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PubMed1997

Spontaneous resolution of rhodococcal pulmonary infection in a liver transplant recipient.

Pulmonary infection by Rhodococcus equi is characterized by indolent infection in an immunocompromised host with a propensity to form cavitary lesions. Mortality can be greater than 50%; treatment involves prolonged therapy with multiple antibiotics and, occasionally, surgical resection. Recurrence is common. We report a case of a liver transplant patient with a pulmonary nodule caused by R equi; the nodule followed a benign clinical course and resolved spontaneously. This case illustrates that the spectrum of disease caused by R equi is not fully appreciated and that significant pitfalls complicate the diagnosis and management of infection by this unusual and probably underrecognized pathogen.

Actinomycetales Infections↗

PubMed1997

A phylogenetic analysis of aerobic polychlorinated biphenyl-degrading bacteria.

Several bacterial isolates were characterized based on their abilities to degrade specific polychlorinated biphenyls (PCBs) and their 16S rRNA gene sequences. The members of one group of bacteria consisting of Alcaligenes species, including the PCB-degrading bacterium Alcaligenes eutrophus H850, had strong abilities to degrade a broad range of PCBs but not the di-para-chlorine-substituted congeners. The members of another group, which included the PCB-degrading bacterium originally classified as Corynebacterium sp. strain MB1, had strong abilities to degrade di-para-chlorine-substituted PCBs. These bacteria were most likely different members of Rhodococcus species.

DNA, Bacterial↗

PubMed1994

Permeabilization of mycolic-acid-containing actinomycetes for in situ hybridization with fluorescently labelled oligonucleotide probes.

The application of whole-cell hybridization using labelled oligonucleotide probes in microbial systematics and ecology is limited by difficulties in permeabilizing many Gram-positive organisms. In this investigation paraformaldehyde treatment, acid methanolysis and acid hydrolysis were evaluated as a means of permeabilizing mycolic-acid-containing actinomycetes prior to hybridization with a fluorescently labelled oligonucleotide probe designed to bind to a conserved sequence of bacterial 16S rRNA. Methods were evaluated on stationary-phase cultures of Gordona bronchialis, Mycobacterium fortuitum, Nocardia asteroides, N. brasiliensis, Rhodococcus equi, R. erythropolis, R. fascians, R. rhodochrous and Tsukamurella paurometabola, none of which could be probed following 4% (w/v) paraformaldehyde fixation. For comparison and to test the general applicability of mild acid pretreatments, Bacillus subtilis, Lactobacillus plantarum, Escherichia coli and Pseudomonas putida were also studied. The data showed that most of the mycolic-acid-containing organisms were successfully permeabilized by mild acid hydrolysis in 1 M HCl at 37 degrees C. Cells were treated for different lengths of time. In general, the mycolic-acid-containing organisms required between 30 and 50 min hydrolysis, whereas B. subtilis, E. coli and P. putida were rendered permeable in only 10 min. Interestingly, L. plantarum could not be permeabilized using acid hydrolysis even after 60 min exposure to 1 M HCl.

Actinomyces↗

PubMed1998

Mycobacterial linear plasmids have an invertron-like structure related to other linear replicons in actinomycetes.

The authors previously identified large plasmids in Mycobacterium xenopi, M. branderi and M. celatum which appeared to have a linear topology. This study has confirmed the presence of such linear plasmids in mycobacteria, including M. avium, and demonstrated that the ends of these replicons are covalently bound with protein(s), suggesting an invertron-like structure. The termini of one 25 kb plasmid, designated pCLP, from M. celatum were cloned and the first 500 bp of each terminus were sequenced. The termini of this plasmid show the characteristic features of invertrons with terminal inverted repeats of 45 bp (with imperfect matches) and several palindromic sequences. Moreover, similarity existed in the structure and terminal nucleotide sequence of pCLP and the termini of linear replicons of Streptomyces and Rhodococcus species, indicating a conservation of these linear extrachromosomal elements within the Actinomycetales.

Base Sequence↗

PubMed2002

The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase alpha subunit.

