Molecular characterization of three 3-ketosteroid-Δ(1)-dehydrogenase isoenzymes of Rhodococcus ruber strain Chol-4.

Rhodococcus ruber strain Chol-4 isolated from a sewage sludge sample is able to grow on minimal medium supplemented with steroids, showing a broad catabolic capacity. This paper reports the characterization of three different 3-ketosteroid-Δ(1)-dehydrogenases (KstDs) in the genome of R. ruber strain Chol-4. The genome of this strain does not contain any homologues of a 3-keto-5α-steroid-Δ(4)-dehydrogenase (Kst4d or TesI) that appears in the genomes of Rhodococcus erythropolis SQ1 or Comamonas testosteroni. Growth experiments with kstD2 mutants, either a kstD2 single mutant, kstD2 double mutants in combination with kstD1 or kstD3, or the triple kstD1,2,3 mutant, proved that KstD2 is involved in the transformation of 4-androstene-3,17-dione (AD) to 1,4-androstadiene-3,17-dione (ADD) and in the conversion of 9α-hydroxy-4-androstene-3,17-dione (9OHAD) to 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD). kstD2,3 and kstD1,2,3 R. ruber mutants (both lacking KstD2 and KstD3) did not grow in minimal medium with cholesterol as the only carbon source, thus demonstrating the involvement of KstD2 and KstD3 in cholesterol degradation. In contrast, mutation of kstD1 does not alter the bacterial growth on the steroids tested in this study and therefore, the role of this protein still remains unclear. The absence of a functional KstD2 in R. ruber mutants provoked in all cases an accumulation of 9OHAD, as a branch product probably formed by the action of a 3-ketosteroid-9α-hydroxylase (KshAB) on the AD molecule. Therefore, KstD2 is a key enzyme in the AD catabolism pathway of R. ruber strain Chol-4 while KstD3 is involved in cholesterol catabolism.

The strengths and weaknesses of Gordonia: a review of an emerging genus with increasing biotechnological potential.

This review about the genus Gordonia provides a current overview of recent research on a young genus that was introduced in the year 1997 ( Stackebrandt et al., 1997 ). This emerging genus has attracted increasing environmental, industrial, biotechnological and medical interest during the last few years, in particular due to the capabilities of its members to degrade, transform, and synthesize organic compounds as well as to the pathogenic effects that have been described in many case studies. The number of publications about Gordonia has increased significantly after the year 2004 (the year of the first Gordonia review published by Arenskötter et al.) describing 13 new validly published species (type strains), many newly described physiological and metabolic capabilities, new patent applications and many new case reports of bacterial infections. Members of the genus Gordonia are widely distributed in nature and it is therefore important to unravel the species richness and metabolic potential of gordoniae in future studies to demonstrate their environmental impact especially on the degradation of persistent organic compounds and their ecological participation in the carbon cycle of organic material in soil and water. This review summarizes mainly the current state of importance and potential of the members of this genus for the environmental and biotechnological industry ("the strengthsâ) and briefly its pathogenic impact to humans ("the weaknessesâ).

ChoG is the main inducible extracellular cholesterol oxidase of Rhodococcus sp. strain CECT3014.

Cholesterol catabolism has been reported in different bacteria and particularly in several Rhodococcus species, but the genetic of this complex pathway is not yet very well defined. In this work we report the isolation and sequencing of a 9.8 kb DNA fragment of Rhodococcus sp. strain CECT3014, a bacterial strain that we here identify as a Rhodococcus erythropolis strain. In this DNA fragment we found several ORF that are probably involved in steroid catabolism, and choG, a gene encoding a putative cholesterol oxidase whose functional characterization we here report. ChoG protein is a class II cholesterol oxidase with all the structural features of the enzymes of this group. The disruption of the choG gene does not alter the ability of strain CECT3014 cells to grow on cholesterol, but it abolishes the production of extracellular cholesterol oxidase. This later effect is reverted when the mutant cells are transformed with a plasmid expressing choG. We conclude that choG is the gene responsible for the inducible extracellular cholesterol oxidase activity of strain CECT3014. This activity distributes between the cellular membrane and the culture supernatant in a way that suggests it is produced by the same ChoG protein that occurs in two different locations. RT-PCR transcript analysis showed a dual scheme of choG expression: a low constitutive independent transcription, plus a cholesterol induced transcription of choG into a polycistronic kstD-hsd4B-choG mRNA.

Morphological, physiological, and molecular characterization of a newly isolated steroid-degrading actinomycete, identified as rhodococcus ruber strain Chol-4.

