Evidence for the pathogenic role of Rhodococcus species in pulmonary diseases

Three hundred expectorated sputa from patients suffering from chest disorders were examined microscopically and cultured on various media. Among other micro-organisms isolated there were four strains of Rhodococcus aurantiacus (Gordona aurantiaca), one each of Rh. erythropolis, Rh. pellegrino, Rh. rubropertinctus and Rh. rhodnii. Suspensions in 5% hog gastric mucin were virulent for immuno-suppressed white mice and the pathology of their lungs was compatible with those produced in experimental nocardial and rhodococcus infections. The in-vitro antibiogram was similar to that of Nocardia asteroides. Two of the patients from whom Rh. aurantiacus and Rh. pellegrino were isolated responded bacteriologically and clinically to treatment with co-trimoxazole. The frequency with which these rhodococci were isolated and the clinical conditions of the patients strongly indicated a pathogenic role for some Rhodococcus species. It is suggested that rhodococci should be sought in chronic pulmonary infections, particularly in those who are immuno-compromised or debilitated, and their aetiologic role determined.     

Differentiation between genera Rhodococcus and Nocardia by susceptibility testing to kanamycin and some other antituberculosis agents

The test for susceptibility to kanamycin is useful for differentiating between the genera Rhodococcus and Nocardia. All rhodococci except R. equi, R. erythropolis, and R. aurantiacus are susceptible to kanamycin, whereas all nocardiae except N. otitidis-caviarum are resistant to kanamycin. Tests for susceptibility to rifampicin, streptomycin, and minocycline also are useful for differentiating among the species of each genus.     

Lysozyme of the mollusk Unio pictorum and the sensitivity of alkanotrophic rhodococci to its effect

A preparation of lysozyme from a freshwater bivalve, Unio pictorum, has been isolated by sorption to chitin, and its physicochemical properties have been studied. An assessment of the sensitivity of 48 strains of rhodococci, belonging to the species Rhodococcus rubber, R. luteus, and R. erythropolis (Specialized Collection of Alkanotrophic Microorganisms of the Institute of Ecology and Genetics of Microorganisms, Ural Division of the Russian Academy of Sciences), which were isolated from diverse natural waters, to lysozyme of the mollusk Unio pictorum demonstrated that the three species differ in their sensitivity to its effects. The high resistance of rhodococci to lysozyme is indicative of their considerable permanence in hydrobiocenoses (and, therefore, ability to maintain self-purification of microbiocenoses from hydrocarbons).     

Metabolism of the herbicide atrazine by Rhodococcus strains.

Rhodococcus strains were screened for their ability to degrade the herbicide atrazine. Only rhodococci that degrade the herbicide EPTC (s-ethyl-dipropylthiocarbamate) metabolized atrazine. Rhodococcus strain TE1 metabolized atrazine under aerobic conditions to produce deethyl- and deisopropylatrazine, which were not degraded further and which accumulated in the incubation medium. The bacterium also metabolized the other s-triazine herbicides propazine, simazine, and cyanazine. The N dealkylation of triazine herbicides by Rhodococcus strain TE1 was associated with a 77-kb plasmid previously shown to be required for EPTC degradation.     

Lysozyme of the Mollusk Unio pictorum and the Sensitivity of Alkanotrophic Rhodococci to Its Effect

A preparation of lysozyme from a freshwater bivalve, Unio pictorum, has been isolated by sorption to chitin, and its physicochemical properties have been studied. An assessment of the sensitivity of 48 strains of rhodococci, belonging to the species Rhodococcus rubber, R. luteus, and R. erythropolis (Specialized Collection of Alkanotrophic Microorganisms of the Institute of Ecology and Genetics of Microorganisms, Ural Division of the Russian Academy of Sciences), which were isolated from diverse natural waters, to lysozyme of the mollusk Unio pictorum demonstrated that the three species differ in their sensitivity to its effects. The high resistance of rhodococci to lysozyme is indicative of their considerable permanence in hydrobiocenoses (and, therefore, ability to maintain self-purification of microbiocenoses from hydrocarbons).     

