Degradation of Delor 103, a technical mixture of polychlorinated biphenyls,by selected bacteria

Ability to utilize a technical mixture of polychlorinated biphenyls (PCB), Delor 103, as the sole carbon source, has been tested in 14 bacterial strains. For the five best growing strains (Alcaligenes latus, Alcalgenes eutrophus, Comamonas testosteroni, Micrococcus varians and Pseudomonas putida), the dependence of the degradation of individual PCB congeners on the number of chlorine substituents is discussed.     

Systematic study of the genus Acetobacter with descriptions of Acetobacter indonesiensis sp. nov., Acetobacter tropicalis sp. nov., Acetobacter orleanensis (Henneberg 1906) comb. nov., Acetobacter lovaniensis (Frateur 1950) comb. nov., and Acetobacter estunensis (Carr 1958) comb. nov

Thirty-one Acetobacter strains obtained from culture collections and 45 Acetobacter strains isolated from Indonesian sources were investigated for their phenotypic characteristics, ubiquinone systems, DNA base compositions, and levels of DNA-DNA relatedness. Of 31 reference strains, six showed the presence of ubiquinone 10 (Q-10). These strains were eliminated from the genus Acetobacter. The other 25 reference strains and 45 Indonesian isolates were subjected to a systematic study and separated into 8 distinct groups on the basis of DNA-DNA relatedness. The known species, Acetobacter aceti, A. pasteurianus, and A. peroxydans are retained for three of these groups. New combinations, A. orleanensis (Henneberg 1906) comb. nov., A. lovaniensis (Frateur 1950) comb. nov., and A. estunensis (Carr 1958) comb. nov. are proposed for three other groups. Two new species, A. indonesiensis sp. nov. and A. tropicalis sp. nov. are proposed for the remaining two. No Indonesian isolates were identified as A. aceti, A. estunensis, and A. peroxydans. Phylogenetic analysis on the basis of 16S rDNA sequences was carried out for representative strains from each of the groups. This supported that the eight species belonged to the genus Acetobacter. Several strains previously assigned to the species of A. aceti and A. pasteurianus were scattered over the different species. It is evident that the value of DNA-DNA relatedness between strains comprising a new species should be determined for the establishment of the species. Thus current bacterial species without data of DNA-DNA relatedness should be reexamined for the stability of bacterial nomenclature.     

Regulation of testosterone degradation in Comamonas testosteroni

Recently, we have identified a gene encoding a LuxR-type factor, TeiR (Testosterone-inducible Regulator), which positively regulates steroid degradation in Comamonas testosteroni. Herein, we demonstrate that TeiR interacts in vivo with steroid catabolic gene promoters. The presence of testosterone induces a significant TeiR protein increase at the early logarithmic phase of growth. Interestingly, it is not until the early stationary phase where the activation of a steroid-inducible gene promoter is observed, indicating that testosterone might not be the true inductor of the steroid degradation pathway. In addition, beta-galactosidase expression driven by a testosterone-inducible promoter is prematurely activated in cells cultured in medium supplemented with ethyl acetate extracts obtained from the early stationary phase cell-free supernatants of C. testosteroni grown in presence of testosterone. Complementation experiments of C. testosteroni wild type performed with teiR deletion constructs indicate that extra-copies of deleted-TeiR exert a dominant negative effect on the wild-type TeiR protein. While, when C. testosteroni teiR mutants were used to carry out complementation assays only the full length gene can overcome the teiR mutant phenotype. Altogether these findings indicate that TeiR regulates steroid catabolic genes interacting with their promoters and suggest that this interaction requires the presence of a testosterone-derived metabolite to induce the system.     

Map of the IncP1beta plasmid pTSA encoding the widespread genes (tsa) for p-toluenesulfonate degradation in Comamonas testosteroni T-2

The catabolic IncP1beta plasmid pTSA from Comamonas testosteroni T-2 was mapped by subtractive analysis of restriction digests, by sequencing outwards from the tsa operon (toluenesulfonate degradation), and by generating overlapping, long-distance-PCR amplification products. The plasmid was estimated to comprise 72 +/- 4 kb. The tsa region was found to be a composite transposon flanked by two IS1071 elements. A cryptic tsa operon was also present in the tsa transposon. Those backbone genes and regions which we sequenced were in the same order as the corresponding genes in resistance plasmid R751, and identities of about 99% were observed. Enrichment cultures with samples from four continents were done to obtain organisms able to utilize p-toluenesulfonate as the sole source of carbon and energy for aerobic growth. Most (15) of the 16 cultures (13 of them isolates) were obtained from contaminated sites and were attributed to three metabolic groups, depending on their metabolism of p-toluenesulfonate. The largest group contained the tsa transposon, usually (six of seven isolates) with negligible differences in sequence from strain T-2.     

