Identification and Characterization of a Plasmid in Strain Aeronomas hydrophila IBRB-36 4CPA Carrying Genes for Catabolism of Chlorophenoxyacetic Acids

Results of studying plasmid pAH36 in strain Aeronomas hydrophila IBRB-36 4CPA are presented. Plasmid pAH36 possesses BamHI, PstI, and HindIII restriction sites and is 5.4 kb in size. The plasmid was shown to contain genes for catabolism of chlor-substituted phenoxyacetic acids.     

Naturally occurring lactococcal plasmid pAH90 links bacteriophage resistance and mobility functions to a food-grade selectable marker

The bacteriophage resistance plasmid pAH90 (26,490 bp) is a natural cointegrate plasmid formed via homologous recombination between the type I restriction-modification specificity determinants (hsdS) of two smaller lactococcal plasmids, pAH33 (6,159 bp) and pAH82 (20,331 bp), giving rise to a bacteriophage-insensitive mutant following phage challenge (D. O'Sullivan, D. P. Twomey, A. Coffey, C. Hill, G. F. Fitzgerald, and R. P. Ross, Mol. Microbiol. 36:866-876; 2000). In this communication we provide evidence that the recombination event is favored by phage infection. The entire nucleotide sequence of plasmid pAH90 was determined and found to contain 24 open reading frames (ORFs) responsible for phenotypes which include restriction-modification, phage adsorption inhibition, plasmid replication, cadmium resistance, cobalt transport, and conjugative mobilization. The cadmium resistance property, encoded by the cadA gene, which has an associated regulatory gene (cadC), is of particular interest, as it facilitated the selection of pAH90 in other phage-sensitive lactococci after electroporation. In addition, we report the identification of a group II self-splicing intron bounded by two exons which have the capacity to encode a relaxase implicated in conjugation in gram-positive bacteria. The functionality of this intron was evident by demonstrating splicing in vivo. Given that pAH90 encodes potent phage defense systems which act at different stages in the phage lytic cycle, the linkage of these with a food-grade selectable marker on a replicon that can be mobilized among lactococci has significant potential for natural strain improvement for industrial dairy fermentations which are susceptible to phage inhibition.     

Plasmid involvement in the formation of a spontaneous bacteriophage insensitive mutant of Lactococcus lactis

Lactococcus lactis subsp. lactis biovar. diacetylactis DPC721 is a spontaneous bacteriophage insensitive mutant of strain DPC220, isolated after challenge with an industrial bacteriophage, phi D1. Plasmid analysis demonstrated that the bacteriophage insensitivity was associated with the absence of two native DPC220 plasmids (pAH82 and pAH33), and the presence of a novel plasmid (pAH90) in DPC721. The plasmids were transferred by conjugative mobilization to a plasmid free background where it was confirmed by restriction mapping that pAH90 is a co-integrate formed by the precise recombination of pAH82 and pAH33. The resistance phenotype encoded by pAH90 was also active against two bacteriophage homologous for the plasmid-free strain. Plasmid pAH90 was shown to encode at least two independent resistance mechanisms, including an adsorption-inhibition mechanism and a restriction and modification system. The adsorption-inhibition mechanism encoded by the co-integrate plasmid was specific for one of the phage used in this study.     

Novel Plasmids from Alkaliphilic Halomonads

Seventeen alkaliphilic halomonads were examined for the presence of plasmids. Of these, eight strains harbored one or more from 5.3 to 33 kb in size, the first plasmids to be identified from an alkaliphilic halomonad source. Restriction and hybridization analysis revealed three strains that maintained an identical 5.9-kb plasmid which we named pAH1, two that had an identical 33-kb plasmid, and three others, of which one carried two plasmids of 5.3 and 15 kb, the former being designated pAH2. The two final strains maintained plasmids of 15 and 20.5 kb. Restriction mapping of both pAH1 and pAH2 indicated that they have a number of unique restriction sites and are of a small enough size to make them suitable for vector construction.     

