Physiology and comparative genomics of the haloalkalitolerant and hydrocarbonoclastic marine strain Rhodococcus ruber MSA14.

Marine hydrocarbonoclastic bacteria can use polycyclic aromatic hydrocarbons as carbon and energy sources, that makes these bacteria highly attractive for bioremediation in oil-polluted waters. However, genomic and metabolic differences between species are still the subject of study to understand the evolution and strategies to degrade PAHs. This study presents Rhodococcus ruber MSA14, an isolated bacterium from marine sediments in Baja California, Mexico, which exhibits adaptability to saline environments, a high level of intrinsic pyrene tolerance (> 5 g L- 1), and efficient degradation of pyrene (0.2 g L- 1) by 30% in 27 days. Additionally, this strain demonstrates versatility by using naphthalene and phenanthrene as individual carbon sources. The genome sequencing of R. ruber MSA14 revealed a genome spanning 5.45 Mbp, a plasmid of 72 kbp, and three putative megaplasmids, lengths between 110 and 470 Kbp. The bioinformatics analysis of the R. ruber MSA14 genome revealed 56 genes that encode enzymes involved in the peripheral and central pathways of aromatic hydrocarbon catabolism, alkane, alkene, and polymer degradation. Within its genome, R. ruber MSA14 possesses genes responsible for salt tolerance and siderophore production. In addition, the genomic analysis of R. ruber MSA14 against 13 reference genomes revealed that all compared strains have at least one gene involved in the alkanes and catechol degradation pathway. Overall, physiological assays and genomic analysis suggest that R. ruber MSA14 is a new haloalkalitolerant and hydrocarbonoclastic strain toward a wide range of hydrocarbons, making it a promising candidate for in-depth characterization studies and bioremediation processes as part of a synthetic microbial consortium, as well as having a better understanding of the catabolic potential and functional diversity among the Rhodococci group.

Genomic sequence of three hydrocarbonoclastic pseudomonadaceae strains isolated from marine sediments of the port of Rosarito, Baja California, Mexico.

We report the draft genome sequence of three marine bacteria belonging to Pseudomonas and Stutzerimonas genera, with hydrocarbonoclastic metabolism for oil and monoaromatic hydrocarbon degradation. The genomic information of these organisms contributes to the knowledge of natural and polluted marine environments with ubiquitous presence of hydrocarbons as a selective pressure.

Surviving in the Brine: A Multi-Omics Approach for Understanding the Physiology of the Halophile Fungus Aspergillus sydowii at Saturated NaCl Concentration.

Although various studies have investigated osmoadaptations of halophilic fungi to saline conditions, only few analyzed the fungal mechanisms occurring at saturated NaCl concentrations. Halophilic Aspergillus sydowii is a model organism for the study of molecular adaptations of filamentous fungi to hyperosmolarity. For the first time a multi-omics approach (i.e., transcriptomics and metabolomics) was used to compare A. sydowii at saturated concentration (5.13 M NaCl) to optimal salinity (1 M NaCl). Analysis revealed 1,842 genes differentially expressed of which 704 were overexpressed. Most differentially expressed genes were involved in metabolism and signal transduction. A gene ontology multi-scale network showed that ATP binding constituted the main network node with direct interactions to phosphorelay signal transduction, polysaccharide metabolism, and transferase activity. Free amino acids significantly decreased and amino acid metabolism was reprogrammed at 5.13 M NaCl. mRNA transcriptional analysis revealed upregulation of genes involved in methionine and cysteine biosynthesis at extreme water deprivation by NaCl. No modifications of membrane fatty acid composition occurred. Upregulated genes were involved in high-osmolarity glycerol signal transduction pathways, biosynthesis of ß-1,3-glucans, and cross-membrane ion transporters. Downregulated genes were related to the synthesis of chitin, mannose, cell wall proteins, starvation, pheromone synthesis, and cell cycle. Non-coding RNAs represented the 20% of the total transcripts with 7% classified as long non-coding RNAs (lncRNAs). The 42% and 69% of the total lncRNAs and RNAs encoding transcription factors, respectively, were differentially expressed. A network analysis showed that differentially expressed lncRNAs and RNAs coding transcriptional factors were mainly related to the regulation of metabolic processes, protein phosphorylation, protein kinase activity, and plasma membrane composition. Metabolomic analyses revealed more complex and unknown metabolites at saturated NaCl concentration than at optimal salinity. This study is the first attempt to unravel the molecular ecology of an ascomycetous fungus at extreme water deprivation by NaCl (5.13 M). This work also represents a pioneer study to investigate the importance of lncRNAs and transcriptional factors in the transcriptomic response to high NaCl stress in halophilic fungi.

