Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630

An oleaginous hydrocarbon-degrading Rhodococcus opacus strain (PD630) was isolated from a soil sample. The cells were able to grow on a variety of substrates and to produce large amounts of three different types of intracellular inclusions during growth on alkanes, phenylalkanes, or non-hydrocarbon substrates. Electron microscopy revealed large numbers of electron-transparent inclusions with a sphere-like structure. In addition, electron-dense inclusions representing polyphosphate and electron-transparent inclusions with an elongated disc-shaped morphology occurred in small amounts. The electron-transparent inclusions of alkane- or gluconate-grown cells were composed of neutral lipids (98%, w/w), phospholipids (1.2%, w/w), and protein (0.8%, w/w). The major component of the cellular inclusions was triacylglycerols; minor amounts of diacylglycerols and probably also some free fatty acids were also present. Free fatty acids and/or fatty acids in acylglycerols in cells of R. opacus amounted up to 76 or 87% of the cellular dry weight in gluconate- or olive-oil-grown cells, respectively. The fatty acid composition of the inclusions depended on the substrate used for cultivation. In cells cultivated on n-alkanes, the composition of the fatty acids was related to the substrate, and intermediates of the β-oxidation pathway, such as hexadecanoic or pentadecanoic acid, were among the acylglycerols. Hexadecanoic acid was also the major fatty acid (up 36% of total fatty acids) occurring in the lipid inclusions of gluconate-grown cells. This indicated that strain PD630 utilized β-oxidation and de novo fatty acid biosynthesis for the synthesis of storage lipids. Inclusions isolated from phenyldecane-grown cells contained mainly the non-modified substrate and phenylalkanoic acids derived from the hydrocarbon oxidation, such as phenyldecanoic acid, phenyloctanoic acid, and phenylhexanoic acid, and approximately 5% (w/w) of diacylglycerols. The lipid inclusions seemed to have definite structures, probably with membranes at their surfaces, which allow them to maintain their shape, and with some associated proteins, probably involved in the inclusion formation. Received: 22 December 1995 / Accepted: 12 March 1996     

Glycogenformation by Rhodococcus species and the effect of inhibition of lipid biosynthesis on glycogen accumulation in Rhodococcus opacus PD630

Members of the genus Rhodococcus were investigated for their ability to produce glycogen during cultivation on gluconate or glucose. Strains belonging to Rhodococcus ruber, Rhodococcus opacus, Rhodococcus fascians, Rhodococcus erythropolis and Rhodococcus equi were able to produce glycogen up to 0.2–5.6% of cellular dry weight (CDW). The glycogen content varied from 0.8% to 3.2% of CDW in cells of R. opacus PD630, which is a well-known oleaginous bacterium, during the exponential growth phase, when cultivated on diverse carbon sources. Maltose and pyruvate promoted glycogen accumulation by cells of strain PD630 to a greater extent than glucose, gluconate, lactose, sucrose or acetate. This strain was able to produce triacylglycerols, polyhydroxyalkanoates and glycogen as storage compounds during growth on gluconate, although triacylglycerols were always the main product under the conditions of this study. Cerulenin, an inhibitor of de novo fatty acid synthesis, inhibited the accumulation of triacylglycerols from gluconate and increased the content of polyhydroxyalkanoates (from 2.0% to 4.2%, CDW) and glycogen (from 0.1% to 3.0%, CDW). An increase of the polyhydroxyalkanoates and glycogen content was also observed in two mutants of R. opacus PD630, which produced reduced amounts of triacylglycerols during cultivation of cells on gluconate.     