Novel archaeal plasmid pAH1 and its interactions with the lipothrixvirus AFV1

At present very little is known about interactions between extrachromosomal genetic elements in Archaea . Here we describe an Acidianus strain which carries naturally a novel 28 kb conjugative plasmid-like element, pAH1, and also serves as a laboratory host for lipothrixvirus AFV1. In an attempt to establish a system for studying plasmid–virus interactions we characterized the genome of pAH1 which closely resembles those of the Sulfolobus conjugative plasmids pARN3 and pARN4. pAH1 integrates site specifically into, and excises from, the host chromosome indicating a dynamic interaction with the latter. Although nucleotide sequence comparisons revealed extensive intergenomic exchange during the evolution of archaeal conjugative plasmids, pAH1 was shown to be stably maintained suggesting that the host system is suitable for studying plasmid–virus interactions. AFV1 infection and propagation leads to a loss of the circular form of pAH1 and this effect correlates positively with the increase in the intracellular quantity of AFV1 DNA. We infer that the virus inhibits plasmid replication since no pAH1 degradation was observed. This mechanism of archaeal viral inhibition of plasmid propagation is not observed in bacteria where relevant bacteriophages either are dependent on a conjugative plasmid for successful infection or are excluded by a resident plasmid.     

Naturally Occurring Lactococcal Plasmid pAH90 Links Bacteriophage Resistance and Mobility Functions to a Food-Grade Selectable Marker

The bacteriophage resistance plasmid pAH90 (26,490 bp) is a natural cointegrate plasmid formed via homologous recombination between the type I restriction-modification specificity determinants (hsdS) of two smaller lactococcal plasmids, pAH33 (6,159 bp) and pAH82 (20,331 bp), giving rise to a bacteriophage-insensitive mutant following phage challenge (D. O'Sullivan, D. P. Twomey, A. Coffey, C. Hill, G. F. Fitzgerald, and R. P. Ross, Mol. Microbiol. 36:866–876; 2000). In this communication we provide evidence that the recombination event is favored by phage infection. The entire nucleotide sequence of plasmid pAH90 was determined and found to contain 24 open reading frames (ORFs) responsible for phenotypes which include restriction-modification, phage adsorption inhibition, plasmid replication, cadmium resistance, cobalt transport, and conjugative mobilization. The cadmium resistance property, encoded by the cadA gene, which has an associated regulatory gene (cadC), is of particular interest, as it facilitated the selection of pAH90 in other phage-sensitive lactococci after electroporation. In addition, we report the identification of a group II self-splicing intron bounded by two exons which have the capacity to encode a relaxase implicated in conjugation in gram-positive bacteria. The functionality of this intron was evident by demonstrating splicing in vivo. Given that pAH90 encodes potent phage defense systems which act at different stages in the phage lytic cycle, the linkage of these with a food-grade selectable marker on a replicon that can be mobilized among lactococci has significant potential for natural strain improvement for industrial dairy fermentations which are susceptible to phage inhibition.     

Novel type I restriction specificities through domain shuffling of HsdS subunits in Lactococcus lactis

This study identifies a natural system in Lactococcus lactis, in which a restriction modification specificity subunit resident on a 6159 bp plasmid (pAH33) alters the specificity of a functional R/M mechanism encoded by a 20.3 kb plasmid, pAH82. The new specificity was identified after phenotypic and molecular analysis of a 26.5 kb co-integrate plasmid (pAH90), which was detected after bacteriophage challenge of the parent strain. Analysis of the regions involved in the co-integration revealed that two novel hybrid hsdS genes had been formed during the co-integration event. The HsdS chimeras had interchanged the C- and N-terminal variable domains of the parent subunits, generating two new restriction specificities. Comparison of the parent hsdS genes with other type I specificity determinants revealed that the region of the hsdS genes responsible for the co-integration event is highly conserved among lactococcal type I hsdS determinants. Thus, as hsdS determinants are widespread in the genus Lactococcus, new restriction specificities may evolve rapidly after homologous recombination between these genes. This study demonstrates that, similar to previous observations in Gram-negative bacteria, a Gram-positive bacterium can acquire novel restriction specificities naturally through domain shuffling of resident HsdS subunits.     