Bioconversion of pyrimidine by resting cells of quinoline-degrading bacteria

Nine quinoline-degrading bacterial strains were tested for their ability to hydroxylate pyrimidine. All strains converted pyrimidine to uracil via pyrimidine-4-one in a cometabolic process. Quinoline 2-oxidoreductases (QuinORs) were the catalysts of fortuitous pyrimidine hydroxylation. Whereas in most strains the activity of the QuinOR towards pyrimidine was very low compared to its activity towards quinoline, QuinOR in crude extracts from Comamonas testosteroni 63 showed a specific activity of 64 (mU mg protein)-1 with pyrimidine as substrate, compared to a specific activity of 237 (mU mg protein)-1 towards the intrinsic substrate quinoline. Resting cells of Comamonas testosteroni 63 rapidly converted pyrimidine almost stoichiometrically to uracil, which accumulated in the cell suspension. Using an adsorbent resin, uracil was prepared from the supernatant of Comamonas testosteroni 63 resting cells with a yield of > 98%.     

Analysis of the catalytic domain of the lysin of the lactococcal bacteriophage Tuc2009 by chimeric gene assembling

Abstract Dye-linked alcohol dehydrogenase from Rhodopseudomonas acidophila strain M402, able to oxidize polyethylene glycols, was purified to homogeneity. The monomeric enzyme, having a molecular mass of 72 kDa, contains one PQQ and one haem c per enzyme molecule. In other respects also, the enzyme is very similar to the type I quinohaemoprotein alcohol dehydrogenases known to occur in Comamonas testosteroni, Comamonas acidovorans , and Pseudomonas putida species. However, dissimilarities exist with respect to the isoelectric points and the substrate specificities. On reinvestigating the substrate specificity of the C. testosteroni enzyme, it also appeared to exhibit good activity towards polyethylene glycols. Based on what has been reported for the polyethylene glycol-oxidizing alcohol dehydrogenase of Sphingomonas macrogoltabidus , this enzyme is quite different from that of R. acidophila . Keywords: Polyethylene glycol dehydrogenase activity; Alcohol dehydrogenase; PQQ; Haem c ; Rhodopseudomonas acidophila     

Alcaligenes faecalis subsp. parafaecalis subsp. nov., a bacterium accumulating poly-beta-hydroxybutyrate from acetone-butanol bioprocess residues

The authors have previously isolated a solvent tolerant bacterium, strain G(T), (T = type strain) capable to convert acetone-butanol bioprocess residues into poly-beta-hydroxybutyrate. Strain G(T) was initially identified as Alcaligenes spp by standard bacteriological tests. In this study the taxonomic position of the bacterium was investigated in detail. The 165 rDNA sequence analysis, the G + C content of DNA (56 mol%) and the presence of ubiquinone Q-8 confirmed strain G(T) as a representative of the genus Alcaligenes. In the polyamine pattern of the bacterium putrescine and cadaverine were detected, but only trace amounts of 2-hydroxyputrescine. The extremely low content of 2-hydroxyputrescine is remarkable, since this unique diamine is a common marker for beta-proteobacteria. Phylogenetic analyses of 16S rDNA demonstrated that Alcaligenes sp. G(T) is most closely related to the species Alcaligenes faecalis (99.6% sequence similarity to A. faecalis HR4 and 98.7% sequence similarity to A. faecalis [ATCC 8750T = DSM 30030T]. On the basis of DNA-DNA relatedness (56% similarity), the unique polyamine pattern, the physiological and biochemical differences strain G(T) could be distinguished from the species A. faecalis. Therefore, a new subspecies for the species Alcaligenes faecalis is proposed; Alcaligenes faecalis subsp. parafaecalis subsp. nov.     

Testosterone-regulated expression of enzymes involved in steroid and aromatic hydrocarbon catabolism in Comamonas testosteroni.

The effect of testosterone as the sole carbon source on protein expression was analyzed in Comamonas testosteroni. Testosterone simultaneously induced the expression of steroid- and aromatic hydrocarbon-catabolizing enzymes and repressed one amino acid-degrading enzyme. It is suggested that steroids play a regulative role in catabolic enzyme synthesis during adaptive growth of C. testosteroni.     