The aerobic degradation of cholesterol, testosterone, androsterone, progesterone, and further steroid compounds as sole carbon source has been observed in the newly isolated bacterial Gram-positive strain Chol-4. The 16S rRNA gene sequence shares the greatest similarity with members of the genus Rhodococcus, with the closest shared nucleotide identities of 98-99% with Rhodococcus ruber (DSM 43338(T)) and Rhodococcus aetherivorans (DSM 44752(T)). Phylogenetic analysis of Rhodococcus 16S rRNA gene sequences consistently places strain Chol-4 in a clade shared with those both type strains within the Rhodococcus rhodochrous subclade. The results of DNA-DNA hybridization against its two phylogenetically closest neighbors as well as the results of morphological, physiological, and biochemical tests allowed genotypic and phenotypic differentiation of strain Chol-4 from Rhodococcus ruber (DSM 43338(T)) on the species level and from the other validly described Rhodococcus species on the genus level. Strain Chol-4 therefore merits recognition as a novel strain of the species Rhodococcus ruber and demonstrates for the first time the capability of this species to utilize a great variety of steroid compounds as growth substrates never shown for other species of this genus so far. The genome of strain Chol-4 harbors at least one gene cluster that may be responsible for the degradation of steroid compounds. This gene cluster was identified in a cloned 5458 bp BamHI-EcoRV DNA fragment and compared to similar genes from other Gram-positive and Gram-negative bacteria described so far.

Gordonia cholesterolivorans sp. nov., a cholesterol-degrading actinomycete isolated from sewage sludge.

The taxonomic position of the cholesterol-degrading strain Chol-3(T), isolated from a sewage sludge sample, was clarified using a polyphasic taxonomic approach. Phylogenetic analysis of its 16S rRNA gene sequence, whole-cell fatty acid profile and mycolic acid composition revealed that this isolate is a member of the genus Gordonia with the species Gordonia sihwensis, G. hydrophobica and G. shandongensis being the nearest phylogenetic neighbours. The results of DNA-DNA hybridization against its phylogenetically closest neighbours as well as the results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain Chol-3(T) from the other Gordonia species with validly published names. Strain Chol-3(T) therefore merits recognition as a member of a novel species within the genus Gordonia, for which the name Gordonia cholesterolivorans sp. nov. is proposed. The type strain is Chol-3(T) (=CECT 7408(T) =DSM 45229(T)).

Genetic analysis of phenylacetic acid catabolism in Arthrobacter oxydans CECT386.

Arthrobacter oxydans CECT386 is a Gram-positive bacterium able to use either phenylacetic acid or phenylacetaldehyde as the sole carbon and energy source for aerobic growth. Genes responsible for the catabolism of these compounds have been located at two chromosomal regions and were organized in one isolated paaN gene and two putative paa operons, one consisting of the paaD, paaF, tetR and prot genes, and one consisting of the paaG, paaH, paaI, paaJ, paaK and paaB genes. The identity of the paaF and paaN genes was supported by functional complementation experiments. A comparison with the paa catabolic genes and/or gene clusters of other bacteria that degrade these aromatic compounds is presented. The results of this study broaden the knowledge regarding the range of metabolic potential of this strain and eventually make it attractive for environmental applications.

In vitro and in vivo sulfate reduction in the gut contents of the termite Mastotermes darwiniensis and the rose-chafer Pachnoda marginata.

Sulfate-reducing bacteria (SRB) from termites have been assigned to the genus Desulfovibrio. Desulfovibrio intestinalis lives in the gut of the Australian termite Mastotermes darwiniensis. For the first time we were able to enrich and identify a sulfate-reducing bacterium from the gut of the rose-chafer Pachnoda marginata, which showed the highest 16S rDNA sequence identity (93%) to Desulfovibrio intestinalis and Desulfovibrio strain STL1. Compared to Mastotermes darwiniensis (1x10(7) cells of SRB per ml gut contents), sulfate-reducing bacteria occurred in higher numbers in the gut contents of Pachnoda marginata reaching cell titers of up to 2x10(8) cells per ml gut contents. In vitro sulfate reduction rates were determined with SRB from the gut contents of the termite Mastotermes darwiniensis and the beetle Pachnoda marginata. Due to the higher cell titer, the sulfate reduction rate of Pachnoda marginata was 10(4) nmolxh-1xml-1 and therefore, 21 times higher than that of Mastotermes darwiniensis. In addition, we detected in vivo sulfate reduction in Mastotermes darwiniensis, which indicates that sulfate reducers play an active role in the sulfur metabolism in the termite gut.

