Genome sequence of Desulfobacterium autotrophicum HRM2, a marine sulfate reducer oxidizing organic carbon completely to carbon dioxide.

Sulfate-reducing bacteria (SRB) belonging to the metabolically versatile Desulfobacteriaceae are abundant in marine sediments and contribute to the global carbon cycle by complete oxidation of organic compounds. Desulfobacterium autotrophicum HRM2 is the first member of this ecophysiologically important group with a now available genome sequence. With 5.6 megabasepairs (Mbp) the genome of Db. autotrophicum HRM2 is about 2 Mbp larger than the sequenced genomes of other sulfate reducers (SRB). A high number of genome plasticity elements (> 100 transposon-related genes), several regions of GC discontinuity and a high number of repetitive elements (132 paralogous genes Mbp(-1)) point to a different genome evolution when comparing with Desulfovibrio spp. The metabolic versatility of Db. autotrophicum HRM2 is reflected in the presence of genes for the degradation of a variety of organic compounds including long-chain fatty acids and for the Wood-Ljungdahl pathway, which enables the organism to completely oxidize acetyl-CoA to CO(2) but also to grow chemolithoautotrophically. The presence of more than 250 proteins of the sensory/regulatory protein families should enable Db. autotrophicum HRM2 to efficiently adapt to changing environmental conditions. Genes encoding periplasmic or cytoplasmic hydrogenases and formate dehydrogenases have been detected as well as genes for the transmembrane TpII-c(3), Hme and Rnf complexes. Genes for subunits A, B, C and D as well as for the proposed novel subunits L and F of the heterodisulfide reductases are present. This enzyme is involved in energy conservation in methanoarchaea and it is speculated that it exhibits a similar function in the process of dissimilatory sulfate reduction in Db. autotrophicum HRM2.

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.

Substrate-dependent regulation of carbon catabolism in marine sulfate-reducing Desulfobacterium autotrophicum HRM2.

Desulfobacterium autotrophicum HRM2 is a metabolically versatile sulfate-reducing bacterium, capable of heterotrophic (e.g. with organic acids and alcohols) and chemolithoautotrophic growth (with H(2)/CO(2)). It employs the Wood-Ljungdahl pathway for complete oxidation of acetyl-CoA to CO(2) and for CO(2) fixation. Here, we investigated substrate-dependent regulation at different levels of anaerobic carbon catabolism in this bacterium. (a) Whole-cell adaptation studies indicated an inducibleutilization of short-chained alcohols, agreeing with a substrate-specific abundance increase (up to 40-fold) of alcohol dehydrogenase Adh4. Simultaneous utilization of lactate and 1-propanol was paralleled by adh4 expression and Adh4 formation, respectively. (b) Degradation of propionate generally involves methylmalonyl-CoA mutase (Sbm). Expression of sbm was upregulated during growth with 1-propanol, but not with a mixture of lactate and 1-propanol. Correspondingly, propionate was excreted during growth with this substrate mixture. (c) CO dehydrogenase, the key enzyme of the Wood-Ljungdahl pathway, is encoded by several genes (cdhC, cdh1 and cdh2) located at different genomic positions. Expression of all of these genes during heterotrophic and autotrophic growth points to a reversible operation of the Wood-Ljungdahl pathway. In summary, the different regulatory patterns displayed by Db. autotrophicum HRM2 at the tested metabolic levels point to a multi-layered regulatory network.

Physiological response to temperature changes of the marine, sulfate-reducing bacterium Desulfobacterium autotrophicum.

The physiological response of bacteria to temperature is critical for the regulation of biogeochemical processes on daily, seasonal, and inter-annual time scales. We investigated the temperature response of the marine sulfate-reducing bacterium Desulfobacterium autotrophicum strain HRM2. Growth experiments in a temperature gradient block demonstrated that D. autotrophicum is psychrotolerant and grows between 0 and 31 degrees C. The normal range of temperature for growth is between 4 and 29 degrees C. The physiological response to temperature changes was studied with three sets of cells that were acclimated at 4, 10, and 28 degrees C, respectively. Sulfate reduction rates were determined in the temperature gradient block with short-term incubations to minimize growth. The rates were similar at the 4 and 10 degrees C acclimation temperature, and exhibited an enhanced response at 28 degrees C. At every acclimation temperature, sulfate reduction rates increased 20-fold from -1.7 to 41 degrees C. The relative proportion of cellular unsaturated fatty acids (e.g. cis16:1) and short-chain fatty acids increased when cells were grown at 4 degrees C compared to 28 degrees C. The proteome of D. autotrophicum strain HRM2 was studied by two-dimensional gel electrophoresis with soluble extracts of cells grown at the three respective acclimation temperatures. Protein patterns were similar with the exception of two proteins showing 5-10-fold lower abundance in the 4 degrees C culture compared to the 28 degrees C culture. In general, D. autotrophicum strain HRM2 responded to low temperatures by reduced metabolic activity rather than by pronounced de novo synthesis of specifically adapted enzymes. Such a strategy agrees well with in situ activities measured in field studies and may reflect a common physiological principle of psychrotolerant marine sulfate-reducing bacteria.

Genes involved in the anaerobic degradation of toluene in a denitrifying bacterium, strain EbN1.

The organization of all genes required for the anaerobic conversion of toluene to benzoyl-CoA was investigated in denitrifying Azoarcus-like strain EbN1. All of these genes are clustered within 25.3 kb of contiguous DNA sequence, which includes only a few intervening sequences. The toluene-catabolic genes are organized in two apparent operons. One contains the genes ( bssCAB) for the three subunits of benzylsuccinate synthase, which initiates anaerobic toluene degradation by converting toluene to ( R)-benzylsuccinate. The BssCAB proteins of strain EbN1 are most similar to those of Thauera aromatica strain K172. The bssCAB genes are part of a larger putative operon ( bssDCABEFGH), which contains the gene bssD, encoding the activase for benzylsuccinate synthase, and four genes ( bssEFGH) encoding proteins of unknown function. RT-PCR experiments showing continuation of transcription over the three largest intergenic regions of the bss operon support the assumed structure. Moreover, BssG was identified as toluene-induced protein. Downstream of the bss genes, another large putative operon ( bbsA- H) was identified that contains all genes required for beta-oxidation of benzylsuccinate to benzoyl-CoA, e.g. bbsEF, encoding succinyl-CoA:( R)-benzylsuccinate CoA-transferase. Immediately upstream of the bss operon, genes for a two-component regulatory system were identified; their products may sense toluene and induce the expression of both catabolic operons. The order and sequences of the bss and bbs genes are highly similar among toluene-degrading denitrifiers. The bss and bbs genes of the Fe(III)-reducing Geobacter metallireducens display less sequence similarity and are organized differently. The genes between the bss and bbs operons and in the flanking regions differ between strain EbN1 and the other strains.