Comparative and functional genomics of Rhodococcus opacus PD630 for biofuels development

The Actinomycetales bacteria Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 bioconvert a diverse range of organic substrates through lipid biosynthesis into large quantities of energy-rich triacylglycerols (TAGs). To describe the genetic basis of the Rhodococcus oleaginous metabolism, we sequenced and performed comparative analysis of the 9.27 Mb R. opacus PD630 genome. Metabolic-reconstruction assigned 2017 enzymatic reactions to the 8632 R. opacus PD630 genes we identified. Of these, 261 genes were implicated in the R. opacus PD630 TAGs cycle by metabolic reconstruction and gene family analysis. Rhodococcus synthesizes uncommon straight-chain odd-carbon fatty acids in high abundance and stores them as TAGs. We have identified these to be pentadecanoic, heptadecanoic, and cis-heptadecenoic acids. To identify bioconversion pathways, we screened R. opacus PD630, R. jostii RHA1, Ralstonia eutropha H16, and C. glutamicum 13032 for growth on 190 compounds. The results of the catabolic screen, phylogenetic analysis of the TAGs cycle enzymes, and metabolic product characterizations were integrated into a working model of prokaryotic oleaginy.     

The Rhodococcus opacus PD630 Heparin-Binding Hemagglutinin Homolog TadA Mediates Lipid Body Formation

Generally, prokaryotes store carbon as polyhydroxyalkanoate, starch, or glycogen. The Gram-positive actinomycete Rhodococcus opacus strain PD630 is noteworthy in that it stores carbon in the form of triacylglycerol (TAG). Several studies have demonstrated that R. opacus PD630 can accumulate up to 76% of its cell dry weight as TAG when grown under nitrogen-limiting conditions. While this process is well studied, the underlying molecular and biochemical mechanisms leading to TAG biosynthesis and subsequent storage are poorly understood. We designed a high-throughput genetic screening to identify genes and their products required for TAG biosynthesis and storage in R. opacus PD630. We identified a gene predicted to encode a putative heparin-binding hemagglutinin homolog, which we have termed tadA (triacylglycerol accumulation deficient), as being important for TAG accumulation. Kinetic studies of TAG accumulation in both the wild-type (WT) and mutant strains demonstrated that the tadA mutant accumulates 30 to 40% less TAG than the parental strain (WT). We observed that lipid bodies formed by the mutant strain were of a different size and shape than those of the WT. Characterization of TadA demonstrated that the protein is capable of binding heparin and of agglutinating purified lipid bodies. Finally, we observed that the TadA protein localizes to lipid bodies in R. opacus PD630 both in vivo and in vitro. Based on these data, we hypothesize that the TadA protein acts to aggregate small lipid bodies, found in cells during early stages of lipid storage, into larger lipid bodies and thus plays a key role in lipid body maturation in R. opacus PD630.While the majority of eubacteria (24, 33), and indeed many archaea (22, 33), store carbon as polyhydroxyalkanoate (PHA), a small subset of organisms, primarily actinomycetes, are capable of storing carbon in the form of triacylglycerol (TAG). TAG biosynthesis and storage has been observed in members of the genera Mycobacterium, Rhodococcus, Streptomyces, Nocardia, and others (4, 6, 11, 12, 19, 20, 36). Of these organisms, TAG biosynthesis and storage has been most extensively studied for the Gram-positive, non-spore-forming actinomycete Rhodococcus opacus, strain PD630 (1-6, 11, 12, 19, 20, 25, 36, 38-41).Several studies have demonstrated that R. opacus PD630 is capable of accumulating up to 76% of its cell dry weight (CDW) as TAG (summarized in reference 3). As is the case for PHA biosynthesis, TAG accumulation occurs during nitrogen starvation when carbon is in excess (1-3, 27, 41). Paralleling PHA biosynthesis further, TAG is stored in R. opacus PD630 in distinct inclusion bodies, termed lipid bodies (2, 3, 25, 38, 40). While several studies have sought to identify the underlying molecular and biochemical mechanisms behind TAG biosynthesis and storage in the form of lipid bodies, very little is known concerning these processes.We sought to identify genes and their products that are essential for lipid metabolism in R. opacus PD630. Utilizing a forward genetic approach, we identified a conserved hypothetical gene, termed herein tadA (triacylglycerol accumulation deficient), which is predicted to encode a protein with sequence similarity to the heparin-binding hemagglutinin (HbhA) family of proteins from the genus Mycobacterium. The tadA::Tn5 mutant accumulates 30 to 40% less TAG than the parental strain. We demonstrate that this deficiency is most likely the result of altered lipid body formation and morphology. Through biochemical studies, we further demonstrate that the predicted heparin-binding activity of this protein is essential for its activity both in vivo and in vitro. To our knowledge, this is the first protein shown to regulate lipid body assembly and maturation in prokaryotes.     