Protein engineering by segment exchange was used to distinguish between regions of major and minor influence on the structure of the substrate-binding pocket of a biphenyl dioxygenase (BDO). Eight chimaeric enzyme systems were generated that each consisted of a hybrid hydroxylase alpha subunit (BphA1) containing segments from Burkholderia sp. strain LB400 and Rhodococcus globerulus P6, and of a hydroxylase beta subunit (BphA2), a ferredoxin (BphA3) and a ferredoxin reductase (BphA4) from strain LB400. All hybrid bphA1 genes were expressed at high levels. Seven of the resulting fusion subunits functionally interacted with the other polypeptides of the dioxygenase system to yield catalytically active enzymes. Changes in the regiospecificity of substrate attack, monitored by the formation of seventeen different dioxygenation products obtained from seven chlorobiphenyls, were used to monitor effects of segment exchanges on the structure of the BDO substrate-binding site. Exchanges of neither the beta subunit nor the N- and C-terminal regions of the alpha subunit exerted significant influences. All BDO regions that showed major effects on the substrate-binding pocket were located between approximately positions 165 and 395 of the alpha subunit. Within this part of the enzyme, in addition to segments identified previously, a subregion which is involved in ligation of the mononuclear iron significantly influenced the regiospecificity of substrate dioxygenation. Moreover, the results indicate that the construction of appropriate hybrid genes may be used as a general strategy to overcome problems in obtaining heterologous BDO activities in Escherichia coli or other host organisms.

Amino Acid Sequence↗

PubMed2004

Crystallization and preliminary X-ray analyses of desulfurization enzyme DszB and its C27S mutant complexed with biphenyl-2-sulfinic acid.

DszB is a hydrolase involved in the biodegradation of dibenzothiophene in the soil bacterium Rhodococcus sp. IGTS8. DszB catalyzes the hydrolysis of 2'-hydroxybiphenyl-2-sulfinic acid to sulfite and biphenyl-2-ol. DszB and DszB C27S mutant complexed with biphenyl-2-sulfinic acid were crystallized and preliminary X-ray crystallographic analyses were conducted. The crystals of DszB were found to belong to the orthorhombic P2(1)2(1)2(1) space group, with unit-cell parameters a = 36.7, b = 82.6, c = 139.6 A, and to contain one molecule of DszB in the asymmetric unit. Crystals of DszB C27S complexed with biphenyl-2-sulfinic acid belong to space group C2, with unit-cell parameters a = 153.4, b = 45.9, c = 112.9 A, beta = 115.93 degrees. The calculated Matthews coefficient V(M) for the C2 crystals is approximately 2.3 A(3) Da(-1) if two molecules of DszB are present in the asymmetric unit.

Biphenyl Compounds↗

PubMed2000

Post-translational modification is essential for catalytic activity of nitrile hydratase.

Nitrile hydratase from Rhodococcus sp. N-771 is an alphabeta heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, alphaCys112 and alphaCys114 are modified to a cysteine sulfinic acid (Cys-SO2H) and a cysteine sulfenic acid (Cys-SOH), respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the nitrile hydratase from recombinant unmodified subunits. The alphabeta complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI-LC/MS analysis showed that the anaerobically reconstituted alphabeta complex did not have the modification of alphaCys112-SO2H and aerobic incubation induced the modification. The activity of the reconstituted alphabeta complex correlated with the amount of alphaCys112-SO2H. Furthermore, ESI-LC/MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that alphaCys114 is modified to Cys-SOH together with the sulfinic acid modification of alphaCys112. These results suggest that alphaCys112 and alphaCys114 are spontaneously oxidized to Cys-SO2H and Cys-SOH, respectively, and alphaCys112-SO2H is responsible for the catalytic activity solely or in combination with alphaCys114-SOH.

Amino Acid Sequence↗

PubMed1987

Possible existence of a novel amphipathic immunostimulator in the phenol-water extracts of Mycobacteriaceae.

The extracts having diverse immunostimulating activities were obtained as a water-phase fraction from four bacterial species representing the 4 genera (Mycobacterium, Nocardia, Gordona, and Rhodococcus) of Mycobacteriaceae by the phenol-water method, which is commonly used for extraction of endotoxic lipopolysaccharides (LPS) from gram-negative bacteria and amphipathic substances from gram-positives. These fractions, especially those of G. aurantiaca and R. terrae, showed strong stimulatory effects on murine splenocytes, macrophages of mice and guinea pigs, the immunoadjuvant activities in guinea pigs and mice, and the distinct activities inducing a tumor necrosis factor and interferons alpha/beta and gamma in primed mice. The fractions from G. aurantiaca and R. terrae exhibited potent pyrogenicity and the ability to activate the clotting enzyme cascade of the horseshoe crab (Tachypleus tridentatus). Some of these biological activities were not very different from the potency of the reference endotoxic LPS derived from Escherichia coli or Fusobacterium nucleatum. But the test fractions neither showed the activity to prepare rabbit skin to the local Shwartzman reaction, nor reacted with anti-lipid A conventional and monoclonal antibodies. Furthermore, unlike LPS, these fractions stimulated the splenocytes of C3H/HeJ mice (LPS-Nonresponder). Although the fractions showing the above biological activities have not yet been adequately purified, they contained polysaccharides, whose main constituent sugar is mannose with a smaller amount of arabinose, fatty acids consisting primarily of palmitic, stearic, and tuberculostearic acids, and small amounts of peptides and amino sugars. Since components characteristic of known immunomodulators of bacterial origin, namely endotoxins (lipid A's), cell wall peptidoglycans, lipoteichoic acids, cord factors (trehalose dimycolates), or deoxyribonucleic acids, were practically not detected in these fractions, the agent responsible for the above bioactivities is considered to be a novel substance different from the known, bacterial immunomodulators.

