Epoxidation of alkenes by alkene-grown Xanthobacter spp.

Summary Newly isolated Xanthobacter spp. were able to grow on the gaseous alkenes like ethene, propene, 1-butene and 1,3-butadiene. Resting-cell suspensions of propene-, 1-butene- or 1,3-butadiene-grown Xanthobacter Py10 accumulated 1,2-epoxyethane from ethene. Ethene-grown Xanthobacter Py10 did not produce any 1,2-epoxyalkane from the alkenes tested. Furthermore, propenegrown Xanthobacter Py2 accumulated 2,3-epoxybutane from trans-butene and cis-butene but did not form epoxides from other substrates tested.     

Isolation and characterization of alkene-utilizing Xanthobacter spp.

Yellow-pigmented bacteria showing typical characteristics of Xanthobacter spp. were isolated from enrichments with propene and 1-butene, using classical techniques. The generation time for growth on propene and 1-butene of these bacteria ranged from 5 to 7h. A NADH-dependent mono-oxygenase was identified in cell-free extract of Xanthobacter Py2. This mono-oxygenase was not influenced by potential inhibitors tested indicating that propene mono-oxygenase is different from other hydrocarbon mono-oxygenases described until now. Nitrogenase activity could be measured using the acetylene reduction assay with propene as energy source, because acetylene did not inhibit the mono-oxygenase activity.     

Identification of an indigenous plasmid carrying a gene for trimethoprim resistance in Pseudomonas syringae pv. glycinea

Summary Pseudomonas syringae pv. glycinea Race 8 strain PgB3 is naturally resistant to trimethoprim (Tp) at concentrations up to 500 g/ml. A genomic library of total PgB3 DNA was constructed by ligating EcoRI-restricted DNA into the EcoRI site of the cosmid vector, pLAFR1, packaging the DNA in vitro into bacteriophage lambda, and transducing E. coli DH1 cells. Of 960 cosmid clones selected for resistance to tetracycline, six were resistant to trimethoprim at 500 g/ml. An insert into pLAFR1 of about 9.4 kb was shown to be consistently present in the tirmethoprim-resistant clones. Southern blot analysis using radioactively labeled insert DNA as probe indicated that the 9.4 kb fragment hybridized only with a 40 kb indigenous plasmid from PgB3 designated pPg2.     

Comparative Study of the Ability of Three Xanthobacter Species To Metabolize Cycloalkanes

The ability of three species of Xanthobacter to metabolize cyclohexane and its derivatives has been compared. Xanthobacter flavus was unable to utilize any of the cycloalkanes under investigation. X. autotrophicus was unable to utilize cyclohexane but was able to grow with a limited range of substituted cycloalkanes, including cyclohexanol and cyclohexanone. Comparison of a previously isolated cyclohexane growing Xanthobacter sp. with X. flavus and X. autotrophicus indicated it to be closely related to X. autotrophicus. Studies with cell-free extracts have indicated that the route of metabolism for cyclohexanol by X. autotrophicus is the same as that shown for the cyclohexane growing Xanthobacter sp., proceeding via cyclohexanol→cyclohexanone→ ε-caprolactone→→ adipic acid. A comparison of the cyclohexanol dehydrogenase found in X. autotrophicus with that found in the cyclohexane-growing Xanthobacter sp. indicated these enzymes to be distinctly different from one another on the basis of substrate specificity, molecular weight, and pH optima. The cyclohexanone monooxygenase enzymes found in the two bacteria were also found to be different when the pH optima and cofactor specificity of the two enzymes were compared. Preliminary genetic studies on the cyclohexane-growing Xanthobacter sp. have indicated that there are no plasmids present in this bacterium. The presence of RP4 in the Xanthobacter sp. can be detected following its conjugation with an RP4-carrying Escherichia coli strain.     

Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid pLAFR1

A genomic library was prepared in Escherichia coli from DNA of wild-type Xanthomonas campestris pv. campestris (aetiological agent of crucifer black rot), partially digested with endonuclease EcoRI, using the mobilisable broad host range cosmid vector pLAFR1. Recombinant plasmids contained inserts ranging in size from 19.1 to 32.3 kb (mean 26.6). Certain of the clones complemented E. coli auxotrophic markers. Using the narrow host range plasmid pRK2013 as a helper the pooled recombinant plasmids were transferred conjugally to X. c. campestris mutants, and clones were identified which restored yellow pigmentation to white mutants, prototrophy to amino acid auxotrophs and pathogenicity towards turnip plants to two non-pathogenic mutants. The lesion in one mutant (8288, complemented by the plasmid pIJ3000) is unknown. However mutant 8237 is defective in production of extracellular protease and polygalacturonate lyase and restoration of pathogenicity by complementation with the plasmid pIJ3020 concomitantly restored both enzyme levels to wild-type values.     

Azospirillum brasilense locus coding for phosphoenolpyruvate:fructose phosphotransferase system and global regulation of carbohydrate metabolism.

Mutants of Azospirillum brasilense unable to grow on fructose include ones affected only on fructose (Fru-) and others impaired on many or all carbohydrates through interference with induction of their specific pathways (Car-). Both types of mutants could be complemented by a cosmid in broad-host-range vector pLAFR1 containing a 27.5-kb genomic insert, Car(-)-complementing activity depending on a 2.2-kb fragment, and Fru(-)-complementing activity depending on an overlapping 9.6-kb fragment.     

Batch cultivation of Xanthobacter Py2 on 1-pentene

Summary The propene isolated strain Xanthobacter Py2 was able to grow on 1-pentene. The biomass yield for growth under 1-pentene limiting conditions was 0.48 (Ceq/Ceq). Upon storage at both +4°C and -20°C no loss of enzymatic epoxide degrading activity in resting cell suspensions was observed after a month. However, activity decay was pronounced during the stationary phase of growth as well as under reaction conditions.     

Propene removal from synthetic waste gas using a hollow-fibre membrane bioreactor

 Hollow-fibre modules containing microporous membrane material were evaluated as bioreactors for waste gas treatment. The reactors were inoculated with the propene-utilizing strain Xanthobacter Py2, which formed a biofilm on the inner side of the fibres. The removal of the poorly soluble volatile propene from synthetic waste gas was monitored for up to 170 days. The maximum removal rates were 70–110 g propene per m³ reactor per hour. A gas residence time of 80 s was required to remove 95% of an initial propene concentration of 0.84 g/m³. The presence of ammonium in the liquid medium resulted in the development of an additional population of nitrifying organisms. Therefore, nitrate was used as the source of nitrogen in later experiments. During long-term operation, the propene removal rates gradually decreased. At low liquid velocities (1–5 cm/s) clogging of individual fibres with excess biomass was observed. Elevation of the liquid velocity in the fibres to 90 cm/s resulted in the formation of a dense biofilm and prevented clogging of the fibres. However, also at this high liquid velocity a gradual decrease in propene removal rate was observed. These results suggest that aging of biofilms is a very important factor in long-term operation of hollow-fibre bioreactors. Received: 24 November 1995 / Received revision: 14 February 1996 / Accepted: 20 February 1996     

Complementation of mutations in Escherichia coli and Bordetella pertussis by B. pertussis DNA cloned in a broad-host-range cosmid vector

A gene library of Bordetella pertussis DNA was constructed in Escherichia coli using the broad-host-range cosmid vector pLAFR1. The average insert size was 24.9 kb. From 500 members of the gene library, clones were identified which complemented trpE, glnA and Thr- mutations in E. coli but none which complemented trpD, trpC, trpB, trpA, proA or Leu- mutations. Four clones were identified which complemented trpE in E. coli. Anthranilate synthase activity was detected in a trpE strain only when it harboured a plasmid from one of these clones; activity was repressed when tryptophan was included in the growth medium. Two clones were identified which complemented glnA of E. coli. A recombinant plasmid from one of these clones also restored some of the nitrogen acquisition functions of glnG and glnL in E. coli. Expression of several B. pertussis virulence-associated products (haemolysin, heat-labile toxin, adenylate cyclase, filamentous haemagglutinin, and the cell-envelope polypeptide of Mr 30,000) was not detected in 500 independent clones. However, by transferring the recombinant plasmids to a mutant of B. pertussis deficient in haemolysin and adenylate cyclase, a plasmid was identified which restored both these activities.     

