosa strains PAO1 and PA103.
Differential gene expression during sporulation in Bacillus subtilis: structure and regulation of the spoIIID gene
Authors: Stevens C. M. , Errington J.
Published: October 2006

The gene spoIIID, which is essential for spore formation in Bacillus subtilis, was cloned and sequenced. It consists of one open reading frame which would encode a 93‐amino‐acid protein with a classic helix‐turn‐helix motif, characteristic of sequence‐specific DNA‐binding proteins. SpoIIID protein is a previously identified transcription factor, capable of altering the specificity of RNA polymerase containing sigma K in vitro (Kroos et al., 1989). The spoIIID83 mutation (by which the locus was originally identified), was sequenced and found to be a single base substitution in the ribosome binding site upstream of the spoIIID open reading frame. A transcriptional fusion to lacZ was constructed and used to examine the regulation of spoIIID. Expression of spoIIID occurred only during sporulation, beginning 1.5 to 2 hours after the initiation of sporulation. The dependence of spoIIID expression on other spo loci suggests that it is mother‐cell‐specific, and that it is transcribed by sigma E‐containing RNA polymerase.
The major subunit of Escherichia coli type 1 fimbriae is not required for D‐mannose‐specific adhesion
Authors: Klemm P. , Krogfelt K. A. , Hedegaard L. , Christiansen G.
Published: October 2006

Type 1 fimbriae are surface organelles on Escherichia coli, which mediate specific binding to D‐mannose‐containing structures. These fimbriae are hetero‐polymers composed of a major building element, the FimA protein, and small amounts of the FimF, FimG and FimH proteins. The FimH protein is uniquely responsible for the D‐mannose receptor binding. In this work data are presented which indicate that the major subunit of type 1 fimbriae is dispensable for D‐mannose‐specific binding. A recombinant strain was studied which harboured an insertional deletion in the fimA gene, and was thereby unable to produce type 1 fimbriae; however, it was still able to express a D‐mannose‐binding phenotype. However, the deletion resulted in a 25‐fold reduction of the adhesive potential, as measured by binding to D‐mannose‐coated Sepharose beads. Serological and specific receptor binding evidence is presented that suggests that the FimH adhesin is capable of being exposed on the bacterial surface without being an integral part of the fimbriae.
Two plasmids other than the nodulation plasmid are necessary for formation of nitrogen‐fixing nodules by Rhizobium leguminosarum
Authors: Hynes M. F. , McGregor N. F.
Published: October 2006

A system which allows direct selection for curing of plasmids in Gram‐negative bacteria was used to generate derivatives of Rhizobium leguminosarum VF39 cured of each of six plasmids present in this strain. Phenotypes could be correlated with the absence of five of the six plasmids. The smallest plasmid, pRIeVF39a, carries genes for the production of a melanin‐like pigment as has been previously reported. Plasmid pRIeVF39d carries nodulation and nitrogen fixation genes. Curing of the plasmids pRleVF39c and pRleVF39e gave rise to strains which formed Fix− nodules on peas, lentils, and faba beans. The nodules formed by the strains cured of pRIeVF39c contained few, if any, bacteria. Analysis of washed cells by SDS‐PAGE showed that this strain is defective in lipopolysaccharide (LPS) production; the defect could be complemented by introducing plasmids from several other R. leguminosarum strains, and by the R. leguminosarum biovar phaseoli LPS gene clones pCos126 and pDel27. The nodules formed by the strain cured of pRleVF39e had a reduced symbiotic zone, an enlarged senescence zone, and an abundance of starch granules. This strain grew at a much slower rate than the wild type, was unable to grow on minimal medium, and no longer produced melanin. These defects could be complemented by at least one other Rhizobium plasmid, pRle336e, a plasmid of strain 336 which is distinct from the nodulation plasmid (pRle336c) and the plasmid (pRle336d) which could complement the LPS defect associated with the loss of pRleVF39c. This demonstrates that genes necessary for symbiosis can be carried on at least three different plasmids in R. leguminosarum.
The identification of the Escherichia coli fts Y gene product: an unusual protein
Authors: Gill O. R. , Salmond G. P. C.
Published: October 2006

