Genetic analysis of mannityl opine catabolism in octopine-type Agrobacterium tumefaciens strain 15955

Summary The genetic organization of functions responsible for mannityl opine catabolism of the Ti plasmid of Agrobacterium tumefaciens strain 15955 was investigated. A partial HindIII digest of pTi15955 was cloned into a broad host range cosmid and the clones obtained were tested for ability to confer mannityl opine degradation upon Agrobacterium. Inserts containing genes for catabolism of mannopinic acid, mannopine, agropine, and agropinic acid were obtained, spanning a segment of 43 kb on the Ti plasmid. Two clones conferring upon Agrobacterium the ability to catabolize the mannityl opines were mobilized to several Rhizobium sp., to Pseudomonas putida and P. fluorescens and to Escherichia coli. The catabolic functions were phenotypically expressed in all Rhizobium sp. tested, and in P. fluorescens, but not in P. putida or in E. coli.     

The molecular cloning of genes specifying some enzymes of the 3,5-xylenol degradative pathway

Enzymes for the degradation of 3,5-xylenol ofPseudomonas putida NCIB9869 are encoded on a transmissible plasmid, pRA500. Genes specifying the inducible synthesis of some enzymes encoded on pRA500, namely, citraconase (G), citramalate coenzyme-A-transferase (H), citramalyl-coenzyme-A lyase (I), maleyl-pyruvate isomerase (J) and fumarylpyruvate hydrolase (K) have been cloned on a 7.9-kbHindIII fragment into the vector pKT231 to give pRA507. Biochemical and restriction analysis of pRA507 and some of its deleted derivatives has enabled preliminary locations to be assigned to the genes encoding these enzymes. The structural genes for enzymes H and I, together with a regulatory gene controlling their expression, are located on a 1.6-kbHindIII/XhoI fragment of pRA507 and the genes for enzymes G, J and K are located within a 4.5-kbXhoI/ClaI fragment of pRA507, which lies immediately adjacent to the former fragment. Biochemical analysis of the strain carrying pRA507, of its plasmid-free derivative and of other plasmid-free derivatives of the wild-typeP. putida has provided indirect evidence for the presence of two isofunctional enzymes at three different steps of the degradative pathway. One set of 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase and maleylpyruvate hydrolase is encoded on pRA500 and these enzymes are inducible only. A second set is encoded on the chromosome and these enzymes are constitutive and not further inducible. The chromosomally located genes appeared to be silent until 3,5-xylenol-negative strains were incubated for prolonged periods with compounds such asm-hydroxybenzoate orp-cresol.     

Macroptilium atropurpureum (siratro) host specificity genes are linked to a nodD-like gene in the broad host range Rhizobium strain NGR234

Summary A 6.7 kb HindIII fragment from the Sym-plasmid of strain NGR234 was found to code a nodD-like gene flanked by two loci which were required for siratro host range. Transfer of the 6.7 kb fragment from NGR234 to R. trifolii strain ANU843 conferred extended host range ability to this strain on siratro plants but not to other plants normally nodulated by strain NGR234. Tn5 mutagenesis of the 6.7 kb fragment showed that insertions located into loci flanking the nodD-like gene abolished the extended host range phenotype. A hybridization probe spanning one of the host specificity loci was shown to hybridize to three specific bands in the NGR234 genome. Complementation and DNA hybridization data showed that the nodD-like gene of strain NGR234 was functionally similar to that in R. trifolii. The introduction to R. trifolii of the 6.7 kb HindIII fragment containing Tn5 insertions located in the nodD-like gene did not abolish the ability to extend the host range of R. trifolii to siratro plants. However, transfer of the 6.7 kb HindIII to R. trifolii derivatives containing Tn5 insertions into either nodA, B or C or other R. trifolii nod genes failed to confer siratro nodulation to these recipients. Reconstruction experiments showed that the 6.7 kb fragment from strain NGR234 and the 14 kb nodulation region of R. trifolii could induce the nodulation of siratro plants when introduced together into Sym-plasmid-cured Rhizobium strains.     