Plant exudates promote PCB degradation by a rhodococcal rhizobacteria

Rhodococcus erythropolis U23A is a polychlorinated biphenyl (PCB)-degrading bacterium isolated from the rhizosphere of plants grown on a PCB-contaminated soil. Strain U23A bphA exhibited 99% identity with bphA1 of Rhodococcus globerulus P6. We grew Arabidopsis thaliana in a hydroponic axenic system, collected, and concentrated the plant secondary metabolite-containing root exudates. Strain U23A exhibited a chemotactic response toward these root exudates. In a root colonizing assay, the number of cells of strain U23A associated to the plant roots (5.7?×?10? CFU g?1) was greater than the number remaining in the surrounding sand (4.5?×?10? CFU g?1). Furthermore, the exudates could support the growth of strain U23A. In a resting cell suspension assay, cells grown in a minimal medium containing Arabidopsis root exudates as sole growth substrate were able to metabolize 2,3,4'- and 2,3',4-trichlorobiphenyl. However, no significant degradation of any of congeners was observed for control cells grown on Luria-Bertani medium. Although strain U23A was unable to grow on any of the flavonoids identified in root exudates, biphenyl-induced cells metabolized flavanone, one of the major root exudate components. In addition, when used as co-substrate with sodium acetate, flavanone was as efficient as biphenyl to induce the biphenyl catabolic pathway of strain U23A. Together, these data provide supporting evidence that some rhodococci can live in soil in close association with plant roots and that root exudates can support their growth and trigger their PCB-degrading ability. This suggests that, like the flagellated Gram-negative bacteria, non-flagellated rhodococci may also play a key role in the degradation of persistent pollutants.     

A comparative study of the 'rhodochrous' complex and related taxa by delayed-type skin reactions on guinea pigs and by polyacrylamide gel electrophoresis.

Cell extracts prepared by ultrasonic disruption of 17 strains of the 'rhodochrous' complex and related taxa were compared by polyacrylamide gel electrophoresis and for immunologic relatedness, by skin test reactions. Two organisms, Jensenia canicruria and Nocardia calcarea, gave similar gel patterns and skin test reactions, and are considered to be identical. Extracts of nocardia rubra showed a strong antigenic relationship with those of three Nocardia pellegrino organisms (N325, N324 and N420) previously assigned to the 'rhodochrous' complex. Two Gordona organisms appeared to be less antigenically related to the 'rhodochrous' complex. Extracts of three of four organisms designated Lspi (Rhodococcus coprophilus Rowbotham & Cross 1976) elicited skin test reactions similar to those of the 'rhodochrous' strains. One Lspi strain, N650, showed striking similarities to the 'rhodochrous' complex strain N420 (Nocardia pellegrino).     

Cloning of genes that have environmental and clinical importance from rhodococci and related bacteria

Generalised and specialised transduction systems were developed for Rhodococcus by means of bacteriophage Q4. The latter was used in conjunction with DNA from an unstable genetic element of R. rhodochrous to construct resistance plasmids which replicate in strains of R. equi, R. erythropolis and R. rhodochrous. One of the plasmids, pDA21, was joined with Erythropolis coli suicide vector pEcoR251 to obtain shuttle plasmids maintained in both rhodococci and E. coli. Conjugation between these rhodococcal strains demonstrated all were interfertile with each other and that some of the determinants for this were located on the unstable genetic element. Plasmids derived from this element, such as pDA21, carried the conjugative and self- incompatibility capacities; deletion analysis revealed that DNA necessary for self-incompatibility overlapped with that for arsenic resistance.Rifampicin is one of the principal chemotherapeutic agents used to treat infections by rhodococci and related organisms. The genes responsible for two types of inactivation have been cloned. The sequence of the R. equi DNA responsible for decomposition of the antibiotic strongly resembled those of monooxygenases acting upon phenolic compounds, consistent with the presence of a naphthalenyl moiety in the rifampicin molecule. Antibiotic resistance conferred by the gene was surprisingly specific to the semisynthetic compounds rifampicin (150-fold increase) and rifapentine (70-fold). Similar specificity was observed with the other inactivation gene cloned, which ribosylates rifampicin at the 23-hydroxyl position. A 60-bp sequence upstream of the monooxygenase and ribosylation genes is strikingly similar suggesting a shared pattern of regulation.Rhodococcal arsenic resistance and azo dye degradation genes have been cloned and characterised.     

