Functional analysis of putative transporters involved in oligotrophic growth of Rhodococcus erythropolis N9T-4.

Rhodococcus erythropolis N9T-4, which is an extremely oligotrophic bacterium, can survive in a completely inorganic medium with no additional carbon source. This bacterium utilizes atmospheric CO2, but does not require any additional energy source such as light and hydrogen gas, required by autotrophic microorganisms. However, its CO2 fixation and energy-acquisition systems in the oligotrophic growth remain unrevealed. We expected N9T-4 to have the transporter(s) that imports essential compound(s) for its oligotrophic growth. Three putative ATP-binding cassette (ABC) transporters were found to be highly upregulated under oligotrophic conditions. We constructed the gene-deletion mutants of a gene encoding the substrate-binding protein for each ABC transporter (∆sbp1, ∆sbp2, and ∆sbp3). Among these mutants, ∆sbp1 showed growth defects on oligotrophic medium without carbon source. We examined the growth of the mutants on the oligotrophic medium containing 1% trehalose as a sole carbon source. The results exhibited worse growth of ∆sbp3 than that of the control strain (∆ligD), whereas intracellular trehalose content of all mutants decreased compared with that of ∆ligD. It was reported that trehalose functions as the mycolate carrier to the arabinogalactan layer in the cell wall of Mycobacterium tuberculosis. Transmission electron microscopic analysis of ∆sbp1 cells showed that an outermost envelope of the ∆sbp1 cell diminished, which was expected to be mycolate layer. From these results, we suggest that the same trehalose-recycling system as that in a Mycobacterium cell functions in the oligotrophic growth of N9T-4, and the ABC transporter comprising Sbp1 as the substrate-binding protein is strongly involved in the oligotrophic growth of N9T-4.

Biodegradation and chemotaxis of polychlorinated biphenyls, biphenyls, and their metabolites by Rhodococcus spp.

Two biphenyl-degrading bacterial strains, SS1 and SS2, were isolated from polychlorinated biphenyl (PCB)-contaminated soil. They were identified as Rhodococcus ruber and Rhodococcus pyridinivorans based on the 16S rRNA gene sequence, as well as morphological, physiological and biochemical characteristics. SS1 and SS2 exhibited tolerance to 2000 and 3000 mg/L of biphenyl. And they could degrade 83.2 and 71.5% of 1300 mg/L biphenyl within 84 h, respectively. In the case of low-chlorinated PCB congeners, benzoate and 3-chlorobenzoate, the degradation activities of SS1 and SS2 were also significant. In addition, these two strains exhibited chemotactic response toward TCA-cycle intermediates, benzoate, biphenyl and 2-chlorobenzoate. This study indicated that, like the flagellated bacteria, non-flagellated Rhodococcus spp. might actively seek substrates through the process of chemotaxis once the substrates are depleted in their surroundings. Together, these data provide supporting evidence that SS1 and SS2 might be good candidates for restoring biphenyl/PCB-polluted environments.

Bacterial lipid droplets bind to DNA via an intermediary protein that enhances survival under stress.

Lipid droplets (LDs) are multi-functional organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer, and exist in organisms ranging from bacteria to humans. Here we study the functions of LDs in the oleaginous bacterium Rhodococcus jostii. We show that these LDs bind to genomic DNA through the major LD protein, MLDS, which increases survival rate of the bacterial cells under nutritional and genotoxic stress. MLDS expression is regulated by a transcriptional regulator, MLDSR, that binds to the operator and promoter of the operon encoding both proteins. LDs sequester MLDSR, controlling its availability for transcriptional regulation. Our findings support the idea that bacterial LDs can regulate nucleic acid function and facilitate bacterial survival under stress.

Biosynthesis of 1α-hydroxycorticosterone in the winter skate Leucoraja ocellata: evidence to suggest a novel steroidogenic route.

