Peculiar citric acid cycle of hydrothermal vent chemolithoautotroph Hydrogenovibrio crunogenus, and insights into carbon metabolism by obligate autotrophs

Abstract

The genome sequence of the obligate chemolithoautotroph Hydrogenovibrio crunogenus paradoxically predicts a complete oxidative citric acid cycle (CAC). This prediction was tested by multiple approaches including whole cell carbon assimilation to verify obligate autotrophy, phylogenetic analysis of CAC enzyme sequences and enzyme assays. Hydrogenovibrio crunogenus did not assimilate any of the organic compounds provided (acetate, succinate, glucose, yeast extract, tryptone). Enzyme activities confirmed that its CAC is mostly uncoupled from the NADH pool. 2-Oxoglutarate:ferredoxin oxidoreductase activity is absent, though pyruvate:ferredoxin oxidoreductase is present, indicating that sequence-based predictions of substrate for this oxidoreductase were incorrect, and that H. crunogenus may have an incomplete CAC. Though the H. crunogenus CAC genes encode uncommon enzymes, the taxonomic distribution of their top matches suggests that they were not horizontally acquired. Comparison of H. crunogenus CAC genes to those present in other 'Proteobacteria' reveals that H. crunogenus and other obligate autotrophs lack the functional redundancy for the steps of the CAC typical for facultative autotrophs and heterotrophs, providing another possible mechanism for obligate autotrophy.

Genomes of ubiquitous marine and hypersaline Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira spp. encode a diversity of mechanisms to sustain chemolithoautotrophy in heterogeneous environments

“…Their genomes are smaller than those from organisms with additional capabilities such as photosynthesis, denitrification or heterotrophic growth (Supporting Information Table S1). The rRNA operon copy numbers (3-4) are at the high range of what is observed in other autotrophic members of the Gammaproteobacteria (Supporting Information Table S1), which may help them to respond more quickly to c. Data from (Kuenen and Veldkamp, 1972;Jannasch et al, 1985;Nishihara et al, 1991;Muyzer et al, 1995;Brinkhoff et al, 1999a,b,c;Rainey et al, 2001;Sorokin et al, 2002;Knittel et al, 2005;Sorokin et al, 2007;2011;Zhang et al, 2016;Ang et al, 2017;Quasem et al, 2017). d.-, no assimilation of organic compounds, and no growth in the absence of reduced sulfur compounds; NG, no growth in the absence of inorganic electron donors; A, organic carbon assimilation.…”

“…Instead, they carry genes less common among members of the 'Proteobacteria':monomeric NADP 1 isocitrate dehydrogenase (EC 1.1.1.42; (Yasutake et al, 2002), and malate:quinone oxidoreductase (E.C. 1.1.5.4; Quasem et al, 2017). It is possible that they have incomplete citric acid cycles (Smith's horseshoe), as is often the case for obligate autotrophs (e.g., Boden et al, 2016;Hutt et al, 2017) that function primarily to provide biosynthetic intermediates (Smith et al, 1967;Wood et al, 2004).…”

“…To identify traits that distinguish members of Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira, these genomes were compared with those of other members of 'Proteobacteria' that use the CBB cycle for carbon fixation. This comparison group was collected based on the presence of form I or form II RubisCO genes in their genomes, and demonstrated ability to grow with CO 2 as the major carbon source, as described in Quasem et al (2017). To compare the distribution of electron transport chain components among these organisms, BLAST queries were undertaken, using genes encoding biochemically characterized enzymes (Brune, 1995;Gregersen et al, 2011;Dahl et al, 2013;Weissgerber et al, 2014).…”

“… c . Data from (Kuenen and Veldkamp, ; Jannasch et al ., ; Nishihara et al ., ; Muyzer et al ., ; Brinkhoff et al ., ; Rainey et al ., ; Sorokin et al ., ; Knittel et al ., ; Sorokin et al ., ; Zhang et al ., ; Ang et al ., ; Quasem et al ., ).…”

Engineering lithoheterotrophy in an obligate chemolithoautotrophic Fe(II) oxidizing bacterium

“…The ability to form colonies on solid medium is advantageous for selection and screening of mutants, while metabolic flexibility allows for viability of mutants missing pathways of interest. However, the vast majority of microorganisms are not readily cultured on solid medium 1 and many exhibit specialist lifestyles using a singular metabolic mode to grow [2][3][4][5][6][7][8] . Our overall hypothesis is that synthetic biology can be leveraged to better understand and domesticate environmental microorganisms with novel metabolic capabilities.…”