Broad Host Range Cloning Research Articles

Genetic tools for tagging Gram-negative bacteria with mCherry for visualization in vitro and in natural habitats, biofilm and pathogenicity studies

Live-cell imaging techniques are essential to gain a better understanding of microbial functioning in natural systems, for example in biofilms. Autofluorescent proteins, such as the green fluorescent protein (GFP) and the red fluorescent protein (DsRed), are valuable tools for studying microbial communities in their natural environment. Because of the functional limitations of DsRed such as slow maturation and low photostability, new and improved variants were created such as mCherry. In this study, we developed genetic tools for labeling Gram-negative bacteria in order to visualize them in vitro and in their natural environment without the necessity of antibiotic pressure for maintenance. mcherry was cloned into two broad host-range cloning vectors and a pBK-miniTn7 transposon under the constitutive expression of the tac promoter. The applicability of the different constructs was shown in Escherichia coli, various Pseudomonas spp. and Edwardsiella tarda. The expression of mcherry was qualitatively analyzed by fluorescence microscopy and quantified by fluorometry. The suitability of the constructs for visualizing microbial communities was shown for biofilms formed on glass and tomato roots. In addition, it is shown that mCherry in combination with GFP is a suitable marker for studying mixed microbial communities.

Improved Expression Vector for Brucella Species

We constructed an improved vector pNSGroE for gene expression stud-ies in Brucella spp. It is derived from the broad host range cloning vector pBBR1MCS (1). This new plasmid has several advantages over pBBR1MCS or its derivatives pBBGroE (2): (i) it is smaller in size, 2.9 kb; (ii) it expresses proteins as His-tagged fusion for easy detection and purification; (iii) it car-ries the groE promoter for constitutive expression that is enhanced under con-ditions of stress in vitro and in vivo; and (iv) it has higher levels of heterologous protein expression. Our expression studies in Brucella abortus strain RB51 indicated that the level of heterologous protein expression is higher with pNS-GroE compared to pBBGroE vector. We have also demonstrated the ability of the new vector to express heterolo-gous fusion proteins stably in Brucella species. The smaller size of the new vector was achieved by combining only the very essential elements for replica-tion and expression [minimum origin of replication, promoter, antibiotic selection marker, and multiple cloning sites (MCS)] and removing all nones-sential elements in the original vector pBBR1MCS or pBBGroE (lacZ gene, mobilization gene, and 1 kb upstream of the groE promoter). The vector was constructed by excision of a 185-bp fragment containing the MCS of pRSETA vector (Invitrogen, Carlsbad, CA, USA) using XbaI and HindIII. This fragment was cloned into XbaI and HindIII sites of pGEM11 vector (Promega, Madison, WI, USA) to form construct A. The groE promoter of Brucella spp. was amplified from the genomic DNA of B. abortus strain 2308 using Platinum

Construction of a Broad Host Range Cloning Vector Conferring Triclosan Resistance

Construction of a Broad Host Range Cloning Vector Conferring Triclosan Resistance

RSF1010-based shuttle vectors for cloning and expression in Pasteurella multocida

The broad host-range cloning vectors, pJRD215 and pMMB67EH, were evaluated for stability and cloning efficiency in Pasteurella multocida. Transformation of P. multocida by electroporation was unreliable and poorly efficient regardless of whether the transforming DNA was isolated from E. coli or P. multocida. Both vectors contain a mob site that enabled transfer by conjugation from E. coli to P. multocida with high efficiency. Kanamycin, streptomycin, and ampicillin resistance encoded by the vectors were expressed in P. multocida. LacZ was cloned in pMMB67EH, an expression vector, and was transferred to P. multocida by conjugation. The transconjugants expressed a functional β-galactosidase as determined by o-nitrophenyl-β- d-galactopyranoside (ONPG) test. We propose the use of these cosmid and expression vectors as a shuttle vectors for cloning in P. multocida.

