HindIII Fragment Research Articles

Genetics of the lipoprotein lipase gene and hypertriglyceridaemia

The aim of this study is to assess whether genetic variation at the lipoprotein lipase (LPL) gene is related to fasting triglyceride levels or to the presence of vascular disease. Hypertriglyceridaemic patients are genotyped for the N291S, G188E, and P207L variants and the HindIII and PvuII restriction fragment length polymorphisms of the LPL gene. Sequence analysis is carried out on exons 1–9 of the LPL gene for patients with severe hypertriglyceridaemia, to search for new gene variants. No differences were found between the patient and control group for the N291S, G188E and P207L variants. The HindIII and PvuII allelic frequencies were found to be similar for patients and controls; however, the frequency of the PvuII P2 allele was higher in patients with vascular disease (allele frequency: 0.56) than patients with no vascular disease (allele frequency, 0.42) (P=0.03). Sequence analysis revealed no exon sequence variants in the LPL gene but two intron sequence variants were found in intron 5 in two patients.

D-Alanine-D-Alanine Ligase Gene (ddl) of Erwinia chrysanthemi Strain EC16 I. Isolation and Gene Dosage Effect on Pectate Lyase Synthesis

New regulatory gene for pectate lyase (Pel) production of Erwinia chrysanthemi strain EC16 was searched by observing the gene dosage effect of each cosmid library in Pel production. From this survey, a cosmid clone, p5A, had reduced in Pel production under both inducing and non-inducing conditions and caused less tissue maceration of potato tubers. The 2.64-kb HindIII fragment from p5A was found to be responsible for this phenotype and to contain one major ORF consisting of 1107 nucleotides, which had homology with ddlA (49.6%) and ddlB (49.6%) of Escherichia coli and with ddlA (44.6%) of Salmonella typhimurium. These genes had been shown to encode D-alanine-D-alanine ligase (Ddl), an enzyme which is involved in the synthesis of peptidoglycan. This ORF encodes 368 amino acid residues and its molecular weight is estimated to be 43 kDa. This ORF of EC16 could complement ddl deficient mutant of Escherichia coli.

Two dnaB genes are associated with the origin of replication of pQBR55, an exogenously isolated plasmid from the rhizosphere of sugar beet

Plasmid pQBR55 (∼149 kb) represents one of five (Groups I–V) genetically distinct transfer proficient, mercury resistance plasmid groups that have been observed in the phytosphere pseudomonad community at a single geographic location in Oxford, UK. A 4.9-kb HindIII fragment was cloned from pQBR55 (a Group III plasmid) that facilitates autonomous replication of a narrow host range cloning vector, pKIL29, in the non-permissive host Pseudomonas putida UWC1. Sequencing revealed that the fragment contains a unique tandem array of dnaB genes (one partial and one complete), a homologue of the traA gene of plasmid RP4 and several potential open reading frames with little homology to any known sequences. The fragment also contains: a short region of direct and indirect repeats, two sequences that resemble the consensus Escherichia coli DnaA box motif, an A+T rich region and evidence of a GC-skew inversion. All features associated with origins of replication. Two specific oligonucleotide primer pairs, one targeted at the tandem dnaB genes and the other at the dnaB–traA arrangement were used for PCR analysis of other plasmids isolated from the same field site. PCR generated amplification products were only amplified from Group III plasmids (defined by RFLP typing) but not from plasmids belonging to Group I, II, IV or V. The nucleotide sequences of the amplified fragments were identical to those of pQBR55, even though the other Group III plasmids had distinct RFLP patterns. The results of a more general PCR screen, using primers to amplify a 389-bp fragment of all described pseudomonad dnaB genes, suggests that closely related dnaB sequences are only associated with Group III plasmids.

