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PubMed1991

Molecular cloning, expression, and characterization of endoglucanase genes from Fibrobacter succinogenes AR1.

A cosmid gene library was constructed in Escherichia coli from genomic DNA isolated from the ruminal anaerobe Fibrobacter succinogenes AR1. Clones were screened on carboxymethyl cellulose, and 8 colonies that produced large clearing zones and 25 colonies that produced small clearing zones were identified. Southern blot hybridization revealed the existence of at least three separate genes encoding cellulase activity. pRC093, which is representative of cosmid clones that produce large clearing zones, was subcloned in pGem-1, and the resulting hybrid pRCEH directed synthesis of endoglucanase activity localized on a 2.1-kb EcoRI-HindIII insert. Activity was expressed from this fragment when it was cloned in both orientations in pGem-1 and pGem-2, indicating that F. succinogenes promoters functioned successfully in E. coli. A high level of endoglucanase activity was detected on acid-swollen cellulose, ball-milled cellulose, and carboxymethyl cellulose; and a moderate level was detected on filter paper, Avicel, lichenan, and xylan. Most activity (80%) was localized in the periplasm of E. coli, with low but significant levels (16%) being detected in the extracellular medium. The periplasmic endoglucanase had an estimated molecular weight of 46,500, had an optimum temperature of 39 degrees C, and exhibited activity over a broad pH range, with a maximum at pH 5.0.

Bacteroides↗
PubMed1993

Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus.

Aflatoxins are extremely potent carcinogens produced by Aspergillus flavus and Aspergillus parasiticus. Cloning of genes in the aflatoxin pathway provides a specific approach to understanding the regulation of aflatoxin biosynthesis and, subsequently, to the control of aflatoxin contamination of food and feed. This paper reports the isolation of a gene involved in aflatoxin biosynthesis by complementation of an aflatoxin-nonproducing mutant with a wild-type genomic cosmid library of A. flavus. Strain 650-33, blocked in aflatoxin biosynthesis at the afl-2 allele, was complemented by a 32-kb cosmid clone (B9), resulting in the production of aflatoxin. The onset and profile of aflatoxin accumulation was similar for the transformed strain and the wild-type strain (NRRL 3357) of the fungus, indicating that the integrated gene is under the same control as in wild-type strains. Complementation analyses with DNA fragments from B9 indicated that the gene resides within a 2.2-kb fragment. Because this gene complements the mutated afl-2 allele, it was designated afl-2. Genetic evidence obtained from a double mutant showed that afl-2 is involved in aflatoxin biosynthesis before the formation of norsolorinic acid, the first stable intermediate identified in the pathway. Further, metabolite feeding studies with the mutant, transformed, and wild-type cultures and enzymatic activity measurements in cell extracts of these cultures suggest that afl-2 regulates gene expression or the activity of other aflatoxin pathway enzymes. This is the first reported isolation of a gene for aflatoxin biosynthesis in A. flavus.

Aflatoxins↗
PubMed1996

Overcoming a defect in generalized recombination in the marine fish pathogen Vibrio anguillarum 775: construction of a recA mutant by marker exchange.

A defect in generalized recombination has prevented the use of marker exchange for the construction of specific chromosomal mutations in the marine fish pathogen Vibrio anguillarum 775. Through the use of large segments of homologous DNA, we were successful in overcoming this defect and used marker exchange to construct a recA mutant of V. anguillarum H775-3. A recombinant cosmid carrying the recA gene of V. anguillarum 775 in the center of a 25-kb cloned DNA insert was isolated by complementation of methyl methanesulfonate (MMS) sensitivity in Escherichia coli HB101. The recA gene was inactivated by inserting a kanamycin resistance gene into recA, and the mutant gene was subsequently introduced into V. anguillarum H775-3 by conjugal mobilization. Isolation of recombinants between cosmid-borne recA::kan sequences and chromosomal DNA was facilitated by the introduction of an incompatible plasmid, and Southern hybridization was used to verify the presence of recA::kan in the chromosomal DNA of the recA mutant. V. anguillarum carrying recA::kan was considerably more sensitive to UV radiation and to MMS than was its parent, and near wild-type levels of resistance to MMS and UV light were restored by introduction of cloned recA genes from both E. coli and V. anguillarum. These results indicate that recA is required for DNA repair in V. anguillarum and demonstrate the utility of this modified marker exchange technique for the construction of mutations in this economically important fish pathogen.