Accumulation and mobilization of storage lipids by Rhodococcus opacus PD630 and Rhodococcus ruber NCIMB 40126

The time course of the accumulation of triacylglycerols (TAGs) in Rhodococcus opacus PD630 or of TAGs plus polyhydroxyalkanoates (PHA) in Rhodococcus ruber NCIMB 40126 with gluconate or glucose as carbon source, respectively, was studied. In addition, we examined the mobilization of these storage compounds in the absence of a carbon source. R. opacus accumulated TAGs only after the exhaustion of ammonium in the medium, and, with a fixed concentration of the carbon source, the amounts of TAGs in the cells increased with decreasing concentrations of ammonium in the medium. When these cells were incubated in the absence of an additional carbon source, about 90% of these TAGs were mobilized and used as endogenous carbon source, particularly if ammonium was available. R. ruber accumulated a copolyester consisting of 3-hydroxybutyrate and 3-hydroxyvalerate already during the early exponential growth phase, whereas TAGs were synthesized and accumulated mainly during the late exponential and stationary growth phases. In the stationary growth phase, synthesis of TAGs continued, whereas PHA was partially mobilized. In the absence of an additional carbon source but in the presence of ammonium, mobilization of TAGs started first and was then paralleled by the mobilization of PHA, resulting in an approximately 90% and 80% decrease of these storage compounds, respectively. During the accumulation phase, interesting shifts in the composition of the two storage compounds occurred, indicating that the substrates of the PHA synthase and the TAG synthesizing enzymes were provided to varying extents, depending on whether the cells were in the early or late exponential or in the stationary growth phase. Received: 12 January 2000 / Received revision: 22 February 2000 / Accepted: 25 February 2000     

The atf2 gene is involved in triacylglycerol biosynthesis and accumulation in the oleaginous Rhodococcus opacus PD630

Rhodococcus opacus PD630 is an oleaginous bacterium able to accumulate large amounts of triacylglycerols (TAG) in different carbon sources. The last reaction for TAG biosynthesis is catalyzed by the bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) enzymes encoded by atf genes. R. opacus PD630 possesses at least 17 putative atf homologous genes in its genome, but only atf1 and atf2 exhibited a significant DGAT activity when expressed in E. coli, as revealed in a previous study. The contribution of atf1 gene to TAG accumulation by strain PD630 has been demonstrated previously, although additional Atfs may also contribute to lipid accumulation, since the atf1-disrupted mutant is still able to produce significant amounts of TAG (Alvarez et al., Microbiology 154:2327–2335, 2008). In this study, we investigated the in vivo role of atf2 gene in TAG accumulation by R. opacus PD630 by using different genetic strategies. The atf2-disrupted mutant exhibited a decrease in TAG accumulation (up to 25–30 %, w/w) and an approximately tenfold increase in glycogen formation in comparison with the wild-type strain. Surprisingly, in contrast to single mutants, a double mutant generated by the disruption of atf1 and atf2 genes only showed a very low effect in TAG and in glycogen accumulation under lipid storage conditions. Overexpression of atf1 and atf2 genes in strain PD630 promoted an increase of approximately 10 % (w/w) in TAG accumulation, while heterologous expression of atf2 gene in Mycobacterium smegmatis caused an increase in TAG accumulation during cultivation in nitrogen-rich media. This study demonstrated that, in addition to atf1 gene, atf2 is actively involved in TAG accumulation by the oleaginous R. opacus PD630.     