Two dnaB genes are associated with the origin of replication of pQBR55, an exogenously isolated plasmid from the rhizosphere of sugar beet

Plasmid pQBR55 ( approximately 149 kb) represents one of five (Groups I-V) genetically distinct transfer proficient, mercury resistance plasmid groups that have been observed in the phytosphere pseudomonad community at a single geographic location in Oxford, UK. A 4.9-kb HindIII fragment was cloned from pQBR55 (a Group III plasmid) that facilitates autonomous replication of a narrow host range cloning vector, pKIL29, in the non-permissive host Pseudomonas putida UWC1. Sequencing revealed that the fragment contains a unique tandem array of dnaB genes (one partial and one complete), a homologue of the traA gene of plasmid RP4 and several potential open reading frames with little homology to any known sequences. The fragment also contains: a short region of direct and indirect repeats, two sequences that resemble the consensus Escherichia coli DnaA box motif, an A+T rich region and evidence of a GC-skew inversion. All features associated with origins of replication. Two specific oligonucleotide primer pairs, one targeted at the tandem dnaB genes and the other at the dnaB-traA arrangement were used for PCR analysis of other plasmids isolated from the same field site. PCR generated amplification products were only amplified from Group III plasmids (defined by RFLP typing) but not from plasmids belonging to Group I, II, IV or V. The nucleotide sequences of the amplified fragments were identical to those of pQBR55, even though the other Group III plasmids had distinct RFLP patterns. The results of a more general PCR screen, using primers to amplify a 389-bp fragment of all described pseudomonad dnaB genes, suggests that closely related dnaB sequences are only associated with Group III plasmids.

Genetic characterization of the fusidic acid and cadmium resistance determinants of Staphylococcus aureus plasmid pUB101.

We report the cloning of the fusidic acid and cadmium resistance determinants from Staphylococcus aureus plasmid pUB101. The pUB101 fusidic acid resistance determinant was located on a 2.9 kb HindIII fragment. Sequencing of this fragment revealed three putative open reading frames (ORFs) of 213 (far1), 152 (orf152) and 170 amino acids (orf170), which are flanked by the right-hand end of insertion sequence IS431/257 (IS431/257RH) and a partial ORF. Far1 and Orf152 demonstrated homology with a chromosomally encoded fibronectin-binding protein of Listeria monocytogenes and the putative protein YosT, found on the SP beta c2 prophage of Bacillus subtilis, respectively. Transformation of S. aureus with a construct containing a 949 bp far1-specific amplicon led to the isolation of a fusidic acid-resistant transformant, thereby identifying the pUB101 fusidic acid resistance structural gene. Between orf152 and far1 we identified a unique 113 bp symmetrical element and other repeat elements that may be involved with the control of orf152 and/or far1 expression. Hybridization of Southern blots revealed that far1 was not located on the chromosome or plasmid content of a limited number of Australian, UK and Hong Kong fusidic acid-resistant isolates. The pUB101 cadmium resistance determinant was located on a 3.6 kb HindIII fragment that carried a cadDX operon, remnants of two putative plasmid replication protein genes and IS431/257RH. Sequence analysis also demonstrated the presence of a single-stranded origin of replication, normally found on rolling circle replicating plasmids, within the putative promoter region of the cadDX operon.

Assembly of Photosystem I: I. INACTIVATION OF THE rubA GENE ENCODING A MEMBRANE-ASSOCIATED RUBREDOXIN IN THE CYANOBACTERIUM SYNECHOCOCCUS SP. PCC 7002 CAUSES A LOSS OF PHOTOSYSTEM I ACTIVITY