A Host–Vector System for a Cellulose-Producing Acetobacter Strain

An indigenous plasmid, named pAH4, was detected in a cellulose-producing Acetobacter strain. This plasmid, consisting of 4002 bp, contained an AT-rich region and encoded several open reading frames, as deduced by the complete nucleotide sequence. One of the putative open reading frames showed homology with replication proteins of other plasmids. A shuttle vector of Escherichia coli and this strain was constructed by connecting pAH4 to pUC18. Electroporation of the shuttle vector into the strain yielded 1.7 × 10⁵ ampicillin resistant transformants per μg DNA. The shuttle plasmid was very stably maintained in the strain.     

The use of cadmium resistance on the phage-resistance plasmid pNP40 facilitates selection for its horizontal transfer to industrial dairy starter lactococci

AIMS: To facilitate the horizontal transfer and selection of phage-resistance plasmids in industrial lactococci. METHODS AND RESULTS: Cadmium-resistance properties similar to those previously identified in Lactococcus were linked to the well-known phage-resistance plasmid pNP40. This finding was exploited to facilitate delivery of the plasmid to an industrial cheese starter Lactococcus lactis DPC4268. Additionally, 25 different cadmium-sensitive cheese starter lactococci were also identified as potential recipients for the phage-resistance plasmid pNP40, and also the plasmids pAH90/pAH82 which also encode cadmium resistance. All three plasmids were successfully conjugated to strain DPC4268. Cheddar cheese was manufactured in industry with the pNP40 phage-resistant transconjugant. SIGNIFICANCE AND IMPACT OF THE STUDY: Food-grade enhancement of phage resistance in industrial starter strains has been made simpler by the use of this selection, especially since the majority of potential recipient starter strains analysed were cadmium sensitive.     

Characterization of rabbit liver cytochrome P-450 (laurate omega-1 hydroxylase) synthesized in transformed yeast cells

Three cDNAs for chimeras between cytochrome P-450s (pHP3 and pHP2-1) were constructed and inserted between the alcohol dehydrogenase promoter and terminator regions of the yeast expression vector pAAH5 to form expression plasmids, pAH3P2, pAH3E2, and pAH3A2. pAH3P2 contained the entire coding sequence of cytochrome P-450 (pHP2-1) except for the 3rd, the 8th, the 36th, and the 42nd residues of the total of 490 amino acids. Nucleotide sequences of pAH3P2 were replaced with those of cytochrome P-450 (pHP3) in the region coding for the NH2-terminal 210 and 262 amino acid residues to yield pAH3E2 and pAH3A2, respectively. The three expression plasmids were introduced into Saccharomyces cerevisiae AH22 cells and cytochrome P-450 s (3P2, 3E2, and 3A2) were purified from the microsomal fractions of the transformed yeast cells. In the oxidized state either of the cytochromes exhibited a low- and high-spin mixed-type spectrum of cytochrome P-450. The reduced CO complex of the cytochromes showed a Soret absorption maximum at 450 nm. When laurate or caprate was added to ferric cytochrome P-450 s (3P2 and 3E2), the spectrum was converted to that of the typical high-spin type, indicating the binding of the fatty acids to the substrate site of the cytochromes. On the other hand, the addition of the fatty acids to ferric cytochrome P-450 (3A2) induced no spectral change. Only chemicals having a carboxyl group caused such spectral conversion of cytochrome P-450 (3P2) among dodecyl compounds examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction of Environmental DNA Libraries in Escherichia coli and Screening for the Presence of Genes Conferring Utilization of 4-Hydroxybutyrate