The present study explores the ability of intracellular bacteria within the renal-inter-renal tissue of the winter skate Leucoraja ocellata to metabolize steroids and contribute to the synthesis of the novel elasmobranch corticosteroid, 1α-hydroxycorticosterone (1α-OH-B). Despite the rarity of C1 hydroxylation noted in the original identification of 1α-OH-B, literature provides evidence for steroid C1 hydroxylation by micro-organisms. Eight ureolytic bacterial isolates were identified in the renal-inter-renal tissue of L. ocellata, the latter being the site of 1α-OH-B synthesis. From incubations of bacterial isolates with known amounts of potential 1α-OH-B precursors, one isolate UM008 of the genus Rhodococcus was seen to metabolize corticosteroids and produce novel products via HPLC analysis. Cations Zn2+ and Fe3+ altered metabolism of certain steroid precursors, suggesting inhibition of Rhodococcus steroid catabolism. Genome sequencing of UM008 identified strong sequence and structural homology to that of Rhodococcus erythropolis PR4. A complete enzymatic pathway for steroid-ring oxidation as documented within other Actinobacteria was identified within the UM008 genome. This study highlights the potential role of Rhodococcus bacteria in steroid metabolism and proposes a novel alternative pathway for 1α-OH-B synthesis, suggesting a unique form of mutualism between intracellular bacteria and their elasmobranch host.

[Morphological, Physiological, and Biochemical Characteristics of a Benzoate-Degrading Strain Rhodococcus opacus 1CP under Stress Conditions].

Ability of actinobacteria Rhodococcus opacus 1CP to survive under unfavorable conditions and retain its biodegradation activity was assessed. The morphological and ultrastructural features of R. opacus 1CP cells degrading benzoate in the presence of oxidants and stress-protecting agents were investigated. The cells of R. opacus 1CP were resistant to oxidative stress caused by up to 100 mM H2O2 or up to 25 µM juglone (5-oxy-1,4-naphthoquinone). After 2 h of stress impact, changes in the fatty acid composition, increased activity of antioxidant enzymes, and changes in cell morphology and ultrastructure were observed. The strain retained its ability to degrade benzoate. Quercetin had a protective effect on benzoate-degrading cells of R. opacus 1CP. The strategy for cells survival under unfavorable conditions was formulated, which included decreased cell size/volume and formation of densely-packed cell conglomerates, in which the cells are embedded into a common matrix. Formation of conglomerates may probably be considered as a means for protecting the cells against aggressive environmental factors. The multicellular conglomerate structure and the matrix material impede the penetration of toxic substances into the conglomerates, promoting survival of the cells located inside.

A unique intracellular compartment formed during the oligotrophic growth of Rhodococcus erythropolis N9T-4.

Rhodococcus erythropolis N9T-4, isolated from stored crude oil, shows extremely oligotrophic features and can grow on a basal medium without any additional carbon, nitrogen, sulfur, and energy sources, but requires CO2 for its oligotrophic growth. Transmission electron microscopic observation showed that a relatively large and spherical compartment was observed in a N9T-4 cell grown under oligotrophic conditions. In most cases, only one compartment was observed per cell, but in some cases, it was localized at each pole of the cell, suggesting that it divides at cell division. We termed this unique bacterial compartment an oligobody. The oligobody was not observed or very rarely observed in small sizes under nutrient rich conditions, whereas additional carbon sources did not affect oligobody formation. Energy dispersive X-ray spectroscopy analysis revealed remarkable peaks corresponding to phosphorus and potassium in the oligobody. The oligobodies in N9T-4 cells could be stained by Toluidine blue, suggesting that the oligobody is composed of inorganic polyphosphate and is a type of acidocalcisome. Two genes-encoding polyphosphate kinases, ppk1 and ppk2, were found in the N9T-4 genome: ppk1 disruption caused a negative effect on the formation of the oligobody. Although it was suggested that the oligobody plays an important role for the oligotrophic growth, both ppk-deleted mutants showed the same level of oligotrophic growth as the wild-type strain.

Drotaverine hydrochloride degradation using cyst-like dormant cells of Rhodococcus ruber.

This work has a focus on adaptive capabilities of the actinobacterium Rhodococcus ruber IEGM 326 to cope with drotaverine hydrochloride (DH), a known pharmaceutical pollutant. Cultivation of R. ruber in a nitrogen-limited medium with incubation at the ambient temperature resulted in the formation of cyst-like dormant cells (CLDCs). They maintained viability for 2-7 months, possessed the undetectable respiratory activity and elevated resistance to heating, and had a specific morphology. CLDCs are regarded to ensure long-term survival in various habitats and may be used as storage formulations. R. ruber IEGM 326 was tolerant to DH (MIC, 200 mg/l) and displayed different abilities to degrade this compound, depending on inoculum, temperature, and the presence of glucose as co-oxidized substrate. Thus, the loss of DH (20 mg/l) over 48 h at the optimal temperature (27 ± 2 °C) was 5-8 % in the absence of glucose after inoculating with vegetative cells. The addition of glucose (5 g/l) increased DH degradation up to 46 %. Noteworthy, CLDCs as inoculum were advantageous over vegetative cells to degrade DH at the non-optimal temperature (35 ± 2 °C) at reduced bulk respiratory activity. The obtained results are promising to improve the biodegrading capabilities of other Rhodococcus strains.