The crystal structure of 3alpha -hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase/reductase family

The crystal structure of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (3alpha-HSDH) as well as the structure of its binary complex with NAD(+) have been solved at 1.68-A and 1.95-A resolution, respectively. The enzyme is a member of the short chain dehydrogenase/reductase (SDR) family. Accordingly, the active center and the conformation of the bound nucleotide cofactor closely resemble those of other SDRs. The crystal structure reveals one homodimer per asymmetric unit representing the physiologically active unity. Dimerization takes place via an interface essentially built-up by helix alphaG and strand betaG of each subunit. So far this type of intermolecular contact has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3alpha-HSDH by the presence of a predominantly alpha-helical subdomain which is missing in all other SDRs of known structure.     

Nucleic Acid Homologies Among Oxidase-Negative Moraxella Species

The deoxyribonucleic acid (DNA) base composition and DNA homologies of more than 40 strains of oxidase-negative Moraxella species were determined. These bacteria have also been identified as belonging to the Mima-Herellea-Acinetobacter group and the Bacterium anitratum group, as well as to several other genera including Achromobacter and Alcaligenes. The DNA base content of these strains ranged from 40 to 46% guanine plus cytosine. DNA-DNA competition experiments distinguished five groups whose members were determined by showing 50% or more homology to one of the reference strains: B. anitratum type B5W, Achromobacter haemolyticus var. haemolyticus, Alcaligenes haemolysans, Achromobacter metalcaligenes, and Moraxella lwoffi. A sixth group comprised those strains showing less than 50% homology to any of the reference strains. Negligible homology was found between strains of oxidase-negative and oxidase-positive Moraxella species in DNA-DNA competition experiments. However, evidence of a distant relationship between the two groups was obtained in competition experiments by using ribosomal ribonucleic acid.     

A model on the regulation of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase expression in Comamonas testosteroni

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic carbonyl compounds. The enzyme has recently been cloned and characterized by our group. A strong induction of enzyme activity is observed in the presence of steroids like testosterone. In the present investigation, two repressor proteins (Rep1 and Rep2) containing 78 and 420 amino acids, respectively, were found to regulate 3alpha-HSD/CR gene (hsdA) expression. Gel shift experiments showed that Rep2 binds to a 10 nucleotide sequence 9 bp upstream of the hsdA promoter. The deletion of this cis-regulating sequence significantly increases hsdA expression. About 1633 bp further upstream, a second ten nucleotide sequence, complementary to the first one, was found, which is also recognized by Rep2 and increases hsdA expression, if deleted. To purify the repressor proteins, the genes encoding each were cloned into His-tag expression vectors and overexpressed in Escherichia coli. Rep1 does not bind to DNA but may bind to 3alpha-HSD/CR mRNA as predicted by its secondary structure. Concluding from our data, induction of 3alpha-HSD/CR in C. testosteroni by steroids in fact appears to be a de-repression, where the steroidal 'inducer' prevents the binding of the two repressor proteins to the hsdA promoter and mRNA, respectively.     

Comamonas testosteroni bacteremia in a patient with perforated acute appendicitis. Short communication

Comamonas testosteroni is an uncommon isolate in the clinical laboratory as a human pathogen. C. testosteroni most commonly emerges in abdominal pathologies especially in perforated appendicitis. In Turkey we report first time a case of bacteremia due to this organism, in a 22-year-old man with perforated acute appendicitis. The organism was shown to be susceptible to routine antibiotics so it was easily eliminated even after having caused a bacteremia.     

Insights into the genetic diversity of initial dioxygenases from PAH-degrading bacteria

Alpha subunit genes of initial polyaromatic hydrocarbon (PAH) dioxygenases were used as targets for the PCR detection of PAH-degrading strains of the genera Pseudomonas, Comamonas and Rhodococcus which were obtained from activated sludge or soil samples. Sequence analysis of PCR products from several Pseudomonas strains showed that alpha subunits (nahAc allele) of this genus are highly conserved. PCR primers for the specific detection of alpha subunit genes of initial PAH dioxygenases from Pseudomonas strains were not suitable for detecting the corresponding genes from the genera Comamonas and Rhodococcus. Southern analysis using a heterologous gene probe derived from the P. putida OUS82 PAH dioxygenase alpha subunit identified segments of the PAH-degradation gene cluster from C. testosteroni strain H. Parts of this gene cluster containing three subunits of the initial PAH dioxygenase were isolated. These three subunits [ferredoxin (pahAb), alpha (pahAc) and beta (pahAd) subunit] were amplified by PCR as one fragment and expressed in Escherichia coli DH5alpha, resulting in an active initial dioxygenase with the ability to transform indole and phenanthrene. The DNA sequence alignment of alpha subunits from C. testosteroni H and various PAH-degrading bacteria permitted the design of new primers and oligonucleotide probes which are useful for the detection of the initial PAH dioxygenases from strains of Pseudomonas, Comamonas and Rhodococcus.     