In vitro effects of sterculic acid on lipid biosynthesis in Rhodococcus opacus strain PD630 and isolation of mutants defective in fatty acid desaturation

The in vivo effects of sterculic acid methyl ester on triacylglycerol fatty acid composition in the oleaginous, hydrocarbon-degrading bacterium R. opacus strain PD630 was investigated. Sterculic acid, a cyclopropene fatty acid and an inhibitor of the stearoyl-CoA desaturase system, strongly inhibited the synthesis of monoenic fatty acids, of saturated fatty acids with more than 16 carbon atoms and of odd-numbered fatty acids when added to the culture medium. In addition, chemical mutagenesis and the application of the penicillin enrichment technique provided mutants, which were more or less completely impaired in the desaturation of long-chain fatty acids and exhibited in some cases a similar fatty acid composition like the wild-type in the presence of sterculic acid methyl ester. The implications of these findings for fatty acid metabolism in R. opacus strain PD630 are discussed.     

Preparative isolation of lipid inclusions from Rhodococcus opacus and Rhodococcus ruber and identification of granule-associated proteins

Triacylglycerol granules synthesized and accumulated by Rhodococcus opacus and Rhodococcus ruber were isolated by glycerol density gradient centrifugation. Whereas only one type of granule could be isolated from R. opacus, two types of granules with different specific densities were isolated from R. ruber. Both types of R. ruber granules showed a similar content of triacylglycerols and poly(3-hydroxybutyrate- co-3-hydroxyvalerate), but the protein profiles of both types were significantly different. The granules with the lower specific density were colorless; the granules with the higher specific density had a deep orange pigmentation. Solubilization studies revealed three different groups of granule-associated proteins: (1) unspecifically bound proteins, (2) relatively weakly associated proteins, and (3) proteins that resisted solubilization by treatment with 2 M NaCl, 2% (w/v) Triton X-114, 6 M guanidinium hydrochloride, up to 8% (w/v) SDS, and proteolytic digestion. The strong association of proteins of the last group suggested that these may play a specific role in the synthesis or mobilization of storage lipids or in the structure of the granules. The N-terminal amino acid sequences of the most tightly bound proteins were obtained. Proteins of low molecular weight with striking sequence similarity to the ribosomal protein L7 from various actinomycetes were always copurified with the granules.     

Establishment of a gene transfer system for Rhodococcus opacus PD630 based on electroporation and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acids)

A gene transfer system for Rhodococcus opacus PD630 based on electroporation was established and optimized employing the Escherichia coli-Rhodococcus shuttle vectors pNC9501 and pNC9503 as well as the E. coli-Corynebacterium glutamicum shuttle vector pJC1 as suitable cloning vectors for R. opacus PD630, resulting in transformation efficiencies up to 1.5 × 10⁵ CFUs/μg plasmid DNA. Applying the optimized electroporation protocol to the pNC9501-derivatives pAK68 and pAK71 harboring the entire PHB synthesis operon from Ralstonia eutropha and the PHA synthase gene phaC1 from Pseudomonas aeruginosa, respectively, recombinant PHA biosynthesis was established in R. opacus PD630 and the TAG-negative mutant ROM34. Plasmid pAK68 enabled synthesis and accumulation of poly(3HB) in R. opacus PD630 and ROM34 during cultivation under storage conditions from 1% (w/v) gluconate, of poly(3HB-co-3HV) from 0.2% (w/v) propionate and of poly(3HV) from 0.1% (w/v) valerate. Under storage conditions, recombinant strains of PD630 and ROM34 harboring pAK71 were able to synthesize and accumulate PHA of the medium chain length hydroxyalkanoic acids 3HHx, 3HO, 3HD and 3HDD from 0.1% (w/v) hexadecane or octadecane and a copolyester composed of 3HHp, 3HN and 3HUD from 0.1% (w/v) pentadecane or heptadecane. In the recombinant strains of PD630 and ROM34, the thiostrepton-induced overexpression of a 20 kDa protein was observed with its N-terminus exhibiting a homology of 60% identical amino acids to TipA from Streptomyces lividans. Received: 13 March 1999 / Received revision: 18 May 1999 / Accepted: 21 May 1999     

