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PubMed1997

Construction of a Helicobacter pylori-Escherichia coli shuttle vector for gene transfer in Helicobacter pylori.

In this study, a Helicobacter pylori-Escherichia coli shuttle vector was constructed for transferring DNA into H. pylori. The smallest cryptic plasmid (1.2 kb), pHP489, among those harbored by 77 H. pylori isolates was selected as a base replicon for constructing vectors. HindIII-digested pHP489 was ligated with a kanamycin resistance gene [aph(3')-III], which originated from Campylobacter jejuni, to produce the recombinant plasmid pHP489K. pHP489K was efficiently transformed into and stably maintained in H. pylori strains. The shuttle vector pBHP489K (3.6 kb) was constructed by the recombination of pHP489, ColE1, and aph(3')-III sequences. pBHP489K was reciprocally transformed into and maintained in both H. pylori and E. coli. Introduction of the shuttle vector clone DNA (pBHP489K/AB; 6.7 kb), containing the ureA and ureB genes of H. pylori, into urease-negative mutants of H. pylori led to the restoration of their urease activity. The transformants were confirmed to contain the incoming plasmid DNA. pBHP489K satisfied the requirements for an H. pylori-E. coli shuttle vector, implying that it might be a useful vector for investigating pathogenicity and restriction-modification systems of H. pylori.

Cloning, Molecular↗
PubMed2002

Cloning of Escherichia coli lacZ and lacY genes and their expression in Gluconobacter oxydans and Acetobacter liquefaciens.

An efficient transformation protocol for Gluconobacter oxydans and Acetobacter liquefaciens strains was developed by preparation of electrocompetent cells grown on yeast extract-ethanol medium. Plasmid pBBR122 was used as broad-host-range vector to clone the Escherichia coli lacZY genes in G. oxydans and A. liquefaciens. Although both lac genes were functionally expressed in both acetic acid bacteria, only a few transformants were able to grow on lactose. However, this ability strictly depended on the presence of a plasmid expressing both lac genes. Mutations in the plasmids and/or in the chromosome were excluded as the cause of growth ability on lactose.

Acetobacter↗
PubMed2003

Identification and application of plasmids suitable for transfer of foreign DNA to members of the genus Gordonia.

Gene transfer systems for Gordonia polyisoprenivorans strains VH2 and Y2K based on electroporation and conjugation, respectively, were established. Several parameters were optimized, resulting in transformation efficiencies of >4 x 10(5) CFU/ micro g of plasmid DNA. In contrast to most previously described electroporation protocols, the highest efficiencies were obtained by applying a heat shock after the intrinsic electroporation. Under these conditions, transfer and autonomous replication of plasmid pNC9503 was also demonstrated to proceed in G. alkanivorans DSM44187, G. nitida DSM44499(T), G. rubropertincta DSM43197(T), G. rubropertincta DSM46038, and G. terrae DSM43249(T). Conjugational plasmid DNA transfer to G. polyisoprenivorans resulted in transfer frequencies of up to 5 x 10(-6) of the recipient cells. Recombinant strains capable of polyhydroxyalkanoate synthesis from alkanes were constructed.

Conjugation, Genetic↗
PubMed2004

Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus.

An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml(-1) in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter(-1) was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml(-1). These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter(-1) when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml(-1) (i.e., 360 mg liter(-1)) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter(-1). In this case, maximal activities were 3,900 and 4,660 nkat ml(-1), respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.

Basidiomycota↗
PubMed2006

Development of a biofilm production-deficient Escherichia coli strain as a host for biotechnological applications.

Bacteria form biofilms by adhering to biotic or abiotic surfaces. This phenomenon causes several problems, including a reduction in the transport of mass and heat, an increase in resistance to antibiotics, and a shortening of the lifetimes of modules in bioindustrial fermentors. To overcome these difficulties, we created a biofilm production-deficient Escherichia coli strain, BD123, by deleting genes involved in curli biosynthesis and assembly, Delta(csgG-csgC); colanic acid biosynthesis and assembly, Delta(wcaL-wza); and type I pilus biosynthesis, Delta(fimB-fimH). E. coli BD123 remained mostly in the form of planktonic cells under the conditions tested and became more sensitive to the antibiotics streptomycin and rifampin than the wild-type E. coli MG1655: the growth of BD123 was inhibited by one-fourth of the concentrations needed to inhibit MG1655. In addition, the transformation efficiency of BD123 was about 20 times higher than that of MG1655, and the production and secretion of recombinant proteins were approximately 16% and approximately 25% greater, respectively, with BD123 than with MG1655. These results indicate that the newly created biofilm production-deficient strain of E. coli displays several key properties that substantially enhance its utility in the biotechnology arena.