Microplastics: Sources and distribution in surface waters and sediments of Todos Santos Bay, Mexico.

Microplastics (MPs) are ubiquitous and a threat to marine and freshwater environments. Effluent waters from secondary wastewater treatment plants (WWTPs) into Todos Santos Bay (TSB) were investigated as sources of MPs. MPs were detected in all analyzed matrices and presented variable morphologies. MPs from surface water samples (n = 18) varied from 0.01 to 0.70 plastic particles/m3 (pp/m3). Fragments (47 ±â€¯23%) and fibers (47 ±â€¯23%) were the most abundant particles found in the surface water samples. In sediment samples (n = 11), MPs varied from 85 to 2494 pp/0.1 m2. Sediment samples showed fragments of 70 ±â€¯19%, fibers 28 ±â€¯18% in mean. The range of MP values from WWTP effluents (n = 24) was 81 to 1556 pp/m3, and fibers (65 ±â€¯28%) were the most abundant MP particles. Several synthetic polymers (polypropylene, polyethylene, polyethylene-propylene, polyvinyl chloride, cellophane), and natural fibers (cotton and wood) were identified. The surface currents and the parameters that modulate them, are the main factors that dominate the distribution of MPs in surface waters. While in the sediments the parameters such as bathymetry and grain size distribution have more influence on their distribution in the marine environment, where the effluent waters from WWTPs only contributes MPs to the TSB.

High-Affinity Chemotaxis to Histamine Mediated by the TlpQ Chemoreceptor of the Human Pathogen Pseudomonas aeruginosa.

Histamine is a key biological signaling molecule. It acts as a neurotransmitter in the central and peripheral nervous systems and coordinates local inflammatory responses by modulating the activity of different immune cells. During inflammatory processes, including bacterial infections, neutrophils stimulate the production and release of histamine. Here, we report that the opportunistic human pathogen Pseudomonas aeruginosa exhibits chemotaxis toward histamine. This chemotactic response is mediated by the concerted action of the TlpQ, PctA, and PctC chemoreceptors, which display differing sensitivities to histamine. Low concentrations of histamine were sufficient to activate TlpQ, which binds histamine with an affinity of 639 nM. To explore this binding, we resolved the high-resolution structure of the TlpQ ligand binding domain in complex with histamine. It has an unusually large dCACHE domain and binds histamine through a highly negatively charged pocket at its membrane distal module. Chemotaxis to histamine may play a role in the virulence of P. aeruginosa by recruiting cells at the infection site and consequently modulating the expression of quorum-sensing-dependent virulence genes. TlpQ is the first bacterial histamine receptor to be described and greatly differs from human histamine receptors, indicating that eukaryotes and bacteria have pursued different strategies for histamine recognition.IMPORTANCE Genome analyses indicate that many bacteria possess an elevated number of chemoreceptors, suggesting that these species are able to perform chemotaxis to a wide variety of compounds. The scientific community is now only beginning to explore this diversity and to elucidate the corresponding physiological relevance. The discovery of histamine chemotaxis in the human pathogen Pseudomonas aeruginosa provides insight into tactic movements that occur within the host. Since histamine is released in response to bacterial pathogens, histamine chemotaxis may permit bacterial migration and accumulation at infection sites, potentially modulating, in turn, quorum-sensing-mediated processes and the expression of virulence genes. As a consequence, the modulation of histamine chemotaxis by signal analogues may result in alterations of the bacterial virulence. As the first report of bacterial histamine chemotaxis, this study lays the foundation for the exploration of the physiological relevance of histamine chemotaxis and its role in pathogenicity.