Animals↗
PubMed1996

Cloning, sequencing, and expression of isopropylbenzene degradation genes from Pseudomonas sp. strain JR1: identification of isopropylbenzene dioxygenase that mediates trichloroethene oxidation.

Pseudomonas sp. strain JR1, recently isolated with isopropylbenzene (IPB) as the inducer substrate for trichloroethene (TCE) oxidation (B. Dabrock, J. Riedel, J. Bertram, and G. Gottschalk, Arch. Microbiol 158:9-13, 1992), is able to degrade IPB via the meta-cleavage pathway. The genes encoding the first three enzymes in the catabolism of isopropylbenzene were isolated from a genomic library with the broad-host-range cosmid vector pWE15. A 7.6-kb fragment from a 37.7-kb primary cosmid clone was subcloned and sequenced. It contained seven complete open reading frames, designated ipbA1A2orf3A3A4BC. ipbA codes for the three subunits of a multicomponent IPB dioxygenase, ipbB codes for 2,3-dihydro-2,3-dihydroxy-IPB dehydrogenase, and ipbC codes for 3-isopropylcatechol 2,3-dioxygenase. The deduced amino acid sequences of ipbA1A2A3A4BC exhibited the highest homologies with the corresponding proteins of biphenyl-degradative pathways in gram-negative and gram-positive bacteria. The gene products of the ipb genes were identified by an in vitro transcription-translation system on the basis of their expected molecular masses. IPB dioxygenase and 3-isopropylcatechol 2,3-dioxygenase expressed in E. coli oxidized a wide range of alkyl aromatic compounds. Incubation of E. coli cells carrying ipbA1A2A3A4 with IPB and 10O2 yielded reaction products containing both atoms of molecular oxygen, which is in accordance with a dioxygenation reaction. E. coli recombinants harboring and expressing the IPB dioxygenase exhibited the ability to degrade TCE. The ipbA1A2A3A4-carrying E. coli strain required neither IPB nor isopropyl-beta-D-thiogalactopyranoside for induction; the rate of TCE degradation was comparable to that by fully induced Pseudomonas strain JR1.

Amino Acid Sequence↗
PubMed2003

Prospecting for novel biocatalysts in a soil metagenome.

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an alpha-amylase (amyA), a 1,4-alpha-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.

Bacteria↗
PubMed2003

Metagenome survey of biofilms in drinking-water networks.

Most naturally occurring biofilms contain a vast majority of microorganisms which have not yet been cultured, and therefore we have little information on the genetic information content of these communities. Therefore, we initiated work to characterize the complex metagenome of model drinking water biofilms grown on rubber-coated valves by employing three different strategies. First, a sequence analysis of 650 16S rRNA clones indicated a high diversity within the biofilm communities, with the majority of the microbes being closely related to the Proteobacteria: Only a small fraction of the 16S rRNA sequences were highly similar to rRNA sequences from Actinobacteria, low-G+C gram-positives and the Cytophaga-Flavobacterium-Bacteroides group. Our second strategy included a snapshot genome sequencing approach. Homology searches in public databases with 5,000 random sequence clones from a small insert library resulted in the identification of 2,200 putative protein-coding sequences, of which 1,026 could be classified into functional groups. Similarity analyses indicated that significant fractions of the genes and proteins identified were highly similar to known proteins observed in the genera Rhizobium, Pseudomonas, and Escherichia: Finally, we report 144 kb of DNA sequence information from four selected cosmid clones, of which two formed a 75-kb overlapping contig. The majority of the proteins identified by whole-cosmid sequencing probably originated from microbes closely related to the alpha-, beta-, and gamma-Proteobacteria: The sequence information was used to set up a database containing the phylogenetic and genomic information on this model microbial community. Concerning the potential health risk of the microbial community studied, no DNA or protein sequences directly linked to pathogenic traits were identified.

