Alkane assimilation ability of Pseudomonas C12B originally isolated for degradation of alkyl sulfate surfactants

Summary Pseudomonas C12B (NCIMB 11753) is able to utilize a broad range of alkyl sulfates. The growth on n-alkanes of different chain lenght (C₆–C₁₆) was tested. Pseudomonas C12B assimilated hydrocarbons from C₉–C₁₆. Growth rate on n-decane (1%) that was chosen as the typical sole source of carbon and energy depended on oxygen supply. The addition of surfactants (Triton X-100 and Tween-80) in a nontoxic concentrations resulted in increased biomass yield. Under optimal growth conditions Pseudomonas C12B exhibited the maximal growth rate and yield with C₁₁ as the sole carbon source.     

Catechol 2,3-Dioxygenase from Pseudomonas sp. Strain ND6: Gene Sequence and Enzyme Characterization

The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5α using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.     

n-Alkane dissimilation by Rhodopseudomonas sphaeroides transferred OCT plasmid

The OCT plasmid from Pseudomonas maltophilia N246-1 was transferred to Rhodopseudomonas sphaeroides M1 with very low frequency (1.4–1.9 × 10^–5 per recipient cell at pH 7–8 for a 3-hour reaction time). P. maltophilia N246-1 was able to utilize C₈-C₁₄ of n-alkanes, whereas R. gas-liquid chromatography determined that the broad range of carbon numbers of n-alkanes in crude oil was remarkably degraded by the transconjugant, R. sphaeroides M1-C1, compared with donor strain N246-1. The fact that donor and transconjugant strains simultaneously lost the capacity to utilize n-alkanes on L-broth medium suggests that the OCT plasmids are unstable. It was found that the OCT plasmid of P. maltophilia N246 was incompatible with the IncP-2 group of P. aeruginosa KCTC 11245. Offprint requests to: K.-H. Min, Sookmyung Women's University.     

Degradation ofn-alkane-1-sulfonates byPseudomonas

We have isolated two strains ofPseudomonas, AJ 1 and AJ 2, growing on the C₄–C₇ and the C₈–C₁₂ n-alkane-1-sulfonates, respectively, as the only source of their carbon and energy. The alkane sulfonates are dissimilated by these strains, ultimately to yield carbon dioxide, water and sulfate. In the primary oxidation reaction(s) the corresponding fatty acid and sulfate are formed. The fatty acid is subsequently degraded by Β-oxidation. For strain AJ 1, growing on the lower alkane sulfonates, we could exclude various pathways of degradation which might have been present in addition to the one mentioned above.     

Alkylsulfonic acids and some S-containing detergents as sulfur sources for growth of Chlorella fusca

Chlorella fusca can utilize the following substances as sole sulfur sources for growth: C₁ to C₈ n-alkane-1-sulfonates, linear alkylbenzenes sulfonates (LAS), -sulfonated fatty acid esters, polyethylene glycol sulfate and alkylsulfates. Good sulfur sources are alkylsulfonic acids, which are comparable to sulfate. Ethanesulfonic acid was used for comparison of the growth on sulfate and on a sulfonic acid, because best growth was achieved on this C₂-sulfonic acid.Growth data of Chlorella on the enviromental important detergents linear alkylbenzene sulfonic acids, -sulfonated fatty acid methylester, Texapon and Sulfopon are presented. So far only microorganisms have been discussed as a source for degradation of sulfonic acids and detergents. It is suggested that green algae could be of similar importance for the biodegradation of these compounds.Abbreviations LAS Linear alkylbenzene sulfonate - ES -sulfonated fatty acid methylester - DTE dithiocrythritol     

Plasmid-encoded dissimilation of condensed tannin in Pseudomonas solanacearum

Pseudomonas solanacearum degrades catechin to phloroglucinolcarboxylic acid, protocatechuic acid, catechol, phloroglucinol, resorcinol and hydroxyquinol, which is plasmid-encoded. This dissimilatory plasmid, designated as pAMB1, was effectively cured by mitomycin C treatment and was transferred to a Pseudomonas sp., with a transfer frequency of 2.2 × 10⁻⁵ transconjugants/donor cell. The cured strains did not utilize catechin or its intermediates, and lacked the plasmid.     