Physiology, biochemistry and taxonomy of deep-sea nitrile metabolising Rhodococcus strains

A collection of nitrile-hydrolysing rhodococci was isolated from sediments sampled from a range of deep coastal, and abyssal and hadal trench sites in the NW Pacific Ocean, as part of our programme on the diversity of marine actinomycetes. Nitrile-hydrolysing strains were obtained by batch enrichments on nitrile substrates with or without dispersion and differential centrifugation pre-treatment of sediments, and were recovered from all of the depths sampled (approximately 1100–6500 m). Two isolates obtained from the Ryukyu (5425 m) and Japan (6475 m) Trenches, and identified as strains of Rhodococcus erythropolis,were chosen for detailed study. Both of the deep-sea isolates grew at in situ temperature (4°C), salinities (0–4% NaCl) and pressures (40–60 MPa), results that suggest, but do not prove, that they may be indigenous marine bacteria. However, the absence of culturable Thermoactinomycespoints to little or no run off of terrestrial microbiota into these particular trench sediments. Nitrile-hydrolysis by these rhodococci was catalysed by a nitrile hydratase–amidase system. The hydratase accommodated aliphatic, aromatic and dinitrile substrates, and enabled growth to occur on a much wider range of nitriles than the only other reported marine nitrile-hydrolysing R. erythropolis which was isolated from coastal sediments. Also unlike the latter strain, the nitrile hydratases of the deep-sea rhodococci were constitutive. The possession of novel growth and enzyme activities on nitriles by these deep-sea R. erythropolisstrains recommends their further development as industrial biocatalysts.     

Bioconversion of beta-sitosterol and its complex esters by Rhodococcus actinobacteria

The ability of pure cultures of Rhodococcus actinobacteria from the Ural specialized collection of alkanotrophic microorganisms (World Federation for Culture Collections accession number 768; http://www.ecology.psu.ru/iegmcol) to convert beta-sitosterol (BSS) and its 3beta-acylated derivatives was studied. Rhodococcus strains with pronounced cholesterol oxidase activity, capable of converting BSS to stigmat-4-ene-3-one in the reaction of cooxidation with n-hexadecane, were selected. The dependence of the activity of cholesterol oxidase of rhodococci on the length of the acyl group in BSS esters was studied. Conditions under which Rhodococcus cells convert BSS to 17beta-hydroxyandrost-4-ene-3-one (testosterone), commonly used in pharmacology, were determined.     

Fluorene degradation by bacteria of the genus Rhodococcus

Of the four investigated Rhodococcus strains (R. rhodochrous 172, R. opacus 4a and 557, and R. rhodnii 135), the first three strains were found to be able to completely transform fluorene when it was present in the medium as the sole source of carbon at a concentration of 12-25 mg/l. At a fluorene concentration of 50-100 mg/l in the medium, the rhodococci transformed 50% of the substrate in 14 days. The addition of casamino acids and sucrose (1-5 g/l) stimulated fluorene transformation, so that R. rhodochrous 172 could completely transform it in 2-5 days. Nine intermediates of fluorene transformation were isolated, purified, and structurally characterized. It was found that R. rhodnii 135 and R. opacus strains 4a and 557 hydroxylated fluorene with the formation of 2-hydroxyfluorene and 2,7-dihydroxyfluorene. R. rhodochrous 172 transformed fluorene via two independent pathways to a greater degree than did the other rhodococci studied.     

