Rhodococcus Species Research Articles

Inferring the evolutionary relationship of 23 Malaysian Rhodococcus isolates with potential as cholesterol degrading bacteria

Abstract Cholesterol is an integral molecule which is used as a building block to numerous biological structures. However, excessive amount of cholesterol in the body causes hypercholesterolemia. Microbial degradation of cholesterol has become a great interest and preferred method to degrade the compound because it is effective and environmental friendly. The genus of Rhodococcus is one of the microorganisms reported to be able to degrade cholesterol by its ability to produce cholesterol oxidase (CHO). Nevertheless, no documentation found so far on the cholesterol degradation by local Rhodococcus species. Thus, this study aims to evaluate the ability of local Rhodococcus isolates in degrading cholesterol. Twenty-three Malaysian Rhodococcus isolates were screened for cholesterol degradation activity using MSC agar which has revealed the ability of all Rhodococcus strains in degrading cholesterol with Rhodococus strain UCC0021 was observed as the best cholesterol degrader. The enzyme activity for CHO was measured and the highest enzyme activity by this bacteria strain was 1.180 U/mL at 72 h, which is 48% higher when compared with other actinobacteria. 16S rRNA sequencing was performed for species identification and phylogenic analysis. The finding in this study has proven the interesting ability of Malaysian Rhodococcus strains to degrade cholesterol and could be used as an alternative tool for cholesterol degradation through the production of cholesterol oxidase. Therefore, future work will be concentrated to enhance the production of enzyme activity through optimization of growth parameters and nutrient conditions by this Rhodococcus strain.

Multiple Non-Species-Specific Pathogens Possibly Triggered the Mass Mortality in Pinna nobilis.

The fan mussel, Pinna nobilis, represents the largest bivalve endemic to the Mediterranean Sea. Since 2016, dramatic mass mortality of this species has been observed in several areas. The first surveys suggested that Haplosporidium pinnae (currently considered species-specific) was the main etiological agent, but recent studies have indicated that a multifactorial disease may be responsible for this phenomenon. In this study, we performed molecular diagnostic analyses on P. nobilis, P. rudis, and bivalve heterologous host species from the island of Sardinia to shed further light on the pathogens involved in the mass mortality. The results support the occurrence of a multifactorial disease and that Mycobacterium spp. and H. pinnae are not necessarily associated with the illness. Indeed, our analyses revealed that H. pinnae is not species-specific for P. nobilis, as it was present in other bivalves at least three years before the mass mortality began, and species of Mycobacterium were also found in healthy individuals of P. nobilis and P. rudis. We also detected the species Rhodococcus erythropolis, representing the first report in fan mussels of a bacterium other than Mycobacterium spp. and Vibrio spp. These results depict a complicated scenario, further demonstrating how the P. nobilis mass mortality event is far from being fully understood.

Biosynthesis and Industrial Production of Androsteroids

Steroids are a group of organic compounds that include sex hormones, adrenal cortical hormones, sterols, and phytosterols. In mammals, steroid biosynthesis starts from cholesterol via multiple steps to the final steroid and occurs in the gonads, adrenal glands, and placenta. This highly regulated pathway involves several cytochrome P450, as well as different dehydrogenases and reductases. Steroids in mammals have also been associated with drug production. Steroid pharmaceuticals such as testosterone and progesterone represent the second largest category of marketed medical products. There heterologous production through microbial transformation of phytosterols has gained interest in the last couple of decades. Phytosterols being the plants sterols serve as inexpensive substrates for the production of steroid derivatives. Various genes and biochemical pathways involved in phytosterol degradation have been identified in many Rhodococcus and Mycobacterium species. Apart from an early investigation in mammals, presence of steroids such as androsteroids and progesterone has also been demonstrated in plants. Their main role is linked with growth, development, and reproduction. Even though plants share some chemical features with mammals, the biosynthesis is different, with the first C22 hydroxylation as an example. This is performed by CYP11A1 in mammals and CYP90B1 in plants. Moreover, the entire plant steroid biosynthesis is not fully elucidated. Knowing this pathway could provide new processes for the industrial biotechnological production of steroid hormones in plants.

A survey of intact low-density polyethylene film biodegradation by terrestrial Actinobacterial species.