Diphenylamine and derivatives in the environment: a review.

Diphenylamine (DPA) is a compound from the third European Union (EU) list of priority pollutants. It was assigned by the EU to Germany to assess and control its environmental risks. DPA and derivatives are most commonly used as stabilizers in nitrocellulose-containing explosives and propellants, in the perfumery, and as antioxidants in the rubber and elastomer industry. DPA is also widely used to prevent post-harvest deterioration of apple and pear crops. DPA is a parent compound of many derivatives, which are used for the production of dyes, pharmaceuticals, photography chemicals and further small-scale applications. Diphenylamines are still produced worldwide by the chemical industries. First reports showed that DPA was found in soil and groundwater. Some ecotoxicological studies demonstrated the potential hazard of various diphenylamines to the aquatic environment and to bacteria and animals. Studies on the biodegradability of DPA and its derivatives are very sparse. Therefore, further investigation is required to determine the complete dimension of the potential environmental hazard and to introduce possible (bio)remediation techniques for sites that are contaminated with this class of compounds. This is the first detailed review on DPA and some derivatives summarizing their environmental relevance as it is published in the literature so far and this review will recommend conducting further research in the future.

Dehalogenation of chlorinated ethenes and immobilization of nickel in anaerobic sediment columns under sulfidogenic conditions.

A sediment column study was carried out to demonstrate the bioremediation of chloroethene- and nickel-contaminated sediment in a single anaerobic step under sulfate-reducing conditions. Four columns (one untreated control column and three experimental columns) with sediment from a chloroethene- and nickel-contaminated site were investigated for 1 year applying different treatments. By stimulating the activity of sulfate-reducing bacteria by the addition of sulfate as supplementary electron acceptor, complex anaerobic communities were maintained with lactate as electron donor (with or without methanol), which achieved complete dehalogenation of tetra- and trichloroethenes (PCE and TCE) to ethene and ethane. A few weeks after sulfate addition, production of sulfide increased, indicating an increasing activity of sulfate-reducing bacteria. The nickel concentration in the effluent of one nickel-spiked column was greatly reduced, likely due to the enhanced sulfide production, causing precipitation of nickel sulfide. At the end of the study, 94% of the initial amount of nickel added to that column was recovered in the sediment As compared to the untreated (nonspiked) control column, all chloroethene-spiked columns ladditions of PCE and TCE) showed a permanent release of small chloride ion quantities (approximately 0.5-0.7 mM chloride), which were detected in the effluents a few weeks after sulfide production was observed for the first time. The formation of ethene and ethane as final products after dechlorination of PCE and TCE was detected in some effluents and in some gas phases of the columns. Other metabolites or intermediates (such as DCE isomers) were only detected sporadically in negligible quantities. The results of this study demonstrated thatmicrobial activity stimulated under sulfate-reducing conditions can have a beneficial effect on both the precipitation of heavy metals and the complete dechlorination of organochlorines. The strongly negative redox potential created by the activity of sulfate-reducing bacteria may be one factor responsible for stimulating the activity of the dehalogenating bacteria in the test columns.

Tetrachloroethene dehalorespiration and growth of Desulfitobacterium frappieri TCE1 in strict dependence on the activity of Desulfovibrio fructosivorans.

Tetrachloroethene (PCE) dehalorespiration was investigated in a continuous coculture of the sulfate-reducing bacterium Desulfovibrio fructosivorans and the dehalorespiring Desulfitobacterium frappieri TCE1 at different sulfate concentrations and in the absence of sulfate. Fructose (2.5 mM) was the single electron donor, which could be used only by the sulfate reducer. With 2.5 mM sulfate, the dehalogenating strain was outnumbered by the sulfate-reducing bacterium, sulfate reduction was the dominating process, and only trace amounts of PCE were dehalogenated by strain TCE1. With 1 mM sulfate in the medium, complete sulfate reduction and complete PCE dehalogenation to cis-dichloroethene (cis-DCE) occurred. In the absence of sulfate, PCE was also completely dehalogenated to cis-DCE, and the population size of strain TCE1 increased significantly. The results presented here describe for the first time dehalogenation of PCE by a dehalorespiring anaerobe in strict dependence on the activity of a sulfate-reducing bacterium with a substrate that is exclusively used by the sulfate reducer. This interaction was studied under strictly controlled and quantifiable conditions in continuous culture and shown to depend on interspecies hydrogen transfer under sulfate-depleted conditions. Interspecies hydrogen transfer was demonstrated by direct H(2) measurements of the gas phase and by the production of methane after the addition of a third organism, Methanobacterium formicicum.