High cell density cultivation of Rhodococcus opacus for lipid production at a pilot-plant scale

The triacylglycerol (TAG)-accumulating bacterium Rhodococcus opacus strain PD630 was investigated with respect to the fermentative production of TAGs consisting of an unusually high fraction of fatty acids with an odd-number of carbon atoms and unsaturated monoenic fatty acids from sugar beet molasses and sucrose. Fed-batch fermentations were optimized at the 30-1 scale in a stirred tank bioreactor at 30 degrees C using a mineral salts medium, which contained sugar beet molasses and sucrose as sole carbon sources. Approximately 37.5 g cell dry matter (CDM) per liter was the highest cell density that was obtained at that scale with a TAG content in the cells of 52%. This fermentative process was also applied to a 500-1 pilot-plant scale. Cell densities as high as 18.4 g CDM per liter were obtained, and 42% of the sucrose present in the medium was converted into cell mass which consisted of 38.4% TAGs.     

Characterization of the naphthalene-degrading bacterium, Rhodococcus opacus M213

Bacterial strain M213 was isolated from a fuel oil-contaminated soil in Idaho, USA, by growth on naphthalene as a sole source of carbon, and was identified as Rhodococcus opacus M213 by 16S rDNA sequence analysis and growth on substrates characteristic of this species. M213 was screened for growth on a variety of aromatic hydrocarbons, and growth was observed only on simple 1 and 2 ring compounds. No growth or poor growth was observed with chlorinated aromatic compounds such as 2,4-dichlorophenol and chlorobenzoates. No growth was observed by M213 on salicylate, and M213 resting cells grown on naphthalene did not attack salicylate. In addition, no salicylate hydroxylase activity was detected in cell free lysates, suggesting a pathway for naphthalene catabolism that does not pass through salicylate. Enzyme assays indicated induction of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase on different substrates. Total DNA from M213 was screened for hybridization with a variety of genes encoding catechol dioxygenases, but hybridization was observed only with catA (encoding catechol 1,2-dioxygenase) from R. opacus 1CP and edoD (encoding catechol 2,3-dioxygenase) from Rhodococcus sp. I1. Plasmid analysis indicated the presence of two plasmids (pNUO1 and pNUO2). edoD hybridized to pNUO1, a very large (approximately 750 kb) linear plasmid.     

Heterologous expression of Rhodococcus opacus L-amino acid oxidase in Streptomyces lividans

L-amino acid oxidase (L-AAO) from Rhodococcus opacus is a highly enantioselective enzyme with a broad substrate specificity that catalyses the oxidation of L-amino acids to keto acids. The lao-gene (AY053450) from R. opacus was cloned into different Escherichia coli and Streptomyces lividans expression vectors. Expression in E. coli resulted in the accumulation of insoluble protein, but S. lividans was a suitable host for the heterologous production of L-AAO. When using the thiostrepton-inducible vector pIlaao, a specific activity of 0.18 Umg(-1) was obtained in the crude extract of S. lividans 1326. For the vector pUlaao, which contains the constitutive ermEp(*) promoter, a specific activity of 0.05 Umg(-1) was reached. Both the wild type and the recombinant L-AAO were purified to homogeneity. The expression systems described here now allow the structural and biochemical analysis of the L-AAO using genetic engineering methods.     