Low-density polyethylene (LDPE) polymer is mainly used in the production of plastic bags and food packaging making up the largest volume of plastic pollutions. These polymers are potential substrates for bacteria in the bioremediation process. In this study, soil samples were collected from different plastic landfills in Iran and subsequently enriched in specific media (polyethylene as carbon source) to increase the population of LDPE-degrading bacteria. Seventeen PE-degrading bacteria, some novel, were isolated from Iranian soil samples and identified using 16S rDNA gene sequencing. These isolates were capable of degrading PE in a limited incubation period without the need for physicochemical pretreatments and comprise mostly of Actinobacteria which include the three genera of Streptomyces, Nocardia, and Rhodococcus. The isolates belonged to 17 different species of gram-positive Actinobacteria. In all, 11 species of the genus Streptomyces, 3 species of the genus Rhodococcus, and 3 species of the genus Nocardia were identified. The isolates with less than 99% 16S rRNA gene similarity to previously known species were suspected to be new species. Various analyses (weight loss, SEM, FTIR, and tensile strength test) to determine polyethylene biodegradation rate were carried out after a 60-day incubation period. Analysis of polyethylene biodegradation elucidates that Actinobacteria have a high ability for biodegradation of polyethylene-based plastics. Streptomyces sp. IR-SGS-T10 showed the highest reduction in weight of the LDPE film (1.58mg/g/day) after 60days of incubation without any pretreatments. Rhodococcus sp. IR-SGS-T11 shows the best reduction in the tensile property of LDPE film, while results from FTIR study for Streptomyces sp. IR-SGS-Y1 indicated a significant change in structural analysis.

Computational-based insights into the phylogeny, structure, and function of Rhodococcus alkane-1-monooxygenase.

Alkane-1-monooxygenase of alkanotrophic Rhodococcus species has been characterized using standard bioinformatics tools to investigate phylogenetic relationships, and three-dimensional structure and functions. Results revealed that activity of the Rhodococcus alkane-1-monooxygenase would be optimum in alkaline pH as their isoelectric points were in the range of 7.5 to 9. Higher aliphatic index (87 to 95) indicated that these enzymes are thermostable. Extinction coefficient of the enzyme varied from 68,793 to 1,25,820M-1cm-1 and average molecular weight was 45kDa. Secondary structures predicted maximum alpha-helical content rather than the other conformations such as sheets or turns. The instability index (II) of most stable query protein was 39.7% which was lowest among all 76 proteins analysed in this study. Predicted 3D structure of query protein revealed that it contains homodimer polypeptides. The suitable template for query protein was Flavin-dependent luciferase-type alkane monooxygenase. The presence of 98.3% amino acid residues in Ramachandran plot was determined in 3-D protein model which confirmed the model feasibility. The predicted model contains 12% more α-helix than template protein which indicated towards membrane localization of the protein. The protein interactome partners of predicted model were determined as FMN-dependent oxidoreductase, molybdopterin, nuclear transport factor, and peroxiredoxin. The predicted tertiary model of R. rhodochrous alkane-1-monooxygenase (OOL33526.1) was deposited in Protein Model Database (Accession No.: PM0083166). The overall report is unique to best of our knowledge, and the importance of this study is to understand the theoretical aspects of structure and functions of alkane-1-monooxygenase of hydrocarbonoclastic strains of Rhodococcus.

Chemical Composition, in vitro Cytotoxic, and Antibacterial Activities of Moroccan Medicinal Plants Euphorbia resinifera and Marrubium vulgare

The purpose of our study was to bring elements of knowledge on the anticancer and antibacterial effects of two plants, widely used in Moroccan traditional pharmacopeia. These plants are: Marrubium vulgare and Euphorbia resinifera. The aerial parts of each plant were extracted successively with Hexane, Dichloromethane, and finally with methanol. The MTT-based method was applied to evaluate the cytotoxicity of the cancer cells: animal cells BSR and Vero and human cell RD. We evidenced an anticancer activity of the extract of the Marrubium vulgare and the dichloromethane extract of Euphorbia resinifera against the studied cells. The antibacterial activity was evaluated for three species of Rhodococcus: Rhodococcus equi, and strains GK1, GK3, grown in a liquid medium, or this medium solidified with agar. In the last test, the method is based on substance diffusion from well throughout the solid medium. The obtained profiles showed that the growth of bacteria is strongly inhibited by the extracts of Marrubium vulgare. However, the extracts of Euphorbia resinifera had no significant effect on bacterial growth. The chemical analysis of the raw extracts of Marrubium vulgare and Euphorbia resinifera by GC-MS analysis showed the presence of several major chemical compounds, mainly: octadecane, 2,6,10,15-tetramethylheptadecane, 2,6,10-trimethyltetradecane, linoleic acid, and deisopropylatrazine. Our observations an encouraging for deepening the studies of the extracts, in order to target better the active molecules, isolate them and to determine their mechanisms of action. The suggested studies would result in the much better valorization of these two medicinal plants.