Organomercurial resistance determinants in Pseudomonas K-62 are present on two plasmids

Pseudomonas strain K-62 was found to contain six plasmids. A mutant derivative cured of the 26-kb plasmid showed a higher sensitivity to mercurials; however, the strain was still able to volatilize them. Loss of the 68-kb plasmid.in addition to the 26-kb plasmid abolished the ability of mercury volatilization in this strain and led to a further decrease in the level of mercurial resistance. These results are the first to demonstrate that the organomercurial resistance of Pseudomonas strain K-62 is plasmid-based, and that both the 26- and 68-kb plasmids are required for full expression of the mercurial resistance. Probes specific for the mer genes merA, merB, and merR strongly hybridized with the 26-kb plasmid, but not with the 68-kb plasmid. Two fragments of the 26-kb plasmid that hybridized with the mer genes were cloned and expressed in Escherichia coli. One recombinant plasmid (pMRA17) inducibly encoded a typical broad-spectrum mercurial resistance, whereas the other recombinant plasmid (pMRB01) constitutively conferred hypersensitivity to phenylmercury in the absence of mercuric reductase activity. The results suggest that the two organomercurial lyases in the cells are transcribed from different operator-promoters.     

Papers of interest in other journals

Forty‐seven manganese‐oxidizing bacterial strains, isolated from manganese nodules, sediment, and sea‐water samples collected from the Pacific Ocean and the Mediterranean Sea, were studied to elucidate the role, if any, of plasmids in bacterial manganese oxidation in the marine environment.

Twenty‐two strains of Pseudomonas and seven unidentified species were found to harbor single plasmids. Seven of the plasmid‐containing Pseudomonas spp. and one of the unidentified strains were selected for curing. Only Pseudomonas strain 57, originally isolated from a manganese nodule collected from the Pacific Ocean, was cured successfully. This strain carried a plasmid (pZPl) of about 9 Mdal, and demonstrated enzymatic oxidation of manganese. Although the function ascribable to pZPl remains cryptic, evidence obtained from the study of Pseudomonas strain 57 (carrying pZPl) and its cured derivative suggests that the plasmid encodes resistance to manganese and copper. It is hypothesized that the plasmid (pZPl) provides an ecologically significant strategy for survival in the deep‐sea nodule environment since it encodes for heavy metal resistance associated with the manganese oxidation process.     

The in vivo construction of 4-chloro-2-nitrophenol assimilatory bacteria

Pseudomonas sp. N31 was isolated from soil using 3-nitrophenol and succinate as sole source of nitrogen and carbon respectively. The strain expresses a nitrophenol oxygenase and can use either 2-nitrophenol or 4-chloro-2-nitrophenol as a source of nitrogen, eliminating nitrite, and accumulating catechol and 4-chlorocatechol, respectively. The catechols were not degraded further. Strains which are able to utilize 4-chloro-2-nitrophenol as a sole source of carbon and nitrogen were constructed by transfer of the haloaromatic degrading sequences from either Pseudomonas sp. B13 or Alcaligenes eutrophus JMP134 (pJP4) to strain N31. Transconjugant strains constructed using JMP134 as the donor strain grew on 3-chlorobenzoate but not on 2,4-dichlorophenoxyacetate. This was due to the non-induction of 2,4-dichlorophenoxyacetate monooxygenase and 2,4-dichlorophenol hydroxylase. Transfer of the plasmid from the 2,4-dichlorophenoxyacetate negative transconjugant strains to a cured strain of JMP134 resulted in strains which also had the same phenotype. This indicates that a mutation has occurred in pJP4 to prevent the expression of 2,4-dichlorophenoxyacetate monooxygenase and 2,4-dichlorophenol hydroxylase.     