Integrated omics study delineates the dynamics of lipid droplets in Rhodococcus opacus PD630.

Rhodococcus opacus strain PD630 (R. opacus PD630), is an oleaginous bacterium, and also is one of few prokaryotic organisms that contain lipid droplets (LDs). LD is an important organelle for lipid storage but also intercellular communication regarding energy metabolism, and yet is a poorly understood cellular organelle. To understand the dynamics of LD using a simple model organism, we conducted a series of comprehensive omics studies of R. opacus PD630 including complete genome, transcriptome and proteome analysis. The genome of R. opacus PD630 encodes 8947 genes that are significantly enriched in the lipid transport, synthesis and metabolic, indicating a super ability of carbon source biosynthesis and catabolism. The comparative transcriptome analysis from three culture conditions revealed the landscape of gene-altered expressions responsible for lipid accumulation. The LD proteomes further identified the proteins that mediate lipid synthesis, storage and other biological functions. Integrating these three omics uncovered 177 proteins that may be involved in lipid metabolism and LD dynamics. A LD structure-like protein LPD06283 was further verified to affect the LD morphology. Our omics studies provide not only a first integrated omics study of prokaryotic LD organelle, but also a systematic platform for facilitating further prokaryotic LD research and biofuel development.

In-situ determination of the mechanical properties of gliding or non-motile bacteria by atomic force microscopy under physiological conditions without immobilization.

We present a study about AFM imaging of living, moving or self-immobilized bacteria in their genuine physiological liquid medium. No external immobilization protocol, neither chemical nor mechanical, was needed. For the first time, the native gliding movements of Gram-negative Nostoc cyanobacteria upon the surface, at speeds up to 900 µm/h, were studied by AFM. This was possible thanks to an improved combination of a gentle sample preparation process and an AFM procedure based on fast and complete force-distance curves made at every pixel, drastically reducing lateral forces. No limitation in spatial resolution or imaging rate was detected. Gram-positive and non-motile Rhodococcus wratislaviensis bacteria were studied as well. From the approach curves, Young modulus and turgor pressure were measured for both strains at different gliding speeds and are ranging from 20±3 to 105±5 MPa and 40±5 to 310±30 kPa depending on the bacterium and the gliding speed. For Nostoc, spatially limited zones with higher values of stiffness were observed. The related spatial period is much higher than the mean length of Nostoc nodules. This was explained by an inhomogeneous mechanical activation of nodules in the cyanobacterium. We also observed the presence of a soft extra cellular matrix (ECM) around the Nostoc bacterium. Both strains left a track of polymeric slime with variable thicknesses. For Rhodococcus, it is equal to few hundreds of nanometers, likely to promote its adhesion to the sample. While gliding, the Nostoc secretes a slime layer the thickness of which is in the nanometer range and increases with the gliding speed. This result reinforces the hypothesis of a propulsion mechanism based, for Nostoc cyanobacteria, on ejection of slime. These results open a large window on new studies of both dynamical phenomena of practical and fundamental interests such as the formation of biofilms and dynamic properties of bacteria in real physiological conditions.

pFiD188, the linear virulence plasmid of Rhodococcus fascians D188.

Rhodococcus fascians is currently the only phytopathogen of which the virulence genes occur on a linear plasmid. To get insight into the origin of this replicon and into the virulence strategy of this broad-spectrum phytopathogen, the sequence of the linear plasmid of strain D188, pFiD188, was determined. Analysis of the 198,917 bp revealed four syntenic regions with linear plasmids of R. erythropolis, R. jostii, and R. opacus, suggesting a common origin of these replicons. Mutational analysis of pFi_086 and pFi_102, similar to cutinases and type IV peptidases, respectively, showed that conserved region R2 was involved in plasmid dispersal and pointed toward a novel function for actinobacterial cutinases in conjugation. Additionally, pFiD188 had three regions that were unique for R. fascians. Functional analysis of the stk and nrp loci of regions U2 and U3, respectively, indicated that their role in symptom development was limited compared with that of the previously identified fas, att, and hyp virulence loci situated in region U1. Thus, pFiD188 is a typical rhodococcal linear plasmid with a composite structure that encodes core functions involved in plasmid maintenance and accessory functions, some possibly acquired through horizontal gene transfer, implicated in virulence and the interaction with the host.

