The Origin Of Acetate Metablism And Photosynthesis-specific Genes From Bathyarchaea

The acetyl-coenzyme A pathway is a set of biochemical reactions used by some bacteria and archaea.This pathway enables these organisms to use hydrogen as an electron donor,and carbon dioxide as an electron acceptor and as a building block for biosynthesis.Acetogens are obligately anaerobic bacteria that use the reductive acetyl-CoA pathway as their main mechanism for energy conservation and for synthesis of acetyl-CoA and produce acetate as the main product of their metabolism.Phosphotransacetylase(Pta)and acetate kinase(Ack)catalyze the last two steps of acetogenesis.The Ack-Pta pathway for acetate production or assimilation is widely distributed in bacteria.In archaea,it has so far only been identified in the methanogenic genus Methanosarcina,which is capable of producing methane from acetate.The ack-pta genes in Methanosarcinaare assumed to have been acquired by a single horizontal gene transfer event,probably from a clade of cellulolytic Clostridia.However,we also found ack-pta genes in some Bathyarchaeota genomic bins.Bathyarchaeota,formerly known as Miscellaneous Crenarchaeotal Group,is a newly defined archaea phylum that may play an important role on global carbon biogeochemical cycling.Phylogenetic analysis of the translated ack andpta sequences of Bathyarchaeota indicated that these genes form a monophylogenetic clade that is distinct from all other known sequences.Thus,the Ack-Pta pathway in Bathyarchaeota appears to have evolved independently from that of Bacteria.Then we cloned and expressed these two genes in E.coli and finally got a large amount of soluble recomninant proteins.Ack catalyzes the reversible transition from acetate to acetyl phosphate,and Pta catalyzes the reversible transition from acetyl phosphate to acetyl-CoA.In vitro assay showed that Ack had a higher affinity to acetyl phosphate(Km=1.69 mM)than to acetate(Km=4.4 mM),while Pta had a higher affinity to acetyl-CoA(Km=0.13 mM)than to acetyl phosphate(Km=0.53 mM),which may indicate that the overall direction of Ack-Pta reaction prefered to the formation of acetate.Photosynthesis is the most important biological process on Earth.This process is carried out by plants on land,and in the oceans by algae and cyanobacteria.Up to now,photosynthesis has only been found in six phyla of bacteria including the cyanobacteria,purple bacteria,chloroflexi,chlorobi,helicobacteria and acidobacteria.Generally,photosynthesis is regarded as most likely having evolved after the divergence of the archaeal-eukaryal and bacterial lineages,as no(bacterio)-chlorophyll((B)Chl)has ever been detected in a member of the domain Archaea.Previously scientists detected a functional bchG gene from Bathyarchaeota,which was a marker gene for Bchl biosynthesis,and only detected in photosynthetic organisms.The finding of a gene encoding functional BchG protein from archaea makes us reconsider the evolution of this protein family and the possibility that the origin of photosynthesis probably predates the divergence of bacteria and archaea.Here we report our studies on archaea-originated putative photosynthesis-related genes.We searched for photosynthesis-related genes,especially those putatively involved in BChl biosynthesis,in the genomes of Bathyarchaeota.Until now we have found some candidate Bchl biosynthesis-related genes such as bchE and bchP.Phylogenetic analysis of the translated bchE and bchP sequences of Bathyarchaeota indicated that these genes have a close relationship with bacteria.Most of proteins involving in bacteriochlorophyll synthesis are membrane associated,which means they are difficult to express in E.coli.As a result,both BchP and BchE formed inclusion bodies when conducting heterologous expression in E.coli.Amino acid alignment shows several mutations in a conseved ADP-binding fold close to the N terminus of bchP sequence of Bathyarchaeota.We didn't detect any reductase acitivities of BchP in vitro.