An improved transconjugation protocol for Bacillus megaterium facilitating a direct genetic knockout

We provide a simple but very efficient transconjugation protocol for Bacillus megaterium. By combining utile attributes of known transconjugation methods (small size of the transferred DNA, close physical contact between donor and recipient cells, and heat treatment of the latter) and by determining the appropriate donor/recipient ratio, mating approaches yielded 5 × 10⁻⁵ transconjugants/recipient. Counter-selection for eliminating Escherichia coli donor cells from the mating mixture was possible by pasteurization in case a wild type sporulation proficient B. megaterium served as the mating partner. For nonsporulating mutants, the sacB gene from Bacillus subtilis coding for levansucrase was successfully employed to select against the E. coli donor. The transfer efficiency, up to 15,000 transconjugants acquirable in a single experiment, sufficed—for the first time in this species—to directly select a gene (uvrA) knockout in a one-step procedure. By making use of a mobilizable B. megaterium suicide vector, ten out of the 40 sampled putative transconjugants displayed the expected UV sensitivity and were found to harbor the suicide vector at the anticipated position. Along with the soon available information arising from current B. megaterium sequencing projects, the possibility to quickly inactivate genetic loci will considerably speed up genetic work with this biotechnologically relevant species.     

Natural transformation of a marineVibrio species by plasmid DNA

Vibrio sp. DI9, recently isolated from Tampa Bay, FL, has been found to be naturally transformed by the broad host range plasmid pKT230 in both filter transformation assays and sterile sediment microcosms. This is the first report of natural transformation by plasmid DNA of aVibrio sp. and of a marine bacterial isolate. Transformation frequencies ranged from 0.3 to 3.1×10^–8 transformants per recipient. Transformants were detected by both plating and by selection for growth in liquid medium in the presence of streptomycin and kanamycin and confirmed by probing of southern transfers. Transformation was enhanced by multimeric forms of the plasmid. A technique using sediment microcosms, mixed populations ofVibrio sp. DI9 and another antibiotic resistant organism, and enrichment in liquid media has been developed which allows detection of transformation at frequencies too low to be detected by plating. This technique may serve as a model for the detection of natural transformation in the environment. These results suggest that natural transformation may be one mechanism of horizontal plasmid transfer in the marine environment, and may provide the methodology with which to detect this process in natural populations of bacteria.     

Heterologous Expression and Purification of Recombinant Allophycocyanin in Marine Streptomyces sp. Isolate M097

Summary Allophycocyanin is one of the most important marine active peptides. Previous studies suggested that recombinant allophycocyanin (rAPC) could remarkably inhibit the S-180 carcinoma in mice, indicating its potential pharmaceutical uses. Based on intergeneric conjugal transfer, heterologous expression of rAPC was first achieved in marine Streptomyces sp. isolate M097 through inserting the apc gene into the thiostrepton-induced vector pIJ8600. The transformation frequency for this system was approximately 10⁻⁴ exconjugants/recipient. In the transformed Streptomyces sp. isolate M097, the yield of purified rAPC could amount to about 38 mg/l using a simple purification protocol, and HPLC analysis showed that the purity of the protein reached about 91.5%. In vitro activity tests also revealed that the purified rAPC had effective scavenging abilities on superoxide and hydroxyl radicals. This would widen the usefulness of the marine Streptomyces as a host to express the rAPC and to offer industrial strain for the production of rAPC.     

Conjugative transfer and autonomous replication of a promiscuous IncQ plasmid in the cyanobacterium Synechocystis PCC 6803

Summary The promiscuous IncQ plasmid pKT210 (Cm^r, Sm^r) is efficiently transferred by transpecific conjugation from Escherichia coli to the facultatively heterotrophic cyanobacterium Synechocystis PCC6803 when mobilized by a helper plasmid coding for IncP transfer functions. The IncQ plasmid is stably maintained in the cyanobacterium as an autonomously replicating multicopy plasmid with no detectable structural alterations and can be recovered by transformation back to E. coli when using a mcrA mcrB host. Thus, the replicative host-range of IncQ plasmids extends beyond purple bacteria to the distinct procaryotic taxon of cyanobacteria, allowing the use of these small plasmids as convenient cloning vectors in Synechocystis PCC6803 and presumably also in cyanobacteria that are not amenable to genetic transformation. In contrast, an IncQ plasmid bearing the TRP1 gene of Saccharomyces cerevisiae failed to replicate when transferred to that yeast by transformation.     