Improved degradation of 4-chlorobiphenyl, 2,3-dihydroxybiphenyl, and catecholic compounds by recombinant bacterial strains

ThepcbC gene encoding (4-chloro-)2,3-dihydroxybiphenyl dioxygenase was cloned from the genomic DNA ofPseudomonas sp. P20 using pKT230 to construct pKK1. A recombinant strain,E. coli KK1, was selected by transforming the pKK1 intoE. coli XL1-Blue. Another recombinant strain,Pseudomonas sp. DJP-120, was obtained by transferring the pKK1 ofE. coli KK1 intoPseudomonas sp. DJ-12 by conjugation. Both recombinant strains showed a 23.7 to 26.5 fold increase in the degradation activity to 2,3-dihydroxybiphenyl compared with that of the natural isolate,Pseudomonas sp. DJ-12. The DJP-120 strain showed 24.5, 3.5, and 4.8 fold higher degradation activities to 4-chlorobiphenyl, catechol, and 3-methylcatechol than DJ-12 strain, respectively. The pKK1 plasmid of both strains and their ability to degrade 2,3-dihydroxybiphenyl were stable even after about 1,200 generations.     

Evidence for plasmid mediated dissimilation of catechol in Azotobacter chroococcum

Catechol dissimilation in A. chroococcum was encoded by plasmid pMSB1. This self transmissible plasmid was effectively cured by mitomycin C. The cured cells did not harbour the plasmid and failed to utilize catechol.     

Oily sludge degradation by bacteria from Ankleshwar, India

Three bacterial strains, Bacillus sp. SV9, Acinetobacter sp. SV4 and Pseudomonas sp., SV17 from contaminated soil in Ankleshwar, India were tested for their ability to degrade the complex mixture of petroleum hydrocarbons (such as alkanes, aromatics, resins and asphaltenes), sediments, heavy metals and water known as oily sludge. Gravimetric analysis showed that Bacillus sp. SV9 degraded approx. 59% of the oily sludge in 5 days at 30 °C whereas Acinetobacter sp. SV4 and Pseudomonas sp. SV17 degraded 37% and 35%. Capillary gas chromatographic analysis revealed that after 5 days the Bacillus strain was able to degrade oily sludge components of chain length C₁₂–C₃₀ and aromatics more effectively than the other two strains. Maximum drop in surface tension (from 70 to 28.4 mN/m) was accompanied by maximum biosurfactant production (6.7 g l⁻¹) in Bacillus sp. SV9 after 72 h, these results collectively indicating that this bacterial strain has considerable potential for bioremediation of oily sludge.     

Loss of Plasmid-Mediated Oligopeptide Transport System in Lactococci: Another Reason for Slow Milk Coagulation

Fast milk-coagulating (Fmc⁺) strains of lactococci are known to segregate slow milk-coagulating (Fmc⁻) variants, which has been attributed to loss of proteinase (Prt) activity encoded by plasmid DNA. It was found that the Fmc⁻phenotype could also be due to loss of a plasmid encoding an oligopeptide permease (Opp) system. InLactococcus lactissubsp.lactis(L. lactis) C2O, lactose metabolism (Lac) and Prt were linked to pJK550 and the Opp system to pJK430. InLactococcus lactissubsp.cremorisSK11, known to possess Prt on a 78-kb plasmid, DNA sequence analysis of a 7.4-kb region from the Lac plasmid, pSK11L, revealed that it possessed the Opp system. The Lac plasmid inL. lactisC2 encoded both the Prt and Opp systems. Fmc⁻derivatives ofL. lactisC2 were missing theprtgenes and had Opp integrated into the chromosome, possibly due to transposition events. Growth studies showed the Opp systems were functional and, in combination with Prt, produced the Fmc⁺phenotype.     