Stability of broad host range cloning vectors in the phototrophic bacterium Rhodospirillum rubrum

The suitability of the broad-host range vectors pRK290, pVK100 and pLAFRI respectively for use with the photosynthetic purple non-sulphur bacterium, Rhodospirillum rubrum has been examined. All vectors are RK2 (RP4) derivatives (incompatibility group P-1) and contain the mob region of RK2. The three vectors could each be transferred from Escherichia coli by triparental mating in the presence of a second E. coli donor containing the helper plasmid pRK2013. The conjugation frequency was approx. 10 −1 in all cases. Selection of transconjugants was performed in the presence of tetracycline (pRK290, pLAFRI, pVK100) or kanamycin (pVK100). Southern-blotting experiments after many generations showed no rearrangements to have occurred. In the absence of antibiotic selection all three vectors were maintained for at least 15 generations under aerobic chemotrophic conditions but were rapidly lost when the culture was grown phototrophically (anaerobic).

Stability of broad host range cloning vectors in the phototrophic bacteriumRhodospirillum rubrum

The suitability of the broad-host range vectors pRK290, pVK100 and pLAFRI respectively for use with the photosynthetic purple non-sulphur bacterium, Rhodospirillum rubrum has been examined. All vectors are RK2 (RP4) derivatives (incompatibility group P-1) and contain the mob region of RK2. The three vectors could each be transferred from Escherichia coli by triparental mating in the presence of a second E. coli donor containing the helper plasmid pRK2013. The conjugation frequency was approx. 10−1 in all cases. Selection of transconjugants was performed in the presence of tetracycline (pRK290, pLAFRI, pVK100) or kanamycin (pVK100). Southern-blotting experiments after many generations showed no rearrangements to have occurred. In the absence of antibiotic selection all three vectors were maintained for at least 15 generations under aerobic chemotrophic conditions but were rapidly lost when the culture was grown phototrophically (anaerobic).

High-efficiency electro-transformation of the plant pathogen Pseudomonas syringae pv. syringae R32

Cells of Pseudomonas syringae pv. syringae R32 were for the first time transformed by electroporation with the BIO-RAD Gene Pulser. Two different types of plasmid, a broad host range cloning vector and a suicide vector for transposon insertion, were used. Different electroporation conditions were tested. Transformation frequencies between 1 × 10 5 and 4.6 × 10 6 per μg DNA could be obtained.

Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria.

A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.

A multipurpose broad host range cloning vector and its use to characterise an extracellular protease gene of Xanthomonas campestris pathovar campestris

A multipurpose broad host range plasmid, pIJ3200, was constructed by inserting the polylinker-containing 445 bp PvuII fragment of Bluescript M13 into the EcoRI site of the cosmid pLAFR1. Using this vector a protease gene of Xanthomonas campestris pathovar campestris, previously cloned in the recombinant plasmid pIJ3070, was located by deletion to a 2.2 kb DNA region. Sequencing of the protease gene revealed an open reading frame encoding a 580 amino acid polypeptide with molecular weight of 57,000. The deduced amino acid sequence showed strong homology with the subtilisin family of serine proteases. This, together with its sensitivity to inhibition by phenylmethylsulphonyl fluoride, suggests that the enzyme belongs to this family of proteases.