A 4.4-kb HindIII fragment, encoding an unusual rubredoxin (denoted RubA), a homolog of the Synechocystis sp. PCC 6803 gene slr2034 and Arabidopsis thaliana HCF136, and the psbEFLJ operon, was cloned from the cyanobacterium Synechococcus sp. PCC 7002. Inactivation of the slr2034 homolog produced a mutant with no detectable phenotype and wild-type photosystem (PS) II levels. Inactivation of the rubA gene of Synechococcus sp. PCC 7002 produced a mutant unable to grow photoautotrophically. RubA and PS I electron transport activity were completely absent in the mutant, although PS II activity was approximately 80% of the wild-type level. RubA contains a domain of approximately 50 amino acids with very high similarity to the rubredoxins of anaerobic bacteria and archaea, but it also contains a region of about 50 amino acids that is predicted to form a flexible hinge and a transmembrane alpha-helix at its C terminus. Overproduction of the water-soluble rubredoxin domain in Escherichia coli led to a product with the absorption and EPR spectra of typical rubredoxins. RubA was present in thylakoid but not plasma membranes of cyanobacteria and in chloroplast thylakoids isolated from spinach and Chlamydomonas reinhardtii. Fractionation studies suggest that RubA might transiently associate with PS I monomers, but no evidence for an association with PS I trimers or PS II was observed. PS I levels were significantly lower than in the wild type ( approximately 40%), but trimeric PS I complexes could be isolated from the rubA mutant. These PS I complexes completely lacked the stromal subunits PsaC, PsaD, and PsaE but contained all membrane-intrinsic subunits. The three missing proteins could be detected immunologically in whole cells, but their levels were greatly reduced, and degradation products were also detected. Our results indicate that RubA plays a specific role in the biogenesis of PS I.

Inhibition of Histone Deacetylases Alters Allelic Chromatin Conformation at the Imprinted U2af1-rs1 Locus in Mouse Embryonic Stem Cells

Most loci that are regulated by genomic imprinting have differentially methylated regions (DMRs). Previously, we showed that the DMRs of the mouse Snrpn and U2af1-rs1 genes have paternal allele-specific patterns of acetylation on histones H3 and H4. To investigate the maintenance of acetylation at these DMRs, we performed chromatin immunoprecipitation on trichostatin-A (TSA)-treated and control cells. In embryonic stem (ES) cells and fibroblasts, brief (6-h) TSA treatment induces global hyperacetylation of H3 and H4. In ES cells only, TSA led to a selective increase in maternal acetylation at U2af1-rs1, at lysine 5 of H4 and at lysine 14 of H3. TSA treatment of ES cells did not affect DNA methylation or expression of U2af1-rs1, but was sufficient to increase DNase I sensitivity along the maternal allele to a level comparable with that of the paternal allele. In fibroblasts, TSA did not alter U2af1-rs1 acetylation, and the parental alleles retained their differential DNase I sensitivity. At Snrpn, no changes in acetylation were observed in the TSA-treated cells. Our data suggest that the mechanisms regulating histone acetylation at DMRs are locus and developmental stage-specific and are distinct from those effecting global levels of acetylation. Furthermore, it seems that the allelic U2af1-rs1 acetylation determines DNase I sensitivity/chromatin conformation.

Molecular characteristics of glnA linked mutations in the nitrogen-fixing cyanobacterium Nostoc muscorum.

The glutamine synthetase (GS)-impaired mutants of Nostoc muscorum, isolated as ethylenediamine (EDA) resistant phenotype, were characterized for glnA linked processes to investigate the molecular mechanism of GS impairment. The GS enzyme was purified to homogeneity from the wild and mutant strains of N. muscorum employing ion-exchange followed by affinity chromatography. The final yield and specific activity of the enzyme were low in the mutant strains as compared to the wild type. The molecular weight of the native enzyme and the subunit size of GS polypeptide in all the strains were identical. Compared to the wild type, the GS purified from the mutant strains showed refractory response to its structural analogues like methionine sulfone (MSO), methionine sulfoximine (MSX), EDA, and metabolic substrates, such as ammonium and glutamine. The Km and Vmax for the substrates of transferase and biosynthetic reactions were altered in the mutant strains. The immunological assays performed with partially purified IgG molecules raised against wild-type GS revealed quantitative and qualitative heterogeneity for cross reactivity among the wild-type and EDA-resistant strains. Southern hybridization of genomic DNA in all the strains of N. muscorum restricted with different enzymes showed identical pattern with Anabaena 7120 glnA probe and localization of complete gene for GS on 7.5 kb HindIII fragment. Northern blot analysis indicated differential levels of GS specific mRNA and abundance of glnA transcripts of 1.7 and 1.9 kb in the mutant strains. Results suggested that point mutation(s) inflicted in glnA gene of N. muscorum are responsible for differential production of catalytically modified GS in EDA-resistant phenotypes.