Routes of phosphoryl group transfer during signal transmission and signal decay in the dimeric sensor histidine kinase ArcB.

The Arc (anoxic redox control) two-component system of Escherichia coli, comprising ArcA as the response regulator and ArcB as the sensor histidine kinase, modulates the expression of numerous genes in response to respiratory growth conditions. Under reducing growth conditions, ArcB autophosphorylates at the expense of ATP, and transphosphorylates ArcA via a His292 → Asp576 → His717 → Asp54 phosphorelay, whereas under oxidizing growth conditions, ArcB catalyzes the dephosphorylation of ArcA-P by a reverse Asp54 → His717 → Asp576 → Pi phosphorelay. However, the exact phosphoryl group transfer routes and the molecular mechanisms determining their directions are unclear. Here, we show that, during signal propagation, the His292 → Asp576 and Asp576 → His717 phosphoryl group transfers within ArcB dimers occur intra- and intermolecularly, respectively. Moreover, we report that, during signal decay, the phosphoryl group transfer from His717 to Asp576 takes place intramolecularly. In conclusion, we present a mechanism that dictates the direction of the phosphoryl group transfer within ArcB dimers and that enables the discrimination of the kinase and phosphatase activities of ArcB.

Organization and mode of action of two component system signaling circuits from the various kingdoms of life.

Two-component system (TCS) signaling circuits regulate numerous cellular processes in response to environmental cues in both prokaryotic and eukaryotic cells. These signaling circuits are all based on phosphoryl-group transfers between histidine and aspartate containing modules of sensor kinase and response regulator proteins. Curiously, the architecture and organization of prokaryotic and eukaryotic two-component systems reveal notable variations, raising the question of whether the input-response specificity that governs the majority of prokaryotic TCSs also governs the eukaryotic ones. In this review, we contrast the TCS architecture and signaling circuits of prokaryotes and eukaryotes, and discuss their possible consequences on signaling specificity.

Multiple signals modulate the activity of the complex sensor kinase TodS.

The reason for the existence of complex sensor kinases is little understood but thought to lie in the capacity to respond to multiple signals. The complex, seven-domain sensor kinase TodS controls in concert with the TodT response regulator the expression of the toluene dioxygenase pathway in Pseudomonas putida F1 and DOT-T1E. We have previously shown that some aromatic hydrocarbons stimulate TodS activity whereas others behave as antagonists. We show here that TodS responds in addition to the oxidative agent menadione. Menadione but no other oxidative agent tested inhibited TodS activity in vitro and reduced PtodX expression in vivo. The menadione signal is incorporated by a cysteine-dependent mechanism. The mutation of the sole conserved cysteine of TodS (C320) rendered the protein insensitive to menadione. We evaluated the mutual opposing effects of toluene and menadione on TodS autophosphorylation. In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione. It was shown by others that menadione increases expression of glucose metabolism genes. The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways. This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules.

Characterization of molecular interactions using isothermal titration calorimetry.

Isothermal titration calorimetry (ITC) is based on a simple titration of one ligand with another and the small heat changes caused by the molecular interaction are detected. From one ITC experiment the complete set of thermodynamic parameters of binding including association and dissociation constants as well as changes in enthalpy, entropy, and free energy can be derived. Using this technique almost any type of molecular interaction can be analyzed. Both ligands are in solution, and there is no need for their chemical derivatization. There are no limits as to the choice of the analysis buffer, and the analysis temperature can be set between 4 and 80 °C. This technique has been primarily applied to study the interaction between various proteins of Pseudomonas with small molecule ligands. In addition, ITC has been used to study the binding of Pseudomonas proteins to target DNA fragments.

The Pseudomonas putida HskA hybrid sensor kinase controls the composition of the electron transport chain.