Adjuvants, Immunologic↗

PubMed1999

Production and partial characterization of antibody to cord factor (trehalose 6,6'-dimycolate) in mice.

Antibody production against the trehalose 6,6'-dimycolate (TDM, cord factor) of Rhodococcus ruber, a non-pathogenic species of the Actinomycetales group, was investigated in mice by repeated intraperitoneal injection of TDM in water-in-oil-in-water micelles without carrier protein. The antigenic TDM was isolated and purified chromatographically from the chloroform-methanol extractable lipids of R. ruber. The hydrophobic moiety of this TDM was composed of two molecules of monoenoic or dienoic alpha-mycolic acids with a carbon chain length ranging from C44 to C48 centering at C46. To detect the antibody, an enzyme-linked immunosorbent assay (ELISA) system was employed using plastic plates coated with TDM. The antibody reacted against the TDM of R. ruber. The antibody was reactive in similar fashion against glycosyl monomycolates differing in the carbohydrate moiety, such as that of glucose mycolate (GM) and mannose mycolate (MM), obtained from R. ruber. Moreover, the antibody reacted against mycolic acid methyl ester itself when it was used as the antigen in ELISA, and trehalose did not absorb the antibody to TDM or inhibit the reaction. These results indicate that the epitope of TDM recognized by the antibody is mycolic acid, an extremely hydrophobic part of the molecule. Next, we prepared monoclonal anti-TDM antibody (moAb) in mice myeloma cells to examine its biological activities and the role of humoral immunity in mycobacterial infection. MoAb reacted against the TDM, glycosyl mycolate, and mycolic acid methyl ester in ELISA in the same manner as our polyclonal antibody did. The administration of moAb suppressed granuloma formation in the lungs, spleen, and liver induced by TDM and inhibited the production of interleukin-1 (IL-1) and chemotactic factor, which is reported to precede granuloma formation.

Absorption↗

PubMed1987

Degradations of 4-cholesten-3-one and 1,4-androstadiene-3,17-dione by cholesterol-degrading bacteria.

Degradations of 4-cholesten-3-one and 1,4-androstadiene-3,17-dione, which are intermediates of microbial conversion of cholesterol, by cholesterol-degrading bacteria (12 strains of the genus Rhodococcus isolated from food of animal origin and 12 culture collection strains) were examined. All strains had the ability to degrade 4-cholesten-3-one without necessarily being able to degrade cholesterol. On the other hand, the bacteria were divided into three groups with little or no (0-10%), intermediate (10-70%) and high (70-100%) degradation abilities for 1,4-androstadiene-3,17-dione.

Androstadienes↗

PubMed1987

Presumptive diagnosis of pulmonary nocardiosis: value of sputum microscopy.

Three hundred samples of sputum from patients suffering from various forms of pulmonary disorders were homogenized by pancreatic digestion, examined microscopically and cultured on brain heart infusion agar, trypticase soy agar, in brain heart infusion broth, trypticase soy broth and McClung's carbon-free broth. Several elements of varying morphological forms, believed to be from aerobic actinomycetes or nocardias, were observed in 16 cases. Six strains of Nocardia asteroides, one N. brasiliensis, eight of Rhodococcus species and one Micromonospora were isolated and studied at various stages of growth. Several nocardial and rhodococcal elements closely resembling those observed in the Gram-stained sputa were found. It is suggested that some of those in the sputum were from nocardias and that their appearance in sputum, as seen from the Gram-stained slide, when combined with clinical symptoms can serve for a presumptive diagnosis of pulmonary nocardiosis pending a more definitive identification by a specialized laboratory.

Actinomycetales Infections↗

PubMed2005

Mechanism of lipid-body formation in prokaryotes: how bacteria fatten up.

