Roles of horizontal gene transfer and gene integration in evolution of 1,3-dichloropropene- and 1,2-dibromoethane-degradative pathways

The haloalkane-degrading bacteria Rhodococcus rhodochrous NCIMB13064, Pseudomonas pavonaceae 170, and Mycobacterium sp. strain GP1 share a highly conserved haloalkane dehalogenase gene (dhaA). Here, we describe the extent of the conserved dhaA segments in these three phylogenetically distinct bacteria and an analysis of their flanking sequences. The dhaA gene of the 1-chlorobutane-degrading strain NCIMB13064 was found to reside within a 1-chlorobutane catabolic gene cluster, which also encodes a putative invertase (invA), a regulatory protein (dhaR), an alcohol dehydrogenase (adhA), and an aldehyde dehydrogenase (aldA). The latter two enzymes may catalyze the oxidative conversion of n-butanol, the hydrolytic product of 1-chlorobutane, to n-butyric acid, a growth substrate for many bacteria. The activity of the dhaR gene product was analyzed in Pseudomonas sp. strain GJ1, in which it appeared to function as a repressor of dhaA expression. The 1,2-dibromoethane-degrading strain GP1 contained a conserved DNA segment of 2.7 kb, which included dhaR, dhaA, and part of invA. A 12-nucleotide deletion in dhaR led to constitutive expression of dhaA in strain GP1, in contrast to the inducible expression of dhaA in strain NCIMB13064. The 1, 3-dichloropropene-degrading strain 170 possessed a conserved DNA segment of 1.3 kb harboring little more than the coding region of the dhaA gene. In strains 170 and GP1, a putative integrase gene was found next to the conserved dhaA segment, which suggests that integration events were responsible for the acquisition of these DNA segments. The data indicate that horizontal gene transfer and integrase-dependent gene acquisition were the key mechanisms for the evolution of catabolic pathways for the man-made chemicals 1, 3-dichloropropene and 1,2-dibromoethane.     

Degradation of 1,2-dibromoethane by Mycobacterium sp. strain GP1

The newly isolated bacterial strain GP1 can utilize 1, 2-dibromoethane as the sole carbon and energy source. On the basis of 16S rRNA gene sequence analysis, the organism was identified as a member of the subgroup which contains the fast-growing mycobacteria. The first step in 1,2-dibromoethane metabolism is catalyzed by a hydrolytic haloalkane dehalogenase. The resulting 2-bromoethanol is rapidly converted to ethylene oxide by a haloalcohol dehalogenase, in this way preventing the accumulation of 2-bromoethanol and 2-bromoacetaldehyde as toxic intermediates. Ethylene oxide can serve as a growth substrate for strain GP1, but the pathway(s) by which it is further metabolized is still unclear. Strain GP1 can also utilize 1-chloropropane, 1-bromopropane, 2-bromoethanol, and 2-chloroethanol as growth substrates. 2-Chloroethanol and 2-bromoethanol are metabolized via ethylene oxide, which for both haloalcohols is a novel way to remove the halide without going through the corresponding acetaldehyde intermediate. The haloalkane dehalogenase gene was cloned and sequenced. The dehalogenase (DhaAf) encoded by this gene is identical to the haloalkane dehalogenase (DhaA) of Rhodococcus rhodochrous NCIMB 13064, except for three amino acid substitutions and a 14-amino-acid extension at the C terminus. Alignments of the complete dehalogenase gene region of strain GP1 with DNA sequences in different databases showed that a large part of a dhaA gene region, which is also present in R. rhodochrous NCIMB 13064, was fused to a fragment of a haloalcohol dehalogenase gene that was identical to the last 42 nucleotides of the hheB gene found in Corynebacterium sp. strain N-1074.     