Sensor kinases play a key role in sensing and responding to environmental and physiological signals in bacteria. In this study we characterized a previously unknown orphan hybrid sensor kinase from Pseudomonas putida, which is conserved in several Pseudomonads. Inactivation of the gene coding for this sensor kinase, which we have named HskA, modified the expression of at least 85 genes in cells growing in a complete medium. HskA showed a strong influence on the composition of the electron transport chain. In cells growing exponentially in a complete medium, the absence of HskA led to a significant reduction in the expression of the genes coding for the bc1 complex and for the CIO and Cbb3-1 terminal oxidases. In stationary phase cells, however, lack of HskA caused a higher expression of the Cyo terminal oxidase and a lower expression of the Aa3 terminal oxidase. The HskA polypeptide shows two PAS (signal-sensing) domains, a transmitter domain containing the invariant phosphorylatable histidine and an ATP binding site, and a receiver domain containing the conserved aspartate capable of transphosphorylation, but lacks an Hpt module. It is therefore a hybrid sensor kinase. Phosphorylation assays showed that purified HskA undergoes autophosphorylation in the presence of ATP.

Analysis of solvent tolerance in Pseudomonas putida DOT-T1E based on its genome sequence and a collection of mutants.

Pseudomonas putida strains are prevalent in a variety of pristine and polluted environments. The genome of the solvent-tolerant P. putida strain DOT-T1E which thrives in the presence of high concentrations of monoaromatic hydrocarbons, contains a circular 6.3 Mbp chromosome and a 133 kbp plasmid. Omics information has been used to identify the genes and proteins involved in solvent tolerance in this bacterium. This strain uses a multifactorial response that involves fine-tuning of lipid fluidity, activation of a general stress-response system, enhanced energy generation, and induction of specific efflux pumps that extrude solvents to the medium. Local and global transcriptional regulators participate in a complex network of metabolic functions, acting as the decision makers in the response to solvents.

Responses of Pseudomonas putida to toxic aromatic carbon sources.

A number of bacteria can use toxic compounds as carbon sources and have developed complex regulatory networks to protect themselves from the toxic effects of these compounds as well as to benefit from their nutritious properties. As a model system we have studied the responses of Pseudomonas putida strains to toluene. Although this compound is highly toxic, several strains are able to use it for growth. Particular emphasis was given to the responses in the context of taxis, resistance and toluene catabolism. P. putida strains analysed showed chemotactic movements towards toluene. Strain DOT-T1E was characterised by an extreme form of chemotaxis, termed hyperchemotaxis, which is mediated by the McpT chemoreceptor encoded by plasmid pGRT1. Close McpT homologs are found in a number of other plasmids encoding degradation pathways of toxic compounds. The pGRT1 plasmid harbours also the genes for the TtgGHI efflux pump which was identified as the primary determinant for the resistance of strain DOT-T1E towards toluene. Pump expression is controlled by the TtgV repressor in response to a wide range of different mono- and biaromatic compounds. Strain DOT-T1E is able to degrade toluene, benzene and ethylbenzene via the toluene dioxygenase (TOD) pathway. The expression of the pathway operon is controlled by the TodS/T two component system. The sensor kinase TodS recognizes toluene with nanomolar affinity, which in turn triggers an increase in its autophosphorylation and consequently transcriptional activation. Data suggest that transcriptional activation of the TOD pathway occurs at very low toluene concentrations whereas TtgV mediated induction of pump expression sets in as the toluene concentration further increases.

Construction of a prototype two-component system from the phosphorelay system TodS/TodT.

Two-component systems (TCSs) play key roles in the adaptation of bacteria to environmental changes. In prototype TCSs a single phosphoryl transfer between the sensor kinase and response regulator occurs, whereas phosphorelay TCSs are characterised by a His1-Asp1-His2-Asp2 phosphorylation cascade. The TodS/TodT TCS controls the expression of a toluene degradation pathway and the TodS sensor kinase operates by a three-step internal phosphorelay. Based on TodS we report the construction of a minimal form of TodS, termed as Min-TodS, that contains only three of the seven TodS domains. Min-TodS is composed of the N-terminal PAS sensor domain as well as the C-terminal dimerisation/phosphotransfer domain and catalytic domain of TodS. We have conducted a comparative analysis of the phosphorelay TCS with its prototypal derivative. We demonstrate that Min-TodS binds effector molecules with affinities comparable with those observed for TodS. Min-TodS forms a TCS with TodT and toluene increases the amount of TodT-P. In contrast to TodS, toluene does not stimulate Min-TodS autophosphorylation. The half-life of Min-TodS-P was significantly increased as compared with TodS. Analysis of TodSD500A revealed that the hydrolysis of the acylphosphate of the receiver domain is responsible for the reduced half-life of TodS. The regulation of P(todX) expression by Min-TodS/TodT and TodS/TodT in response to different effectors are compared. The Min-TodS/TodT system was characterized by a higher basal activity but a lower magnitude of response. Data will be discussed in the context that the phosphorelay system appears to be better suited for the control of a degradation pathway for toxic compounds.

