Gordon R. Stewart

Limiting factors for hydrocarbon biodegradation at low temperature in Arctic soils

Hydrocarbon fuel spills are common in the Arctic. But, little is known about hydrocarbon-degrading microflora in Arctic tundra soils or the potential for bioremediation of these soils. We examined mineralization of radiolabeled hydrocarbons in microcosms containing soils collected from sites across the Canadian Arctic. The soils all contained psychrotolerant microorganisms which mineralized dodecane and substantially removed total petroleum hydrocarbons (TPH) at 7°C. Dodecane mineralization was severely limited by both N and P. Dodecane mineralization kinetics varied greatly among different soils. Multiple regression analysis showed that soil N and TPH concentrations together accounted for 73% of the variability of the lag time preceding dodecane mineralization. Soil characteristics were less effective as predictors of mineralization kinetic parameters other than lag time. High total C concentrations were associated with high mineralization rate constants, and high sand contents were associated with long times for half-maximal dodecane mineralization. Very high concentrations of TPH (100 mg g−1 of dry soil) and heavy metals (e.g., 1.4 mg Pb g−1 of dry soil) did not prevent dodecane mineralization. Inoculation of soils with indigenous or non-indigenous hydrocarbon-degrading microorganisms stimulated dodecane mineralization. Bioremediation of hydrocarbon-contaminated Arctic tundra soils appears to be feasible, and various engineering strategies, such as heating or inoculating the soil, can accelerate hydrocarbon biodegradation.

The Actinobacterial mce4 Locus Encodes a Steroid Transporter

Bioinformatic analyses have suggested that Mce proteins in diverse actinobacteria are components of complex ATP-binding cassette transporter systems, comprising more than eight distinct proteins. In Mycobacterium tuberculosis, these proteins are implicated in interactions of this deadly pathogen with its human host. Here, we provide direct evidence that the Mce4 system of Rhodococcus jostii RHA1 is a steroid uptake system. Transcriptional analyses indicate that the system is encoded by an 11-gene operon, up-regulated 4.0-fold during growth on cholesterol versus on pyruvate. Growth of RHA1 on cholesterol and uptake of radiolabeled cholesterol both required expression of genes in the mce4 operon encoding two permeases plus eight additional proteins of unknown function. Cholesterol uptake was ATP-dependent and exhibited Michaelis-Menten kinetics with a Km of 0.6 ± 0.1 μm. This uptake system was also essential for growth of RHA1 on β-sitosterol, 5-α-cholestanol, and 5-α-cholestanone. Bioinformatic analysis revealed that all mce4 loci in sequenced genomes are linked to steroid metabolism genes. Thus, we predict that all Mce4 systems are steroid transporters. The transport function of the Mce4 system is consistent with proposed roles of cholesterol and its metabolism in the pathogenesis of M. tuberculosis.

Mycobacterial cytochrome P450 125 (Cyp125) catalyzes the terminal hydroxylation of C27 steroids

Cyp125 (Rv3545c), a cytochrome P450, is encoded as part of the cholesterol degradation gene cluster conserved among members of the Mycobacterium tuberculosis complex. This enzyme has been implicated in mycobacterial pathogenesis, and a homologue initiates cholesterol catabolism in the soil actinomycete Rhodococcus jostii RHA1. In Mycobacterium bovis BCG, cyp125 was up-regulated 7.1-fold with growth on cholesterol. A cyp125 deletion mutant of BCG did not grow on cholesterol and accumulated 4-cholesten-3-one when incubated in the presence of cholesterol. Wild-type BCG grew on this metabolite. By contrast, a parallel cyp125 deletion mutation of M. tuberculosis H37Rv did not affect growth on cholesterol. Purified Cyp125 from M. tuberculosis, heterologously produced in R. jostii RHA1, bound cholesterol and 4-cholesten-3-one with apparent dissociation constants of 0.20 ± 0.02 μm and 0.27 ± 0.05 μm, respectively. When reconstituted with KshB, the cognate reductase of the ketosteroid 9α-hydroxylase, Cyp125 catalyzed the hydroxylation of these steroids. MS and NMR analyses revealed that hydroxylation occurred at carbon 26 of the steroid side chain, allowing unambiguous classification of Cyp125 as a steroid C26-hydroxylase. This study establishes the catalytic function of Cyp125 and, in identifying an important difference in the catabolic potential of M. bovis and M. tuberculosis, suggests that Cyp125 may have an additional function in pathogenesis.