Discrimination and taxonomy of geographically diverse strains of nitrile-metabolizing actinomycetes using chemometric and molecular sequencing techniques

Mycolic acid-containing actinomycetes capable of metabolizing nitriles were recovered from deep-sea sediments and terrestrial soils by enrichment culture on acetonitrile, benzonitrile, succinonitrile or bromoxynil. A total of 43 nitrile-degrading strains were isolated and, together with previously recovered nitrile-degrading rhodococci, were identified by a polyphasic taxonomic approach, which included mycolic acid profiles, pyrolysis mass spectrometry (PyMS), genomic fingerprinting based on sequence variability of the 16S ribosomal RNA gene using polymerase chain reaction-restriction fragment length polymorphism-single-strand conformational polymorphism, and 16S rRNA gene sequence comparison. Isolates phylogenetically related to Rhodococcus erythropolis dominated the culturable microorganisms from most marine and terrestrial samples. These isolates clustered together in a major pyrogroup that showed high congruence with PRS profiles of the 16S rRNA gene. Such high congruence also was obtained for other recovered isolates that were assigned to species of Rhodococcus and Gordonia. Sequencing data validated the results obtained by PRS analysis and enabled phylogenetic relationships to be established. Some of the recovered bacteria probably represent novel microbial species. The fact that nitrile-metabolizing microorganisms were recovered from a wide range of habitat types suggests that nitrile transforming enzymatic activity is geographically widely distributed in nature.     

A consortium of immobilized rhodococci for oilfield wastewater treatment in a column bioreactor

The possible use of a consortium of actinobacteria from the genus Rhodococcus immobilized on a polymeric carrier has been investigated for oilfield wastewater treatment in a bioreactor. It has been found that Rhodococcus opacus IEGM 263 and Rhodococcus ruber IEGM 231 cells remain viable at high concentrations of mineral salts in water and are able to oxidize oil hydrocarbons up to 62–81%. It has been shown that the consortium of rhodococci was more efficient in the elimination of hydrocarbons from wastewater than monocultures.     

Complete nucleotide sequence and genetic organization of the 210-kilobase linear plasmid of Rhodococcus erythropolis BD2

The complete nucleotide sequence of the linear plasmid pBD2 from Rhodococcus erythropolis BD2 comprises 210,205 bp. Sequence analyses of pBD2 revealed 212 putative open reading frames (ORFs), 97 of which had an annotatable function. These ORFs could be assigned to six functional groups: plasmid replication and maintenance, transport and metalloresistance, catabolism, transposition, regulation, and protein modification. Many of the transposon-related sequences were found to flank the isopropylbenzene pathway genes. This finding together with the significant sequence similarities of the ipb genes to genes of the linear plasmid-encoded biphenyl pathway in other rhodococci suggests that the ipb genes were acquired via transposition events and subsequently distributed among the rhodococci via horizontal transfer.     

Applied aspects of Rhodococcus genetics

Eubacteria of the genus Rhodococcus are a diverse group of microorganisms commonly found in many environmental niches from soils to seawaters and as plant and animal pathogens. They exhibit a remarkable ability to degrade many organic compounds and their economic importance is becoming increasingly apparent. Although their genetic organisation is still far from understood, there have been many advances in recent years. Reviewed here is the current knowledge of rhodococci relating to gene transfer, recombination, plasmid replication and functions, cloning vectors and reporter genes, gene expression and its control, bacteriophages, insertion sequences and genomic rearrangements. Further fundamental studies of Rhodococcus genetics and the application of genetic techniques to the these bacteria will be needed for their continued biotechnological exploitation.     

Detection of genes for alkane and naphthalene catabolism in Rhodococcus sp. strain 1BN

Rhodococcus sp. 1BN was isolated from a contaminated site and showed various biodegradative capabilities. Besides naphthalene, strain 1BN degraded medium- (C6) and long-chain alkanes (C16-C28), benzene and toluene, alone or when the hydrocarbons were mixed in equal proportions. The nucleotide sequence of an alk polymerase chain reaction (PCR) fragment revealed a 59% nucleotide homology to the Pseudomonas oleovorans alkB gene. The nar fragments were highly homologous to genes coding for large and small subunits of cis-naphthalene 1,2-dioxygenase (narAa and narAb) and to cis-naphthalene dihydrodiol dehydrogenase (narB) from other rhodococci. The oxidation of indene to cis-(1S,2R)-1,2-dihydroxyindan by toluene-induced cells allows to hypothesize that strain 1BN also carries a toluene dioxygenase-like system.     