Nucleotide homology and organization of chlorocatechol oxidation genes of plasmids pJP4 and pAC27

Summary The 2,4-dichlorophenoxyacetate (2,4-D) catabolic plasmid pJP4 of Alcaligenes eutrophus JMP134 contains two sets of nonidentical chlorocatechol oxidation gene sequences physically separated by a 7 kb DNA region. We determined the nucleotide sequence of the 1.6 kb HindIII fragment containing the known genes tfdC and tfdD (Don et al. 1985) which encode pyrocatechase and cycloisomerase, respectively. The 1.3 kb BglII-HindIII segment of recombinant plasmid pDC25 containing at least three chlorocatechol (clc) oxidation genes of the pAC27 plasmid in Pseudomonas putida AC868 (Ghosal et al. 1985a; Frantz and Chakrabarty 1986), was also sequenced. When the tfdC gene of the pJP4 plasmid was compared with gene clcA of plasmid pAC27, which encodes the chlorocatechol specific pyrocatechase (pyrocatechase II), the two genes showed 63% nucleotide sequence homology with 60% homology in their amino acid sequences. In both plasmid pJP4 and pAC27, the two genes encoding the pyrocatechase and the cycloisomerase showed a 4 bp overlap spanning the initiation codon of the cycloisomerase gene and the termination codon of the pyrocatechase gene. The sizes of the polypeptides encoded by the isofunctional genes tfdC and clcA are very similar and thus reflect their functional homology.     

Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6

Summary DNA fragments generated by the EcoRI or HindIII endonucleases from the low copy number antibiotic resistance plasmids R6 and R6-5 were separately cloned using the high copy number ColEl or pML21 plasmid vectors and the insertional inactivation procedure. The hybrid plasmids that were obtained were used to determine the location of the EcoRI and HindIII cleavage sites on the parent plasmid genomes by means of electron microscope heteroduplex analysis and agarose gel electrophoresis. Ultracentrifugation of the cloned fragments in caesium chloride gradients localized the high buoyant density regions of R6-5 to fragments that carry the genes for resistance to streptomycin-spectinomycin, sulfonamide, and mercury and a low buoyant density region to fragments that carry the tetracycline resistance determinant. Functional analysis of hybrid plasmids localized a number of plasmid properties such as resistances to antibiotics and mercury and several replication functions to specific regions of the R6-5 genome. Precise localisation of the genes for resistance to chloramphenicol, kanamycin, fusidic acid and tetracycline was possible due to the presence of identified restriction endonuclease cleavage sites within these determinants.Only one region competent for autonomous replication was identified on the R6-5 plasmid genome and this was localized to EcoRI fragment 2 and HindIII fragment 1. However, two additional regions of replication activity designated RepB and RepC, themselves incapable of autonomous replication but capable of supporting replication of a linked ColE1 plasmid in polA^– bacteria, were also identified.     

Construction of a bacterial artificial chromosome library containing large Eco RI and Hin dIII genomic fragments of lettuce

 Existing bacterial artificial chromosome (BAC) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5 and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLP^TM markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA. Received: 5 August 1996 / Accepted: 23 August 1996     

A plasmid cloning vector for KpnI-cleaved DNA

A plasmid cloning vector containing a single site for KpnI has been generated by insertion of a 3.5-kb EcoRI/HindIII fragment of pCR1 into the EcoRI/HindIII sites of pBR322. KpnI cleavage yields 3′ rather than 5′ “sticky ends” which allows reconstitution of the recognition site after cloning by a homopolymer joining procedure. This is an advantage shared with only one or two other commercially available restriction enzymes.     

Molecular cloning in streptococci: Physical mapping of the vehicle plasmid pSM10 and demonstration of intergroup DNA transfer

Summary The streptococcal resistance plasmid pSM10 (8.3 kb), a deletion derivative of pSM10419 (22.9 kb) determining constitutive erythromycin and lincomycin resistance, was physically mapped with the restriction endonucleases AvaI, AvaII, EcoRI, HpaI, KpnI, PvuII (one site each), HindIII, HaeII (three sites each), HincII (four sites), and HhaI (five sites). Using the cryptic plasmid pVA318 as cloning vehicle, the largest HindIII fragment of pSM10 (3.3 kb) was shown to contain the erythromycin/lincomycin resistance gene(s) of the plasmid. The AvaII site of pSM10 proved to be suitable as a site for cloning AvaII-generated chromosomal DNA fragments from a group C streptococcal strain in the Challis strain of Streptococcus sanguis (group) H. A detailed physical map of the chimeric plasmid pSM10221 (12.8 kb), a fusion product of pSM10 and the staphylococcal chloramphenicol resistance plasmid pC221 (4.5 kb), is also presented. The plasmid chimera has properties making it potentially useful in development of a doubly selective streptococcal cloning vehicle by searching for insertional inactivation.     