Plasmid-encoded enzymatic degradation of dimethoate

Dimethoate-degrading enzymatic activity in Bacillus licheniformis, Pseudomonas aeruginosa, Aeromonas hydrophila, Proteus mirabilis and Bacillus pumilus was found to be 6.4, 1.760, 4.09, 1.196 and 0.505 units/mg protein, respectively. The Escherichia coli C600 transconjugants of the isolated bacterial strains also exhibited dimethoate-degrading enzymatic activities. The cured derivatives did not show any decrease in the amount of dimethoate substrate and did not harbour plasmid as found in the original and transconjugant strains. Thus, the ability of enzymatic degradation of dimethoate was plasmid-mediated in B. licheniformis, Ps. aeruginosa, A. hydrophila, P. mirabilis and B. pumilus.     

The atzABC Genes Encoding Atrazine Catabolism Are Located on a Self-Transmissible Plasmid in Pseudomonas sp. Strain ADP

Pseudomonas sp. strain ADP initiates atrazine catabolism via three enzymatic steps, encoded by atzA, -B, and -C, which yield cyanuric acid, a nitrogen source for many bacteria. In-well lysis, Southern hybridization, and plasmid transfer studies indicated that the atzA, -B, and -C genes are localized on a 96-kb self-transmissible plasmid, pADP-1, in Pseudomonas sp. strain ADP. High-performance liquid chromatography analyses showed that cyanuric acid degradation was not encoded by pADP-1. pADP-1 was transferred to Escherichia coli strains at a frequency of 4.7 × 10⁻². This suggests a potential molecular mechanism for the dispersion of the atzABC genes to other soil bacteria.     

Use of sodium dodecyl sulphate for stimulation of biodegradation of n-alkanes without residual contamination by the surfactant

The capacity of a range of aliphatic alkanes (C₆–C₁₆), intermediates of n-decane oxidation and sodium dodecyl sulphate (SDS) to induce decane-mineralization activity in the cells of Pseudomonas C12B was compared with that for n-decane. The comparison on quantitative basis had two serious limitations: low solubility of tested inducers in aqueous solutions and their toxicity to bacterial cells. Carbon chain length and the presence of hydroxyl group were the important factors for induction activity. However, presence of hydroxyl groups at both ends of alkyl chain prevented the induction of decane-mineralization activity. Good induction activity by SDS was caused either by the presence of free end of alkyl chain, or by bacterial hydrolysis of sulphate group to yield alcohol, which in turn served as true inducer. The presence of SDS in the culture medium with n-decane as main source of carbon and energy accelerated the growth of Pseudomonas C12B. SDS disappeared from the culture medium in early stages of cultivation suggesting preferential degradation by the bacterium, while the consumption of n-decane was accelerated. This may be associated with the capacity of SDS to induce decane-mineralization system in Pseudomonas C12B and/or with the ability of SDS to stimulate the surface attachment of competent bacteria resulting in the close proximity of the cells with alkane droplets, and thus, enhanced breakdown of the hydrocarbon pollutant.     

Evaluation of the biological containment system based on the Escherichia coli gef gene in Pseudomonas aeruginosa W51D

The cell-killing gef gene was introduced, under the control of the lac promoter and as part of a transposon, into Pseudomonas aeruginosa W51D, a strain able to degrade branched-chain alkylbenzene sulfonates. Only 1% of the cells that inherited the transposon (Tngef) showed a conditional lethal phenotype, and this phenotype was lost at a high frequency without apparent loss of the tetracycline resistance encoded by the transposon. Southern blot analysis of two W51D::Tngef derivatives that expressed the cell-killing function showed multiple insertions of the transposon. These data suggest that Gef protein is able to kill P. aeruginosa W51D, but it seems that the level of resistance to Gef toxin in this stain is higher than that of previously reported bacteria, and that the expression of multiple copies of the gef gene is necessary to attain cell death. The higher level of resistance does not seem to be particular to strain W51D since two other P. aeruginosa strains analyzed (PAO2003 and ATCC 9027) also presented a small proportion of cells expressing the conditional lethal phenotype when they inherited the Tngef transposon. Received: 11 December 1995 / Received revision: 15 July 1996 / Accepted: 5 August 1996     