Genome sequence of Comamonas testosteroni ATCC 11996, a representative strain involved in steroid degradation

Comamonas testosteroni strains belong to the family of Comamonadaceae and are known for their ability to utilize steroid compounds as carbon source. Here, we present the draft genome sequence of strain ATCC 11996, with a G+C content of 61.48%.     

Conjugative plasmids and the degradation of arylsulfonates in Comamonas testosteroni.

Comamonas testosteroni T-2 degrades p-toluenesulfonate (TSA) via p-sulfobenzoate (PSB) and protocatechuate and degrades toluenecarboxylate via terephthalate (TER) and protocatechuate. The appropriate genes are expressed in at least five regulatory units, some of which are also found in C. testosteroni PSB-4 (F. Junker, R. Kiewitz, and A. M. Cook, J. Bacteriol. 179:919-927, 1997). C. testosteroni T-2 was found to contain two plasmids, pTSA (85 kbp) and pT2T (50 kbp); a TSA- mutant (strain TER-1) contained only plasmid pT2T. C. testosteroni PSB-4, which does not degrade TSA, contained one plasmid, pPSB (85 kbp). The type strain contained no plasmids. Conjugation experiments showed that plasmid pTSA (possibly in conjunction with pT2T) was conjugative, and the single copy of the TSA operon (tsaMBCD) with its putative regulator gene (tsaR) in strain T-2 was found on plasmid pTSA, which also carried the PSB genes (psbAC) and presumably transport for both substrates. Plasmid pTSA was assigned to the IncP1 beta group and was found to carry two copies of insertion element IS1071. Plasmid pPSB (of strain PSB-4), which could be maintained in strains with plasmid pTSA or pT2T, was also conjugative and was found to carry the PSB genes as well as to contain two copies of IS1071. In attempted conjugations with the type strain, no plasmid was recovered, but the PSB+ transconjugant carried two copies of IS1071 in the chromosome. We presume the PSB genes to be located in a composite transposon. The genes encoding the putative TER operon and degradation of protocatechuate, with the meta cleavage pathway, were attributed a chromosomal location in strains T-2 and PSB-4.     

Isolation of bacteria capable of growth with 2-methylisoborneol and geosmin as the sole carbon and energy sources

Using a relatively simple enrichment technique, geosmin and 2-methylisoborneol (MIB)-biodegrading bacteria were isolated from a digestion basin in an aquaculture unit. Comparison of 16S rRNA gene sequences affiliated one of the three isolates with the Gram-positive genus Rhodococcus, while the other two isolates were found to be closely related to the Gram-negative family Comamonadaceae (Variovorax and Comamonas). Growth rates and geosmin and MIB removal rates by the isolates were determined under aerated and nonaerated conditions in mineral medium containing either of the two compounds as the sole carbon and energy source. All isolates exhibited their fastest growth under aerobic conditions, with generation times ranging from 3.1 to 5.7 h, compared to generation times of up to 19.1 h in the nonaerated flasks. Incubation of the isolates with additional carbon sources caused a significant increase in their growth rates, while removal rates of geosmin and MIB were significantly lower than those for incubation with only geosmin or MIB. By fluorescence in situ hybridization, members of the genera Rhodococcus and Comamonas were detected in geosmin- and MIB-enriched sludge from the digestion basin.     

Conjugal transfer of plasmid pNB2 to activated sludge bacteria leads to 3-chloroaniline degradation in enrichment cultures

AIMS: The involvement of the aniline-degradative plasmid pNB2 in degradation of 3-chloroaniline (3-CA) was investigated. METHODS AND RESULTS: Plate matings of a Pseudomonas putida strain containing pNB2 with a mixed bacterial culture derived from activated sludge was carried out. After inoculation of the mating mixtures into batch cultures containing 3-CA, degradation of the compound was observed. A total of five different transconjugant strains could be isolated from one of the batch cultures and two of them were able to degrade 3-CA. These two isolates were identified as Comamonas testosteroni by partial 16S rDNA sequencing. CONCLUSIONS: It can be assumed that pNB2 carries a part of the genes involved in the catabolism of 3-CA, but that completion of the pathway must be provided by chromosomal genes in the host strain. SIGNIFICANCE AND IMPACT OF THE STUDY: pNB2 is a candidate plasmid which can be used in plasmid-mediated bioaugmentation of wastewater bacteria involved in degradation of chlorinated anilines.