Alterations of thyroid hormone receptor α gene: frequency and association with Nm23 protein expression and metastasis in gastric cancer

Thyroid hormone receptor genes ( c-erbA, or TR) have been implicated as tumor suppressor genes. We investigated whether two subtypes of TR genes ( TRα and TRβ) are altered in human gastric cancer, as well as the role of such alterations in tumor metastasis. As a preliminary survey ( n=18), Southern blot analysis was conducted with full-length TRα or TRβ cDNA probes, after digestion with restriction enzyme ( EcoRI, BamHI, or HindIII). The survey showed the most frequent alteration was in TRα gene by a loss of two HindIII fragments (7.1 and 10.1 kb). Subsequent study focused at this TRα alteration by increasing patient number up to a total of 42. TRα alteration was noted in 19 (49%) of the 39 patients with informative results. The TRα alteration was associated with the development of distant (hematogenous) metastasis ( P=0.0084) and high expression level of Nm23 protein ( P=0.020). These findings suggest that TRα might be involved in tumorigenesis, and that interactions between the TRα and nm23 genes might take part in hematogenous metastasis of gastric cancer.

Molecular cloning of a new crystal protein gene cry1Af1 of Bacillus thuringiensis NT0423 from Korean sericultural farms.

A new cry1Ab-type gene encoding the 130 kDa protein of Bacillus thuringiensis NT0423 bipyramidal crystals was cloned, sequenced, and expressed in a crystal-negative B. thuringiensis host. Hybridization experiments revealed that the crystal protein gene is located on a 44 MDa plasmid of B. thuringiensis NT0423. A strong positive signal detected on the 6.6 kb HindIII fragment from B. thuringiensis NT0423 plasmid DNA was cloned and sequenced. The cry1Ab-type gene, designated cry1Af1, consisted of open reading frame of 3453 bp, encoding a protein of 1151 amino acid residues. The polypeptide has the deduced amino acid sequences predicting molecular masses of 130,215 Da. With both Bt I and Br II promoter sequences were found, the B. thuringiensis NT0423 crystal protein gene promoter closely aligned with those of cry1A-type crystal protein gene. When compared with known sequences of other Cry and Cyt proteins, the Cry1Af1 protein showed maximum 93% sequence identity to Cry1Ab protein of B. thuringiensis subsp. kurstaki. The expressed Cry1Af1 protein in a crystal-negative B. thuringiensis host appears to have strong insecticidal activity against lepidopteran larvae (Plutella xylostella). Crystals containing Cry1Af1 were about six times more toxic than the wild-type crystals of B. thuringiensis NT0423.

Isolation of ftsI and murE genes involved in peptidoglycan synthesis from Corynebacterium glutamicum.

Corynebacterium glutamicum is known to excrete large amounts of L-glutamic acid upon treatment by penicillin. However, the mechanism of L-glutamate overproduction by penicillin treatment is still unknown. A 5.3-kb HindIII fragment was isolated by directly introducing the C. glutamicum (Brevibacterium lactofermentum) ATCC 13869 gene library into the temperature-sensitive Escherichia coli murE mutant and selecting temperature resistant clones. Two open reading frames (ORFs) were found in this fragment: (1) murE, encoding UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase, and (2)ftsI, encoding septum-peptidoglycan synthetase, one of the targets of penicillin (penicillin-binding protein 3). Both ORFs were involved in peptidoglycan synthesis. Proteins were synthesized from the C. glutamicum murE and ftsI genes, 55 kDa and 73 kDa respectively, in an in vitro protein synthesis system, using E. coli S30 extracts.