Bacterial Proteins↗
PubMed2006

A pleiotropic regulator, Frp, affects exopolysaccharide synthesis, biofilm formation, and competence development in Streptococcus mutans.

Exopolysaccharide synthesis, biofilm formation, and competence are important physiologic functions and virulence factors for Streptococcus mutans. In this study, we report the role of Frp, a transcriptional regulator, on the regulation of these traits crucial to pathogenesis. An Frp-deficient mutant showed decreased transcription of several genes important in virulence, including those encoding fructosyltransferase (Ftf), glucosyltransferase B (GtfB), and GtfC, by reverse transcription and quantitative real-time PCR. Expression of Ftf was decreased in the frp mutant, as assessed by Western blotting as well as by the activity assays. Frp deficiency also inhibited the production of GtfB in the presence of glucose and sucrose as well as the production of GtfC in the presence of glucose. As a consequence of the effects on GtfB and -C, sucrose-induced biofilm formation was decreased in the frp mutant. The expression of competence mediated by the competence-signaling peptide (CSP) system, as assessed by comC gene transcription, was attenuated in the frp mutant. As a result, the transformation efficiency was decreased in the frp mutant but was partially restored by adding synthetic CSP. Transcription of the frp gene was significantly increased in the frp mutant under all conditions tested, indicating that frp transcription is autoregulated. Furthermore, complementation of the frp gene in the frp mutant restored transcription of the affected genes to levels similar to those in the wild-type strain. These results suggest that Frp is a novel pleiotropic effector of multiple cellular functions and is involved in the modulation of exopolysaccharide synthesis, sucrose-dependent biofilm formation, and competence development.

Bacterial Proteins↗
PubMed1982

Protoplast fusion permits high-frequency transfer of a Streptomyces determinant which mediates actinomycin synthesis.

Prototrophic recombinants and heterocaryotic colonies developed at high frequency when protoplasts of nutritionally complementary actinomycin-producing and nonproducing strains of Streptomyces antibioticus were fused in the presence of polyethylene glycol and plated on minimal regeneration medium. Of the spores obtained from aerial hyphae of a single heterocaryotic colony, 99% carried the act+ character regardless of whether the nutritional markers of the spore were derived from the act+ or the act parent. Similarly, a high-frequency transfer (68% in S. antibioticus, 48% in Streptomyces parvulus) of act+ determinant(s) to act was achieved by protoplast fusion. Protoplasts of a doubly auxotrophic act strain of S. parvulus were efficiently transformed in the presence of polyethylene glycol with respect to the auxotrophic markers by DNA of an act+ auxotrophic strain with complementary nutritional requirements. The transformation frequency of the nutritional (chromosomal) markers was 17%. In contrast, the transformation frequency for actinomycin synthesis was less than 1%.

Chromosomes, Bacterial↗
PubMed1982

Helper plasmid cloning in Streptococcus sanguis: cloning of a tetracycline resistance determinant from the Streptococcus mutans chromosome.

A model system for testing the helper plasmid cloning system of Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980) was devised for the Streptococcus sanguis (Challis) host-vector system. In this system, linearized pVA736 plasmid efficiently transformed an S. sanguis (Challis) host containing a homologous plasmid, pVA380-1, but did not transform a plasmidless host or a host containing a nonhomologous plasmid, pVA380. In addition, whereas monomeric circular pVA736 transformed a plasmidless host with two-hit kinetics, it transformed a pVA380-1-containing host with one-hit kinetics. This helper plasmid cloning system was used to isolate two HindIII fragments (5.0 megadaltons [Mdal] and 1.9 Mdal in size) from the chromosome of Streptococcus mutans V825 which conferred high-level tetracycline resistance. One tetracycline-resistant clone was examined and found to contain three plasmids which were sized and designated pVA868 (9.0 Mdal), pVA869 (9.5 Mdal), and pVA870 (9.8 Mdal). Results of Southern blot hybridization and restriction endonuclease digestion confirmed that all three chimeras were composed of two HindIII fragments of the S. mutans V825 chromosome, as well as a large portion, varying in size for each chimera, of the 2.8 Mdal cloning vector, pVA380-1. Incompatibility observed between pVA380-1 and each of the chimeras indicated that replication of the chimeras was governed by the pVA380-1 replicative origin. Southern blotting experiments revealed that the chimeras hybridized to Tn916, providing the first evidence that transposon-related genes of enteric streptococcal origin are disseminated among oral streptococci.