Environmental DNA libraries from three different soil samples were constructed. The average insert size was 5 to 8 kb and the percentage of plasmids with inserts was approximately 80%. The recombinant Escherichia coli strains (approximately 930,000) were screened for 4-hydroxybutyrate utilization. Thirty-six positive E. coli clones were obtained during the initial screen, and five of them contained a recombinant plasmid (pAH1 to pAH5) which conferred a stable 4-hydroxybutyrate-positive phenotype. These E. coli clones were studied further. All five were able to grow with 4-hydroxybutyrate as sole carbon and energy source and exhibited 4-hydroxybutyrate dehydrogenase activity in crude extracts. Sequencing of pAH5 revealed a gene homologous to the gbd gene of Ralstonia eutropha, which encodes a 4-hydroxybutyrate dehydrogenase. Two other genes (orf1 and orf6) conferring utilization of 4-hydroxybutyrate were identified during subcloning and sequencing of the inserts of pAH1 and pAH3. The deduced orf1 gene product showed similarities to members of the DedA family of proteins. The sequence of the deduced orf6 gene product harbors the fingerprint pattern of enoyl-coenzyme A hydratases/isomerases. The other sequenced inserts of the plasmids recovered from the positive clones revealed no significant similarity to any other gene or gene product whose sequence is available in the National Center for Biotechnology Information databases.     

Construction of a neo fusion gene for expression in both prokaryotic and eukaryotic cells

A high-copy-number plasmid, pLink, was constructed to allow the direct selection in Escherichia coli of a neo fusion gene capable of conferring Geneticin (G418) resistance on mouse L cells. pLink was derived from pdMmtneo by insertion of a KpnI linker within the 5'-coding region of the neo gene. This created a minus-one frameshift mutation resulting in a translational termination within the N-terminal region of the protein. The Neo activity was restored by insertion into the modified neo gene of a piece of coding sequence derived from human HPRT cDNA. The resulting plasmid, pAH, was microinjected into mouse A9 cells and shown to confer resistance to G418.     

Transformation of the Acidophilic Heterotroph Acidiphilium facilis by Electroporation

We constructed a cloning vector for use in the acidophilic heterotroph Acidiphilium facilis. The vector pAH1O1 (8.8kb) was constructed from a 6.1 kb restriction fragment of the Acidiphilium plasmid pAU1 and a pUC19 carrying a β-lactamase gene. The antibiotic resistance gene was efficiently expressed in A. facilis. Several factors which influenced the transformation efficiency were optimized, resulting in a transformation efficiency of up to 3 X 10³ transformants per μg of plasmid DNA at a field strength of 10kV/cm with a 7.0ms pulse.     

Production of D-p-hydroxyphenylglycine by N-carbamoyl-D-amino acid amidohydrolase-overproducing Escherichia coli strains.

The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene from Agrobacterium radiobacter NRRL B11291 has been successfully cloned and expressed in Escherichia coli. Subcloning of the D-carbamoylase gene into different types of vectors and backgrounds of E. coli strains showed that the optimal expression level of D-carbamoylase was achieved in a ColE1-derived plasmid with a 150-fold increase in specific enzyme activity compared to that in a pSC101-derived plasmid. In addition, the recombinant plasmids were very stable in the E. coli strain ATCC11303 but not in JCL1258 tested here. Employing the recombinant E. coli strain DH5alpha/pAH61 for D-p-hydroxyphenylglycine production showed that the cell was capable of transforming N-carbamoyl-D-hydroxylphenylglycine to D-p-hydroxyphenylglycine with a molar conversion yield of 100% and a production rate of 1.9 g/(L h). In comparison with A. radiobacter NRRL B11291, this productivity approximates a 55-fold increase in D-hydroxyphenylglycine production. This result suggests the potential application of recombinant E. coli strains for the transformation reaction.     

Conjugative transfer of the Lactococcus lactis chromosomal sex factor promotes dissemination of the Ll.LtrB group II intron