Poly(cis-1,4-isoprene)-cleavage enzymes from natural rubber-utilizing bacteria.

Natural rubber and synthetic poly(cis-1,4-isoprene) are used industrially in the world. Microbial utilization for the isoprene rubbers has been reported in gram-positive and gram-negative bacteria. Poly(cis-1,4-isoprene)-cleavage enzymes that are secreted by rubber-utilizing bacteria cleave the poly(cis-1,4-isoprene) chain to generate low-molecular-weight oligo(cis-1,4-isoprene) derivatives containing aldehyde and ketone groups. The resulting products are converted to the compounds including carboxyl groups, which could then be further catabolized through β-oxidation pathway. One of poly(cis-1,4-isoprene)-cleavage enzymes is latex-clearing protein (Lcp) that was found in gram-positive rubber degraders including Streptomyces, Gordonia, Rhodococcus, and Nocardia species. The other one is rubber oxygenase A and B (RoxA/RoxB) which have been identified from gram-negative rubber degraders such as Steroidobacter cummioxidans and Rhizobacter gummiphilus. Recently, the transcriptional regulation mechanisms for Lcp-coding genes in gram-positive bacteria have been characterized. Here, the current knowledge of genes and enzymes for the isoprene rubber catabolism were summarized.

Bacterial Separation and Community Diversity Analysis of Petroleum Contaminated Soil in Yumen Oilfield

The problem of environmental pollution caused by the development and use of petroleum is increasingly obvious, which is a serious threat to human health. The use of microbial degradation to treat oil pollution is one of the environmentally effective, economical and practical methods.In order to explore the soil microbial diversity in the desert area of Northwest China, this paper analyzes the soil bacterial diversity of soil samples collected from different oil-contaminated areas in Yumen Oilfield for the oil pollution problem in the Yumen Oilfield in the northwest desert area, and selects the high efficiency through pure culture technology. Petroleum degradation bacteria, and research on the biological characteristics of degrading bacteria. The composition, abundance and diversity of bacterial communities in oil-contaminated soil in Yumen Oilfield were analyzed. The culturable bacteria in western oil-contaminated desert soil were separated by coating plate method. The bacterial morphology and 16S rRNA gene system development analysis were studied. The structure and diversity of bacterial community could be cultured, and the oil utilization and degradation ability of the strain could be analyzed. The microbial diversity of Yumen oil-contaminated desert soil was analyzed by Illumina Miseq high-throughput sequencing. Through research, it is found that there are abundant bacterial groups in the oil-contaminated desert soil, and there are obvious diversity. The genetic material in the variable regions of the six soil samples detected a total of 3943 0TU at 97% similarity level, and obtained the soil microbial community. Doors, 48 classes, 78 orders, 179 families and 471 genera, including most common high-efficiency petroleum-degrading bacteria. Petroleum hydrocarbon pollution can change the microbial diversity and community structure of the original soil. The size of microbial diversity in the six soil samples is B2]A1]B1]A2]C1]C2, the diversity of B2 is the highest, the diversity of C2 is the lowest, and the microbial diversity differed greatly between groups, and there was no difference in the group. Among the dominant bacteria isolated from contaminated soil, 8 strains of oil have a degradation rate of more than 30%, including the species of the genus Rhodococcus and Pseudomonas. Soil desertification in western China has a great impact on the local ecological environment. Studying the microbial diversity of desert soils and separating high-efficiency petroleum-degrading strains is of great significance for strengthening the ecological restoration of oil-contaminated environment in desert areas.

Application of Thin-Layer Chromatography-Flame Ionization Detection (TLC-FID) to Total Lipid Quantitation in Mycolic-Acid Synthesizing Rhodococcus and Williamsia Species.