Degradation of 1,3-Dichloropropene by Pseudomonas cichorii 170

The gram-negative bacterium Pseudomonas cichorii 170, isolated from soil that was repeatedly treated with the nematocide 1,3-dichloropropene, could utilize low concentrations of 1,3-dichloropropene as a sole carbon and energy source. Strain 170 was also able to grow on 3-chloroallyl alcohol, 3-chloroacrylic acid, and several 1-halo-n-alkanes. This organism produced at least three different dehalogenases: a hydrolytic haloalkane dehalogenase specific for haloalkanes and two 3-chloroacrylic acid dehalogenases, one specific for cis-3-chloroacrylic acid and the other specific for trans-3-chloroacrylic acid. The haloalkane dehalogenase and the trans-3-chloroacrylic acid dehalogenase were expressed constitutively, whereas the cis-3-chloroacrylic acid dehalogenase was inducible. The presence of these enzymes indicates that 1,3-dichloropropene is hydrolyzed to 3-chloroallyl alcohol, which is oxidized in two steps to 3-chloroacrylic acid. The latter compound is then dehalogenated, probably forming malonic acid semialdehyde. The haloalkane dehalogenase gene, which is involved in the conversion of 1,3-dichloropropene to 3-chloroallyl alcohol, was cloned and sequenced, and this gene turned out to be identical to the previously studied dhaA gene of the gram-positive bacterium Rhodococcus rhodochrous NCIMB13064. Mutants resistant to the suicide substrate 1,2-dibromoethane lacked haloalkane dehalogenase activity and therefore could not utilize haloalkanes for growth. PCR analysis showed that these mutants had lost at least part of the dhaA gene.     

Isolation of Rhodococcus rhodochrous NCIMB13064 derivatives with new biodegradative abilities

Abstract Rhodococcus rhodochrous NCIMB 13064 can dehalogenate and utilise a number of halogenated aliphatic compounds as sole carbon and energy source. Mutants of NCIMB13064 can be easily isolated with an enlarged range of 1-chloroalkane utilising ability. Dehalogenation of 1-chlorononane, 1-chlorodecane and short-chain 1-chloroalkanes (C₃-C₈) is encoded by the same plasmid pRTL1. However, a different genetic element(s) is required for the dehalogenation of 3-chloropropionic acid. Two derivatives (P200 and P400) of R. rhodochrous NCIMB 13064 were isolated which had acquired the ability to utilise naphthalene as sole carbon and energy source. Both strains lost the ability to utilise short-chain 1-chloroalkanes and underwent some rearrangements associated with pRTLl plasmid.     

Catalytic mechanism of the maloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26

Haloalkane dehalogenases are bacterial enzymes capable of carbon-halogen bond cleavage in halogenated compounds. To obtain insights into the mechanism of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), we studied the steady-state and presteady-state kinetics of the conversion of the substrates 1-chlorohexane, chlorocyclohexane, and bromocyclohexane. The results lead to a proposal of a minimal kinetic mechanism consisting of three main steps: (i) substrate binding, (ii) cleavage of the carbon-halogen bond with simultaneous formation of an alkyl-enzyme intermediate, and (iii) hydrolysis of the alkyl-enzyme intermediate. Release of both products, halide and alcohol, is a fast process that was not included in the reaction mechanism as a distinct step. Comparison of the kinetic mechanism of LinB with that of haloalkane dehalogenase DhlA from Xantobacter autotrophicus GJ10 and the haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 shows that the overall mechanisms are similar. The main difference is in the rate-limiting step, which is hydrolysis of the alkylenzyme intermediate in LinB, halide release in DhlA, and liberation of an alcohol in DhaA. The occurrence of different rate-limiting steps for three enzymes that belong to the same protein family indicates that extrapolation of this important catalytic property from one enzyme to another can be misleading even for evolutionary closely related proteins. The differences in the rate-limiting step were related to: (i) number and size of the entrance tunnels, (ii) protein flexibility, and (iii) composition of the halide-stabilizing active site residues based on comparison of protein structures.     

Utilization of trihalogenated propanes by Agrobacterium radiobacter AD1 through heterologous expression of the haloalkane dehalogenase from Rhodococcus sp. strain M15-3.

Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp. strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better. Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 under the control of the heterologous promoters P(lac), P(dhlA), and P(trc). The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes.     