Phenylacetate Catabolism in Rhodococcus sp. Strain RHA1: a Central Pathway for Degradation of Aromatic Compounds

In gram-negative bacteria, a pathway for aerobic degradation of phenylacetic acid (PAA) that proceeds via phenylacetyl-coenzyme A (CoA) and hydrolytic ring fission plays a central role in the degradation of a range of aromatic compounds. In contrast, the PAA pathway and its role are not well characterized in gram-positive bacteria. A cluster including 13 paa genes encoding enzymes orthologous to those of gram-negative bacteria was identified on the chromosome of Rhodococcus sp. strain RHA1. These genes were transcribed during growth on PAA, with 11 of the genes apparently in an operon yielding a single transcript. Quantitative proteomic analyses revealed that at least 146 proteins were more than twice as abundant in PAA-grown cells of RHA1 than in pyruvate-grown cells. Of these proteins, 29 were identified, including 8 encoded by the paa genes. Knockout mutagenesis indicated that paaN, encoding a putative ring-opening enzyme, was essential for growth on PAA. However, paaF, encoding phenylacetyl-CoA ligase, and paaR, encoding a putative regulator, were not essential. paaN was also essential for growth of RHA1 on phenylacetaldehyde, phenylpyruvate, 4-phenylbutyrate, 2-phenylethanol, 2-phenylethylamine, and l-phenylalanine. In contrast, growth on 3-hydroxyphenylacetate, ethylbenzene, and styrene was unaffected. These results suggest that the range of substrates degraded via the PAA pathway in RHA1 is somewhat limited relative to the range in previously characterized gram-negative bacteria.

Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp.

Bile acids are highly abundant steroids with important functions in vertebrate digestion. Their catabolism by bacteria is an important component of the carbon cycle, contributes to gut ecology, and has potential commercial applications. We found that Rhodococcus jostii RHA1 grows well on cholate, as well as on its conjugates, taurocholate and glycocholate. The transcriptome of RHA1 growing on cholate revealed 39 genes upregulated on cholate, occurring in a single gene cluster. Reverse transcriptase quantitative PCR confirmed that selected genes in the cluster were upregulated 10-fold on cholate versus on cholesterol. One of these genes, kshA3, encoding a putative 3-ketosteroid-9α-hydroxylase, was deleted and found essential for growth on cholate. Two coenzyme A (CoA) synthetases encoded in the cluster, CasG and CasI, were heterologously expressed. CasG was shown to transform cholate to cholyl-CoA, thus initiating side chain degradation. CasI was shown to form CoA derivatives of steroids with isopropanoyl side chains, likely occurring as degradation intermediates. Orthologous gene clusters were identified in all available Rhodococcus genomes, as well as that of Thermomonospora curvata. Moreover, Rhodococcus equi 103S, Rhodococcus ruber Chol-4 and Rhodococcus erythropolis SQ1 each grew on cholate. In contrast, several mycolic acid bacteria lacking the gene cluster were unable to grow on cholate. Our results demonstrate that the above-mentioned gene cluster encodes cholate catabolism and is distinct from a more widely occurring gene cluster encoding cholesterol catabolism.

Involvement of a Novel ABC Transporter and Monoalkyl Phthalate Ester Hydrolase in Phthalate Ester Catabolism by Rhodococcus jostii RHA1