Role of amine oxidase expression to maintain putrescine homeostasis in Rhodococcus opacus

While applications of amine oxidases are increasing, few have been characterised and our understanding of their biological role and strategies for bacteria exploitation are limited. By altering the nitrogen source (NH₄Cl, putrescine and cadaverine (diamines) and butylamine (monoamine)) and concentration, we have identified a constitutive flavin dependent oxidase (EC 1.4.3.10) within Rhodococcus opacus. The activity of this oxidase can be increased by over two orders of magnitude in the presence of aliphatic diamines. In addition, the expression of a copper dependent diamine oxidase (EC 1.4.3.22) was observed at diamine concentrations > 1 mM or when cells were grown with butylamine, which acts to inhibit the flavin oxidase. A Michaelis–Menten kinetic treatment of the flavin oxidase delivered a Michaelis constant (K_M) = 190 μM and maximum rate (k_cat) = 21.8 s^?1 for the oxidative deamination of putrescine with a lower K_M (=60 μM) and comparable k_cat (=18.2 s^?1) for the copper oxidase. MALDI–TOF and genomic analyses have indicated a metabolic clustering of functionally related genes. From a consideration of amine oxidase specificity and sequence homology, we propose a putrescine degradation pathway within Rhodococcus that utilises oxidases in tandem with subsequent dehydrogenase and transaminase enzymes. The implications of PUT homeostasis through the action of the two oxidases are discussed with respect to stressors, evolution and application in microbe-assisted phytoremediation or bio-augmentation.     

Enzymes of a New Modified ortho-Pathway Utilizing 2-Chlorophenol in Rhodococcus opacus 1CP

Chlorocatechol 1,2-dioxygenase (CC 1,2-DO), chloromuconate cycloisomerase (CMCI), chloromuconolactone isomerase (CMLI), and dienolactone hydrolase (DELH), the key enzymes of a new modified ortho-pathway in Rhodococcus opacus 1CP cells utilizing 2-chlorophenol via a 3-chlorocatechol branch of a modified ortho-pathway, were isolated and characterized. CC 1,2-DO showed the maximum activity with 3-chlorocatechol; its activity with catechol and 4-chlorocatechol was 93 and 50%, respectively. The enzyme of the studied pathway had physicochemical properties intermediate between the pyrocatechase of ordinary and chlorocatechase of modified pathways described earlier for this strain. In contrast to the enzymes investigated earlier, CMCI of the new pathway exhibited high substrate specificity. The enzyme had K _m for 2-chloromuconate of 142.86 M, V _max = 71.43 U/mg, pH optimum around 6.0, and temperature optimum at 65°C. CMCI converted 2-chloromuconate into 5-chloromuconolactone. CMLI converted 5-chloromuconolactone into cis-dienolactone used as a substrate by DELH; this enzyme did not convert trans-dienolactone. DELH had Km for cis-dienolactone of 200 M, V _max = 167 U/mg, pH optimum of 8.6, and temperature optimum of 40°C. These results confirm the existence of a new modified ortho-pathway for utilization of 2-chlorophenol by R. opacus 1CP.     

Identification of fluoropyrogallols as new intermediates in biotransformation of monofluorophenols in Rhodococcus opacus 1cp

The transformation of monofluorophenols by whole cells of Rhodococcus opacus 1cp was investigated, with special emphasis on the nature of hydroxylated intermediates formed. Thin-layer chromatography, mass spectrum analysis, and (19)F nuclear magnetic resonance demonstrated the formation of fluorocatechol and trihydroxyfluorobenzene derivatives from each of three monofluorophenols. The (19)F chemical shifts and proton-coupled splitting patterns of the fluorine resonances of the trihydroxyfluorobenzene products established that the trihydroxylated aromatic metabolites contained hydroxyl substituents on three adjacent carbon atoms. Thus, formation of 1,2, 3-trihydroxy-4-fluorobenzene (4-fluoropyrogallol) from 2-fluorophenol and formation of 1,2,3-trihydroxy-5-fluorobenzene (5-fluoropyrogallol) from 3-fluorophenol and 4-fluorophenol were observed. These results indicate the involvement of fluoropyrogallols as previously unidentified metabolites in the biotransformation of monofluorophenols in R. opacus 1cp.     