Nonconventional hydrolytic dehalogenation of 1-chlorobutane by dehydrated bacteria in a continuous solid-gas biofilter

Rhodococcus erythropolis NCIMB 13064 and Xanthobacter autotrophicus GJ10 are able to catalyze the conversion of halogenated hydrocarbons to their corresponding alcohols. These strains are attractive biocatalysts for gas phase remediation of polluted gaseous effluents because of their complementary specificity for short or medium and for mono-, di-, or trisubstituted halogenated hydrocarbons (C2-C8 for Rhodococcus erythropolis and C1-C4 for Xanthobacter autotrophicus).After dehydration, these bacteria can catalyze the hydrolytic dehalogenation of 1-chlorobutane in a nonconventional gas phase system under a controlled water thermodynamic activity (a(w)). This process makes it possible to avoid the problems of solubility and bacterial development due to the presence of water in the traditional biofilters.In the aqueous phase, the dehalogenase activity of Rhodococcus erythropolis is less sensitive to thermal denaturation and the apparent Michaelis-Menten constants at 30 degrees C were 0.4 mM and 2.40 micromol min(-1) g(-1) for Km and Vmax, respectively. For Xanthobacter autotrophicus they were 2.8 mM and 0.35 micromol min(-1) g(-1). In the gas phase, the behavior of dehydrated Xanthobacter autotrophicus cells is different from that observed with Rhododcoccus erythropolis cells. The stability of the dehalogenase activity is markedly lower. It is shown that the HCl produced during the reaction is responsible for this low stability. Contrary to Rhodococcus erythropolis cells, disruption of cell walls does not increase the stability of the dehalogenase activity.The activity and stability of lyophilized Xanthobacter autotrophicus GJ10 cells are dependant on various parameters. Optimal dehalogenase activity was determined for water thermodynamic activity (a(w)) of 0.85. A temperature of 30 degrees C offers the best compromise between activity and stability. The pH control before dehydration plays a role in the ionization state of the dehalogenase in the cells. The apparent Michaelis-Menten constants Km and Vmax for the dehydrated Xanthobacter autotrophicus cells were 0.07 (1-chlorobutane thermodynamic activity) and 0.08 micromol min(-1) g(-1) of cells, respectively. A maximal transformation capacity of 1.4 g of 1-chlorobutane per day was finally obtained using 1g of lyophilized Xanthobacter autotrophicus GJ10 cells.     

Degradation of 1,2-dichloroethane by Ancylobacter aquaticus and other facultative methylotrophs.

Cultures of the newly isolated bacterial strains AD20, AD25, and AD27, identified as strains of Ancylobacter aquaticus, were capable of growth on 1,2-dichloroethane (DCE) as the sole carbon and energy source. These strains, as well as two other new DCE utilizers, were facultative methylotrophs and were also able to grow on 2-chloroethanol, chloroacetate, and 2-chloropropionate. In all strains tested, DCE was degraded by initial hydrolytic dehalogenation to 2-chloroethanol, followed by oxidation by a phenazine methosulfate-dependent alcohol dehydrogenase and an NAD-dependent aldehyde dehydrogenase. The resulting chloroacetic acid was converted to glycolate by chloroacetate dehalogenase. The alcohol dehydrogenase was induced during growth on methanol or DCE in strain AD20, but no activity was found during growth on glucose. However, in strain AD25 the enzyme was synthesized to a higher level during growth on glucose than on methanol, and it reached levels of around 2 U/mg of protein in late-exponential-phase cultures growing on glucose. The haloalkane dehalogenase was constitutively produced in all strains tested, but strain AD25 synthesized the enzyme at a level of 30 to 40% of the total cellular protein, which is much higher than that found in other DCE degraders. The nucleotide sequences of the haloalkane dehalogenase (dhlA) genes of strains AD20 and AD25 were the same as the sequence of dhlA from Xanthobacter autotrophicus GJ10 and GJ11. Hybridization experiments showed that the dhlA genes of six different DCE utilizers were all located on an 8.3-kb EcoRI restriction fragment, indicating that the organisms may have obtained the dhlA gene by horizontal gene transmission.     

Degradation of 1,2-dichloroethane by Ancylobacter aquaticus and other facultative methylotrophs.