Ammonia assimilation enzymes in a thermophilicBacillus sp. of marine origin

The physiology of ammonia assimilation enzymes was examined inBacillus sp. FE-1, a thermophilic marine bacterium. Glutamine synthetase (GS) and glutamate synthase (GOGAT) activities varied with the nitrogen source present in the medium, ranging as much as 10-fold for the former and 2.5-fold for the latter. Glutamate dehydrogenase (GDH) was detected but, under the growth conditions studied, levels were not affected by the nitrogen source. Anaerobic growth in the presence of nitrate yielded enzyme levels that were not significantly different from those measured under aerobic growth. Partially purified GS exhibited a temperature optimum between 65° and 75°C. The enzyme's Mn²⁺-dependent reverse transferase activity was stimulated by K₂SO₄ and demonstrated some tolerance to NaCl. Hyperbolic kinetics were observed for ammonium, with an apparentK _M of 1.0mm.     

Mercury Resistance in Bacterial Strains Isolated from Tailing Ponds in a Gold Mining Area Near El Callao (Bolívar State,Venezuela)

Bacterial resistance to mercury (Hg) was investigated in strains isolated from Hg-contaminated tailing ponds located in the gold mining area of El Callao (Bolívar State, Venezuela). High frequencies of resistance were detected to both inorganic-Hg and organomercurials among these strains. A broad range of resistance levels was observed when determining minimal inhibitory concentrations of Hg²⁺. Some strains were able to grow in liquid medium containing 25 μM Hg²⁺, whereas others grew at 300 μM Hg²⁺. Of 190 Hg-resistant strains tested, 58.2% were additionally shown to be resistant to ampicillin (40 mg/L), 33.3% to chloramphenicol (30 mg/L), 24.9% to streptomycin (30 mg/L), 23.3% to tetracycline (30 mg/L), and 1.6% to kanamycin (30 mg/L). Furthermore, we found that 20% of the Hg-resistant strains were simultaneously resistant to as many as four of these antibiotics, at the concentrations tested. The presence of large plasmids in 62.9% of 53 Hg-resistant strains screened prompted us to investigate the horizontal transfer of resistance determinants. Mating experiments were performed using Escherichia coli and Pseudomonas aeruginosa as recipient strains. The results obtained confirmed that indigenous Hg-resistant bacteria colonizing the tailing ponds can effectively transfer the phenotype to potentially pathogenic species.     

Trehalose-Producing Enzymes MTSase and MTHase in <Emphasis Type="Italic">Anabaena</Emphasis> 7120 Under NaCl Stress

Salt tolerance, a multigenic trait, necessitates knowledge about biosynthesis and function of candidate gene(s) at the cellular level. Among the osmolytes, trehalose biosynthesis in cyanobacteria facing NaCl stress is little understood. Anabaena 7120 filaments exposed to 150 mm NaCl fragmented and recovered on transfer to –NaCl medium with the increased heterocysts frequency (7%) over the control (4%). Cells failed to retain Na⁺ beyond a threshold [2.19 mm/cm³ (PCV)]. Whereas NaCl-stressed cells exhibited a marginal rise in K⁺ (1.1-fold) only at 30 h, for Na⁺ it was 130-fold at 48 h over cells in control. A time-course study (0–54 h) revealed reduction in intracellular Na⁺ beyond 48 h [0.80 mm/cm³ (PCV)] suggestive of ion efflux. The NaCl-stressed cells showed differential expression of maltooligosyltrehalose synthase (MTSase; EC 5.4.99.15) and maltooligosyltrehalose trehalohydrolase (MTHase; EC 3.2.1.141) depending on the time and the extent of intracellular Na⁺ buildup.     

Bioactive hydroxyphenylpyrrole-dicarboxylic acids from a new marine Halomonas sp.: production and structure elucidation

The new marine Halomonas sp. strain GWS-BW-H8hM (DSM 17996) was found to produce 3-(4′-hydroxyphenyl)-4-phenylpyrrole-2,5-dicarboxylic acid (HPPD-1) and 3,4-bis(4′-hydroxy- phenyl)pyrrole-2,5-dicarboxylic acid (HPPD-2). In initial cultivations using marine broth, only low contents of these compounds have been isolated. Improving the conditions and growing the strain on artificial seawater supplemented with tryptone 10 g l⁻¹, yeast extract 5 g l⁻¹, l-tyrosine 0.6 g l⁻¹, glycine 1 g l⁻¹, and glucose 6 g l^-1, the growth-associated HPPD-1 and HPPD-2 production of a 40-l batch cultivation reached the amounts of 47 mg l⁻¹ and 116 mg l⁻¹, respectively, after 65 h. Both compounds showed potent anti-tumor-promoting activities.     

Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.     

Transformation of freshwater and marine caulobacters by electroporation.

We performed plasmid electrotransformation of Caulobacter crescentus strains and obtained up to 3 x 10(8) transformants per micrograms of pKT230. The presence and integrity of the paracrystalline protein surface (S) layer influenced electroporation; caulobacters lacking the S layer were electrotransformed 10 times more efficiently than caulobacters possessing the S layers. A procedure yielding 1,500 transformants per micrograms of pKT230 was developed for a marine caulobacter. Electroporation was used in combination with several genetic techniques, including introduction of ligation mixtures, suicide transposon mutagenesis, gene replacement, and plasmid electrotransfer from Escherichia coli to caulobacters.     

Characterization of Candida sp. NY7122, a novel pentose-fermenting soil yeast

Yeasts that ferment both hexose and pentose are important for cost-effective ethanol production. We found that the soil yeast strain NY7122 isolated from a blueberry field in Tsukuba (East Japan) could ferment both hexose and pentose (d-xylose and l-arabinose). NY7122 was closely related to Candida subhashii on the basis of the results of molecular identification using the sequence in the D1/D2 domains of 26S rDNA and 5.8S-internal transcribed spacer region. NY7122 produced at least 7.40 and 3.86 g l⁻¹ ethanol from 20 g l⁻¹ d-xylose and l-arabinose within 24 h. NY7122 could produce ethanol from pentose and hexose sugars at 37°C. The highest ethanol productivity of NY7122 was achieved under a low pH condition (pH 3.5). Fermentation of mixed sugars (50 g l⁻¹ glucose, 20 g l⁻¹ d-xylose, and 10 g l⁻¹ l-arabinose) resulted in a maximum ethanol concentration of 27.3 g l⁻¹ for the NY7122 strain versus 25.1 g l⁻¹ for Scheffersomyces stipitis. This is the first study to report that Candida sp. NY7122 from a soil environment could produce ethanol from both d-xylose and l-arabinose.     

Transfer of the chromosomally encoded tetracycline resistance gene <Emphasis Type="Italic">tet</Emphasis>(M) from marine bacteria to <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Enterococcus faecalis</Emphasis>

The transferability of the tetracycline (TC) resistance gene tet(M) from marine bacteria to human enteric bacteria was examined by a filter-mating method. Vibrio spp., Lactococcus garvieae, Bacillus spp., Lactobacillus sp., and Paenibacillus sp. were used as donors, and Escherichia coli JM109 and Enterococcus faecalis JH2-2 were used as recipients. The combination of Vibrio spp. and E. coli resulted in 5/68 positive transconjugants with a transfer rate of 10⁻⁷ to 10⁻³; however, no transfer was observed with E. faecalis. In case of L. garvieae and E. faecalis, 6/6 positive transconjugants were obtained with a transfer rate of 10⁻⁶ to 10⁻⁵; however, no transfer was observed with E. coli. The tet(M) gene of Bacillus, Lactobacillus, and Paenibacillus were not transferred to either E. coli or E. faecalis. tet(M) transfer was confirmed in positive E. coli and E. faecalis transconjugants by polymerase chain reaction (PCR) and Southern hybridization. All the donor strains did not harbor plasmids, while they all harbored transposon Tn916. In the transconjugants, the transposon was not detected by PCR, suggesting the possible transfer of tet(M) from the marine bacterial chromosome to the recipient chromosome. This is the first report to show that tet(M) can be transferred from marine bacteria to human enteric bacteria in a species-specific manner.     

Ornithinibacillus scapharcae sp. nov., isolated from a dead ark clam

A novel Gram-positive, aerobic, motile, hemolytic, endospore-forming and rod-shaped bacterium TW25^T was isolated from a dead ark clam during a mass mortality event on the South coast of Korea. The strain grew optimally at 30°C, at pH 8–9, and with 1% (w/v) NaCl. The 16S rRNA gene sequence analysis indicated that strain TW25^T was associated with the genus Ornithinibacillus and that it was most closely related to the type strain of Ornithinibacillus californiensis (98.5% similarity). The dominant cellular fatty acids were iso-C15:0, anteiso-C15:0 and C16:0. The peptidoglycan amino acid type was A4β, containing l-ornithine and d-aspartic acid. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, four unidentified phospholipids, two unidentified aminolipids and two unidentified lipids. The major respiratory quinone was menaquinone-7 (MK-7). The G + C content of genomic DNA was 36.7 mol%. DNA–DNA hybridization experiments with related strains revealed lower than 11 ± 3% relatedness. Based on this polyphasic taxonomic study, strain TW25^T represents a novel species in the genus Ornithinibacillus, for which the name Ornithinibacillus scapharcae sp. nov. is proposed. The type strain is TW25^T (=KACC 15116^T = JCM 17314^T).     