Bacteria↗
PubMed2005

Isolation and characterization of o-xylene oxygenase genes from Rhodococcus opacus TKN14.

o-Xylene is one of the most difficult-to-degrade environmental pollutants. We report here Rhodococcus genes mediating oxygenation in the first step of o-xylene degradation. Rhodococcus opacus TKN14, isolated from soil contaminated with o-xylene, was able to utilize o-xylene as the sole carbon source and to metabolize it to o-methylbenzoic acid. A cosmid library from the genome of this strain was constructed in Escherichia coli. A bioconversion analysis revealed that a cosmid clone incorporating a 15-kb NotI fragment had the ability to convert o-xylene into o-methylbenzyl alcohol. The sequence analysis of this 15-kb region indicated the presence of a gene cluster significantly homologous to the naphthalene-inducible dioxygenase gene clusters (nidABCD) that had been isolated from Rhodococcus sp. strain I24. Complementation studies, using E. coli expressing various combinations of individual open reading frames, revealed that a gene (named nidE) for rubredoxin (Rd) and a novel gene (named nidF) encoding an auxiliary protein, which had no overall homology with any other proteins, were indispensable for the methyl oxidation reaction of o-xylene, in addition to the dioxygenase iron-sulfur protein genes (nidAB). Regardless of the presence of NidF, the enzyme composed of NidABE was found to function as a typical naphthalene dioxygenase for converting naphthalene and various (di)methylnaphthalenes into their corresponding cis-dihydrodiols. All the nidABEF genes were transcriptionally induced in R. opacus TKN14 by the addition of o-xylene to a mineral salt medium. It is very likely that these genes are involved in the degradation pathways of a wide range of aromatic hydrocarbons by Rhodococcus species as the first key enzyme.

Base Sequence↗
PubMed1989

Cloning and temperature-dependent expression in Escherichia coli of a Legionella pneumophila gene coding for a genus-common 60-kilodalton antigen.

All Legionella species express a 60-kilodalton (kDa) protein which contains a genus-specific epitope recognized by murine monoclonal antibody GW2X4B8B2H6. A genomic cosmid library of Legionella pneumophila chromosomal DNA was constructed in pHC79 and screened for 60-kDa antigen-expressing clones with the monoclonal antibody. A 3.2-kilobase EcoRI fragment from cosmid 14B11 expressing a 60-kDa protein was subcloned into pUC19 (pSH16), and deletion of a 1.2-kilobase HindIII fragment (pSH16A) generated a 33-kDa truncated polypeptide no longer reactive with the monoclonal antibody. Southern blot analysis of chromosomal DNA from selected Legionella species restricted with EcoRI and probed with the 1.2-kilobase fragment coding for the carboxyl region of the protein revealed DNA homology which was not observed with DNA from Escherichia coli. Maxicell analysis of pSH16 identified a second polypeptide of approximately 15 kDa expressed from a gene (htpA) upstream of the gene coding the 60-kDa protein (htpB). Both proteins were preferentially synthesized by L. pneumophila following heat shock (temperature shift from 25 to 42 degrees C), and under steady-state growth conditions the relative level of 60-kDa protein was unaffected by temperature. In E. coli, expression of a 60-kDa protein from pSH16 also increased following heat shock (25 to 42 degrees C), but under steady-state conditions expression was temperature dependent. Temperature-dependent expression from pSH16 was not observed in an rpoH (htpR) mutant strain of E. coli. The Legionella 60-kDa protein appears to be a heat shock protein which shares cross-reactive epitopes with the GroEL homolog of E. coli. In addition, a region of htpB encoding the 27-kDa carboxyl portion of the protein containing the monoclonal antibody-reactive epitope also contains DNA sequences unique to and conserved within the genus.

Antigens, Bacterial↗
PubMed1997

Pathogenicity island sequences of pyelonephritogenic Escherichia coli CFT073 are associated with virulent uropathogenic strains.