Two-dimensional screening of the Wageningen chicken BAC library

We have constructed a Bacterial Artificial Chromosome (BAC) library that provides 5.5-fold redundant coverage of the chicken genome. The library was made by cloning partial HindIII-digested high-molecular-weight (HMW) DNA of a female White Leghorn chicken into the HindIII site of the vector pECBAC1. Several modifications of standard protocols were necessary to clone efficiently large partial HindIII DNA fragments. The library consists of 49,920 clones arranged in 130 384-well plates. An average insert size of 134 kb was estimated from the analysis of 152 randomly selected BAC clones. The average number of NotI restriction sites per clone was 0.77. After individual growth, DNA was isolated of the pooled clones of each 384-well plate, and subsequently DNA of each plate was isolated from the individual row and column pools. Screening of the Wageningen chicken BAC library was performed by two-dimensional PCR with 125 microsatellite markers. For 124 markers at least one BAC clone was obtained. FISH experiments of 108 BAC clones revealed chimerism in less than 1%. The number of different BAC clones per marker present in the BAC library was examined for 35 markers which resulted in a total of 167 different BAC clones. Per marker the number of BAC clones varied from 1 to 11, with an average of 4.77. The chicken BAC library constitutes an invaluable tool for positional cloning and for comparative mapping studies. Received: 26 October 1999 / Accepted: 6 January 2000     

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Grass Carp

Bacterial artificial chromosome (BAC) library is an important tool in genomic research. We constructed two libraries from the genomic DNA of grass carp (Ctenopharyngodon idellus) as a crucial part of the grass carp genome project. The libraries were constructed in the EcoRI and HindIII sites of the vector CopyControl pCC1BAC. The EcoRI library comprised 53,000 positive clones, and approximately 99.94% of the clones contained grass carp nuclear DNA inserts (average size, 139.7 kb) covering 7.4× haploid genome equivalents and 2% empty clones. Similarly, the HindIII library comprised 52,216 clones with approximately 99.82% probability of finding any genomic fragments containing single-copy genes; the average insert size was 121.5 kb with 2.8% insert-empty clones, thus providing genome coverage of 6.3× haploid genome equivalents of grass carp. We selected gene-specific probes for screening the target gene clones in the HindIII library. In all, we obtained 31 positive clones, which were identified for every gene, with an average of 6.2 BAC clones per gene probe. Thus, we succeeded in constructing the desired BAC libraries, which should provide an important foundation for future physical mapping and whole-genome sequencing in grass carp.     

Characterization of ard B and ard C actin gene loci of Physarum polycephalum

Summary Physarum polycephalum (strain M₃CVIII) contains four unlinked actin gene loci, each with two alleles (ard A1, ard A2, ard B1, ard B2, ard C1, ard C2, ard D1 and ard D2). The 4.8 kbp HindIII component of the ard C2 locus was isolated as a recombinant phage-, after HindIII fragments of Physarum DNA ranging from 4.3 kbp to 5.5 kbp were cloned into phage- NM1149. The fraction of Physarum DNA cloned contained the ard C locus, and no other actin locus. Small inserts were favoured to reduce the probability of cloning a complete repetitive element, because such elements have been found to adversely affect the stability of recombinants.The coding sequences of the actin gene (approximately 1.1 kbp) spanned more than 3 kbp indicating the presence of introns. A 1.6 kbp HindIII/EcoRI fragment of the ard C locus, which contained some coding sequences, hybridized extensively with HindIII fragments of genomic DNA indicating the presence of repetitive sequences. A 2.3 kbp HindIII/EcoRI fragment containing most of the coding sequences of the C2 allele of the ard C locus hybridized with the C1, allele and both alleles of the ard B locus, but not with the ard A locus or ard D locus. This distinction was used to establish for the ard B and ard C loci the relationship between the EcoRI and HindIII fragments that define an ard locus. The ability to distinguish between ard loci may facilitate studies of the expression of particular actin loci.     