Yeast Mps1p Phosphorylates the Spindle Pole Component Spc110p in the N-terminal Domain

The yeast spindle pole body (SPB) component Spc110p (Nuf1p) undergoes specific serine/threonine phosphorylation as the mitotic spindle apparatus forms, and this phosphorylation persists until cells enter anaphase. We demonstrate that the dual-specificity kinase Mps1p is essential for the mitosis-specific phosphorylation of Spc110p in vivo and that Mps1p phosphorylates Spc110p in vitro. Phosphopeptides generated by proteolytic cleavage were identified and sequenced by mass spectrometry. Ser(60), Thr(64), and Thr(68) are the major sites in Spc110p phosphorylated by Mps1p in vitro, and alanine substitution at these sites abolishes the mitosis-specific isoform in vivo. This is the first time that phosphorylation sites of an SPB component have been determined, and these are the first sites of Mps1p phosphorylation identified. Alanine substitution for any one of these phosphorylated residues, in conjunction with an alanine substitution at residue Ser(36), is lethal in combination with alleles of SPC97, which encodes a component of the Tub4p complex. Consistent with a specific dysfunction for the alanine substitution mutations, simultaneous mutation of all four serine/threonine residues to aspartate does not confer any defect. Sites of Mps1p phosphorylation and Ser(36) are located within the N-terminal globular domain of Spc110p, which resides at the inner plaque of the SPB and binds the Tub4p complex.

A primordial tRNA modification required for the evolution of life?

The evolution of reading frame maintenance must have been an early event, and presumably preceded the emergence of the three domains Archaea, Bacteria and Eukarya. Features evolved early in reading frame maintenance may still exist in present-day organisms. We show that one such feature may be the modified nucleoside 1-methylguanosine (m(1)G37), which prevents frameshifting and is present adjacent to and 3' of the anticodon (position 37) in the same subset of tRNAs from all organisms, including that with the smallest sequenced genome (Mycoplasma genitalium), and organelles. We have identified the genes encoding the enzyme tRNA(m(1)G37)methyltransferase from all three domains. We also show that they are orthologues, and suggest that they originated from a primordial gene. Lack of m(1)G37 severely impairs the growth of a bacterium and a eukaryote to a similar degree. Yeast tRNA(m(1)G37)methyltransferase also synthesizes 1-methylinosine and participates in the formation of the Y-base (yW). Our results suggest that m(1)G37 existed in tRNA before the divergence of the three domains, and that a tRNA(m(1)G37)methyltrans ferase is part of the minimal set of gene products required for life.

Identification and phylogeny of a non-specific endonuclease gene of white spot syndrome virus of shrimp.

White spot syndrome virus (WSSV) is a taxonomically unclassified virus which causes a disease in shrimps worldwide. A 936 bp long open reading frame (ORF) was found on a 7.2 kb HindIII fragment of the DNA genome of WSSV located adjacent to the ribonucleotide reductase small subunit gene. This putative ORF showed homology to prokaryotic and eukaryotic endonucleases, which contain a non-specific endonuclease motif. Alignment with viral and eukaryotic endonuclease ORFs revealed that most catalytically and structurally important amino acid residues were present in the putative WSSV non-specific endonuclease gene. An unrooted parsimonous phylogenetic tree of non-specific endonucleases indicated that the WSSV ORF was located in a well bootstrap supported clade containing only arthopods, including one of WSSV's natural hosts, Penaeus japonicus. A similar conjunction was found for the only other viral homologue, present in Fowlpox virus, which was also found in a well bootstrap-supported clade with its natural host, Gallus gallus. This clustering of virus and host suggests that both WSSV and Fowlpox virus may have acquired their nuclease genes from their respective natural hosts. Because the motif for non-specific nucleases is found in only two viruses, this gene cannot be used to clarify the taxonomic position of WSSV. However, the presence of this type of nuclease rarely found in viruses adds a novel feature to WSSV.