A transposon vector for broad host range cloning

The genetic analysis of many procaryotes is hindered by the lack of suitable broad host range cloning vectors. These vectors are often relatively large with limited cloning possibilities and frequently exhibit instability in recipient cells (1). Furthermore, the antibiotic resistance markers are not always well expressed in the recipient. We have constructed a 6.9 kb vector, based on transposon Tn5, which allows transposition of cloned DNA onto any broad host range conjugative (BHRC) plasmid and subsequent gene transfer. This flexible approach alleviates the aforementioned problems. Plasmid pJP5011 consists of pJP5010 carrying transposon Tn5-lacZ' in the ampicillin resistance (Amr/) gene. Plasmid pJP5010 is pUC19 (2) with the lacZ' fragment deleted. Tn5-lacZ' is Tn5 carrying the lacZ' fragment of pUC19. HaeU was used for deletion and insertion of the lacZ' fragment. Plasmid pJP5011 has unique Sail, Accl, Xbal, BamHl, Xmal, Smal, Kpnl, Sacl and EcoRI restriction sites available for a-complementation cloning. This was confirmed by performing clonings without insert DNA and noting the absence of white colonies on selective MacConkey (Oxoid) or X-gal media. Using pJP5011, a number of DNA fragments have been transferred to Rhodobacter sphaeroides giving the expected resultant phenotype. Routinely, a BHRC plasmid is mated into the recombinant cells followed by mating with the recipient. Growth on an appropriate contraselective antibiotic and kanamycin indicates recombinant transfer via transposition. In some cases, kanamycin resistance is not well expressed in the recipient or the recipient mutates frequently to kanamycin resistance. In such situations, transposition can first be selected by transfer to another E. coli strain followed by the use of a BHRC plasmid marker to detect BHRC plasmid: :Tn5-insert transfer to the recipient. Selection of transposition in E. coli is also advisable in situations where conjugal transfer frequency to the recipient is low due to restriction or other factors. Some advantages of this system are: a) The selection marker employed can be varied with the use of alternate BHRC plasmids. b) BHRC plasmid: :Tn-5 insert DNA constructs are very stable in a wide variety of organisms. For example, the 28 kb carotenoid gene cluster, cloned in Tn5 and expressed in a number of gram negative organisms (3), has almost never shown loss or alteration of colour due to DNA rearrangement. This experience has been repeated with other cloned pathways (unpublished data), c) Inserts in Tn5 as large as 40 kb can be transposed to BHRC plasmids (3). d) The kanamycin resistance (KanO gene of Tn5 is well expressed in many organisms (4). e) The carrier replicon (BHRC, mobilizable, suicidal, viral etc.) can be varied according to need. For instance, stable chromosomal insertions of recombinant DNA could be obtained in recipients by using mobilizable suicide vectors. For transformable recipients, pJP5011 itself could be B-y~t J|!D

Dissimilative Iron Reduction by the Marine Eubacterium Alteromonas putrefaciens Strain 200

The ability of microorganisms to mediate the transformation of iron coupled with the participation of iron as a reactive redox species in natural waters and sediments has prompted recent speculation that such bacteria play an important role in such activities as global iron cycling, biologically induced mineral formation, and localized competition for available organic substrates. Microaerophilic soil bacteria dominate the list of microorganisms capable of iron reduction. However, screening experiments have shown that the marine eubacterium Alteromonas putrefaciens strain 200 is one of the most efficient iron reducers yet isolated. Respiratory inhibitor studies have suggested that a branched respiratory chain and two ferri-reductases (one constitutive and one induced by growth under microaerophilic conditions (2 µM O2)) are involved in iron reduction. Further complementary biochemical and genetic studies have concentrated on characterizing the components of the induced iron reduction system. Ethyl methane sulfonate (EMS) has been used to develop mutagenesis procedures for A.putrefaciens 200; under optimal conditions, the frequency of total auxotrophy for EMS-treated cultures is approximately 0.4%. Tests for specific auxotrophic requirements have indicated the presence of a complex distribution of loci required for biosynthetic pathways. Mutants in both high-rate and low-rate ferri-reductase activity have been identified via a newly developed technique for screening EMS-treated A.putrefaciens 200 cultures. Ferri-reductase-deficient mutants generated by these techniques have been shown to contain reduced cytochrome content. Preliminary results indicate that the broad host range cloning vector pVK100 (Inc P) is mobilizable into A.putrefaciens 200. Current work is aimed at construction of an A.putrefaciens 200 clone bank, and complementation of the ferri-reductase mutants.

Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker

The non-nitrogen-fixing (Nif −) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif +); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif + transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif −. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif + phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

The Avirulence GeneavrBs1fromXanthomonas campestrispv.vesicatoriaEncodes a 50-kD Protein

A gene cloned from Xanthomonas campestris pv. vesicatoria race 2, avrBs1, specified avirulence on pepper cultivars containing the resistance gene Bs1. A series of exonuclease III deletions were made on a 3.2-kbp DNA fragment that determined full avirulence activity, observed as hypersensitive response (HR) induction. The deletion products were subcloned into the broad host range cloning vector pLAFR3, conjugated into a virulent X. c. pv. vesicatoria race 1 strain, 82-8, and scored for their ability to induce a HR on a pepper cultivar (ECW10R) containing the resistance gene Bs1. A span of approximately 1.8 kbp of DNA was necessary for full induction of the HR. The nucleotide sequence revealed two open reading frames (ORFs) capable of encoding proteins of 12.3 and 49.8 kD, designated ORF1 and ORF2, respectively. Deletions into ORF1 altered the HR-inducing activity to give an intermediate phenotype. Deletions into ORF2 completely destroyed activity. When the ORF2 coding region was driven by the lacZ promoter on plasmid pLAFR3 (placD), full avirulence activity was restored, indicating that ORF2 alone can induce the HR. Antisera raised to a beta-galactosidase-ORF2 fusion protein reacted with a 50-kD protein in X. c. pv. vesicatoria race 1 (placD) transconjugants. The deduced amino acid sequence of ORF2 had approximately 47% overall homology to the carboxyl terminus of the avirulence gene, avrA, isolated from Pseudomonas syringae pv. glycinea race 6, and 86% homology over a region of 49 amino acids. P. s. pv. glycinea, however, did not induce an HR on ECW10R plants.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of marine methanotrophs.

Four new methane-oxidizing bacteria have been isolated from marine samples taken at the Hyperion sewage outfall, near Los Angeles, CA. These bacteria require NaCl for growth. All exhibit characteristics typical of Type I methanotrophs, except they contain enzyme activities of both the ribulose monophosphate pathway and the serine cycle. All four strains are characterized by rapid growth in liquid culture and on agar plates, and all have temperature optima above 35 degrees C. One strain, chosen for further study, has been shown to maintain broadhost range cloning vectors and is currently being used for genetic studies.

Enzymatic Hydrolysis of Organophosphates: Cloning and Expression of a Parathion Hydrolase Gene from Pseudomonas diminuta

Pseudomonas diminuta strain MG hydrolyzes parathion to diethylthiophosphoric acid and p-nitrophenol. The esterase responsible for this reaction is encoded by a gene located on a plasmid termed pCMS1. The gene was cloned into plasmid pBR322 and the broad host range cloning vector pKT230. Enzyme activity was detected in Escherichia coli strains that contained the recombinant plasmids. A 1.5 kilobase BamHI fragment with single restriction sites for SalI, PstI and XhoI was shown to direct the synthesis of the enzyme. The 1.5 kilobase BamHI fragment was inserted into the high expression vector pUC7, and the resulting recombinant, pCMS40, was used to construct pKT230 derivatives containing the parathion hydrolase gene. Subsequent transfer of the recombinant plasmids into a cured derivative of P. diminuta MG and a p-nitrophenol-utilizing strain of Pseudomonas resulted in the isolation of transconjugants that exhibited parathion hydrolase activity. The highest enzyme activity was observed with P. diminuta MG.

Construction of broad host range cloning vectors for gram-negative bacteria.

A new cloning vector, pMFY31, has been constructed based on the high-copy-number, broad-host-range plasmid RSF1010. The plasmid has a size of 13.2 kb and carries the Apr, Cmr, and Tcr genes. It contains unique PstI, EcoRI, HindIII, BamHI, and SalI sites, all of which are located within the antibiotic resistance genes, therefore all sites are applicable to insertional inactivation. We also constructed pMFY40, a 11.6 kb derivative of pMFY31, by the elimination of the Cmr gene. Plasmid pMFY31 has been efficiently introduced into a Pseudomonas putida strain not only by plasmid-DNA transformation but also by conjugal co-transfer with the helper plasmid, and was maintained stably in the strain.