Neutral lipid accumulation is frequently observed in some Gram-negative prokaryotes like Acinetobacter sp. and most actinomycetes, including the pathogenic Mycobacterium tuberculosis and antibiotic producing streptomycetes. We examined the formation of wax ester- and triacylglycerol (TAG)-bodies in Acinetobacter calcoaceticus and Rhodococcus opacus using microscopic, immunological and biophysical methods. A general model for prokaryotic lipid-body formation is proposed, clearly differing from the current models for the formation of lipid inclusions in eukaryotes and of poly(hydroxyalkanoic acid) (PHA) inclusions in prokaryotes. Formation of lipid-bodies starts with the docking of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) to the cytoplasm membrane. Both, analyses of in vivo and in vitro lipid-body synthesis, demonstrated the formation of small lipid droplets (SLDs), which remain bound to the membrane-associated enzyme. SLDs conglomerated subsequently to membrane-bound lipid-prebodies which are then released into the cytoplasm. The formation of matured lipid-bodies in the cytoplasm occurred by means of coalescence of SLDs inside the lipid prebodies, which are surrounded by a half-unit membrane of phospholipids.

Acinetobacter calcoaceticus↗

PubMed1992

NAD-linked, factor-dependent formaldehyde dehydrogenase or trimeric, zinc-containing, long-chain alcohol dehydrogenase from Amycolatopsis methanolica.

NAD-linked, factor-dependent formaldehyde dehydrogenase (FD-FA1DH) of the Gram-positive methylotrophic bacterium, Amycolatopsis methanolica, was purified to homogeneity. It is a trimeric enzyme with identical subunits (molecular mass 40 kDa) containing 6 atoms Zn/enzyme molecule. The factor is a heat-stable, low-molecular-mass compound, which showed retention on an Aminex HPX-87H column. Inactivation of the factor occurred during manipulation, but activity could be restored by incubation with dithiothreitol. The identity of the factor is still unknown. It could not be replaced by thiol compounds or cofactors known to be involved in metabolism of C1 compounds. Of the aldehydes tested, only formaldehyde was a substrate. However, the enzyme showed also activity with higher aliphatic alcohols and the presence of the factor was not required for this reaction. Methanol was not a substrate, but high concentrations of it could replace the factor in the conversion of formaldehyde. Presumably, a hemiacetal of formaldehyde is the genuine substrate, which, in the case of methanol, acts as a factor leading to methylformate as the product. This view is supported by the fact that formate could only be detected in the reaction mixture after acidification. Inhibition studies revealed that the enzyme contains a reactive thiol group, being protected by the binding of NAD against attack by heavy-metal ions and aldehydes. Studies on the effect of the order of addition of coenzyme and substrate suggested that optimal catalysis required NAD as the first binding component. Substrate specificity and the induction pattern clearly indicate a role of the enzyme in formaldehyde oxidation. However, since FD-FA1DH was also found in A. methanolica grown on n-butanol, but not on ethanol, it may have a role in the oxidation of higher aliphatic alcohols as well. FD-FA1DH and the factor from A. methanolica are very similar to a combination already described for Rhodococcus erythropolis [Eggeling, L. & Sahm, H. (1985) Eur. J. Biochem. 150, 129-134]. NAD-linked, glutathione-dependent formaldehyde dehydrogenase (GD-FA1DH) resembles FD-FA1DH in many respects. Since glutathione has so far not been detected in Gram-positive bacteria, FD-FA1DH could be the counterpart of this enzyme in Gram-positive bacteria. Alignment of the N-terminal sequence (31 residues) of FD-FA1DH with that of GD-FA1DH from rat liver indeed showed similarity (30% identical positions). However, comparable similarity was found with class I alcohol dehydrogenase from this organism and with cytosolic alcohol dehydrogenase from Saccharomyces cerevisiae, isozyme 1.(ABSTRACT TRUNCATED AT 400 WORDS)

Actinomycetales↗

PubMed1992

Hemolytic interactions of Dermatophilus congolensis.

The strains of Dermatophilus congolensis grew on blood agar with washed sheep erythrocytes with marked total hemolysis. In testing for hemolytic interactions they gave a significant synergistic effect of a characteristic shape with Rhodococcus equi and Streptococcus agalactiae, whereas with Staphylococcus aureus producing beta hemolysin and with Staphylococcus aureus producing delta hemolysin a simultaneous synergistic as well as antagonistic effect were observed. First of all a conspicuous inhibition of in the beta hemolysin zone began and then the hemolytic effect of D. congolensis was enhanced. A similar double reaction was also observed with Listeria ivanovii. With delta hemolysin there was an inhibition of the hemolytic effect of D. congolensis and at the same time a synergistic effect could be observed. Also D. congolensis gave a weak synergistic effect with Micrococcus lylae and Listeria monocytogenes, and a further weak antagonistic effect with alpha hemolysin of Staphylococcus aureus, Staphylococcus hyicus, Staphylococcus chromogenes and Micrococcus luteus. No interaction of D. congolensis was established with Corynebacterium pseudotuberculosis.