The ftsYEX operon in Escherichia coli encodes three proteins, two of which (FtsE and FtsX) are known to be required for cell division. Although FtsE and FtsX have been identified using SDS‐PAGE, the FtsY protein has not. We have used in vitro insertion mutagenesis to identify FtsY as a 92kD polypeptide in maxicell experiments, although predictions from the DNA sequence estimated FtsY to be 54kD. Results suggest that this disparity could be due to the unusually high percentage of acidic residues within the protein. Complementation tests indicated the presence of a promoter within ftsY required for expression of ftsE and ftsX. The FtsY protein exhibits sequence homology with the SRα protein of eukaryotes which is involved in protein secretion. The essential nature of the ftsY gene was also demonstrated.
Developmental regulation of cell‐type‐enriched mRNAs in Dictyostelium discoideum
Authors: Corney A. J. , Richards A. J. , Phillpots T. , Hames B. D.
Published: October 2006

We describe sixteen new families of cDNA clones representing mRNAs that are expressed preferentially in either prespore or prestalk cells during developmentm of Dictyostelium discoideum and two new mRNAs that are expressed in a non‐cell‐type‐specific manner. None of the prespore‐enriched mRNAs are detectable in Dictyostelium cells until 13–15h of development but then they increase dramatically and peak at 18–22h. Upon dissociation of developing aggregates, all these mRNAs rapidly decay to low levels. In marked contrast to data presented for prespore genes by other workers, cyclic AMP either has no effect on the mRNA levels in dissociated cells or is only weakly effective in restoring normal expression. A prestalk‐enriched mRNA examined, 5G mRNA, is similarly expressed late in development but is also expressed in vegetative cells. The level of 5G mRNA is only moderately affected by cell disaggregation.
Serratia marcescens rpr gene sensitizes Escherichia coli wild‐type, xth, and nfo strains to methyl methanesulphonate
Authors: Murphy K. E. , Braymer H. D.
Published: October 2006

It is reported here that the rpr DNA repair gene of Serratia marcescens does not complement an Escherichia coli xth nfo AP endonuclease mutation for resistance to methyl methanesulphonate (MMS). Rather, rpr sensitized Escherichia coli wild‐type, xth, and nfo strains to MMS. Also, it was found that rpr could not complement a triple tag alkA recA mutation in E. coli, indicating that there are limits to rpr complementing capabilities. It was determined that rpr gene dosage was not a factor in recA complementation. MMS sensitization of an E. coli wild‐type strain, however, was directly related to rpr copy number. These data indicate that Rpr does not have an associated AP endonuclease activity, and that it is incapable of substituting for Tag I, Tag II, and RecA in a tag alkA recA background.
Genetic instability and hypervariability in Streptomyces ambofaciens: towards an understanding of a mechanism of genome plasticity
Authors: Leblond P. , Demuyter P. , Simonet J. M. , Decaris B.
Published: October 2006

Many Streptomyces species exhibit a very high degree of genetic instability which is usually manifested as genomic rearrangements such as large deletions. In Streptomyces ambofaciens DSM40697, two levels of genetic instability were previously described: (i) a basic genetic instability similar to that reported for other strains, and (ii) hypervariability, a phenomenon that we believe to be a new aspect of instability closely associated with DNA amplification. A large DNA region undergoes deletions, amplifications and large genomic changes strictly associated with both aspects of genetic instability. The genetic and molecular analyses of the different aspects of genetic instability allow us to propose that they result from a cascade of molecular events and to investigate the relationships between genetic instability phenomena and genome fluidity.
Transposition of Tn916 to different sites in the chromosome of Neisseria meningitidis: a genetic tool for meningococcal mutagenesis
Authors: Kathariou S. , Stephens D. S. , Spellman P. , Morse S. A.
Published: October 2006

The difficulty in obtaining mutants in pathogenic Neisseria has limited the ability to genetically define determinants responsible for virulence as well as the ability to generate a genetic map. We show that the 16.5kb conjugative transposon Tn916 can be introduced into Neisseria meningitidis on the suicide vectors pAM120 and pAM170. After introduction, Tn916 transposed to different sites in the chromosome of recipient meningococci, apparently at random, and was stably incorporated. Following its integration into the meningococcal chromosome, Tn916 did not appear to readily express its conjugative and transpositional functions. However, chromosomal DNA from Tn916‐carrying meningococci could be used to transform other meningococcal strains to tetracycline resistance. These studies indicate that Tn916 may be an important tool for genetic analysis of N. meningitidis.
Pulmonary dehydration and infection in cystic fibrosis: evidence that ethanol activates alginate gene expression and induction of mucoidy in Pseudomonas aeruginosa
Authors: DeVault J. D. , Kimbara K. , Chakrabarty A. M.
Published: October 2006