Duplicate Copies of Genes Encoding Methanesulfonate Monooxygenase in Marinosulfonomonas methylotropha Strain TR3 and Detection of Methanesulfonate Utilizers in the Environment

Marinosulfonomonas methylotropha strain TR3 is a marine methylotroph that uses methanesulfonic acid (MSA) as a sole carbon and energy source. The genes from M. methylotropha strain TR3 encoding methanesulfonate monooxygenase, the enzyme responsible for the initial oxidation of MSA to formaldehyde and sulfite, were cloned and sequenced. They were located on two gene clusters on the chromosome of this bacterium. A 5.0-kbp HindIII fragment contained msmA, msmB, and msmC, encoding the large and small subunits of the hydroxylase component and the ferredoxin component, respectively, of the methanesulfonate monooxygenase, while a 6.5-kbp HindIII fragment contained duplicate copies of msmA and msmB, as well as msmD, encoding the reductase component of methanesulfonate. Both sets of msmA and msmB genes were virtually identical, and the derived msmA and msmB sequences of M. methylotropha strain TR3, compared with the corresponding hydroxylase from the terrestrial MSA utilizer Methylosulfonomonas methylovora strain M2 were found to be 82 and 69% identical. The msmA gene was investigated as a functional gene probe for detection of MSA-utilizing bacteria. PCR primers spanning a region of msmA which encoded a unique Rieske [2Fe-2S] binding region were designed. These primers were used to amplify the corresponding msmA genes from newly isolated Hyphomicrobium, Methylobacterium, and Pedomicrobium species that utilized MSA, from MSA enrichment cultures, and from DNA samples extracted directly from the environment. The high degree of identity of these msmA gene fragments, compared to msmA sequences from extant MSA utilizers, indicated the effectiveness of these PCR primers in molecular microbial ecology.     

The nickel resistance determinant cloned from the enterobacterium Klebsiella oxytoca: conjugational transfer,expression, regulation and DNA homologies to various nickel-resistant bacteria

Klebsiella oxytoca strain CCUG 15788, isolated from a mineral oil emulsion tank in Göteborg, Sweden, was found to be nickel-resistant (tolerating 10 mm NiCl₂ in non-complexing mineral-gluconate media; inducible resistance). The nickel resistance determinants were transferred by helper-assisted conjugation to various strains of Escherichia coli and Citrobacter freundii and expressed to between 5 and 10 mm NiCl₂. A 4.3 kb HindIII fragment was cloned from the genomic DNA of K. oxytoca. Ligated into the vector pSUP202, the fragment caused constitutive nickel resistance (of up to 3 or 10 mm Ni²⁺) in various E. coli strains. After cloning into the broad host range vector pVDZ'2 the fragment even expressed low nickel resistance in the transconjugant of Alcaligenes eutrophus AE104. With the 4.3 kb HindIII fragment as a biotinylated DNA probe it was shown by DNA-DNA hybridization that the nickel resistance determinant resides on the chromosome of K. oxytoca and not on its circular plasmid pKO1 (160 kb) or linear plasmid pKO2 (50 kb). Nickel resistance strongly correlated with the presence of the 4.3 kb HindIII fragment in the transconjugants. No homologies were detected when the nickel resistance determinants of other well-known nickel-resistant bacteria, such as A. eutrophus CH34 or A. denitrificans 4a-2, were used as target DNA. Among the 60 strains examined, positive signals only appeared with the 3.1 kb DNA fragment from A. xylosoxydans 31A and the genomic DNA of two enterobacterial strains (5-1 and 5–5) isolated from nickel-rich soil in New Caledonia.     

Post-transformational rearrangement of an in vitro reconstructed group-A streptococcal erythromycin resistance plasmid

Cleavage of the group-A streptococcal macrolide, lincosamide, and streptogramin B (MLS) resistance plasmid pSM19035 yields 2 fragments [13 and 4 megadaltons (MD)] with EcoRI, and 15 fragments with HindIII, 12 of which are 6 pairs of identical fragments derived from the inverted repeats that comprise about 80% of the pSM19035 genome. The large EcoRI fragment was isolated, ligated, and used to transform the Challis strain of Streptococcus sanguis to erythromycin resistance. Plasmids (pDB101, pDB102, and pDB103) isolated from three different transformants had lower molecular masses than the original large EcoRI fragment. HindIII digestion of these molecules and subsequent analysis of fragment radioactivity distributions indicated the loss of plasmid segments of various sizes. The deletions, all of which occurred in the palindrome, did not affect the level and the inducible nature of pSM19035-determined antibiotic resistance. Only pDB101 retained the unique EcoRI cleavage site. The results of this analysis allowed the construction of an EcoRI and HindIII cleavage-site map of pSM19035 and promise to simplify future studies of genetic functions specified by streptococcal MLS resistance plasmids.     