Production of 3-hydroxypropionic acid from acrylic acid by newly isolated rhodococcus erythropolis LG12.

A novel microorganism, designated as LG12, was isolated from soil based on its ability to use acrylic acid as the sole carbon source. An electron microscopic analysis of its morphological characteristics and phylogenetic classification by 16S rRNA homology showed that the LG12 strain belongs to Rhodococcus erythropolis. R. erythropolis LG12 was able to metabolize a high concentration of acrylic acid (up to 40 g/l). In addition, R. erythropolis LG12 exhibited the highest acrylic acid-degrading activity among the tested microorganisms, including R. rhodochrous, R. equi, R. rubber, Candida rugosa, and Bacillus cereus. The effect of the culture conditions of R. erythropolis LG12 on the production of 3-hydroxypropionic acid (3HP) from acrylic acid was also examined. To enhance the production of 3HP, acrylic acid-assimilating activity was induced by adding 1 mM acrylic acid to the culture medium when the cell density reached an OD600 of 5. Further cultivation of R. erythropolis LG12 with 40 g/l of acrylic acid resulted in the production of 17.5 g/l of 3HP with a molar conversion yield of 44% and productivity of 0.22 g/I/h at 30 degrees after 72 h.

Analysis of force interactions between AFM tips and hydrophobic bacteria using DLVO theory.

Microbial adhesion to surfaces and interfaces is strongly influenced by their structure and physicochemical properties. We used atomic force microscopy (AFM) to measure the forces between chemically functionalized AFM tips and two bacterial species exhibiting different cell surface hydrophobicities, measured as the oil/water contact angle (theta): Acinetobacter venetianus RAG-1 (theta = 56.4 degrees ) and Rhodococcus erythropolis 20S-E1-c (theta = 152.9 degrees ). The forces were measured as the AFM tips, coated with either hydrophobic (octadecane) or hydrophilic (undecanol) groups, approached the bacterial cells in aqueous buffer. The experimental force curves between the two microbial cells and functionalized AFM probes were not successfully described by the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory of colloid stability. To reconcile the discrepancy between theory and experiments, two types of extended DLVO models were proposed. The first modification considers an additional acid-base component that accounts for attractive hydrophobic interactions and repulsive hydration effects. The second model considers an additional exponentially decaying steric interaction between polymeric brushes in addition to the acid-base interactions. These extended DLVO predictions agreed well with AFM experimental data for both A. venetianus RAG-1, whose surface consists of an exopolymeric capsule and pili, and R. erythropolis 20S-E1-c, whose surface is covered by an exopolymeric capsule. The extended models for the bacteria-AFM tip force-distance curves were consistent with the effects of steric interactions.

Biodegradation of dimethyl phthalate, diethyl phthalate and di-n-butyl phthalate by Rhodococcus sp. L4 isolated from activated sludge.

In this study, an aerobic bacterial strain capable of utilizing dimethyl phthalate (DMP), diethyl phthalate (DEP) and di-n-butyl phthalate (DBP) as sole carbon source and energy was isolated from activated sludge collected from a dyeing plant. According to its morphology, physiochemical characteristics and 16S rDNA sequence, the strain was identified as Rhodococcus ruber. The biodegradation batch tests of DMP, DEP and DBP by the Rhodococcus sp. L4 showed the optimal pH value, temperature and substrate concentration: pH 7.0-8.0, 30-37 degrees C and PAEs concentration

Laboratory-scale biofiltration of acrylonitrile by Rhodococcus rhodochrous DAP 96622 in a trickling bed bioreactor.