Actinomycetales↗

PubMed1995

Rapid growth and increased biomass yield of Mycobacterium farcinogenes and some related taxa in broth and agar media.

A broth medium containing yeast extract (4 g/l), glucose (15 g/l), magnesium sulphate (0.5 g/l), tri-sodium citrate (1.5 g/l), potassium sulphate (0.5 g/l), ammonium ferric citrate (trace) and buffered with potassium di-hydrogen phosphate (5 g/l) was formulated and found to support a luxuriant growth of Mycobacterium farcinogenes strains that was superior to the conventional media used before. Similar results were obtained with M. senegalense strains and with representatives of the genera Gordona, Mycobacterium, Nocardia, Rhodococcus and Tsukamurella. Out of 13 diverse agar-based media, M. farcinogenes was found to grow particularly well on Mueller Hinton's medium, followed, in order of decreasing growth rate, by modified Bennett's, DST, tryptic soya and glucose yeast extract agars.

Animals↗

PubMed2004

Temporal dynamics and degradation activity of an bacterial inoculum for treating waste metal-working fluid.

In order for established bioreactors to be effective for treating chemically mixed wastes such as metal working fluids (MWF) it is essential that they harbour microbial populations that can maintain sufficient active biomass and degrade each of the chemical constituents present. In this study we investigated the effectiveness of a bacterial consortium composed of four species (Clavibacter michiganensis, Methylobacterium mesophilicum, Rhodococcus erythropolis and Pseudomonas putida), assembled on the basis of their apparent ubiquity in waste MWF, degradation ability and tolerance to fluctuating chemistry of the waste. The temporal dynamics of the inoculum and its effects on the fate of individual chemical components of the waste were studied, by regular sampling, over 400 h. Using a complementary approach of culture with chemotaxonomic (FAME) analysis and applying group specific probes (FISH), the inoculum was found to represent a significant component of the community in bioreactors with and without presence of indigenous MWF populations. In addition, the reduction in the COD by the consortium was approximately 85% of the total pollution load, and 30-40% more effectively than any other treatment (indigenous MWF community alone or activated sludge). Furthermore, all the chemical constituents, including the biocide (a formaldehyde release agent) demonstrated > 60% reduction. Many chemical components of the MWF proved to be recalcitrant in the other treatments. The results of this study confirm that assemblage of an inoculum, based on a comprehensive knowledge of the indigenous microbial community, in the target habitat, is a highly effective way of selecting microbial populations for bioaugmentation of bioreactors.

Actinomycetales↗

PubMed2006

Study of a hexane-degrading consortium in a biofilter and in liquid culture: biodiversity, kinetics and characterization of degrading strains.

A gasoline-degrading consortium, originating from a Mexican soil, was used to study its hexane-degradation kinetics in liquid culture and in a biofilter with mineral support. The biodiversity of the consortium depending on the culture conditions and electron and energy source (gasoline, hexane in liquid or hexane in the biofilter) was analyzed using a 16S rRNA-based approach. Significant differences between the populations were observed, indicating a probable adaptation to the substrate. Two strains, named SP2B and SP72-3, isolated from the consortium, belonged to Actinomycetes and demonstrated a high metabolic potential in hexane degradation. Even though the SP2B strain was related to Rhodococcus ruber DSM 43338(T) by phylogenetic studies, it displayed enlarged metabolic properties in hexane and other short-alkane degradation compared with the collection strain.

Actinobacteria↗

PubMed2002

Reductive deamination as a new step in the anaerobic microbial degradation of halogenated anilines.

In this paper we report the isolation and characterization of an anaerobic enrichment culture as well as of a Rhodococcus sp. strain 2 capable of degrading 3,4-dihaloanilines under nitrate reducing conditions. Using mass spectrometry several of the intermediates formed in the process of 3,4-dichloroaniline conversion were identified. Most interesting is the observation of reductive deamination and the formation of 1,2-dichlorobenzene as one of the intermediates. Using 19F NMR and fluorinated 3,4-dihaloaniline model substrates it was corroborated that reductive deamination of the anilines to give dihalobenzene intermediates represents a new initial step in the anaerobic microbial degradation of these halogenated anilines.

Anaerobiosis↗