Urinary tract infection is the most frequently diagnosed kidney and urologic disease, and Escherichia coli is by far the most common etiologic agent. Defined blocks of DNA termed pathogenicity islands have been found in uropathogenic strains to carry genes not generally found in fecal strains. We have identified one of these regions of DNA within the chromosome of the highly virulent E. coli CFT073, isolated from the blood and urine of a woman with acute pyelonephritis. This strain, which is cytotoxic for cultured renal cells and causes acute pyelonephritis in transurethrally infected CBA mice, contains two distinct copies of the pap operon and is hemolytic. One pap operon was localized on a cosmid clone which was used to identify three overlapping cosmid clones. By using restriction mapping, DNA hybridization, sequencing, and PCR amplification, a region of approximately 50 kb was found to be present in this uropathogenic strain and to have no corresponding sequences in E. coli K-12. This gene block also carries hemolysin genes hlyCABD. The pathogenicity island begins 7 bp downstream of dadX (catabolic alanine racemase; 26.55 min) and ends at a position in the K-12 genome 75 bp downstream of the metV tRNA gene (62.74 min); this suggests that a chromosomal rearrangement has occurred relative to the K-12 linkage map. The junctions of the pathogenicity island were verified by PCR amplification directly from the genomic DNA of strain CFT073. DNA sequencing within the boundaries of the junctions revealed genes not previously identified in E. coli or in some cases bearing no known homologs. When used as probes for DNA hybridization, these sequences were found significantly more often in strains associated with the clinical syndromes of cystitis (82%) and acute pyelonephritis (79%) than in fecal strains (19%; P < 0.001).

Adult↗
PubMed1986

Molecular cloning and analysis of genes for production of K5, K7, K12, and K92 capsular polysaccharides in Escherichia coli.

With a DNA fragment from within the region encoding the transport functions for K1 production as a hybridization probe in Southern blot experiments, homologous DNA sequences were detected in the DNA from Escherichia coli strains producing K5, K7, K92, and K100 capsular polysaccharides. No homology with the laboratory strain LE392 was detected. The same DNA probe was used to prescreen cosmid libraries in LE392 by colony hybridization, as a rapid method to isolate clones encoding the genes for K5, K7, K12, and K92 antigen production. Clones carrying sequences homologous to the probe that also produced capsular material were identified by using polyclonal and monoclonal antibodies raised against the K antigen in question and K antigen-specific phages. By restriction enzyme mapping of the appropriate cosmid clones it was possible to align the genes for the production of different K antigens in terms of common restriction endonuclease cleavage sites. A DNA fragment encoding the postulated transport functions for K7 antigen production could complement deletion mutations in the transport functions for K1 antigen production. Thus the transport to the cell surface of chemically distinct polysaccharides may be by a common process. Analysis in E. coli of the proteins produced by plasmids carrying the likely transport functions for K1, K5, and K7 antigen production revealed that each region coded for a similar polypeptide.

Antigens, Bacterial↗
PubMed1987

Pseudomonas solanacearum genes controlling both pathogenicity on tomato and hypersensitivity on tobacco are clustered.

A pLAFR3 cosmid clone designated pVir2 containing a 25-kilobase (kb) DNA insert was isolated from a wild-type Pseudomonas solanacearum GMI1000 genomic library. This cosmid was shown to complement all but one of the nine Tn5-induced mutants which have been isolated after random mutagenesis and which have lost both pathogenicity toward tomato and ability to induce hypersensitive reaction (HR) on tobacco (hrp mutants). The insert is colinear with the genome and provides restoration of the HR-inducing ability when transferred into several Tn5-induced hrp mutants, but failed to complement deletion mutants extending on both sides of the pVir2 region. Localized mutagenesis demonstrated that the hrp genes are clustered within a 17.5-kb region of pVir2 and that this cluster probably extends on the genomic region adjacent to the pVir2 insert. A 3-kb region adjacent to the hrp cluster modulates aggressiveness toward tomato but does not control HR-inducing ability. Sequences within the hrp cluster of pVir2 have homology with the genomic DNA of Xanthomonas campestris strains representing eight different pathovars, suggesting that a set of common pathogenicity functions could be shared by P. solanacearum and X. campestris.

Cloning, Molecular↗
PubMed1987

Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea.