Mortality among cystic fibrosis (CF) patients is most commonly attributed to pulmonary infection by mucoid, alginate‐producing Pseudomonas aeruginosa. The initial infecting P. aeruginosa are typically non‐mucoid; however, upon continued exposure to the CF lung environment, they become highly mucoid. The CF lung is an osmotically high environment because of the presence of substantial concentrations of electrolytes and dehydrated mucus. In this report we demonstrate that ethanol (a commonly used dehydrating agent) activates transcription from a critical alginate promoter, algD, and show that prolonged exposure to ethanol allows switching to the mucoid form. This activation appears to be dependent on DNA gyrase. Analysis of alginate gene activation, and the subsequent reversal of the activation process by bacterial DNA gyrase inhibitors, should aid the development of treatment strategies for CF patients infected with this organism.
The complete nucleotide sequence of the Pasteurella multocida toxin gene and evidence for a transcriptional repressor, TxaR
Authors: Petersen S. K.
Published: October 2006

The osteolytic toxin of Pasteurella multocida induces bone resorption in vivo and in vitro (Foged et al., 1988; Kimman et al., 1987). In this report the toxin‐encoding toxA gene is sequenced, and the deduced primary structure of the toxin shows a protein of 1285 amino acids, containing a striking homology to a metal‐binding motif. Evidence that expression of the toxA gene is repressed at a transcriptional level in Escherichia coli is presented. Repression could be abolished either by deletion of a region upstream of toxA, or by a putative frame‐shift mutation in the same region. The repressor protein encoded within this region was efficient in trans, and was named TxaR.
Notes High‐efficiency transformation system for the biocontrol agents, Trichoderma spp.
Authors: Herrera‐Estrella A. , Goldman G. H. , Montagu M. Van
Published: October 2006

We have developed an efficient transformation system based on the use of polyethylene glycol and CaCl2 for the biocontrol agents, Trichoderma spp. Trans formation was obtained with the plasmid pAN7‐1, carrying a bacterial hygromycin‐resistance gene as a selectable marker, under the control of Aspergillus nidulans heterologous expression signals. The system described here yielded 200‐800 transformants per μg of DNA. Transformants contained several copies of the plasmid integrated into their genome, apparently at the same site in the different transformants analysed. Stability of the transformants was achieved by inserting a 2.4kb homologous DNA fragment into pAN7‐1. Southern blot analysis indicated that integration in the stable transformants occurs through non‐homologous recombination.
Wolinella succinogenes fumarate reductase contains a dihaem cytochrome b
Authors: Körtner C. , Lauterbach F. , Tripier D. , Unden G. , Kröger A.
Published: October 2006

The fumarate reductase operon of Wolinella succinogenes is made up of three structural genes (frd‐CAB). The frdC gene was located next to the promoter region and identified as the cytochrome b structural gene encoding 256 amino acid residues. The N‐terminal amino acid sequences of seven fragments derived from the cytochrome b moiety of the enzyme all mapped within the frdC gene. This suggested that the enzyme contained only one species of cytochrome b. Re‐evaluation of earlier measurements of subunit composition, haem B content and molecular weight led to the conclusion that the enzyme contained one molecule of cytochrome b with two haem B groups. The hydropathy plot of the amino acid sequence predicted five membrane‐spanning hydrophobic segments, the first four of which contained a single histidine residue each. These residues could form the axial ligands to the two haem B groups. FrdC was found to be homologous with the cytochrome b (SdhC) of the Bacillus subtilis succinate dehydrogenase, but not with the hydrophobic subunits of the fumarate reductase or succinate dehydrogenase of Escherichia coli.
Intron‐associated splicing reactions in bacteriophage T4
Authors: Maley F. , Chu F. K. , Maley G. F.
Published: October 2006

Group I introns are present in at least three bacteriophage T4 genes: td, nrdB and sunY. The transcription products of these three genes have similar intron consensus regions and secondary structures, which render them capable of guanosine‐mediated in vitro autocatalytic splicing reactions. Moreover, it has been shown that the 245‐amino‐acid protein encoded in the td intron expresses an endonuclease that cleaves near the joining site for the two exons in the intron‐deleted thymidylate synthase gene. The intron‐containing td gene is resistant to the enzyme. As in the case of other group I intron‐containing genes that have been described in eukaryotes, which also encode site‐specific endonucleases, the td intron is highly mobile and can insert into the intronless td gene by a process initiated by endonuclease cleavage near the insertion site. Whether intron transposition reactions have any physiological significance to the phage, or represent an early imprint on the evolution of introns, remains to be determined.