Denitrifying activity of Xanthobacter autotrophicus strains isolated from a submerged fixed-film reactor

Xanthobacter autotrophicus strains with the ability to reduce nitrate and nitrite to either nitrous oxide or molecular nitrogen were isolated from submerged fixed-film reactors. Isolated strains were Gram-negative rods able to grow on methanol, ethanol and sucrose. The yellow cellular pigmentation, pleomorphic appearance, and the presence of poly-β-hydroxybutyrate granules suggest that the organisms might belong to the genus Xanthobacter. Comparison of 16S rDNA gene sequences demonstrated the affiliation of the strains to X. autotrophicus species. The results show that X. autotrophicus may play a role in inorganic nitrogen removal from a denitrifying submerged filter used for the treatment of contaminated groundwater. To our knowledge, no data on denitrifying activity in X. autotrophicus strains have been reported previously.     

Cloning of genes involved in the biosynthesis of pseudobactin, a high-affinity iron transport agent of a plant growth-promoting Pseudomonas strain

A gene bank of DNA from plant growth-promoting Pseudomonas sp. strain B10 was constructed using the broad host-range conjugative cosmid pLAFR1. The recombinant cosmids contained insert DNA averaging 21.5 kilobase pairs in length. Nonfluorescent mutants of Pseudomonas sp. strain B10 were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, or UV light and were defective in the biosynthesis of its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin. No yellow-green, fluorescent mutants defective in the production of pseudobactin were identified. Nonfluorescent mutants were individually complemented by mating the gene bank en masse and identifying fluorescent transconjugants. Eight recombinant cosmids were sufficient to complement 154 nonfluorescent mutants. The pattern of complementation suggests that a minimum of 12 genes arranged in four gene clusters is required for the biosynthesis of pseudobactin. This minimum number of genes seems reasonable considering the structural complexity of pseudobactin.     

Population Dynamics of Two Toluene Degrading Bacterial Species in a Contaminated Stream

Toluene uptake by a benthic biofilm community was previously shown to vary seasonally from 0.03 m hr⁻¹ in winter to 0.2 m hr⁻¹ in summer in a solvent-contaminated stream of the Aberjona watershed. We used quantitative PCR to estimate the population dynamics of previously isolated species of toluene-degrading Xanthobacter autotrophicus and Mycobacterium sp. in both toluene-contaminated and uncontaminated reaches of the stream, and to estimate their relative roles in overall biodegradation rate. Quantification using specific 16S rDNA primers for X. autotrophicus and Mycobacterium sp. showed that populations of both species were much larger in the toluene-contaminated than the toluene-free reach, in agreement with earlier culture-based investigations. A relatively brief bloom of X. autotrophicus occurred in the contaminated reach in the summer, while Mycobacterium sp. populations occurred at elevated densities for more than 5 months. Calculations showed that Mycobacterium, previously thought to be less important than Xanthobacter in annual toluene degradation based on single time-point CFU estimates, appears actually more important because of this longer persistence.     

The alkene monooxygenase from Xanthobacter Py2 is a binuclear non-haem iron protein closely related to toluene 4-monooxygenase

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter Py2 have been sequenced. The predicted amino acid sequence of the first ORF shows homology with the iron binding subunits of binuclear non-haem iron containing monooxygenases including benzene monooxygenase, toluene 4-monooxygenase (>60% sequence similarity) and methane monooxygenase (>40% sequence similarity) and that the necessary sequence motifs associated with iron co-ordination are also present. Secondary structure prediction based on the amino acid sequence showed that the predominantly α-helical structure that surrounds the binuclear iron binding site was conserved allowing the sequence to be modelled on the co-ordinates of the methane monooxygenase α-subunit. Significant differences in the residues forming the hydrophobic cavity which forms the substrate binding site are discussed with reference to the differences in reaction specificity and stereospecificity of binuclear non-haem iron monooxygenases.     