Cultures of the newly isolated bacterial strains AD20, AD25, and AD27, identified as strains of Ancylobacter aquaticus, were capable of growth on 1,2-dichloroethane (DCE) as the sole carbon and energy source. These strains, as well as two other new DCE utilizers, were facultative methylotrophs and were also able to grow on 2-chloroethanol, chloroacetate, and 2-chloropropionate. In all strains tested, DCE was degraded by initial hydrolytic dehalogenation to 2-chloroethanol, followed by oxidation by a phenazine methosulfate-dependent alcohol dehydrogenase and an NAD-dependent aldehyde dehydrogenase. The resulting chloroacetic acid was converted to glycolate by chloroacetate dehalogenase. The alcohol dehydrogenase was induced during growth on methanol or DCE in strain AD20, but no activity was found during growth on glucose. However, in strain AD25 the enzyme was synthesized to a higher level during growth on glucose than on methanol, and it reached levels of around 2 U/mg of protein in late-exponential-phase cultures growing on glucose. The haloalkane dehalogenase was constitutively produced in all strains tested, but strain AD25 synthesized the enzyme at a level of 30 to 40% of the total cellular protein, which is much higher than that found in other DCE degraders. The nucleotide sequences of the haloalkane dehalogenase (dhlA) genes of strains AD20 and AD25 were the same as the sequence of dhlA from Xanthobacter autotrophicus GJ10 and GJ11. Hybridization experiments showed that the dhlA genes of six different DCE utilizers were all located on an 8.3-kb EcoRI restriction fragment, indicating that the organisms may have obtained the dhlA gene by horizontal gene transmission.     

Plasmid pRTL1 Controlling 1-Chloroalkane Degradation by Rhodococcus rhodochrous NCIMB13064

Rhodococcus rhodochrous NCIMB13064 can dehalogenate and use a wide range of 1-haloalkanes as sole carbon and energy source. The 1-chloroalkane degradation phenotype may be lost by cells spontaneously or after treatment with Mitomycin C. Two laboratory derivatives of the original strain exhibited differing degrees of stability of the chloroalkane degradation marker. Plasmids of approximately 100 kbp (pRTL1) and 80 kbp (pRTL2) have been found in R. rhodochrous NCIMB13064. pRTL1 was shown to be carrying at least some genes for the dehalogenation of 1-chloroalkanes with short chain lengths (C₃ to C₉). However, no connection was found between the utilization of 1-chloroalkanes with longer chain lengths (C₁₂ to C₁₈) and the presence of pRTL1. Three separate events were observed to lead to the inability of NCIMB13064 to dehalogenate the short-chain 1-chloroalkanes; the complete loss of pRTL1, the integration of pRTL1 into the chromosome, or the deletion of a 20-kbp fragment in pRTL1. High-frequency transfer of the 1-chloroalkane degradation marker associated with pRTL1 has been demonstrated in bacterial crosses between different derivatives of R. rhodochrous NCIMB13064.     

Bacillus methanolicus sp. nov., a new species of thermotolerant, methanol-utilizing, endospore-forming bacteria.

The generic position of 14 strains of gram-positive bacteria able to use methanol as a growth substrate was determined. All are obligately aerobic, thermotolerant organisms that are able to grow at temperatures of 35 to 60 degrees C. Nine of the strains produce oval spores at a subterminal-to-central position in slightly swollen rod-shaped cells. DNA-DNA hybridization studies, 5S rRNA sequence analysis, and physiological characteristics revealed that all 14 strains cluster as a well-defined group and form a distinct new genospecies. Analysis of the 16S and 5S rRNA sequences indicated that this new species is distinct from Bacillus brevis but closely related to B. firmus and B. azotoformans. The name proposed for this new species is B. methanolicus. The type strain, PB1, has been deposited in the National Collection of Industrial and Marine Bacteria as NCIMB 13113.     

Localization and physical mapping of a plasmid-borne 23-kb nif gene cluster from Enterobacter agglomerans showing homology to the entire nif gene cluster of Klebsiella pneumoniae M5a1

A physical and genetical map of the plasmid pEA3 indigenous to Enterobacter agglomerans is presented. pEA3 is a 111-kb large plasmid containing a 23-kb large cluster of nif genes which shows extensive homology (Southern hybridization and heteroduplex analysis) to the entire nif gene cluster of Klebsiella pneumoniae (Kp) M5a1. All the nif genes on pEA3 are organized in the same manner as in K. pneumoniae, except nifJ, which is located on the left end of pEA3 nif gene cluster (near nifQB). A BamHI restriction map of pEA3 and a detailed restriction map of the 23-kb nif region on pEA3 is also presented. The nif genes of pEA3 showed a low level of acetylene reduction in Escherichia coli, demonstrating that these genes are functional and contain the whole genetic information required to fix nitrogen. The origin of vegetative replication (OriV) of pEA3 was localized about 5.5 kb from the right end of the nif gene cluster. In addition to pEA3, large plasmids from four other strains of E. agglomerans showed homology to all the Kp nif genes tested, indicating that in diazotrophic strains of E. agglomerans nif genes are usually located on plasmids. In contrast, in most of the free-living, nitrogen-fixing bacteria the nif genes are on chromosome.     