Generation of glyoxylate in methylotrophic bacteria

The mode of glyoxylate production from acetyl-CoA was investigated in three strains of methylotrophic bacteria,Pseudomonas MA,Pseudomonas AM1 and organism PAR. This investigation was prompted by the recently reported discovery of a homoisocitrate lyase in methylotrophic bacteria and the suggested involvement of this novel enzyme in assimilation of C₁ and C₂ compounds as part of a homoisocitrate-glyoxylate cycle. We were unable to detect cleavage of any of the four stereoisomers of homoisocitric acid by cell-free extracts of C₁-or C₂-grown bacteria. Extracts of C₁-grown bacteria did not catalyze condensation of glyoxylate with glutarate or production of glyoxylate from acetyl-CoA and 2-ketoglutarate. Extracts of C₁-grownPseudomonas MA catalyzed cleavage of isocitrate;threo-homoisocitrate was a potent competitive inhibitor of this reaction. These results indicate that homoisocitrate cleavage does not occur in any of the methylotrophs tested. The pathway for oxidation of acetyl-CoA to glyoxylate inPseudomonas AM1 and organism PAR therefore remains obscure.     

A general method for identification of polyhydroxyalkanoic acid synthase genes from pseudomonads belonging to the rRNA homology group I

Using a 30-mer oligonucleotide probe highly specific for polyhydroxyalkanoic acid (PHA) synthase genes, the respective genes of Pseudomonas citronellolis, P. mendocina, Pseudomonas sp. DSM 1650 and Pseudomonas sp. GP4BH1 were cloned from genomic libraries in the cosmid pHC79. A 19.5-kbp and a 22.0-kbp EcoRI restriction fragment of P. citronellolis or Pseudomonas sp. DSM 1650, respectively, conferred the ability to accumulate PHA of medium-chain-length 3-hydroxyalkanoic acids (HA _mcl ) from octanoate as well as from gluconate to the PHA-negative mutant P. putida GPp104. An 11.0-kbp EcoRI fragment was cloned from P. mendocina, which restored in GPp104 the ability to synthesize PHA from octanoate but not from gluconate. From Pseudomonas sp. GP4BH1 three different genomic fragments encoding PHA synthases were cloned. This indicated that strain GP4BH1 possesses three different functionally active PHA synthases. Two of these fragments (6.4 kbp and 3.8 kbp) encoded for a PHA synthase, preferentially incorporating hydroxyalkanoic acids of short chain length (HA _scl ), and the synthases were expressed in either GPp104 and Alcaligenes eutrophus H16-PHB^–4, respectively. The PHA synthase encoded by the third fragment (6.5 kbp) led to the incorporation of HA _mcl and was expressed in GPp104 but not in PHB^–4. Correspondence to: A. Steinbüchel     

Isolation of transfer-negative nif-plasmids (pCE1) and their integration into the chromosome of Escherichia coli with the help of phage Mu

Summary Strain JC5466 of Escherichia coli K12 harbouring the nitrogen fixation plasmid pCE1 was lysogenized with bacteriophage Mu cts, followed by partial induction and infection with bacteriophage PRD1. This made it possible to obtain transfer-defective derivatives of pCE1, carrying Mu prophage. These derivatives could be mobilized by using the helper plasmid pME400 and it was possible to segregate the helper plasmid from the donor plasmid in the transconjugants.By incubating the strains 302 and 328 at 42°C, for induction of Mu prophage, derivatives with different plasmid contents could be obtained such as strains without plasmids, some with smaller or larger plasmids and others possessing plasmids without any visible alteration in size. Integration of the nitrogen-fixation (nif) genes into the chromosomes of the strains without plasmids and those containing a smaller plasmid, was confirmed by Southern hybridization using radioactive nifKDH DNA. Conjugation assays have shown that the plasmid is integrated into the chromosome as a unit but that it can also be excised.