ABSTRACT Phthalate esters (PEs) are important environmental pollutants. While the biodegradation of the parent compound, phthalate (PTH), is well characterized, the biodegradation of PEs is not well understood. In particular, prior to this study, genes involved in the uptake and hydrolysis of these compounds were not conclusively identified. We found that Rhodococcus jostii RHA1 could grow on a variety of monoalkyl PEs, including methyl, butyl, hexyl, and 2-ethylhexyl PTHs. Strain RHA1 could not grow on most dialkyl PEs, but suspensions of cells grown on PTH transformed dimethyl, diethyl, dipropyl, dibutyl, dihexyl and di-(2-ethylhexyl) PTHs. The major products of these dialkyl PEs were PTH and the corresponding monoalkyl PEs, and minor products resulted from the shortening of the alkyl side chains. RHA1 exhibited an inducible, ATP-dependent uptake system for PTH with a Km of 22 μM. The deletion and complementation of the patB gene demonstrated that the ATP-binding cassette (ABC) transporter encoded by patDABC is required for the uptake of PTH and monoalkyl PEs by RHA1. The hydrolase encoded by patE of RHA1 was expressed in Escherichia coli. PatE specifically hydrolyzed monoalkyl PEs to PTH but did not transform dialkyl PEs or other aromatic esters. This investigation of RHA1 elucidates key processes that are consistent with the environmental fate of PEs.

Apparent Contradiction: Psychrotolerant Bacteria from Hydrocarbon-Contaminated Arctic Tundra Soils That Degrade Diterpenoids Synthesized by Trees

ABSTRACT Resin acids are tricyclic terpenoids occurring naturally in trees. We investigated the occurrence of resin acid-degrading bacteria on the Arctic tundra near the northern coast of Ellesmere Island (82°N, 62°W). According to most-probable-number assays, resin acid degraders were abundant (103 to 104propagules/g of soil) in hydrocarbon-contaminated soils, but they were undetectable (<3 propagules/g of soil) in pristine soils from the nearby tundra. Plate counts indicated that the contaminated and the pristine soils had similar populations of heterotrophs (106to 107 propagules/g of soil). Eleven resin acid-degrading bacteria belonging to four phylogenetically distinct groups were enriched and isolated from the contaminated soils, and representative isolates of each group were further characterized. Strains DhA-91, IpA-92, and IpA-93 are members of the genusPseudomonas. Strain DhA-95 is a member of the genusSphingomonas. All four strains are psychrotolerant, with growth temperature ranges of 4°C to 30°C (DhA-91 and DhA-95) or 4°C to 22°C (IpA-92 and IpA-93) and with optimum temperatures of 15 to 22°C. Strains DhA-91 and DhA-95 grew on the abietanes, dehydroabietic and abietic acids, but not on the pimaranes, isopimaric and pimaric acids. Strains IpA-92 and IpA-93 grew on the pimaranes but not the abietanes. All four strains grew on either aliphatic or aromatic hydrocarbons, which is unusual for described resin acid degraders. Eleven mesophilic resin acid degraders did not use hydrocarbons, with the exception of two Mycobacterium sp. strains that used aliphatic hydrocarbons. We conclude that hydrocarbon contamination in Arctic tundra soil indirectly selected for resin acid degraders, selecting for hydrocarbon degraders that coincidentally use resin acids. Psychrotolerant resin acid degraders are likely important in the global carbon cycle and may have applications in biotreatment of pulp and paper mill effluents.

Isolation of a Substantial Proportion of Forest Soil Bacterial Communities Detected via Pyrotag Sequencing

ABSTRACT We isolated 1,264 bacterial strains from forest soils previously surveyed via pyrosequencing of rRNA gene amplicons. Conventional culturing techniques recovered a substantial proportion of the community, with isolates representing 22% of 98,557 total pyrotags. Growth characteristics of isolates indicated that ecological traits were associated with relative abundances of corresponding pyrotag operational taxonomic units.

The mycobacterial P55 efflux pump is required for optimal growth on cholesterol

Cholesterol catabolism is thought to be a key factor contributing to the pathogenesis of Mycobacterium tuberculosis. Previous epistasis and mutant screening studies predicted that the P55 efflux pump (Rv1410c) positively interacts with the Mce4 transporter, a major cholesterol import system of M. tuberculosis and is needed for optimal growth in vitro, in macrophages, and in vivo. Using a combination of cell growth kinetic techniques, cholesterol consumption, and [4–14C]cholesterol uptake studies, we demonstrated that the Mycobacterium bovis BCG rv1410c gene indeed is needed for optimal in vitro growth on cholesterol and other carbon sources. Our data, together with previous predictions, support hypotheses that the P55 efflux pump functions in maintaining general metabolism or as a subunit of the Mce4 transport apparatus (catalyzing its assembly or providing cell wall integrity) to allow more efficient cholesterol uptake.