Experimental studies on fermentative firmicutes from anoxic environments: isolation, evolution, and their geochemical impacts

Fermentative microorganisms from the bacterial phylum Firmicutes are quite ubiquitous in subsurface environments and play an important biogeochemical role. For instance, fermenters have the ability to take complex molecules and break them into simpler compounds that serve as growth substrates for other organisms. The research presented here focuses on two groups of fermentative Firmicutes, one from the genus Clostridium and the other from the class Negativicutes. Clostridium species are well-known fermenters. Laboratory studies done so far have also displayed the capability to reduce Fe(III), yet the mechanism of this activity has not been investigated further. We studied three clostridial organisms, Clostridium sp. FGH that was previously enriched from Oak Ridge, TN; Clostridium beijerinckii; and Clostridium acetobutylicum to determine how Fe(III) oxide reduction occurs. The results indicate that Fe(III) oxide reduction by fermentative clostridial species does not require direct cell-to-mineral contact, meaning that it is an extracellular process mediated by a soluble molecule. Fermentation oft leads to the accumulation of acidic by-products, which can act as Fe(III)-chelating ligands. But during glucose fermentation, soluble Fe(III) accumulation did not occur, nor did supplementation of organic acids to fermenting cultures stimulate either the rate or extent of Fe(III) reduction even though rapid Fe(III) solubilization was observed. This extracellular process may be mediated by flavins, and supplementation of flavin adenine dinucleotide (FAD), a flavin molecule that serves as an electron carrier intracellularly, stimulated Fe(III) oxide reduction. These results indicate the potential for Clostridium species to perform Fe(III) reduction in contaminated environments and may be relevant towards bioremediation. Clostridum sp. FGH was enriched as a co-culture with another spore-forming Firmicutes organism that we isolated and named Anaerosporomusa subterranea strain RU4. This organism is likewise an obligate fermenter but with a very narrow metabolic niche. Surprisingly, this organism in pure culture showed no ability to reduce Fe(III), despite the fact that it was isolated from an Fe(III)-reducing enrichment culture. Furthermore, the addition of typical terminal electron acceptors were not utilized by A. subterranea. However, this organism was interesting because it was a Gram-negative (or didermic, since they have two membranes in their cell wall structure) organism within a phylum that was previously thought to be only Gram-positive (or monodermic, since these microorganisms have only one membrane as part of their cell wall). We investigated how this organism could have evolved through comparative phylogenomics of outer membrane biosynthesis genes. We identified 46 potential protein sequences involved in lipopolysaccharide and outer membrane biosynthesis in the genome of A. subterranea. Specifically out of these proteins, we focused on the proteins LptACD that are responsible for transporting mature lipopolysaccharide from the inner membrane, across the periplasmic space, and to the outer membrane. This is because most Gram-negative or didermic bacteria require these proteins to synthesize an outer membrane. Our results demonstrated that A. subterranea shared approximately 50% sequence similarity for the LptACD proteins with other Negativicutes. When the alignment was expanded to other Gram-negative or didermic bacterial clades, on average 25% similarity was reported. This suggested significant homology within the Negativicutes and with other microorganisms, such that these genes in A. subterranea were ancestrally derived. Furthermore, phylogenetic comparisons using the LptACD protein sequences showed comparable tree topology to the widely-accepted 16S phylogenetic tree, illustrating vertical gene transfer and that recent horizontal gene transfer did not occur. In conclusion, our data suggests that the ancestor to the Firmicutes phylum was originally didermic and that A. subterranea may represent an ancestral lineage.