Study of the TmoS/TmoT two-component system: towards the functional characterization of the family of TodS/TodT like systems.

The two-component system TmoS/TmoT controls the expression of the toluene-4-monooxygenase pathway in Pseudomonas mendocina RK1 via modulation of P(tmoX) activity. The TmoS/TmoT system belongs to the family of TodS/TodT like proteins. The sensor kinase TmoS is a 108 kDa protein composed of seven different domains. Using isothermal titration calorimetry we show that purified TmoS binds a wide range of aromatic compounds with high affinities. Tightest ligand binding was observed for toluene (K(D) = 150 nM), which corresponds to the highest affinity measured between an effector and a sensor kinase. Other compounds with affinities in the nanomolar range include benzene, the 3 xylene isomers, styrene, nitrobenzene or p-chlorotoluene. We demonstrate that only part of the ligands that bind to TmoS increase protein autophosphorylation in vitro and consequently pathway expression in vivo. These compounds are referred to as agonists. Other TmoS ligands, termed antagonists, failed to increase TmoS autophosphorylation, which resulted in their incapacity to stimulate gene expression in vivo. We also show that TmoS saturated with different agonists differs in their autokinase activities. The effector screening of gene expression showed that promoter activity of P(tmoX) and P(todX) (controlled by the TodS/TodT system) is mediated by the same set of 22 compounds. The common structural feature of these compounds is the presence of a single aromatic ring. Among these ligands, toluene was the most potent inducer of both promoter activities. Information on the TmoS/TmoT and TodS/TodT system combined with a sequence analysis of family members permits to identify distinct features that define this protein family.

Bacterial sensor kinases: diversity in the recognition of environmental signals.

Bacteria sense and respond to a wide range of physical and chemical signals. Central to sensing and responding to these signals are two-component systems, which have a sensor histidine kinase (SK) and a response regulator (RR) as basic components. Here we review the different molecular mechanisms by which these signals are integrated and modulate the phosphorylation state of SKs. Apart from the basic mechanism, which consists of signal recognition by the SK that leads to an alteration of its autokinase activity and subsequently a change in the RR phosphorylation state, a variety of alternative modes have evolved. The biochemical data available on SKs, particularly their molecular interactions with signals, nucleotides, and their cognate RRs, are also reviewed.

Catabolite repression of the TodS/TodT two-component system and effector-dependent transphosphorylation of TodT as the basis for toluene dioxygenase catabolic pathway control.

The TodS/TodT two-component system of Pseudomonas putida regulates the expression of the toluene dioxygenase (tod) operon for the metabolism of toluene, benzene, and ethylbenzene. The sensor kinase TodS has a complex domain arrangement containing two functional modules, each harboring a sensor and an autokinase domain separated by a receiver domain. The TodT protein is the cognate response regulator that activates transcription of the toluene dioxygenase (TOD) pathway genes at the P(todX) promoter. We report in this study that the todST operon is transcribed from a main promoter and that the +1 initiation point is located 31 nucleotides upstream from the A of the first ATG codon and is preceded by a -10/-35 canonical promoter. Expression from P(todS) is under catabolite control, and in cells growing with glucose, the level of expression from this promoter is reduced, which in turn translates to low levels of the TodS/TodT regulators and results in a decrease of transcription from the P(todX) promoter. Thus, the main underlying regulatory mechanisms of the tod structural genes are at the levels of catabolite repression control from P(todS) and transcription activation, mediated by the TodT response regulator through a regulatory cascade in which the effector enhances autophosphorylation of TodS by ATP, with subsequent transphosphorylation of TodT.

