6‐Oxocyclohex‐1‐ene‐1‐carbonyl‐coenzyme A hydrolases from obligately anaerobic bacteria: characterization and identification of its gene as a functional marker for aromatic compounds degrading anaerobes

Abstract

In anaerobic bacteria, most aromatic growth substrates are channelled into the benzoyl-coenzyme A (CoA) degradation pathway where the aromatic ring is dearomatized and cleaved into an aliphatic thiol ester. The initial step of this pathway is catalysed by dearomatizing benzoyl-CoA reductases yielding the two electron-reduction product, cyclohexa-1,5-diene-1-carbonyl-CoA, to which water is subsequently added by a hydratase. The next two steps have so far only been studied in facultative anaerobes and comprise the oxidation of the 6-hydroxyl-group to 6-oxocyclohex-1-ene-1-carbonyl-CoA (6-OCH-CoA), the addition of water and hydrolytic ring cleavage yielding 3-hydroxypimelyl-CoA. In this work, two benzoate-induced genes from the obligately anaerobic bacteria, Geobacter metallireducens (bamA(Geo)) and Syntrophus aciditrophicus (bamA(Syn)), were heterologously expressed in Escherichia coli, purified and characterized as 6-OCH-CoA hydrolases. Both enzymes consisted of a single 43 kDa subunit. Some properties of the enzymes are presented and compared with homologues from facultative anaerobes. An alignment of the nucleotide sequences of bamA(Geo) and bamA(Syn) with the corresponding genes from facultative anaerobes identified highly conserved DNA regions, which enabled the discrimination of genes coding for 6-OCH-CoA hydrolases from those coding for related enzymes. A degenerate oligonucleotide primer pair was deduced from conserved regions and applied in polymerase chain reaction reactions. Using these primers, the expected DNA fragment of the 6-OCH-CoA hydrolase genes was specifically amplified from the DNA of nearly all known facultative and obligate anaerobes that use aromatic growth substrates. The only exception was the aromatic compound-degrading Rhodopseudomonas palustris, which uniquely uses a modified benzoyl-CoA degradation pathway. Using the oligonucleotide primers, the expected DNA fragment was also amplified in a toluene-degrading and a m-xylene-degrading enrichment culture demonstrating its potential use in less defined bacterial communities. The gene probe established in this work provides for the first time a general tool for the detection of a central functionality in aromatic compound-degrading anaerobes.

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…Recently, the characterization of the ring-opening hydrolases from G. metallireducens (BamA) and S. aciditrophicus (BamA Syn ) as well as the BamQ dehydrogenase from G. metallireducens further confirmed that the Thauera-type ␤-oxidation pathway is present in both facultative and obligate anaerobes ( Fig. 2) (206). The observation that the Rhodopseudomonas type is restricted to photosynthetic anaerobes may reflect an evolutionary adaptation of phototrophs to carry out the catabolism of aromatic and alicyclic acids through the same ␤-oxidation pathway (see below).…”

“…Sequence comparison analyses of the dienoyl-CoA hydratases and ring-opening hydrolases suggested the existence of two phylogenetic groups, one including the enzymes from T. aromatica and G. metallireducens and the deduced gene products from Magnetospirillum strains and a second group that includes the enzymes from S. aciditrophicus, the deduced gene products from Azoarcus species, and the hydrolase from the sulfate-reducing bacterium D. multivorans (206,279). This observation suggests that these two groups represent neither the phylogenetic relationship nor a common overall energy metab-olism of the organisms (facultative versus obligate anaerobes) but rather points to the fact that they might have been acquired by the corresponding bacteria through horizontal gene transfer events (206,279).…”

“…The genome of S. aciditrophicus contains four different genes (syn1638, syn2417, syn2896, and syn2898) that are predicted to encode AMP-forming ligases that could activate aromatic or alicyclic compounds (237). Out of these four genes, syn1638 is the only one that is located in a gene cluster (Sa2) that also contains genes which have been shown to be involved in the reaction steps after benzoylCoA reduction (206,277,279), and therefore, it is assumed to be the bamY ortholog (bamY Syn ) in S. aciditrophicus (Fig. 2B).…”

“…Interestingly, one of these gene clusters, Sa2, also contains the putative benzoate-CoA ligase gene (bamY Syn ) and genes that have been shown to be involved in the reaction steps after benzoyl-CoA reduction ( Fig. 2) (206,279). The presence of these genes in the genome of S. aciditrophicus suggests that, like in G. metallireducens, the membrane potential rather than ATP hydrolysis may drive the electron transfer needed for benzoyl-CoA ring reduction by as-yet-unknown membrane components (237).…”

Sulfuritalea hydrogenivorans gen. nov., sp. nov., a facultative autotroph isolated from a freshwater lake

“…Following these results, three primer pairs were tested to detect genes involved in degradation of monoaromatic hydrocarbons. The primer pairs used were bssA_f/bssA_r to detect the bssA gene (Botton et al, 2007), BamA-SP9/BamA-ASP1 to detect the bamA gene (Kuntze et al, 2008) and bzAQ4R/ bzAQ4R to detect the bcrA gene (Song & Ward, 2005). With these primer pairs, however, no PCR product was obtained from the genomic DNA of strain sk43H T (Thauera aromatica K172 T was used as a positive control and fragments of the expected sizes were obtained in all cases).…”