Broad host range cloning vectors for Gram-negative bacteria

A series of cloning vectors has been constructed based on the broad-host-range plasmid R300B. One of these vectors, pGSS33, has a size of 13.4kb and carries four antibiotic resistance genes [ampicillin (Ap r), chloramphenicol (Cm r), streptomycin (Sm r) and tetracycline (Tc r) L all of which have restriction sites for insertional inactivation. The derivation, structure and uses of the plasmids are described.

Construction and Characterization of a Versatile Broad Host Range DNA Cloning System for Gram–Negative Bacteria

A set of broad host range cloning vectors has been constructed from the IncW plasmid pSa. These vectors have been constructed from the transfer defective deletion derivative pSa151, which encodes resistance to kanamycin and spectinomycin–streptomycin. Two of the vectors also contain the chloramphenicol resistance gene of Tn9, and a third contains the chloramphenicol resistance gene of pSa. One of the vectors contains the cos sequence of bacteriophage λ and can be used with in vitro packaging systems in the construction of large recombinant plasmids. Two of these vectors can be mobilized and transferred in the presence of a pBR322 derivative containing the transfer genes of pSa. Together these vectors contain cloning sites for SstII, HindIII, EcoRI, KpnI, PvuII, BamHI, SmaI, and Bg1II, and recombinants at certain of these sites can be detected by insertional inactivation of a drug resistance phenotype. The broad host range properties of the origin of replication of pSa allow the use of these vectors in a variety of gram–negative bacteria.

Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants

We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK.290 (Ditta et al., 1980) by inserting a 1.6-kb BglII fragment containing λ cos into the unique BglII site in pRK.290. The new vector, pLAFRl, is 21.6 kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts. We constructed a clone bank of Rhizobium meliloti DNA in pLAFRl using a partial EcoRI digest. The mean insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti auxotrophic mutants, tetracycline-resistant (Tc r) transconjugants were obtained at frequencies ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001 to 0.007. pLAFRl cosmids were mobilized from R. meliloti prototrophic colonies into E. coli and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tc r transconjugants were prototrophic.

Perspectives for the genetic engineering of hydrocarbon oxidizing bacteria.

Hydrocarbons have many different structures, but these microbial substrates have two general characteristics which influence the nature of oxidation pathways. (1) Hydrocarbons are usually hydrophobic molecules, and oxidizing activities are consequently located in cellular membranes. (2) Hydrocarbons are composed of several basic structural units (such as aromatic rings and aliphatic chains). In order to break down these composite substrates, oxidation pathways are organized into segments that are conveniently labeled “throats” and “stomachs”. Throats contain the initial activities which convert a specific hydrocarbon substrate into one of a few common intermediates, such as fatty acids or catechols. Stomachs contain the subsequent activities which transform the intermediates into the organic acid substrates of central metabolic pathways. Typical stomachs include β-oxidation and catechol ring fission pathways. Detailed genetic analysis has been carried out for a few hydrocarbon oxidation pathways, including the plasmidborne alk and xyl systems for oxidation of n-alkanes and of toluene or xylenes. In both cases, there is duplication of plasmic-and chromosome-encoded activities at certain steps of the oxidation pathway. Induction specificities in both systems are often more important than enzyme specificities in determining growth phenotypes on different substrates. Oxidation of halogenated aromatic substrates, such as chlorobenzoic acid or the pesticide 24D, has been the subject of less detailed genetic study, but haloaromatic pathways illustrate the same basic principles as those for non-halogenated substrates: (i) the need to match throats and stomachs, and (ii) the importance of proper pathway regulation to ensure substrate catabolism. The use of broad host-range cloning vectors means that recombinant DNA methods are available for use with hydrocarbon-oxidizing gram-negative bacteria. However, detailed consideration of the possible rate-limiting steps in hydrocarbon oxidation indicates that cloning and overproduction of particular enzymes may often not be successful in strain improvement for specific biodegradations or bioconversions. A particular problem can be the toxicity of membrane hydrocarbon-oxidizing proteins when they are produced at very high levels.