Acrylonitrile (ACN), a volatile component of the waste generated during the production of acrylamide, also is often associated with aromatic contaminants such as toluene and styrene. Biofiltration, considered an effective technique for the treatment of volatile hydrocarbons, has not been used to treat volatile nitriles. An experimental laboratory-scale trickling bed bioreactor using cells of Rhodococcus rhodochrous DAP 96622 supported on granular activated carbon (GAC) was developed and evaluated to assess the ability of biofiltration to treat ACN. In addition to following the course of treatability of ACN, kinetics of ACN biodegradation during both recycle batch and open modes of operation by immobilized and free cells were evaluated. For fed-batch mode bioreactor with immobilized cells, almost complete ACN removal (>95%) was achieved at a flow rate of 0.1 microl/min ACN and 0.8 microl/min toluene (TOL) (for comparative purposes this is equivalent to 6.9 mg l(-1) h(-1) ACN and 83.52 mg l(-1) h(-1) TOL). In a single-pass mode bioreactor with immobilized cells, at ACN inlet loads of 100-200 mg l(-1) h(-1) and TOL inlet load of approximately 400 mg l(-1) h(-1), with empty bed retention time (EBRT) of 8 min, ACN removal efficiency was approximately 90%. The three-dimensional structure and characteristics of the biofilm were investigated using confocal scanning laser microscopy (CSLM). CLSM images revealed a robust and heterogeneous biofilm, with microcolonies interspersed with voids and channels. Analysis of the precise measurement of biofilm characteristics using COMSTAT agreed with the assumption that both biomass and biofilm thickness increased along the carbon column depth.

Analysis of benzene-induced effects on Rhodococcus sp. 33 reveals that constitutive processes play a major role in conferring tolerance.

Most studies investigating mechanisms that confer microorganisms with tolerance to solvents have often focused on adaptive responses following exposure, while less attention has been given to inherent, or constitutive, processes that prevail at the onset of exposure to a toxic solvent. In this study, we investigated several properties of the highly solvent-tolerant bacterium Rhodococcus sp. 33 that confer it with a tolerance to high concentrations of benzene. When challenged with liquid benzene, the growth of both nonadapted and adapted cells was decreased by 0.25-0.30% (v/v) liquid benzene, and higher concentrations (> or =0.35% v/v) produced a complete cessation in the growth of only nonadapted cells. When exposed to presolubilized benzene, nonadapted cells tolerated < or =1000 mg/l, whereas adapted cells tolerated >1400 mg/l. Measuring the cell membrane fluidity of the cells during these exposure experiments showed that at the onset of exposure, the membranes of adapted cells were less affected by benzene compared to nonadapted cells, although these effects were insignificant in the long term. Several benzene-sensitive mutants were generated from this Rhodococcus, two of which were unable to degrade benzene, yet they still tolerated 500-800 mg/l. This confirmed our earlier work suggesting that the benzene-degradation pathway of this organism plays a minor role in tolerance. Under the phase and transmission electron microscope, the mutants were found to have lost the ability to produce extracellular polymers, and many cells appeared pleomorphic, containing intracellular membrane invaginations and mesosome-like structures. As will be discussed, these results identify important functions of the cell membrane, the cell wall, and extracellular polymer in their native state (i.e., before exposure) in conferring this organism with tolerance to benzene.

Biofilm formation and partial biodegradation of polystyrene by the actinomycete Rhodococcus ruber: biodegradation of polystyrene.

Polystyrene, which is one of the most utilized thermoplastics, is highly durable and is considered to be non-biodegradable. Hence, polystyrene waste accumulates in the environment posing an increasing ecological threat. In a previous study we have isolated a biofilm-producing strain (C208) of the actinomycete Rhodococcus ruber that degraded polyethylene films. Formation of biofilm, by C208, improved the biodegradation of polyethylene. Consequently, the present study aimed at monitoring the kinetics of biofilm formation by C208 on polystyrene, determining the physiological activity of the biofilm and analyzing its capacity to degrade polystyrene. Quantification of the biofilm biomass was performed using a modified crystal violet (CV) staining or by monitoring the protein content in the biofilm. When cultured on polystyrene flakes, most of the bacterial cells adhered to the polystyrene surface within few hours, forming a biofilm. The growth of the on polystyrene showed a pattern similar to that of a planktonic culture. Furthermore, the respiration rate, of the biofilm, exhibited a pattern similar to that of the biofilm growth. In contrast, the respiration activity of the planktonic population showed a constant decline with time. Addition of mineral oil (0.005% w/v), but not non-ionic surfactants, increased the biofilm biomass. Extended incubation of the biofilm for up to 8 weeks resulted in a small reduction in the polystyrene weight (0.8% of gravimetric weight loss). This study demonstrates the high affinity of C208 to polystyrene which lead to biofilm formation and, presumably, induced partial biodegradation.

Identification of alkane hydroxylase genes in Rhodococcus sp. strain TMP2 that degrades a branched alkane.