Chimera↗
PubMed1985

Repair of cis-platinum-DNA adducts by ABC excinuclease in vivo and in vitro.

cis-Platinum compounds, which are used in cancer chemotherapy, are thought to exert their effect by damaging DNA. It is known that this damage is partially repaired in Escherichia coli. Using cis-Pt-treated pBR322 DNA as a probe, we investigated the role of nucleotide excision repair in the removal of Pt-DNA adducts. We found that the nucleotide excision pathway was the major mechanism for repairing Pt adducts in transforming plasmid DNA but that a recA-dependent pathway also contributed to plasmid survival. When cis-Pt-damaged pBR322 was treated with the purified nucleotide excision enzyme ABC excinuclease in vitro, a fraction of the adducts was removed by the enzyme; this removal resulted in a corresponding increase in transformation efficiency.

Cisplatin↗
PubMed1987

Localization of a genetic region involved in McrB restriction by Escherichia coli K-12.

A 5,500-base-pair BglII-EcoRI fragment proximal to the hsd genes of Escherichia coli K-12 has been cloned in the plasmid vector pUC9. The resultant hybrid plasmid was shown to complement the mcrB mutation of E. coli K802. The presence of the hybrid plasmid in strain K802 caused an 18.3-fold drop in transformation efficiency with AluI-methylated pACYC184 relative to unmethylated pACYC184. These results indicate that the cloned DNA is involved in the McrB system restriction of 5-methylcytosine DNA.

5-Methylcytosine↗
PubMed1988

Localization of Bacillus subtilis sacU(Hy) mutations to two linked genes with similarities to the conserved procaryotic family of two-component signalling systems.

Mutations in the sacU region have a pleiotropic phenotype. Certain mutations designated sacU(Hy), for example, express degradative enzymes at high levels, are able to sporulate in the presence of glucose, have severely reduced transformation efficiencies, and are nonmotile. We isolated and sequenced the sacU gene region of Bacillus subtilis. Two open reading frames were found in the sacU region, and sacU(Hy) mutations were localized to both of these open reading frames. The two open reading frames have similarities to two widespread families of proteins that mediate responses to environmental stimuli.

Amino Acid Sequence↗
PubMed1989

Participation of Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in P1 plasmid replication.

Low-copy-number plasmids, such as P1 prophage and the fertility factor F, require a plasmid-encoded replication protein and several host products for replication. Stable maintenance also depends on active partitioning of plasmids into daughter cells. Mini-P1 par+ and par plasmids were found to be destabilized by mutations in the dnaJ, dnaK, and grpE genes of Escherichia coli. The transformation efficiency and stability of mini-F plasmids were also reduced in the mutant strains. These results indicate that heat shock proteins DnaJ, DnaK, and GrpE play roles in the replication of plasmid P1 and probably also in of F.

DNA Replication↗
PubMed1989

Physically associated enzymes produce and metabolize 2-hydroxy-2,4-dienoate, a chemically unstable intermediate formed in catechol metabolism via meta cleavage in Pseudomonas putida.

The meta-cleavage pathway of catechol is a major mechanism for degradation of aromatic compounds. In this pathway, the aromatic ring of catechol is cleaved by catechol 2,3-dioxygenase and its product, 2-hydroxymuconic semialdehyde, is further metabolized by either a hydrolytic or dehydrogenative route. In the dehydrogenative route, 2-hydroxymuconic semialdehyde is oxidized to the enol form of 4-oxalocrotonate by a dehydrogenase and then further metabolized to acetaldehyde and pyruvate by the actions of 4-oxalocrotonate isomerase, 4-oxalocrotonate decarboxylase, 2-oxopent-4-enoate hydratase, and 4-hydroxy-2-oxovalerate aldolase. In this study, the isomerase, decarboxylase, and hydratase encoded in the TOL plasmid pWW0 of Pseudomonas putida mt-2 were purified and characterized. The 28-kilodalton isomerase was formed by association of extremely small identical protein subunits with an apparent molecular weight of 3,500. The decarboxylase and the hydratase were 27- and 28-kilodalton polypeptides, respectively, and were copurified by high-performance-liquid chromatography with anion-exchange, hydrophobic interaction, and gel filtration columns. The structural genes for the decarboxylase (xylI) and the hydratase (xylJ) were cloned into Escherichia coli. The elution profile in anion-exchange chromatography of the decarboxylase and the hydratase isolated from E. coli XylI+XylJ- and XylI-XylJ+ clones, respectively, were different from those isolated from XylI+ XylJ+ bacteria. This suggests that the carboxylase and the hydratase form a complex in vivo. The keto but not the enol form of 4-oxalocrotonate was a substrate for the decarboxylase. The product of decarboxylation was 2-hydroxypent-2,4-dienoate rather than its keto form, 2-oxopent-4-enoate. The hydratase acts on the former but not the latter isomer. Because 2-hydroxypent-2,4-dienoate is chemically unstable, formation of a complex between the decarboxylase and the hydratase may assure efficient transformation of this unstable intermediate in vivo.