Transposon-mediated insertional mutagenesis of the D-alanyl-lipoteichoic acid ( dlt) operon raises methicillin resistance in Staphylococcus aureus

Two independent mutants of methicillin-resistant Staphylococcus aureus (MRSA), KAN96H1 and KAN96H2, were isolated by insertional mutagenesis with conjugative transposon Tn 918. In both, the minimal inhibitory concentration (MIC) of methicillin was increased to 128 compared to 16 mg/L for the parental strain KAN96. By transduction experiments, we verified that the insertion of Tn 918 conferred higher methicillin resistance on KAN96H1, but not on KAN96H2. In KAN96H1, the integration site of Tn 918 was located in the 6.1-kb HindIII fragment of the KAN96 chromosomal DNA. We identified a novel D-alanyl-lipoteichoic acid ( dlt) operon of S. aureus in this fragment. The amino acid sequences of four open reading frames of this operon were highly homologous to those of the dlt operon genes of Bacillus subtilis. The nucleotide sequence of the staphylococcal dlt operon is under the accession number D86240 in the DDBJ/GenBank/EMBL databases. In KAN96H1, Tn 918 was inserted in the 5'-terminal region of the putative dltB gene which encoded a hypothetical membrane transporter. dlt transcripts of 4.7 kb were detected in KAN96, but were truncated to 2.3 kb in KAN96H1. No corresponding transcripts were observed in KAN96H2. Our results clearly demonstrated that defects in functions of the putative dlt operon resulted in increased methicillin resistance in MRSA.

Architecture of the replication fork stalled at the 3' end of yeast ribosomal genes.

Every unit of the rRNA gene cluster of Saccharomyces cerevisiae contains a unique site, termed the replication fork barrier (RFB), where progressing replication forks are stalled in a polar manner. In this work, we determined the positions of the nascent strands at the RFB at nucleotide resolution. Within an HpaI-HindIII fragment essential for the RFB, a major and two closely spaced minor arrest sites were found. In the majority of molecules, the stalled lagging strand was completely processed and the discontinuously synthesized nascent lagging strand was extended three bases farther than the continuously synthesized leading strand. A model explaining these findings is presented. Our analysis included for the first time the use of T4 endonuclease VII, an enzyme recognizing branched DNA molecules. This enzyme cleaved predominantly in the newly synthesized homologous arms, thereby specifically releasing the leading arm.

Phage-related DNA polymorphism in dairy and probiotic Lactobacillus

Various DNA-based methods are presently being applied for identification of industrial bacterial cultures including dairy starter and probiotic strains of Lactobacillus. The success of strain-specific identification depends on the power of the DNA-based methods to reveal intraspecies DNA polymorphism. This study reveals that all eleven arbitrarily chosen Lactobacillus rhamnosus starter, laboratory and probiotic strains contain Lb. rhamnosus phage Lc-Nu related nucleotide sequences. One of these highly homologous regions in the genome of phage Lc-Nu was the 2.4kb HindIII fragment, which has been sequenced. Nucleotide sequence analysis suggested that one side of the 2.4kb HindIII fragment encodes a phage Lc-Nu helicase and accordingly represents an early gene region of phage Lc-Nu genome. Five forward and five reverse primers were derived from the nucleotide sequence of the 2.4kb HindIII fragment of phage Lc-Nu DNA for PCR-based identification of the eleven Lb. rhamnosus strains included in this study. Six different types of PCR product patterns were obtained. Among the patterns three were unique to particular Lb. rhamnosus strains. The results suggest that phage-related DNA sequences are, surprisingly, distributed widely among the Lb. rhamnosus strains, and that these sequences could also be a source of DNA polymorphism to apply for DNA-based identification of bacterial strains. Phage Lc-Nu related DNA homology was also found in the chromosome of Lb. casei, the species closely related to Lb. rhamnosus.