In addition to cell membrane phospholipids, Actinobacteria in the order Corynebacteriales possess a waxy cell envelope containing mycolic acids (MA). In optimized culture condition, some species can also accumulate high concentrations of intracellular triacylglycerols (TAG), which are a potential source of biodiesel. Bacterial lipid classes and composition alter in response to environmental stresses, including nutrient availability, thus understanding carbon flow into different lipid classes is important when optimizing TAG synthesis. Quantitative and qualitative analysis of lipid classes normally requires combinations of different extraction, derivatization, chromatographic and detection methods. In this study, a single-step thin-layer chromatography-flame ionization detection (TLC-FID) technique was applied to quantify lipid classes in six sub-Antarctic Corynebacteriales strains identified as Rhodococcus and Williamsia species. A hexane:diethyl-ether:acetic acid solvent system separated the total cellular lipids extracted from cells lysed by bead beating, which released more bound and unbound MA than sonication. Typical profiles included a major broad non-polar lipid peak, TAG and phospholipids, although trehalose dimycolates, when present, co-eluted with phospholipids. Ultra-performance liquid chromatography-tandem mass-spectrometry and nuclear magnetic resonance spectroscopy detected MA signatures in the non-polar lipid peak and indicated that these lipids were likely bound, at least in part, to sugars from cell wall arabinogalactan. Waxy esters were not detected. The single-solvent TLC-FID procedure provides a useful platform for the quantitation and preliminary screening of cellular lipid classes when testing the impacts of growth conditions on TAG synthesis.

A new Rhodococcus aetherivorans strain isolated from lubricant-contaminated soil as a prospective phenol-biodegrading agent

Microbe-based decontamination of phenol-polluted environments has significant advantages over physical and chemical approaches by being relatively cheaper and ensuring complete phenol degradation. There is a need to search for commercially prospective bacterial strains that are resistant to phenol and other co-pollutants, e.g. oil hydrocarbons, in contaminated environments, and able to carry out efficient phenol biodegradation at a variable range of concentrations. This research characterizes the phenol-biodegrading ability of a new actinobacteria strain isolated from a lubricant-contaminated soil environment. Phenotypic and phylogenetic analyses showed that the novel strain UCM Ac-603 belonged to the species Rhodococcus aetherivorans, and phenol degrading ability was quantitatively characterized for the first time. R. aetherivorans UCM Ac-603 tolerated and assimilated phenol (100% of supplied concentration) and various hydrocarbons (56.2–94.4%) as sole carbon sources. Additional nutrient supplementation was not required for degradation and this organism could grow at a phenol concentration of 500 mg L−1 without inhibition. Complete phenol assimilation occurred after 4 days at an initial concentration of 1750 mg L−1 for freely-suspended cells and at 2000 mg L−1 for vermiculite-immobilized cells: 99.9% assimilation of phenol was possible from a total concentration of 3000 mg L−1 supplied at daily fractional phenol additions of 750 mg L−1 over 4 days. In terms of phenol degradation rates, R. aetherivorans UCM Ac-602 showed efficient phenol degradation over a wide range of initial concentrations with the rates (e.g. 35.7 mg L−1 h−1 at 500 mg L−1 phenol, and 18.2 mg L−1 h−1 at 1750 mg L−1 phenol) significantly exceeding (1.2–5 times) reported data for almost all other phenol-assimilating bacteria. Such efficient phenol degradation ability compared to currently known strains and other beneficial characteristics of R. aetherivorans UCM Ac-602 suggest it is a promising candidate for bioremediation of phenol-contaminated environments.

Characterization of a New Rhamnolipid Biosurfactant Complex from Pseudomonas Isolate DYNA270.