Carboxy-Lyases↗
PubMed1989

Cloning and characterization of araA, araB, and araD, the structural genes for L-arabinose utilization in Bacillus subtilis.

Two recombinant plasmids, pSNL1 and pSNL2, carrying structural genes for L-arabinose utilization were isolated from a Bacillus subtilis gene library. Both plasmids complemented araD mutations in a Rec- B. subtilis strain and in Escherichia coli. Moreover, pSNL1 also complemented araB mutations in both species and efficiently transformed araA Rec+ B. subtilis strains to Ara+. Detailed physical mapping of both plasmids in addition to transformation experiments involving defined restriction fragments from the pSNL1 insert unambiguously determined the gene order to be araD, araB, and araA, an order different from that found in E. coli.

Arabinose↗
PubMed1990

An expression vector for the archaebacterium Haloferax volcanii.

The recent development of an efficient transformation method and shuttle vectors for Haloferax volcanii has set the stage for rapid progress in archaebacterial molecular biology. We describe a shuttle-expression vector that can be selected for and maintained in either H. volcanii or Escherichia coli and permits the expression of cloned genes in H. volcanii. The vector, pWL204, was constructed by incorporating an H. volcanii tRNA(Lys) gene promoter into a derivative of the H. volcanii-E. coli shuttle vector pWL102. The vector has been used to express a modified, intron-containing, H. mediterranei tRNA(Trp) gene (tRNA(Trp)-O167). Transcription from the tRNA(Lys) gene promoter in vivo was detected by Northern (RNA) analysis with an oligonucleotide probe complementary to the unique intron sequence of tRNA(Trp)-O167. Dependence of transcription on the tRNA(Lys) promoter was demonstrated by the absence of transcription when the promoter sequence was deleted from the vector and by mapping the transcription initiation site by primer extension.

Archaea↗
PubMed1996

The sre gene (ORF469) encodes a site-specific recombinase responsible for integration of the R4 phage genome.

The sre gene (ORF469) of the R4 phage encodes a protein similar to the resolvase-DNA invertase family proteins. Insertional gene disruption of sre prevented a lysogen from entering the lytic cycle, implying that Sre protein is a site-specific recombinase needed for excision of the R4 prophage genome (M. Matsuura, T. Noguchi, T. Aida, M. Asayama, H. Takahashi, and M. Shirai, J. Gen. Appl. Microbiol. 41:53-61, 1995). To determine whether this sre gene is also necessary for the integration reaction, we studied its function by integration plasmid analysis. When deletions, frameshifts, and site-directed mutations that caused an amino acid substitution of Ser-17 for Ala were introduced into the sre structural gene, transformation efficiency of Streptomyces parvulus 2297 with these plasmid DNAs was severely reduced. However, an adenine insertion just before the possible initiation codon of the sre gene did not significantly decrease the efficiency. These data suggest that the Sre protein is a site-specific recombinase responsible for integration of the R4 phage genome.

Bacteriophages↗
PubMed1996

Plasmid-amplified comS enhances genetic competence and suppresses sinR in Bacillus subtilis.

The establishment of genetic competence in Bacillus subtilis is controlled by a vast signal transduction network involving the products of genes that function in several postexponential-phase processes. Two of these proteins, SinR and DegU, serve as molecular switches that influence a cell's decision to undergo either sporulation or genetic competence development. In order to determine the roles of SinR and DegU in competence control, multicopy suppression experiments with plasmid-amplified comS, SinR, and degU genes were undertaken. Multicopy comS was found to elevate competence gene transcription and transformation efficiency in both wild-type and sinR mutant cells but not in degU mutant cells. Multicopy degU failed to suppress comS or sinR mutations. No suppression of comS or degU by multicopy sinR was observed. The expression of a comS'::'lacZ translational fusion and srf-lacZ operon fusion was examined in sinR cells and cells bearing plasmid-amplified sinR. The expression of comS'::'lacZ gene fusion was reduced by the sinR mutation, but both comS'::'lacZ and srf-lacZ were repressed by multicopy sinR. Cells bearing plasmid-amplified sinR were poorly competent. These results suggest that sinR is required for optimal comS expression but not transcription from the srf promoter and that SinR at high concentrations represses srf transcription initiation.

Bacillus subtilis↗