Rhodococcus sp. TMP2 is an alkane-degrading strain that can grow with a branched alkane as a sole carbon source. TMP2 degrades considerable amounts of pristane at 20 degrees C but not at 30 degrees C. In order to gain insights into microbial alkane degradation, we characterized one of the key enzymes for alkane degradation. TMP2 contains at least five genes for membrane-bound, non-heme iron, alkane hydroxylase, known as AlkB (alkB1-5). Phylogenetical analysis using bacterial alkB genes indicates that TMP2 is a close relative of the alkane-degrading bacteria, such as Rhodococcus erythropolis NRRL B-16531 and Q15. RT-PCR analysis showed that expressions of the genes for AlkB1 and AlkB2 were apparently induced by the addition of pristane at a low temperature. The results suggest that TMP2 recruits certain alkane hydroxylase systems to utilize a branched alkane under low temperature conditions.

Atomic force microscopy measurement of heterogeneity in bacterial surface hydrophobicity.

The structure and physicochemical properties of microbial surfaces at the molecular level determine their adhesion to surfaces and interfaces. Here, we report the use of atomic force microscopy (AFM) to explore the morphology of soft, living cells in aqueous buffer, to map bacterial surface heterogeneities, and to directly correlate the results in the AFM force-distance curves to the macroscopic properties of the microbial surfaces. The surfaces of two bacterial species, Acinetobacter venetianus RAG-1 and Rhodococcus erythropolis 20S-E1-c, showing different macroscopic surface hydrophobicity were probed with chemically functionalized AFM tips, terminating in hydrophobic and hydrophilic groups. All force measurements were obtained in contact mode and made on a location of the bacterium selected from the alternating current mode image. AFM imaging revealed morphological details of the microbial-surface ultrastructures with about 20 nm resolution. The heterogeneous surface morphology was directly correlated with differences in adhesion forces as revealed by retraction force curves and also with the presence of external structures, either pili or capsules, as confirmed by transmission electron microscopy. The AFM force curves for both bacterial species showed differences in the interactions of extracellular structures with hydrophilic and hydrophobic tips. A. venetianus RAG-1 showed an irregular pattern with multiple adhesion peaks suggesting the presence of biopolymers with different lengths on its surface. R. erythropolis 20S-E1-c exhibited long-range attraction forces and single rupture events suggesting a more hydrophobic and smoother surface. The adhesion force measurements indicated a patchy surface distribution of interaction forces for both bacterial species, with the highest forces grouped at one pole of the cell for R. erythropolis 20S-E1-c and a random distribution of adhesion forces in the case of A. venetianus RAG-1. The magnitude of the adhesion forces was proportional to the three-phase contact angle between hexadecane and water on the bacterial surfaces.

Isolation and characterization of a carbendazim-degrading Rhodococcus sp. djl-6.

Bacterium djl-6, capable of degrading carbendazim, was isolated by continuous enrichment culture originating from carbendazim-treated soil. The isolate was identified as Rhodococcus sp. according to its phenotypic features, physiologic and biochemical characteristics, and phylogenetic analysis. The strain could use carbendazim as sole carbon or nitrogen source. It showed a high average degradation rate of 55.56 mg . L(-1) . d(-1) in M9 medium amended with carbendazim. High-pressure liquid chromatography-mass spectrometry (HPLC-MS) analysis showed the presence of 2-aminobenzimidazole, benzimidazole, and an unknown metabolite with molecular ions (M(+)) of m/z 104.8 and 118.5. The degradation in the isolate djl-6 seems to be initiated with the cleavage of the methyl carbemate side chain, resulting in the formation of 2-aminobenzimidazole and benzimidazole. This is the first report of the intermediates benzimidazole and 2-aminobenzimidazole found together in the culture filtrate of pure bacterium.

Biofilm development of the polyethylene-degrading bacterium Rhodococcus ruber.

We have recently isolated a biofilm-producing strain (C208) of Rhodococcus ruber that degraded polyethylene at a rate of 0.86% per week (r2=0.98). Strain C208 adheres to polyethylene immediately upon exposure to the polyolefin. This initial biofilm differentiates (in a stepwise process that lasts about 20 h) into cell-aggregation-forming microcolonies. Further organization yields "mushroom-like" three-dimensional structures on the mature biofilm. The ratio between the population densities of the biofilm and the planktonic C208 cells after 10 days of incubation was about 60:1, indicating a high preference for the biofilm mode of growth. Analysis of extracellular polymeric substances (EPS) in the biofilm of C208 revealed that the polysaccharides level was up to 2.5 folds higher than that of the protein. The biofilm showed a high viability even after 60 days of incubation, apparently due to polyethylene biodegradation.