Intracellular distribution of fatty alcohol oxidase activity in Mucor circinelloides YR-1 isolated from petroleum contaminated soils.

In previous studies, Mucor circinelloides YR-1 isolated from petroleum-contaminated soils grown in decane as sole carbon source, showed fatty alcohol oxidase (FAO) activities in either particulate or soluble fractions from a cell-free extract. One is associated to internal membranes (mFAO) and the other one is soluble (sFAO). Both activities appear to be located in the cells in specific compartments other than peroxisomes. Results suggested that mFAO could be located on the inner face of the membrane of these compartments, and sFAO could be in the lumen of the specific compartments. This study reports on the intracellular distribution of FAO activity and the purification of sFAOs and mFAO after several different procedures for release from the membranous fraction using the mixed membrane fraction (MMF) after cellular homogenization as enzymatic source. Results with the purified mFAO show, by molecular weight criteria, that the enzyme has only one type of subunit with molecular mass of 46 kDa, with two isoelectric point components: 6.0 and 6.3. We found that mFAO is strongly associated to the MMF, possibly in a transitory fashion. Using non-denaturating gels, we suggest that sFAO and mFAO have the same subunits in their native structures, and, due to their native molecular weight of approximately 350 kDa, each could be natively structured as an octameric complex.

The enigma of cytosolic two-component systems: a hypothesis.

One-component systems (OCSs) and cytosolic two-component regulatory systems (TCSs) appear to share the same biological function, which consists in the transcriptional control in response to the cellular concentration of signal molecules. However, cytosolic TCSs as compared with OCSs represent a genetic and metabolic burden to the cell: the genetic information encoding a TCS is significantly larger than that of an OCS, two or more proteins instead of one need to be synthesized for a TCS and operation of the latter system requires the expense of ATP which is not the case for most OCSs. The evolutionary advantages of cytosolic TCSs over OCSs are thus not obvious. We hypothesize here that the increased capacity of cytosolic TCSs to respond to multiple signals is a major advantage over OCSs. Different mechanisms for the incorporation of additional signals into the regulatory circuit are discussed. The inclusion of several signals into the definition of the final regulatory response is proposed to result in a better adaptation of the host to given environmental conditions.

Intracellular fate of hydrocarbons: possible existence of specific compartments for their biodegradation.

In previous work, purification procedures and zymogram analysis conducted with supernatants of crude extracts from aerobic mycelium of the YR-1 strain of Mucor circinelloides isolated from petroleum-contaminated soils indicated the existence of only one soluble alcohol oxidase (sAO) activity. In the present work enzymatic activity of alcohol oxidase (AO) was also detected in the mixed membrane fraction (MMF) of a high-speed centrifugation procedure after drastic ballistic cellular homogenization to break the mycelium from strain YR-1. When mycelial cells were gently broken by freezing the mycelium with liquid nitrogen, smashing in a mortar, and submitting the samples to an isopycnic sucrose gradients (10-60% sucrose), AO activity was detected in particular and discrete fractions of the gradient, showing specific density values quite different from the density of peroxisomes. The results suggest that there could be a different intracellular pattern of distribution of the microsomal fraction in aerobically grown mycelium depending on the carbon source used in the culture media, including alcohols and hydrocarbons, but not in glucose. In working with particulate fractions, we found two AO activities: a new membrane alcohol oxidase (mAO) activity and the sAO. Both activities appear to be located in the inner of the cells in specific compartments different from the peroxisomes, so mAO could be in the membrane of these compartments and sAO in the lumen of the vesicles. We also assayed other enzymatic activities involved in hydrocarbon biodegradation to establish its intracellular location and other enzymatic activities such as peroxidase to use them as intracellular markers of different organelles. In the case of monooxygenase, the first enzymatic step in the hydrocarbon biodegradation pathway, its location was in the same fractions where AOs were located, suggesting the existance of a specific organelle that contains the enzymatic activities involved in hydrocarbon biodegradation.

Detection of NAD+-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils.

Different soluble NAD+-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleum- contaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD+-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (approximately 7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD+-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol- dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.