Catalysis of the biodegradation of unusable medicines by alkanotrophic rhodococci

Experiments on the biodegradation of unusable medicines containing a phenolic hydroxy group by actinobacteria of the genus Rhodococcus were performed. Six species and sixty-four strains were tested. It was found that rhodococci could degrade paracetamol, and some R. ruber strains showed high levels of its degradation. An efficient method for the identification and quantification of paracetamol and the products of its conversion (p-aminophenol, pyrocatechol, and hydroquinone) immediately in the culture liquid was developed. Conditions for the complete biodegradation of paracetamol dosage forms (pills) were optimized. The experimental results can be applied for the development of biotechnological methods for degrading medicines: faked, rejected, or those that are expired.     

Biodegradation of drotaverine hydrochloride by free and immobilized cells of Rhodococcus rhodochrous IEGM 608

Drotaverine [1-(3,4-diethoxybenzylidene)-6,7-diethoxy-1,2,3,4-tetrahydroisoquinoline] hydrochloride, an antispasmodic drug derived from benzylisoquinoline was evaluated for its biodegradability using a bacterial strain Rhodococcus rhodochrous IEGM 608. The experiments were performed under aerobic conditions with rhodococci cultures able to degrade drotaverine. In the presence of glucose, the removal efficiency of drotaverine by free Rhodoccocus cells pre-grown with isoquinoline was above 80?% (200?mg/l, initial concentration) after 25?days. Rhodococcus immobilization on hydrophobized sawdust enhanced the biodegradation process, with the most marked drotaverine loss being observed during the first 5?days of fermentation. High metabolic activity of rhodococcal cells towards drotaverine was confirmed respirometrically. GC-MS analysis of transformation products resulting from drotaverine biodegradation revealed 3,4-diethoxybenzoic acid, 3,4-diethoxybenzaldehyde and 3,4-diethoxybenzoic acid ethyl ester which were detected in the culture medium until drotaverine completely disappeared. Based on these major and other minor metabolites, putative pathways for drotaverine biodegradation were proposed. The obtained data broadened the spectrum of organic xenobiotics oxidized by Rhodoccocus bacteria and proved their potential in decontamination of natural ecosystems from pharma pollutants.     

Construction of random transposition mutagenesis system in Rhodococcuserythropolis using IS1415

Recent studies on the metabolic activities of genus Rhodococcus have shown rhodococci to be of important use in industrial, pharmaceutical and environmental biotechnology. The increasing economic significance of Rhodococcus encourages renewed efforts to characterize their genetic systems, as Rhodococcus genetics are still poorly understood. The goal of this study is to adapt a transposon system for use in creating random mutagenesis in Rhodococcus erythropolis. A plasmid carrying IS1415, a member of IS21 family identified from Rerythropolis, has been constructed and designated as pTNR. pTNR is a non-replicating transposon tool introduced into target cells by electroporation. During its transposition, the transposable-marker gene is separated from the open reading frames (istAB) of IS1415, which should avoid secondary transposition. Transposition of pTNR into wild-type R. erythropolis created mutagenesis with a high efficiency of 1.23x10(6)mutants per microgram plasmid DNA. However, it could also be transposed into other Rhodococcus spp. at lower frequencies in comparison with that of R. erythropolis. It has been indicated by Southern hybridization that the generated kanamycin-resistant mutants were resulted from single transposition event of pTNR. The results also revealed that the transposable-marker gene of pTNR was randomly inserted into the chromosomal DNA of R. erythropolis. The affected DNA regions carrying the transposed DNA element could be conveniently recovered for further characterization using a plasmid rescue procedure. Sequence data of the insertion sites of 40 random mutants analyzed indicated that transposition of pTNR generated 6-bp direct target duplications in 36 cases, while in the remaining four mutants; it generated 5- or 7-bp target duplications (two cases each). This study concluded that pTNR could be served as an efficient genetic tool for construction of random mutagenesis system in Rhodococcus species.