The Ll.LtrB group II intron from the low-G+C gram-positive bacterium Lactococcus lactis was the first bacterial group II intron shown to splice and mobilize in vivo. This retroelement interrupts the relaxase gene (ltrB) of three L. lactis conjugative elements: plasmids pRS01 and pAH90 and the chromosomal sex factor. Conjugative transfer of a plasmid harboring a segment of the pRS01 conjugative plasmid including the Ll.LtrB intron allows dissemination of Ll.LtrB among L. lactis strains and lateral transfer of this retroelement from L. lactis to Enterococcus faecalis. Here we report the dissemination of the Ll.LtrB group II intron among L. lactis strains following conjugative transfer of the native chromosomally embedded L. lactis sex factor. We demonstrated that Ll.LtrB dissemination is highly variable and often more efficient from this integrative and conjugative element than from an engineered conjugative plasmid. Cotransfer among L. lactis strains of both Ll.LtrB-containing elements, the conjugative plasmid and the sex factor, was detected and shown to be synergistic. Moreover, following their concurrent transfer, both mobilizable elements supported the spread of their respective copies of the Ll.LtrB intron. Our findings explain the unusually high efficiency of Ll.LtrB mobility observed following conjugation of intron-containing plasmids.     

三个方便实用的植物表达载体构建与验证

为满足植物功能基因组学研究及转基因安全性需要,本研究根据一些国内外引进或商业化的植物表达载体及其相关元件,构建了3个适合于植物,尤其是单子叶植物转化的表达载体,即p AH006、p WMB022和p WMB025。p AH006载体包含由玉米泛素ubi启动子调控的GUS基因和bar基因的完整T-DNA区域,此区段能够被酶切回收,可用于单子叶植物农杆菌介导转化效率评价及基因枪介导线状DNA转化效果研究;p WMB022载体携带由双35S启动子调控的玉米色素基因Lc和C1,可用作基因枪介导的共转化筛选标记,直观筛选含目标基因转基因材料;p WMB025载体携带由ubi启动子调控的、商业化转基因植物中广泛利用的EPSPS基因,可用于禾谷类作物农杆菌或基因枪介导的遗传转化,载体多克隆位点可通过酶切方式更换目标基因。酶切鉴定结合农杆菌或基因枪介导的小麦幼胚愈伤组织或叶片转化验证此3个载体表明,载体构建正确,其标记基因、可视化基因和报告基因均能正常表达。这3个载体的构建对于小麦等植物转化效率提升、安全型转基因作物获得和植物功能基因组学研究等具有重要意义。     

Vector-mediated overexpression of catalase A in the yeast Saccharomyces cerevisiae induces inclusion body formation.

To study the morphological effects of overexpression of catalase A in yeast, the gene coding for catalase A was introduced into Saccharomyces cerevisiae on a multicopy vector. After induction of microbody biogenesis and catalase A expression by growth on oleic acid as sole carbon source, cells were analyzed by immunofluorescence and immunoelectron microscopy. In addition, overexpression of catalase A was studied by quantitative immunoblotting and by activity measurement. Quantitative immunoblotting resulted in a 16-fold difference between immunoreactive material from transformed and non-transformed cells. An 18-fold increase of enzyme activity was measured in transformed cells due to overexpression of catalase A from plasmid pAH521. Immunofluorescent staining of semithin sections of Lowicryl HM20-embedded cells with anti-catalase localized peroxisomes and--at a low percentage--larger particles. By immunoelectron microscopy, these larger structures could be identified as agranular, electron-dense aggregates which are morphologically clearly distinct from the cytoplasm and not bounded by a membrane. These structures, which have been named inclusion bodies, contain catalase A but not other peroxisomal enzymes like thiolase. These findings suggest that cells are capable of compensating for overproduced proteins by formation of particular types of structures.     

1.5 kb mRNA abundantly expressed in rat tumors encodes a 37 kilodalton protein in vitro

A cDNA clone, pAH34, corresponding to a 1.5 kb mRNA present abundantly in various rat tumors was examined for its protein coding capacity. Hybridization-selected RNAs from both poly(A)+ RNAs of a rat ascites hepatoma cell line, AH60C and of normal liver produced a polypeptide of 37 kilo daltons in vitro, but at much higher levels in the AH60C than in the normal rat liver. Two dimensional electrophoresis of the translation product revealed that the pI of this protein was 7.1. Nucleotide sequence analysis of pAH34 showed that the insert of the clone consisted of 462bp and contained the 3' portion of mRNA, including poly(A) stretch with AATAAA signal sequence centered 16 nucleotides upstream, a short untranslated region and an open reading frame corresponding to possibly 67 amino acids of the C-terminal portion.