Molecular cloning of a gene which regulates the adaptive response to alkylating agents in Escherichia coli

Summary The ada ⁺ gene of E. coli is a regulatory gene of the adaptive response to simple alkylating agents. ada mutants are sensitive to both the mutagenicity and toxicity of alkylating agents, and are unable to induce O⁶-methylguanine DNA methyltransferase and 3-methyladenine DNA glycosylase II. The ada ⁺ gene was cloned from wild type E. coli B by ligating bacterial DNA partially digested with Sau3A into the cosmid vector pJB8. The hybrid cosmid, pCS33, conveyed N-methyl-N-nitro-N-nitrosoguanidine resistance to ada mutants of E. coli B and E. coli K12, and resulted in the constitutive synthesis of the two DNA repair enzymes at high levels. An alk mutation, which results in a deficiency of only the DNA glycosylase, was not complemented by this cosmid. It was concluded that the product of the ada ⁺ gene is a positive regulator of the adaptive response. The cosmid insert DNA was subcloned into the plasmid vector pAT153, and the ada ⁺ plasmids pCS42 and pCS58 selected. The ada ⁺ gene located in PCS58 by transposon mutagenesis and subcloning. Two polypeptides of Mr 37,000 and 27,000, were identified in maxicells as products of the ada ⁺ gene(s). It is as yet unclear whether they represent different forms of the same gene product, or are encoded by separate ada ⁺ genes within the same operon.     

Functional expression of Aquaspirillum magnetotacticum genes in Escherichia coli K12

Summary Gene libraries from the magnetotactic bacterium, Aquaspirillum magnetotacticum were constructed in Escherichia coli with cosmids pLAFR3 and c2RB as vectors. Recombinant cosmids able to complement the thr-1, leuB, and proA mutations of the host were identified. The Pro⁺ recombinant cosmid restored wild-type phenotype in proA and proB but not in the proC mutants of E. coli. The results of restriction endonuclease digestion and Southern hybridization analysis indicate that the relevent leu and pro biosynthetic genes of A. magnetotacticum are not closely linked on the chromosome.     

The alkene monooxygenase from Xanthobacter strain Py2 is closely related to aromatic monooxygenases and catalyzes aromatic monohydroxylation of benzene, toluene, and phenol

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2. Sequencing and analysis of the predicted amino acid sequences indicate that the components of Xamo are homologous to those of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and benzene monooxygenase, and that the gene order is identical. The genes and predicted polypeptides are aamA, encoding the 497-residue oxygenase alpha-subunit (XamoA); aamB, encoding the 88-residue oxygenase gamma-subunit (XamoB); aamC, encoding the 122-residue ferredoxin (XamoC); aamD, encoding the 101-residue coupling or effector protein (XamoD); aamE, encoding the 341-residue oxygenase beta-subunit (XamoE); and aamF, encoding the 327-residue reductase (XamoF). A sequence with >60% concurrence with the consensus sequence of sigma54 (RpoN)-dependent promoters was identified upstream of the aamA gene. Detailed comparison of XamoA with the oxygenase alpha-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase from Rhodococcus rhodochrous B276 showed that, despite the overall similarity to the aromatic monooxygenases, XamoA has some distinctive characteristics of the oxygenases which oxidize aliphatic, and particularly alkene, substrates. On the basis of the similarity between Xamo and the aromatic monooxygenases, Xanthobacter strain Py2 was tested and shown to oxidize benzene, toluene, and phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did not. Benzene was oxidized to phenol, which accumulated transiently before being further oxidized. Toluene was oxidized to a mixture of o-, m-, and p-cresols (39.8, 18, and 41.7%, respectively) and a small amount (0.5%) of benzyl alcohol, none of which were further oxidized. In growth studies Xanthobacter strain Py2 was found to grow on phenol and catechol but not on benzene or toluene; growth on phenol required a functional alkene monooxygenase. However, there is no evidence of genes encoding steps in the metabolism of catechol in the vicinity of the aam gene cluster. This suggests that the inducer specificity of the alkene monooxygenase may have evolved to benefit from the naturally broad substrate specificity of this class of monooxygenase and the ability of the host strain to grow on catechol.     

