Genes encoding the candidate enzyme for anaerobic activation of n-alkanes in the denitrifying bacterium, strain HxN1.

Strain HxN1, a member of the Betaproteobacteria, can grow anaerobically by denitrification with n-alkanes. n-Alkanes are apparently activated by subterminal carbon addition to fumarate yielding (1-methylalkyl)succinates, the postulated enzyme being (1-methylalkyl)succinate synthase (Mas). Genes encoding this enzyme (mas) were searched for via proteins that were specifically formed in n-hexane-grown cells (in comparison with caproate-grown cells), as revealed by two-dimensional gel electrophoresis. Partial amino acid sequencing and subsequent probe development for hybridization of restricted DNA led to the identification of a gene cluster. Deduced proteins are similar to the subunits of benzylsuccinate synthase (Bss), the toluene-activating enzyme in other anaerobic bacteria and its activase. The tentative (1-methylalkyl)succinate synthase is presumably a heterotrimer (MasDEC) which, like benzylsuccinate synthase, contains a motif (in MasD, the large subunit) characteristic of glycyl radical-bearing sites. Based on amino acid sequence comparison, the tentative (1-methylalkyl)succinate synthase branches outside of the phylogenetic cluster of benzylsuccinate synthases from different organisms and represents a separate line of descent within glycyl radical enzymes. n-Hexane-induced co-transcription of the mas genes and additional genes of an apparent operon was demonstrated by Northern hybridization experiments.

Alkane degradation under anoxic conditions by a nitrate-reducing bacterium with possible involvement of the electron acceptor in substrate activation.

Microorganisms can degrade saturated hydrocarbons (alkanes) not only under oxic but also under anoxic conditions. Three denitrifying isolates (strains HxN1, OcN1, HdN1) able to grow under anoxic conditions by coupling alkane oxidation to CO(2) with NO(3) (-) reduction to N(2) were compared with respect to their alkane metabolism. Strains HxN1 and OcN1, which are both Betaproteobacteria, utilized n-alkanes from C(6) to C(8) and C(8) to C(12) respectively. Both activate alkanes anaerobically in a fumarate-dependent reaction yielding alkylsuccinates, as suggested by present and previous metabolite and gene analyses. However, strain HdN1 was unique in several respects. It belongs to the Gammaproteobacteria and was more versatile towards alkanes, utilizing the range from C(6) to C(30). Neither analysis of metabolites nor analysis of genes in the complete genome sequence of strain HdN1 hinted at fumarate-dependent alkane activation. Moreover, whereas strains HxN1 and OcN1 grew with alkanes and NO(3) (-), NO(2) (-) or N(2)O added to the medium, strain HdN1 oxidized alkanes only with NO(3) (-) or NO(2) (-) but not with added N(2)O; but N(2)O was readily used for growth with long-chain alcohols or fatty acids. Results suggest that NO(2) (-) or a subsequently formed nitrogen compound other than N(2)O is needed for alkane activation in strain HdN1. From an energetic point of view, nitrogen-oxygen species are generally rather strong oxidants. They may enable enzymatic mechanisms that are not possible under conditions of sulfate reduction or methanogenesis and thus allow a special mode of alkane activation.

Microbial nitrate-dependent cyclohexane degradation coupled with anaerobic ammonium oxidation.

An anaerobic nitrate-reducing enrichment culture was established with a cyclic saturated petroleum hydrocarbon, cyclohexane, the fate of which in anoxic environments has been scarcely investigated. GC-MS showed cyclohexylsuccinate as a metabolite, in accordance with an anaerobic enzymatic activation of cyclohexane by carbon-carbon addition to fumarate. Furthermore, long-chain cyclohexyl-substituted cell fatty acids apparently derived from cyclohexane were detected. Nitrate reduction was not only associated with cyclohexane utilization but also with striking depletion of added ammonium ions. Significantly more ammonium was consumed than could be accounted for by assimilation. This indicated the occurrence of anaerobic ammonium oxidation (anammox) with nitrite from cyclohexane-dependent nitrate reduction. Indeed, nitrite depletion was stimulated upon further addition of ammonium. Analysis of 16S rRNA genes and subsequent cell hybridization with specific probes showed that approximately 75% of the bacterial cells affiliated with the Geobacteraceae and approximately 18% with Candidatus 'Brocadia anammoxidans' (member of the Planctomycetales), an anaerobic ammonium oxidizer. These results and additional quantitative growth experiments indicated that the member of the Geobacteraceae reduced nitrate with cyclohexane to nitrite and some ammonium; the latter two and ammonium added to the medium were scavenged by anammox bacteria to yield dinitrogen. A model was established to quantify the partition of each microorganism in the overall process. Such hydrocarbon oxidation by an alleged 'denitrification' ('pseudo-denitrification'), which in reality is a dissimilatory loop through anammox, can in principle also occur in other microbial systems with nitrate-dependent hydrocarbon attenuation.

Anaerobic degradation of n-hexane in a denitrifying bacterium: further degradation of the initial intermediate (1-methylpentyl)succinate via C-skeleton rearrangement.

The anaerobic degradation pathway of the saturated hydrocarbon n-hexane in a denitrifying strain (HxN1) was examined by gas chromatography-mass spectrometry of derivatized extracts from cultures grown with unlabeled and deuterated substrate; several authentic standard compounds were included for comparison. The study was focused on possible reaction steps that follow the initial formation of (1-methylpentyl)succinate from n-hexane and fumarate. 4-Methyloctanoic, 4-methyloct-2-enoic, 2-methylhexanoic, 2-methylhex-2-enoic and 3-hydroxy-2-methylhexanoic acids (in addition to a few other methyl-branched acids) were detected in n-hexane-grown but not in n-hexanoate-grown cultures. Labeling indicated preservation of the original carbon chain of n-hexane in these acids. Tracing of the deuterium label of 3- d1-(1-methylpentyl)succinate in tentative subsequent products indicated a deuterium/carboxyl carbon exchange in the succinate moiety. This suggests that the metabolism of (1-methylpentyl)succinate employs reactions analogous to those in the established conversion of succinyl-CoA via methylmalonyl-CoA to propionyl-CoA. Accordingly, a pathway is proposed in which (1-methylpentyl)succinate is converted to the CoA-thioester, rearranged to (2-methylhexyl)malonyl-CoA and decarboxylated (perhaps by a transcarboxylase) to 4-methyloctanoyl-CoA. The other identified fatty acids match with a further degradation of 4-methyloctanoyl-CoA via rounds of conventional beta-oxidation. Such a pathway would also allow regeneration of fumarate (for n-hexane activation) from propionyl-CoA formed as intermediate and hence present a cyclic process.