The chemical and physical properties of extracellular rhamnolipid synthesized by a nonfluorescent Pseudomonas species soil isolate, identified as DYNA270, is described, along with characteristics of rhamnolipid production under varying growth conditions and substrates. The biosurfactant is determined to be an anionic, extracellular glycolipid consisting of two major components, the rhamnopyranoside β-1-3-hydroxydecanoyl-3-hydroxydecanoic acid (GU-6) and rhamnopyranosyl β→β2-rhamnopyranoside-β1-3-hydroxydecanoyl-3-hydroxydecanoic acid (GL-2), of molecular weight 504 and 649 daltons, respectively. These glycolipids are produced in a stoichiometric ratio of 1:3, respectively. The purified rhamnolipid mixture exhibits a critical micelle concentration of 20 mg/L, minimum surface (air/water interface) tension of 22 mN/m, and minimum interfacial tension values of 0.005 mN/m (against hexane). The pH optimum, critical micelle concentration, and effective alkane carbon number were established for Pseudomonas species DYNA270 and compared to those of rhamnolipid produced by Pseudomonas aeruginosa PG201. Significant differences are documented in the physical properties of extracellular rhamnolipids derived from these two microorganisms. The surface properties of this rhamnolipid are unique in that ultra-low surface and interfacial tension values are present in both purified rhamnolipid and culture broth containing the rhamnolipid complex (GU6 and GL2). We are not aware of prior studies reporting surface activity values this low for rhamnolipids. An exception is noted for an extracellular trehalose glycolipid produced by Rhodococcus species H13-A, which measured 0.00005 mN/m in the presence of the co-agent pentanol (Singer et al. 1990). Similar CMC values of 20 mg/L have been reported for rhamnolipids, a few being recorded as 5–10 mg/L for Pseudomonas species DSM2874 (Lang et al. 1984).

Structural diversity in the Mycobacteria DUF3349 superfamily.

A protein superfamily with a "Domain of Unknown Function,", DUF3349 (PF11829), is present predominately in Mycobacterium and Rhodococcus bacterial species suggesting that these proteins may have a biological function unique to these bacteria. We previously reported the inaugural structure of a DUF3349 superfamily member, Mycobacterium tuberculosis Rv0543c. Here, we report the structures determined for three additional DUF3349 proteins: Mycobacterium smegmatis MSMEG_1063 and MSMEG_1066 and Mycobacterium abscessus MAB_3403c. Like Rv0543c, the NMR solution structure of MSMEG_1063 revealed a monomeric five α-helix bundle with a similar overall topology. Conversely, the crystal structure of MSMEG_1066 revealed a five α-helix protein with a strikingly different topology and a tetrameric quaternary structure that was confirmed by size exclusion chromatography. The NMR solution structure of a fourth member of the DUF3349 superfamily, MAB_3403c, with 18 residues missing at the N-terminus, revealed a monomeric α-helical protein with a folding topology similar to the three C-terminal helices in the protomer of the MSMEG_1066 tetramer. These structures, together with a GREMLIN-based bioinformatics analysis of the DUF3349 primary amino acid sequences, suggest two subfamilies within the DUF3349 family. The division of the DUF3349 into two distinct subfamilies would have been lost if structure solution had stopped with the first structure in the DUF3349 family, highlighting the insights generated by solving multiple structures within a protein superfamily. Future studies will determine if the structural diversity at the tertiary and quaternary levels in the DUF3349 protein superfamily have functional roles in Mycobacteria and Rhodococcus species with potential implications for structure-based drug discovery.

Microaerobic conditions caused the overwhelming dominance of Acinetobacter spp. and the marginalization of Rhodococcus spp. in diesel fuel/crude oil mixture-amended enrichment cultures

The aim of the present study was to reveal how different microbial communities evolve in diesel fuel/crude oil-contaminated environments under aerobic and microaerobic conditions. To investigate this question, aerobic and microaerobic bacterial enrichments amended with a diesel fuel/crude oil mixture were established and analysed. The representative aerobic enrichment community was dominated by Gammaproteobacteria (64.5%) with high an abundance of Betaproteobacteriales (36.5%), followed by Alphaproteobacteria (8.7%), Actinobacteria (5.6%), and Candidatus Saccharibacteria (4.5%). The most abundant alkane monooxygenase (alkB) genotypes in this enrichment could be linked to members of the genus Rhodococcus and to a novel Gammaproteobacterium, for which we generated a high-quality draft genome using genome-resolved metagenomics of the enrichment culture. Contrarily, in the microaerobic enrichment, Gammaproteobacteria (99%) overwhelmingly dominated the microbial community with a high abundance of the genera Acinetobacter (66.3%), Pseudomonas (11%) and Acidovorax (11%). Under microaerobic conditions, the vast majority of alkB gene sequences could be linked to Pseudomonas veronii. Consequently, results shed light on the fact that the excellent aliphatic hydrocarbon degrading Rhodococcus species favour clear aerobic conditions, while oxygen-limited conditions can facilitate the high abundance of Acinetobacter species in aliphatic hydrocarbon-contaminated subsurface environments.