Molecular cloning of menaquinone biosynthetic genes of Escherichia coli K12

Summary A tranducing phage carrying some of the genes (men) defining the early stages of menaquinone biosynthesis was isolated from a pool of recombinant lambda phages that had been constructed from R.HindIII digests of E. coli DNA and the corresponding insertion vector. The lesions of menB and menC mutants were complemented by the phage but menD mutants were transduced either at low frequencies or not at all. This indicates that the transducing phage contains functional menB and menC genes but that only part of the menD gene had been cloned. The phage (G68) was accordingly disignated menCB(D). Studies with the transducing phage enabled earlier mapping data (Guest 1979) to be reinterpreted in favour of the gene order nalA.... menC..menB..menD.... purF. Restriction analyses established the presence of a bacterial DNA fragment (11.5 kb) linked by a R.HindIII target to the right arm of the genome but fused to the left arm of the vector. Hybridization studies confirmed that the cloned DNA was derived from a larger R.HindIII fragment (21 kb). A physical map of the men region was constructed and some flanking and overlapping fragments were identified.     

A restriction enzyme cleavage map of the histidine utilization (hut) genes of Klebsiella aerogenes and deletions lacking regions of hut DNA

Summary The histidine utilization (hut) operons of Klebsiella aerogenes were cloned into pBR322. The hut genes are wholly contained on a 7.9 kilobase pair fragment bounded by HindIII restriction sites and expression of hut is independent of the orientation of the fragment with respect to pBR322. A restriction map locating the 27 cleavage sites within hut for the enzymes, HindIII, PvuII, SalI, BglII, KpnI, PstI, SmaI, AvaI, and BamHI was deduced. Several of the cleavage sites for the enzymes HaeIII and HinfI were also mapped. A set of deletion plasmids was isolated by removing various restriction fragments from the original plasmid. These deletions were characterized and were used to assist in mapping restriction sites. This physical characterization of hut DNA opens the way for genetic and molecular analysis of the regulation of hut gene expression in vitro as well as in vivo.     

A plasmid cloning vector for Kpnl-cleaved DNA

A plasmid cloning vector containing a single site for KpnI has been generated by insertion of a 3.5-kb EcoRI/HindIII fragment of pCR1 into the EcoRI/HindIII sites of pBR322. KpnI cleavage yields 3′ rather than 5′ “sticky ends” which allows reconstitution of the recognition site after cloning by a homopolymer joining procedure. This is an advantage shared with only one or two other commercially available restriction enzymes.     

Host range,RFLP, and antigenic relationships betweenRhizobium fredii strains andRhizobium sp. NGR234

Rhizobium fredii is a nitrogen-fixing symbiont from China that combines broad host range for nodulation of legume species with cultivar specificity for nodulation of soybean. We have compared 10R. fredii strains withRhizobium sp. NGR234, a well known broad host range strain from Papua New Guinea. NGR234 nodulated 16 of 18 tested lugume species, and nodules on 14 of the 16 fixed nitrogen. TheR. fredii strains were not distinguishable from one another. They nodulated 13 of the legumes, and in only nine cases were nodules effective. All legumes nodulated byR. fredii were included within the host range of NGR234. Restriction fragment length polymorphisms (RFLPs) were detected with four DNA hybridization probes: the regulatory and commonnod genes,nodDABC; the soybean cultivar specificity gene,nolC; the nitrogenase structural genes, nifKDH; and RFRS1, a repetitive sequence fromR. fredii USDA257. A fifth locus, corresponding to a second set of soybean cultivar specificity genes,nolBTUVWX, was monomorphic. Using antisera against whole cells of threeR. fredii strains and NGR234, we separated the 11 strains into four serogroups. The anti-NGR234 sera reacted with a singleR. fredii strain, USDA191. Only one serogroup, which included USDA192, USDA201, USDA217, and USDA257, lacked cross reactivity with any of the others. Although genetic and phenotypic differences amongR. fredii strains were as great as those between NGR234 andR. fredii, our results confirm that NGR234 has a distinctly wider host range thanR. fredii.