Cloning of genes involved in the production of bacteriocin in Cicer–Rhizobium PR 2109a

The molecular basis of bacteriocin production by a Cicer–Rhizobium strain PR2109a was studied. The bacterial strain showed in vitro growth inhibition of non-bacteriocin producing strain of Cicer–Rhizobium PR2005b. Tn5 mutagenesis of the wild-type strain helped in the isolation of the bacteriocin-defective mutant JN365. A genomic library of the wild-type strain was constructed in the cosmid vector pLAFR1 and maintained in Escherchia coli background. Complementation analysis with the cosmid library resulted in the isolation of a cosmid clone which complemented the defective character in the mutant JN365. The size of the complementary DNA fragment was found to be 23 kb.     

Synthesis of 5-Alkyl-5-methyl-2-hydroxy-2-cyclopentenones

A hydrogen bacterium strain, N34, and its oxygen-resistant segregant strain, Y38, were subjected to a taxonomical study. Since both strains were capable of N₂-fixation, N₂-fixing facultative hydrogen autotrophs listed in “Bergey’s Manual of Systematic Bacteriology” were used for comparison. Both strains produced a water-insoluble carotenoid pigment, zeaxanthin dirhamnoside, indicating that both should be classified into the genus Xanthobacter. Then, the differential characteristics of the two species of the genus Xanthobacter, X. autotrophicus and X. flavus, were investigated as to both strains. The vitamin requirement, the sensitivity to oxygen under autotrophic conditions, the inducibility of hydrogenase, the substrate range of carbohydrates and N₂-fixing growth characteristics of both strains were almost completely opposite to those of X. flavus. Moreover, both strains coincided exactly with X. autotrophicus in morphological and other physiological characteristics. From these results both strains were identified as Xanthobacter autotrophicus.     

Cloning of DNA fragments carrying hydrogenase genes of Rhodopseudomonas capsulata

A cosmid library of Rhodopseudomonas capsulata DNA was constructed in Escherichia coli HB101 using the broad-host-range cosmid vector pLAFR1. More than ninety per cent of the clones in the bank contained cosmids with DNA inserts averaging 20 kilobase pairs in length. Mutants deficient in uptake hydrogenase (Hup-) were obtained from R. capsulata strain B10 by ethylmethylsulfonate (EMS) mutagenesis. The content of hydrogenase protein in Hup- mutant cells was tested by rocket immunoelectrophoresis. Hup- mutants (Rifr) were complemented with the clone bank by conjugation and, from the transconjugants selected by rifampicin and tetracycline resistance, Hup+ transconjugants were screened for the ability to grow photoautotrophically and to reduce methylene blue in a colony assay. The recombinant plasmid pAC57 restored hydrogenase activity in the Hup- mutants RCC8, RCC10, RCC12 and ST410 whereas pAG202 restored that of IR4. The cloned R. capsulata DNA insert of pAC57 gave 5 restriction fragments by cleavage with EcoRI endonuclease. Fragment 1 (7 kb) restored hydrogenase activity in Hup- mutant strains RCC12 and ST410 and fragment 5 (1.3 kb) in strains RCC8 and RCC10. Since the 2 cosmids pAC57 and pAG202 are different cosmids, as indicated by restriction analyses and absence of cross hybridization, it is concluded that at least two hup genes are required for the expression of hydrogenase activity in R. capsulata.     

Cloning of Protocatechuate 3,4-Dioxygenase Genes from Bradyrhizobium japonicum USDA110

A heterologous gene probe encoding the α and β subunits of the Pseudomonas cepacia protocatechuate 3,4-dioxygenase (PCD) was used to detect its homolog in the genome of Bradyrhizobium japonicum USDA110. Three cosmid clones carrying a 2.2-kb BamHI insert showed high levels of PCD activity. SacI digestion of one of the genomic clones, pBjG17, produced a 2.5-kb insert DNA that complemented a PCD mutant of P. cepacia.