Biological active metabolite cyclo (l-Trp-l-Phe) produced by South China Sea sponge Holoxea sp. associated fungus Aspergillus versicolor strain TS08

Sponge-associated fungi represent the single most prolific source of novel natural products from marine fungi. Cyclo (l-Trp-l-Phe) exhibits biological functions such as plant growth regulation, moderate cytotoxicity and thus has the application potential in pharmaceutical and agricultural biotechnologies. In this study, a fungal strain TS08 was isolated from sponge Holoxea sp. in the South China Sea and identified as A. versicolor according to its 18S rRNA gene and morphological, physiological, and biochemical characteristics. Meanwhile, cyclo (l-Trp-l-Phe) was found to be produced by A. versicolor strain TS08 mainly in the exponential growth phase. The highest yield of cyclo (l-Trp-l-Phe), 13.24 mg/g (per crude extract of EtOAc), 2.51% of cell dry weigh, was obtained on the tenth day of the fungal cultivation. It was the first time to find the biological active cyclo (l-Trp-l-Phe) in sponge-associated microorganism.     

Characteristics and transformation of Zymomonas mobilis with plasmid pKT230 by electroporation

Transformation of Zymomonas mobilis with plasmid pKT230 by electroporation was achieved with a transformation efficiency of 9.0?±?1.8?×?10³ per μg plasmid DNA. The growing state of the host cells before transformation, the RC time constant for pulsing at the optimal electric field strength (7.5?kV/cm), the plasmid concentration and the post-incubation time prior to outgrowth in RM medium were the sensitive factors influencing the efficiency of the transformation. The data from batch cultures revealed that the plasmid-harboring cells, Z. mobilis (pKT230), had the same growth pattern as plasmid-free cells. The yield factors of biomass production and ethanol formation by Z. mobilis were nearly unchanged after being transformed and grown in the selective medium where the gene for antibiotic resistance was expressed. The results suggested that the plasmid pKT230 was stable in Z. mobilis and qualified for being a cloning vector in the construction of a recombinant ethanol-producer.     

Bioconversion of L-tyrosine to L-DOPA by a novel bacterium Bacillus sp. JPJ

l-DOPA is an amino acid derivative and most potent drug used against Parkinson’s disease, generally obtained from Mucuna pruriens seeds. In present communication, we have studied the in vitro production of l-DOPA from l-tyrosine by novel bacterium Bacillus sp. JPJ. This bacterium produced 99.4% of l-DOPA from l-tyrosine in buffer (pH 8) containing 1 mg ml⁻¹ cell mass incubated at 40°C for 60 min. The combination of CuSO₄ and l-ascorbic acid showed the inducing effect at concentrations of 0.06 and 0.04 mg ml⁻¹, respectively. The activated charcoal 2 mg ml⁻¹ was essential for maximum bioconversion of l-tyrosine to l-DOPA and the crude tyrosinase activity was 2.7 U mg⁻¹ of tyrosinase. Kinetic studies showed significant values of Y _p/s (0.994), Q _s (0.500) and q _s (0.994) after optimization of the process. The production of l-DOPA was confirmed by analytical techniques such as HPTLC, HPLC and GC–MS. This is the first report on rapid and efficient production of l-DOPA from l-tyrosine by bacterial source which is more effective than the plant, fungal and yeast systems.     