A wide-host-range cosmid cloning vector, pLAFR3, was constructed and used to make cosmid libraries of partially digested Sau3A DNA from race 0 and race 1 of Pseudomonas syringae pv. glycinea. Two avirulence genes, avrB0 and avrC, cloned from race 0, elicited the hypersensitivity reaction (HR) on specific cultivars of soybean. Race 4 transconjugants containing avrB0 induced a dark brown necrotic HR within 24 h on the soybean cultivars Harosoy and Norchief, whereas race 4 transconjugants containing avrC induced a light brown necrotic HR within 48 h on the soybean cultivars Acme, Peking, Norchief, and Flambeau. An additional avirulence gene, avrB1, cloned from race 1, appeared to be identical to avrB0 from race 0. The avrB0 and avrC genes from race 0 were characterized by restriction enzyme mapping, Southern blot analysis, Tn5 transposon mutagenesis, and site-directed gene replacements. The effects of these three genes on the in planta bacterial growth of race 4 transconjugants have also been examined. The identification and cloning of avrB1 provides genetic evidence for a gene-for-gene interaction in the bacterial blight disease of soybean, as avrB1 from race 1 interacts with the soybean disease resistance locus, Rpg1.

Cloning, Molecular↗
PubMed1987

Molecular cloning of copper resistance genes from Pseudomonas syringae pv. tomato.

A cosmid library of copper-resistant (Cur) Pseudomonas syringae pv. tomato PT23 plasmid DNA was constructed and mobilized into the copper-sensitive recipient P. syringae pv. syringae PS61. One resultant cosmid clone, pCOP1 (46 kilobases), conferred copper resistance. The PT23 Cur gene(s) was located on pCOP1 by subcloning PstI restriction endonuclease fragments of pCOP1 in the broad-host-range vector pRK404. A subclone containing a 4.4-kilobase PstI fragment conferred Cur on PS61. The Cur gene(s) was further located by insertional inactivation with Tn5. A subcloned fragment internal to the Cur determinant on pCOP2 was probed to plasmid and chromosomal DNA of four copper-resistant and three copper-sensitive strains of P. syringae pv. tomato. The probe hybridized to plasmids in resistant strains, but showed no detectable homology to copper-sensitive strains.

Cloning, Molecular↗
PubMed1987

A genetic locus essential for formate-dependent growth of Bradyrhizobium japonicum.

A genetic locus essential for the formate-dependent growth of Bradyrhizobium japonicum was isolated by complementation of ethyl methanesulfonate-induced mutants with a cosmid gene library of B. japonicum DNA. Three related cosmids containing 18.7 kilobase pairs of B. japonicum DNA in common were identified as being able to restore formate-dependent growth capability to mutants lacking either ribulosebisphosphate carboxylase or both ribulosebisphosphate carboxylase and phosphoribulokinase activities. To further localize the complementing gene(s), a series of four deletions spanning a total of 16.1 kilobase pairs were introduced into the B. japonicum chromosome. Each resulting deletion mutant lacked formate dehydrogenase activity and lacked ribulosebisphosphate carboxylase activity and immunologically detectable protein. Three of the four also lacked phosphoribulokinase activity. Two other mutants in which the deletion-bearing recombinant plasmid had integrated into the chromosome also lacked ribulosebisphosphate carboxylase activity and protein and phosphoribulokinase activities. The genetic locus defined by these mutants could contain the structural genes for these enzymes or a regulatory gene(s) controlling their expression or both.

Cloning, Molecular↗
PubMed1988

Deletion of a 55-kilobase-pair DNA element from the chromosome during heterocyst differentiation of Anabaena sp. strain PCC 7120.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 produces terminally differentiated heterocysts in response to a lack of combined nitrogen. Heterocysts are found approximately every 10th cell along the filament and are morphologically and biochemically specialized for nitrogen fixation. At least two DNA rearrangements occur during heterocyst differentiation in Anabaena sp. strain PCC 7120, both the result of developmentally regulated site-specific recombination. The first is an 11-kilobase-pair (kb) deletion from within the 3' end of the nifD gene. The second rearrangement occurs near the nifS gene but has not been completely characterized. The DNA sequences found at the recombination sites for each of the two rearrangements show no similarity to each other. To determine the topology of the rearrangement near the nifS gene, cosmid libraries of vegetative-cell genomic DNA were constructed and used to clone the region of the chromosome involved in the rearrangement. Cosmid clones which spanned the DNA separating the two recombination sites that define the ends of the element were obtained. The restriction map of this region of the chromosome showed that the rearrangement was the deletion of a 55-kb DNA element from the heterocyst chromosome. The excised DNA was neither degraded nor amplified, and its function, if any, is unknown. The 55-kb element was not detectably transcribed in either vegetative cells or heterocysts. The deletion resulted in placement of the rbcLS operon about 10 kb from the nifS gene on the chromosome. Although the nifD 11-kb and nifS 55-kb rearrangements both occurred under normal aerobic heterocyst-inducing conditions, only the 55-kb excision occurred in argon-bubbled cultures, indicating that the two DNA rearrangements can be regulated differently.