Restriction Fragment Length Polymorphism (RFLP) for protein disulfide isomerase (PDI) gene sequences in Triticum and Aegilops species

RFLP variation revealed by protein disulfide isomerase (PDI) coding gene sequences was assessed in 170 accessions belonging to 23 species of Triticum and Aegilops. PDI restriction fragments were highly conserved within each species and confirmed that plant PDI is encoded either by single-copy sequences or by small gene families. The wheat PDI probe hybridized to single EcoRI or HindIII fragments in different diploid species and to one or two fragments per genome in polyploids. Four Aegilops species in the Sitopsis section showed complex patterns and high levels of intraspecific variation, whereas Ae. searsii possessed single monomorphic fragments. T. urartu and Ae. squarrosa showed fragments with the same mobility as those in the A and D genomes of Triticum polyploid species, respectively, whereas differences were observed between the hybridization patterns of T. monococcum and T. boeoticum and that of the A genome. The single fragment detected in Ae. squarrosa was also conserved in most accessions of polyploid Aegilops species carrying the D genome. The five species of the Sitopsis section showed variation for the PDI hybridization fragments and differed from those of the B and G genomes of emmer and timopheevi groups of wheat, although one of the Ae. speltoides EcoRI fragments was similar to those located on the 4B and 4G chromosomes. The similarity between the EcoRI fragment located on the 1B chromosome of common and emmer wheats and one with a lower hybridization intensity in Ae. longissima, Ae. bicornis and Ae. sharonensis support the hypothesis of a polyphyletic origin of the B genome.

Purification of Dual-Tagged Intact Recombinant Proteins

Large-scale purification of recombinant proteins has been used extensively to assist numerous protein studies, including investigation of function, substrate identification and protein–protein interaction of low abundance proteins. Genetic fusion of affinity tags to these proteins has also been widely used for ease of purification by affinity chromatography. However, this technique sometimes yields unstable and degraded protein products limiting its application. In this study, we show a facile and straightforward method of dual-tagged recombinant protein purification that eliminates contamination by degraded protein products. A 6His-containing BamHI–HindIII fragment from pQE12 was ligated into the pGEX-KG BamHI–HindIII fragment and the protein of interest (p25nck5a, which is highly susceptible to proteolytic degradation when expressed and purified from bacteria) was cloned into the BamHI site without a termination codon. The resulting plasmid construct, designated as pGST-p25nck5a-6His, with GST at the N-terminal and 6His at the C-terminal was expressed in Escherichia coli DH5α and purified using a two-step procedure. We show that using Ni2+-NTA chromatography as a first purification step and GSH-agarose chromatography as a second step, rather than vice-versa, yields a highly purified intact protein that is free of any contaminating degraded protein product. The purified fusion protein is soluble and fully active.

Vibrio vulnificus has the transmembrane transcription activator ToxRS stimulating the expression of the hemolysin gene vvhA.

In an attempt to dissect the virulence regulatory mechanism in Vibrio vulnificus, we tried to identify the V. cholerae transmembrane virulence regulator toxRS (toxRS(Vc)) homologs in V. vulnificus. By comparing the sequences of toxRS of V. cholerae and V. parahaemolyticus (toxRS(Vp)), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kb BglII-HindIII fragment and a 1.2-kb HindIII fragment containing two complete open reading frames and one partial open reading frame attributable to toxR(Vv), toxS(Vv), and htpG(Vv) were cloned. ToxR(Vv) shared 55.0 and 63.0% sequence homology with ToxR(Vc) and ToxR(Vp), respectively. ToxS(Vv) was 71.5 and 65.7% homologous to ToxS(Vc) and ToxS(Vp), respectively. The amino acid sequences of ToxRS(Vv) showed transmembrane and activity domains similar to those observed in ToxRS(Vc) and ToxRS(Vp). Western blot analysis proved the expression of ToxR(Vv) in V. vulnificus. ToxRS(Vv) enhanced, in an Escherichia coli background, the expression of the V. vulnificus hemolysin gene (vvhA) fivefold. ToxRS(Vv) also activated the ToxR(Vc)-regulated ctx promoter incorporated into an E. coli chromosome. A toxR(Vv) null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. The toxR(Vv) mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxR(Vv) may regulate the virulence expression of V. vulnificus.