Identification of Fluoropyrogallols as New Intermediates in Biotransformation of Monofluorophenols in Rhodococcus opacus 1cp

The transformation of monofluorophenols by whole cells of Rhodococcus opacus 1cp was investigated, with special emphasis on the nature of hydroxylated intermediates formed. Thin-layer chromatography, mass spectrum analysis, and ¹⁹F nuclear magnetic resonance demonstrated the formation of fluorocatechol and trihydroxyfluorobenzene derivatives from each of three monofluorophenols. The ¹⁹F chemical shifts and proton-coupled splitting patterns of the fluorine resonances of the trihydroxyfluorobenzene products established that the trihydroxylated aromatic metabolites contained hydroxyl substituents on three adjacent carbon atoms. Thus, formation of 1,2,3-trihydroxy-4-fluorobenzene (4-fluoropyrogallol) from 2-fluorophenol and formation of 1,2,3-trihydroxy-5-fluorobenzene (5-fluoropyrogallol) from 3-fluorophenol and 4-fluorophenol were observed. These results indicate the involvement of fluoropyrogallols as previously unidentified metabolites in the biotransformation of monofluorophenols in R. opacus 1cp.     

The role of formate for the growth of Rhodococcus opacus UFZ B 408 on pyridine

R. opacus UFZ B 408 is able to use pyridine, a potentially growth-inhibiting substrate, as the sole source of carbon, energy and nitrogen. In a previous publication [1] we reported that with the simultaneous utilization of a second carbon and energy source in carbon-substrate-limited chemostat culture, stable steady states could be achieved at higher dilution rates than with growth on pyridine as the sole substrate. Owing to the higher growth yield during growth on such a substrate mixture, both the specific pyridine consumption rates and the residual pyridine concentrations were lower at similar dilution rates than with growth on pyridine alone. Therefore, the critical growth-inhibitory pyridine concentration was only achieved at a higher dilution rate. With the investigations presented here in carbon-substrate-limited continuous culture, the simultaneous utilization of pyridine and formate by R. opacus UFZ B 408 was studied. The yield coefficient during growth on pyridine as the sole substrate amounted to about 0.55 g dry mass/g pyridine. Theoretically, however, the carbon-metabolism-determined yield coefficient should have been about 0.915 g dry mass/g pyridine. Because of the difference between these two values the conclusion was drawn that pyridine is energetically deficient. That means that during growth on pyridine a part of the substrate was dissimilated to supply the energy required for the incorporation of the pyridine carbon into biomass. Formate cannot be used as a carbon source for growth by R. opacus UFZ B 408. However, with growth on pyridine, formate was oxidized simultaneously. During growth on pyridine/formate mixtures, the yield coefficient could be enhanced up to 0.7 g dry mass/g pyridine. That means that biologically usable energy, generated in the course of the formate oxidation, was used for the assimilation of pyridine carbon. The increase in the yield coefficient was related to the utilization ratio of formate to pyridine in a linear manner. However, the carbon-metabolism-determined yield coefficient of 0.915 g dry mass/g pyridine could not be achieved. That can be put down to the fact that R. opacus UFZ B 408 possesses only a limited capacity to oxidize externally supplied formate. Because of the limited formate oxidation capacity the probability is low that, with simultaneous utilization of formate, stable steady states could be achieved at substantially higher dilution rates than with growth on pyridine alone. Enzymatic studies revealed the induction of both NAD(P)⁺-linked glutaric dialdehyde dehydrogenase and isocitrate lyase during growth on pyridine. Therefore, the conclusion was drawn that pyridine is metabolized by R. opacus UFZ B 408 via the same pathway described for the utilization of pyridine by Nocardia Z1 [2]. This conclusion implies that the ability to oxidize formate represents a metabolic performance which seems not to be directly related to the pyridine metabolism of R. opacus UFZ B 408.