Rhodococcus subtropicus sp. nov., a new actinobacterium isolated from a cave

A novel Gram-stain-positive actinobacterial strain, designated C9-28T, was isolated from soil sampled in a natural cave on Jeju Island, Republic of Korea. Strain C9-28T morphologically exhibited a rod-coccus life cycle and grew at 10-37 °C (optimum, 30 °C), pH 6-9 (optimum, pH 7) and 0-3 % (optimum, absence of NaCl). In the maximum-likelihood tree based on 16S rRNA gene sequences, strain C9-28T formed a sublineage between a Rhodococcus equi-Rhodococcus soli-Rhodococcus agglutinans clade and the type strain of Rhodococcus defluvii. The closest relatives of strain C9-28T were the type strains of R. defluvii (98.88 % 16S rRNA gene sequence similarity), R. equi (98.88 %) and R. soli (98.60 %). The phylogenomic tree based on whole genome sequences supported the distinct position of the novel strain within the genus Rhodococcus. The following chemotaxonomic characteristics also supported the assignment to the genus: meso-diaminopimelic acid; arabinose and galactose in whole-cell hydrolysates; the predominant menaquinone of MK-8(H2); and polar lipids including diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, three unidentified glycolipids and two unidentified lipids. The predominant cellular fatty acids were C16 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C18 : 1ω9c and C14 : 0. Based on the values of average nucleotide identity and digital DNA-DNA hybridization from whole genome sequences, and in vitro DNA-DNA hybridization between the isolate and the closest relatives, strain C9-28T (=KACC 19823T=DSM 107559T) represents a novel species of the genus Rhodococcus, for which the name Rhodococcussubtropicus sp. nov. is proposed.

Preliminary Evaluation: Hydrocarbons, Cobalt, & Urea Enhance Bacteria Benefits to Plants

Aim: Develop a method to induce PGPB to biosynthesize nitrile compounds that may act as a reliable and repeatable means to increase seed germination in plant species.
 Study Design: The germination experiment was conducted in a completely randomized design with two replications and 40 pots (80 seedlings) per experimental unit, following a 1x1 factorial design for each culture, treated or untreated soil and 1 germination period for both conditions. The nitrile hydratase experiment was conducted in a completely randomized design with 3 replications and 3 soil samples per experimental unit, following a 1x1 factorial design for each cultivar, induced or non-induced soil and 1 cultivation period.
 Place and Duration of Study: Germination work was executed at G & A Innovative Solutions, LLC, GA., April - May 2017. Nitrile Hydratase Assay work was executed at Georgia State University, Applied Environmental Microbiology Dept., Atlanta, GA. November 2010-August 2011. Methodology: Rhodococcus and Bacillus species were induced with short chained-hydrocarbons, cobalt, and urea in a triphasic system for 3 d to potentially make nitrile compounds to benefit seed germination. Increased NHase activity has been previously correlated to production of these nitrile compounds and an increased ability to affect plant development. NHase activity was measured after bacteria were suspended in soil for 6-7 d.
 Results: The induction method sustained and increased NHase activity by 200%, during suspension in soil. Induced Bacillus increased germination by 34%, shoot & root length by 67% & 10%.
 Conclusion: Enhancing biosynthesis of nitriles in PGPB may enhance bacteria ability increase seed germination rates. Measuring NHase activity may indirectly measure efficacy of PGPB in soil. The results are preliminary and require additional studies to confirm results.

Biodegradation of 4-nitrophenol by a Rhodococcus species and a preliminary insight into its toxicoproteome based on mass spectrometry analysis

Biodegradation of 4-nitrophenol by a Rhodococcus species and a preliminary insight into its toxicoproteome based on mass spectrometry analysis

No evidence that gut microbiota impose a net cost on their butterfly host.