Chromosome Deletion↗
PubMed1988

Physical and functional analyses of the syrA and syrB genes involved in syringomycin production by Pseudomonas syringae pv. syringae.

The syrA and syrB genes involved in syringomycin production in Pseudomonas syringae pv. syringae B301D were identified from an EcoRI-pLAFR3 cosmid library and then physically and functionally analyzed in relation to plant pathogenicity. Homologous recombination of the genes required for syringomycin production from cosmids pGX183 (syrA) and pGX56 (syrB), respectively, introduced into nontoxigenic (Tox-) Tn5 mutants W4S2545 and W4S770 resulted in the concomitant restoration of toxin production and full virulence. The disease indices of the Tox+ strains obtained by recombination of the cloned, homologous DNA into the corresponding Tn5 mutant were essentially equivalent to that of strain B301D-R and significantly higher than those of W4S2545 and W4S770. A 12-kilobase (kb) EcoRI fragment from pGX183 was subcloned (i.e., pGX15) and found to contain the sequences necessary for syringomycin production. A map of pGX15 prepared by a combination of restriction endonuclease digestions and Tn5 mutagenesis showed that the syrA sequence was 2.3 to 2.8 kb. Marker exchange of syrA::Tn5 from pGX15 into B301D-R yielded nonpathogenic phenotypes, indicating that syrA is a regulatory gene since it is necessary for both syringomycin production and pathogenicity. The 4.9-kb EcoRI fragment from pGX56 was subcloned (i.e., pGX4) and shown to carry the syrB sequence which was 2.4 to 3.3 kb. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of protein extracts from B301D-R associated five proteins, ranging from approximately 130,000 to approximately 470,000 in molecular weight, with syringomycin production. The syrA and syrB genes were required for the formation of proteins SR4 (approximately 350,000) and SR5 (approximately 130,000), which are believed to be components of the syringomycin synthetase complex.

Bacterial Proteins↗
PubMed1988

Rhizobium meliloti genes required for C4-dicarboxylate transport and symbiotic nitrogen fixation are located on a megaplasmid.

A mutant of Rhizobium meliloti unable to transport C4 dicarboxylates (dct) was isolated after Tn5 mutagenesis. The mutant, 4F6, could not grow on aspartate or the tricarboxylic acid cycle intermediates succinate, fumarate, or malate. It produced symbiotically ineffective nodules on Medicago sativa in which bacteroids appeared normal, but the symbiotic zone was reduced and the plant cells contained numerous starch granules at their peripheries. Cosmids containing the dct region were obtained by selecting those which restored the ability of 4F6 to grow on succinate. The Tn5 insertion in 4F6 was found to be within a 5.9-kilobase (kb) EcoRI fragment common to the complementing cosmids. Site-specific Tn5-mutagenesis revealed dct genes in a segment of DNA about 4 kb in size extending from within the 5.9-kb EcoRI fragment into an adjacent 2.9-kb EcoRI fragment. The 4F6 mutation was found to be in a complementation group in which mutations yielded a Fix- phenotype, whereas other dct mutations in the region resulted in mutants which produced effective nodules in most, although not all, plant tests (partially Fix-). The dct region was found to be located on a megaplasmid known to carry genes required for exopolysaccharide production.

Cosmids↗
PubMed1988

Cloning of the Rhodobacter capsulatus hemA gene.

Portions of the Rhodobacter capsulatus hemA gene have been cloned from a hemA::Tn5 insertion strain into the lambda bacteriophage derivative EMBL3. A cosmid containing the wild-type R. capsulatus hemA gene was isolated by complementation of the hemA::Tn5 mutant. The cosmid contains a 1.4-kilobase EcoRI fragment that spans the hemA::Tn5 insertion site. The entire hemA gene is contained in this fragment and the adjacent 0.6-kilobase EcoRI fragment.

5-Aminolevulinate Synthetase↗