Occurrence of Tn4371-related mobile elements and sequences in (chloro)biphenyl-degrading bacteria

Tn4371, a 55-kb transposable element involved in the degradation and biphenyl or 4-chlorobiphenyl identified in Ralstonia eutropha A5, displays a modular structure including a phage-like integrase gene (int), a Pseudomonas-like (chloro)biphenyl catabolic gene cluster (bph), and RP4- and Ti-plasmid-like transfer genes (trb) (C. Merlin, D. Springael, and A. Toussaint, Plasmid 41:40-54, 1999). Southern blot hybridization was used to examine the presence of different regions of Tn4371 in a collection of (chloro)biphenyl-degrading bacteria originating from different habitats and belonging to different bacterial genera. Tn4371-related sequences were never detected on endogenous plasmids. Although the gene probes containing only bph sequences hybridized to genomic DNA from most strains tested, a limited selection of strains, all beta-proteobacteria, displayed hybridization patterns similar to the Tn4371 bph cluster. Homology between Tn4371 and DNA of two of those strains, originating from the same area as strain A5, extended outside the catabolic genes and covered the putative transfer region of Tn4371. On the other hand, none of the (chloro)biphenyl degraders hybridized with the outer left part of Tn4371 containing the int gene. The bph catabolic determinant of the two strains displaying homology to the Tn4371 transfer genes and a third strain isolated from the A5 area could be mobilized to a R. eutropha recipient, after insertion into an endogenous or introduced IncP1 plasmid. The mobilized DNA of those strains included all Tn4371 homologous sequences previously identified in their genome. Our observations show that the bph genes present on Tn4371 are highly conserved between different (chloro)biphenyl-degrading hosts, isolated globally but belonging mainly to the beta-proteobacteria. On the other hand, Tn4371-related mobile elements carrying bph genes are apparently only found in isolates from the environment that provided the Tn4371-bearing isolate A5.     

Identification of the fire blight pathogen, Erwinia amylovora, by PCR assays with chromosomal DNA.

Erwinia amylovora, the causative agent of fire blight, was identified independently from the common plasmid pEA29 by three different PCR assays with chromosomal DNA. PCR with two primers was performed with isolated DNA and with whole cells, which were directly added to the assay mixture. The oligonucleotide primers were derived from the ams region, and the PCR product comprised the amsB gene, which is involved in exopolysaccharide synthesis. The amplified fragment of 1.6 kb was analyzed, and the sequence was found to be identical for two E. amylovora strains. The identity of the PCR products was further confirmed by restriction analysis. The 1.6-kb signal was also used for detection of the fire blight pathogen in the presence of other plant-associated bacteria and in infected plant tissue. For further identification of isolated strains, the 16S rRNA gene of E. amylovora and other plant-associated bacteria was amplified and the products were digested with the restriction enzyme HaeIII. The pattern obtained for E. amylovora was different from that of other bacteria. The sequence of the 16S rRNA gene was determined from a cloned fragment and was found to be closely related to the sequences of Escherichia coli and other Erwinia species. Finally, arbitrarily primed PCR with a 17-mer oligonucleotide derived from the sequence of transposon Tn5 produced a unique banding pattern for all E. amylovora strains investigated. These methods expand identification methods for E. amylovora, which include DNA hybridization and a PCR technique based on plasmid pEA29.     

Involvement of a large plasmid in the degradation of 1,2-dichloroethane by Xanthobacter autotrophicus.

Xanthobacter autotrophicus GJ10 is a bacterium that can degrade short-chain halogenated aliphatic compounds such as 1,2-dichloroethane. A 200-kb plasmid, pXAU1, was isolated from this strain and shown to contain the dhlA gene, which codes for haloalkane dehalogenase, the first enzyme in the degradation pathway of 1,2-dichloroethane by GJ10. Loss of pXAU1 resulted in loss of haloalkane dehalogenase activity, significantly decreased chloroacetaldehyde dehydrogenase activity, and loss of resistance to mercuric chloride but did not affect the activity level of haloalkanoate dehalogenase, the second dehalogenase in the degradation of 1,2-dichloroethane.     