Gut microbes are believed to play a critical role in most animal life, yet fitness effects and cost–benefit trade‐offs incurred by the host are poorly understood. Unlike most hosts studied to date, butterflies largely acquire their nutrients from larval feeding, leaving relatively little opportunity for nutritive contributions by the adult's microbiota. This provides an opportunity to measure whether hosting gut microbiota comes at a net nutritional price. Because host and bacteria may compete for sugars, we hypothesized that gut flora would be nutritionally neutral to adult butterflies with plentiful food, but detrimental to semistarved hosts, especially when at high density. We held field‐caught adult Speyeria mormonia under abundant or restricted food conditions. Because antibiotic treatments did not generate consistent variation in their gut microbiota, we used interindividual variability in bacterial loads and operational taxonomic unit abundances to examine correlations between host fitness and the abdominal microbiota present upon natural death. We detected strikingly few relationships between microbial flora and host fitness. Neither total bacterial load nor the abundances of dominant bacterial taxa were related to butterfly fecundity, egg mass or egg chemical content. Increased abundance of a Commensalibacter species did correlate with longer host life span, while increased abundance of a Rhodococcus species correlated with shorter life span. Contrary to our expectations, these relationships were unchanged by food availability to the host and were unrelated to reproductive output. Our results suggest the butterfly microbiota comprises parasitic, commensal and beneficial taxa that together do not impose a net reproductive cost, even under caloric stress.

Analysis of the Alkane Hydroxylase Gene and Long-Chain Cyclic Alkane Degradation in <i>Rhodococcus</i>

To characterize the long-chain cyclic alkane (c-alkane) degradation of bacteria in Rhodococcus, we analyzed the relationship between the alkane hydroxylase gene (alkB) and long-chain c-alkane degradation in 19 species. Eleven strains which were isolated from nature using long-chain c-alkane as a substrate were identified as R. erythropolisc, and all were shown to carry the alkB [alkB R2 type]. This gene type was also carried by two other species, R. rhodochrous and R. baikonurensis. In total, 17 species of the genus Rhodococcus carried alkB, but the gene types differed from each other. The two species R. rhodnii and R. coprophilus did not carry alkB, and their long-chain c-alkane degradation levels were low.

Rhodococcus and Yarrowia-Based Lipid Production Using Lignin-Containing Industrial Residues.

Improvement in biorefining technologies coupled with development of novel fermentation strategies and analysis will be paramount in establishing supplementary and sustainable biofuel pathways. Oleaginous microorganisms that are capable of accumulating triacylglycerides (TAGs) and fatty acid methyl esters (FAMEs), such as Rhodococcus and Yarrowia species, can be used to produce second-generation biofuels from non-food competing carbon sources. These "microbiorefineries" provide a pathway to upgrade agricultural and industrial waste streams to fungible fuels or precursors to chemicals and materials. Here we provide a general overview on cultivating Rhodococcus and Yarrowia on agro-waste/industrial biomass pretreatment waste streams to produce single-cell oils/lipids and preparing samples for FAME detection.

Characterization of Rhodococcus wratislaviensis, a New Gram-Positive Facultative Methylotroph, and Properties of Its C1 Metabolism

A facultative methylotroph, strain 2AzMo (VKM Ac-2782), was isolated from the coastal zone of the Sea of Azov. The cells of the isolate are aerobic gram-positive nonmotile rods. Optimal growth occurs at 1% NaCl, 28°C, and pH 7.5 with 1% СH3OH or 0.3% methylamine as the carbon and energy sources. A broad spectrum of polycarbon substrates is also utilized. Sequencing of the 16S rRNA gene of the strain revealed its similarity to Rhodococcus species: 99.9% to R. wratislaviensis IEGM 1112T (=NCIMB 13082T), 99.4% to R. imtechensis IEGM 940T (=RKJ300T), and 99.2% to R. koreensis IEGM 962T (=DNP505T). The level of DNA–DNA homology of strain 2AzMo with R. wratislaviensis IEGM 1112T (=NCIMB 13082T) was 76%, supporting its identification as a strain of this species. However, unlike strain 2AzMo, the type strain R. wratislaviensis IEGM 1112T, as well as other members of this genus (R. imtechensis IEGM 940T, R. koreensis IEGM 962T, and R. opacus IEGM 716T), do not grow on methanol and methylamine. Methanol oxidation by R.wratislaviensis 2AzMo is catalyzed by alcohol dehydrogenase, which uses 4-nitroso-N,N-dimethylaniline as an artificial electron acceptor. Methylamine is oxidized by methylamine dehydrogenase and the enzymes of the N-methylglutamate pathway. Formaldehyde is then assimilated via the fructose bisphosphate aldolase variant of the ribulose monophosphate pathway of C1 metabolism. Ammonium is assimilated by α-ketoglutarate reductive amination and via the glutamate cycle.