Zhihengliuella salsuginis sp. nov., a moderately halophilic actinobacterium from a subterranean brine

A novel moderately halophilic, alkaliphilic, non-motile, non-sporulating, catalase-positive, oxidase-negative, aerobic, coccus-shaped, Gram-positive bacterium, designated strain JSM 071043^T, was isolated from a subterranean brine sample collected from a salt mine in Hunan Province, China. Growth occurred with 0.5–20% (w/v) NaCl (optimum 5–10%) at pH 6.5–10.5 (optimum pH 8.5) and at 10–40°C (optimum 25–30°C). Good growth also occurred in the presence of 0.5–20% (w/v) KCl (optimum 5–8%) or 0.5–25% (w/v) MgCl₂·6H₂O (optimum 5–10%). The peptidoglycan type was A4α (l-Lys–l-Ala–l-Glu) and major cell-wall sugars were tyvelose and mannose. The major cellular fatty acids were anteiso-C_15:0, iso-C_16:0 and anteiso-C_17:0. Strain JSM 071043^T contained MK-9 and MK-8 as the predominant menaquinones and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the major polar lipids. The DNA G + C content was 67.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JSM 071043^T was a member of the suborder Micrococcineae, and was most closely related to Zhihengliuella halotolerans YIM 70185^T (sequence similarity 98.9%) and Zhihengliuella alba YIM 90734^T (98.2%), and the three strains formed a distinct branch in the phylogenetic tree. The combination of phylogenetic analysis, DNA–DNA relatedness values, phenotypic characteristics and chemotaxonomic data supports the proposal that strain JSM 071043^T represents a novel species of the genus Zhihengliuella, for which the name Z. salsuginis sp. nov. is proposed. The type strain is JSM 071043^T (= DSM 21149^T = KCTC 19466^T).     

Cloning and Sequencing Analysis of Alginate Lyase Genes from the Marine Bacterium Vibrio sp. O2

We isolated a new marine bacteria, which displayed alginate-depolymerizing activity in plate assays, from seawater in Mihonoseki Harbor, Japan. Analysis of the 16S ribosomal RNA gene sequence of one of the isolates proved that this alginate-depolymerizing bacterium belonged to the genus Vibrio and it was named Vibrio sp. O2. The alginate lyase genes of Vibrio sp. O2 were cloned and expressed in Escherichia coli. Two alginate lyase-producing clones, pVOA-A4 and pVOA-B5, were obtained. The alginate lyase gene alyVOA from pVOA-A4 was composed of an 858-bp open reading frame (ORF) encoding 285 amino acid residues, while alyVOB from pVOA-B5 was composed of an 828-bp ORF encoding 275 amino acid residues. The degree of identity between the deduced amino acid sequences of AlyVOA or AlyVOB and Photobacterium sp. ATCC43367 alginate poly(ManA)lyase AlxM was 92.3% or 32.6%, respectively. Alginate lyase consensus regions corresponding to the sequences YFKAGXYXQ and RXELR were observed in all three of these sequences. AlyVOA and AlyVOB both degraded polymannuronate in plate assays and were therefore confirmed to be poly(β-D-mannuronate)lyases.     

Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803

Glutaminase is widely distributed among microorganisms and mammals with important functions. Little is known regarding the biochemical properties and functions of the deamidating enzyme glutaminase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Escherichia coli. The purified protein possessed glutaminase activity, validating the functional assignment of the genomic annotation. The apparent K _m value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na⁺. Moreover, the K _m value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na⁺. These data demonstrate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na⁺ through increasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synechocystis by targeted mutagenesis and the Δslr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between Δslr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, Δslr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosynthetic oxygen evolution rate of Δslr2079 cells was higher than that of the wild-type. To further characterize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in Δslr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimilation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in Δslr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress response by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis. Supported by the National Natural Sciences Foundation of China (Grant No. 30500108) and Hundred Talents Program of Chinese Academy of Sciences.     

Expression of an Erwinia sp. gene encoding diphenyl ether cleavage in Escherichia coli and an isolated Acinetobacter strain PE7

Summary An Acinetobacter strain PE7 with the ability to grow on salicylic acid and to degrade diphenyl ethers was isolated from a petroleum waste pit in Louisiana. A cloned Erwinia sp. dpe gene encoding diphenyl ether cleavage was introduced into PE7 in order to enhance its degradative ability. A broad-host-range expression plasmid, pDPE2388, was constructed by inserting an SspI-HpaI fragment from a dpe gene-containing plasmid, pDPE7321, into the kanamycin resistance gene of plasmid pKT230. The DNA fragment contained the dpe gene flanked between sp6 and T7 promoters. Transconjugants of pDPE2388 plasmid into PE7 were isolated. Expression of the dpe gene in Escherichia coli or PE7 displayed a degradative ability to cleave the following diphenyl ethers: 4-chlorodiphenyl ether, 4-nitrodiphenyl ether, and 4-hydroxydiphenyl ether.Offprint requests to: V. R. Srinivasan