Involvement of a large plasmid in the degradation of 1,2-dichloroethane by Xanthobacter autotrophicus

Xanthobacter autotrophicus GJ10 is a bacterium that can degrade short-chain halogenated aliphatic compounds such as 1,2-dichloroethane. A 200-kb plasmid, pXAU1, was isolated from this strain and shown to contain the dhlA gene, which codes for haloalkane dehalogenase, the first enzyme in the degradation pathway of 1,2-dichloroethane by GJ10. Loss of pXAU1 resulted in loss of haloalkane dehalogenase activity, significantly decreased chloroacetaldehyde dehydrogenase activity, and loss of resistance to mercuric chloride but did not affect the activity level of haloalkanoate dehalogenase, the second dehalogenase in the degradation of 1,2-dichloroethane.     

rRNA gene restriction patterns as taxonomic tools for the genus Aeromonas.

In the genus Aeromonas there are at least 13 DNA hybridization groups, which are difficult to differentiate biochemically. We investigated the usefulness of rRNA gene restriction patterns for characterization and identification of the various groups. Genomic DNA was digested with restriction endonuclease SmaI, transferred to a nylon membrane, and hybridized with biotinylated plasmid pKK3535 containing the rrnB operon of Escherichia coli. The SmaI bands at 0.8 to 4 kb but not those at positions corresponding to sizes larger than 4 kb showed a good correlation with hybridization groups, allowing identification of strains to the level of genetic species. We demonstrated that the 567-bp fragment localized between positions 80 and 647 of the 16S ribosomal gene of E. coli was essential for hybridization to the low-molecular-weight fragments, whereas the remainder of the operon did not hybridize to these fragments. On the basis of these results, we concluded that the Aeromonas chromosome contains multiple rRNA operons which may be used for species identification.     

Physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1403.

A combined physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1403 was determined. We constructed a restriction map for the NotI, ApaI, and SmaI enzymes. The order of the restriction fragments was determined by using the randomly integrative plasmid pRL1 and by performing indirect end-labeling experiments. The strain IL1403 chromosome was found to be circular and 2,420 kb in size. A total of 24 chromosomal markers were mapped on the chromosome by performing hybridization experiments with gene probes for L. lactis and various other bacteria. Integration of pRC1-derived plasmids via homologous recombination allowed more precise location of some lactococcal genes and allowed us to determine the orientation of these genes on the chromosome. Recurrent sequences, such as insertion elements and rRNA gene (rrn) clusters, were also mapped. At least seven copies of IS1076 were present and were located on 50% of the chromosome. In contrast, no copy of ISS1RS was detected. Six ribosomal operons were found on the strain IL1403 chromosome; five were located on 16% of the chromosome and were transcribed in the same direction. A comparison of the physical maps of L. lactis subsp. lactis IL1403 and DL11 showed that these two strains are closely related and that the variable regions are located mainly near the rrn gene clusters. In contrast, despite major restriction pattern dissimilarities between L. lactis IL1403 and MG1363, the overall genetic organization of the genome seems to be conserved between these two strains.     

Cloning of 1,2-dichloroethane degradation genes of Xanthobacter autotrophicus GJ10 and expression and sequencing of the dhlA gene.

A gene bank from the chlorinated hydrocarbon-degrading bacterium Xanthobacter autotrophicus GJ10 was prepared in the broad-host-range cosmid vector pLAFR1. By using mutants impaired in dichloroethane utilization and strains lacking dehalogenase activities, several genes involved in 1,2-dichloroethane metabolism were isolated. The haloalkane dehalogenase gene dhlA was subcloned, and it was efficiently expressed from its own constitutive promoter in strains of a Pseudomonas sp., Escherichia coli, and a Xanthobacter sp. at levels up to 30% of the total soluble cellular protein. A 3-kilobase-pair BamHI DNA fragment on which the dhlA gene is localized was sequenced. The haloalkane dehalogenase gene was identified by the known N-terminal amino acid sequence of its product and found to encode a 310-amino-acid protein of molecular weight 35,143. Upstream of the dehalogenase gene, a good ribosome-binding site and two consensus E. coli promoter sequences were present.