Diversity in CO ₂ -Concentrating Mechanisms among Chemolithoautotrophs from the Genera Hydrogenovibrio , Thiomicrorhabdus , and Thiomicrospira , Ubiquitous in Sulfidic Habitats Worldwide

Abstract

Members of the generaHydrogenovibrio,Thiomicrospira, andThiomicrorhabdusfix carbon at hydrothermal vents, coastal sediments, hypersaline lakes, and other sulfidic habitats. The genome sequences of these ubiquitous and prolific chemolithoautotrophs suggest a surprising diversity of mechanisms for the uptake and fixation of dissolved inorganic carbon (DIC); these mechanisms are verified here. Carboxysomes are apparent in the transmission electron micrographs of most of these organisms but are lacking inThiomicrorhabdussp. strain Milos-T2 andThiomicrorhabdus arctica, and the inability ofThiomicrorhabdussp. strain Milos-T2 to grow under low-DIC conditions is consistent with the absence of carboxysome loci in its genome. For the remaining organisms, genes encoding potential DIC transporters from four evolutionarily distinct families (Tcr_0853 and Tcr_0854, Chr, SbtA, and SulP) are located downstream of carboxysome loci. Transporter genes collocated with carboxysome loci, as well as some homologs located elsewhere on the chromosomes, had elevated transcript levels under low-DIC conditions, as assayed by reverse transcription-quantitative PCR (qRT-PCR). DIC uptake was measureable via silicone oil centrifugation when a representative of each of the four types of transporter was expressed inEscherichia coli. The expression of these genes in the carbonic anhydrase-deficientE. colistrain EDCM636 enabled it to grow under low-DIC conditions, a result consistent with DIC transport by these proteins. The results from this study expand the range of DIC transporters within the SbtA and SulP transporter families, verify DIC uptake by transporters encoded byTcr_0853andTcr_0854and their homologs, and introduce DIC as a potential substrate for transporters from the Chr family.IMPORTANCEAutotrophic organisms take up and fix DIC, introducing carbon into the biological portion of the global carbon cycle. The mechanisms for DIC uptake and fixation by autotrophicBacteriaandArchaeaare likely to be diverse but have been well characterized only for “Cyanobacteria.” Based on genome sequences, members of the generaHydrogenovibrio,Thiomicrospira, andThiomicrorhabdushave a variety of mechanisms for DIC uptake and fixation. We verified that most of these organisms are capable of growing under low-DIC conditions, when they upregulate carboxysome loci and transporter genes collocated with these loci on their chromosomes. When these genes, which fall into four evolutionarily independent families of transporters, are expressed inE. coli, DIC transport is detected. This expansion in known DIC transporters across four families, from organisms from a variety of environments, provides insight into the ecophysiology of autotrophs, as well as a toolkit for engineering microorganisms for carbon-neutral biochemistries of industrial importance.

Functional reconstitution of a bacterial CO2 concentrating mechanism in Escherichia coli

“…pCB encodes 10 carboxysome genes ( Bonacci et al, 2012 ; Cai et al, 2008 ), including rubisco large and small subunits, along with prk . The remaining H. neapolitanus genes, including putative rubisco chaperones ( Aigner et al, 2017 ; Mueller-Cajar, 2017 ; Wheatley et al, 2014 ) and an inorganic carbon transporter ( Desmarais et al, 2019 ; Scott et al, 2019 ), were cloned into the second plasmid, pCCM.…”

“…We chose to include csos1D with the carboxysome because its inclusion was previously observed to result in production of carboxysomes with more regular icosahedral morphology ( Bonacci et al, 2012 ), which we hypothesized to be a correlate of proper assembly. Moreover, when we began this work, the transporters associated with proteobacterial CCMs had not yet been identified ( Desmarais et al, 2019 ; USF MCB4404L et al, 2017 ; Scott et al, 2019 ) and we planned to express cyanobacterial transporters as in Du et al, 2014 . As such, it seemed sensible to include all known carboxysome components on a single plasmid, a design we retained for convenience even after the inorganic carbon transporters were identified.…”

“…Using a genome-wide screen in the CO 2 -fixing proteobacterium Halothiobacillus neapolitanus , we recently demonstrated that a 20-gene cluster encodes all activities required for the CCM, at least in principle ( Desmarais et al, 2019 ). These genes are detailed in Supplementary file 1 and include rubisco large and small subunits, the carboxysomal carbonic anhydrase, seven structural proteins of the ɑ-carboxysome ( Bonacci et al, 2012 ), an energy-coupled inorganic carbon transporter ( Desmarais et al, 2019 ; USF MCB4404L et al, 2017 ; Scott et al, 2019 ), three rubisco chaperones ( Aigner et al, 2017 ; Feiz et al, 2014 ; Mueller-Cajar, 2017 ; Wheatley et al, 2014 ), and four genes of uncertain function ( Figure 1C ). We aimed to test whether these genes are sufficient to establish a functioning CCM in E. coli .…”

Thiomicrorhabdus streamers and sulfur cycling in perennial hypersaline cold springs in the Canadian high Arctic

“…While the RuBisCO gene is located near a carbonic anhydrase gene, the MAG lacks a carboxysomal locus and many typical carboxysomal genes like those found in other Thiomicrorhabdus spp. (Scott et al ., 2019). Thiomicrorhabdus arctica and Milos‐T2 are the only Thiomicrorhabdus , Thiomicrospira and Hydrogenovibrio spp.…”

Effect of CO2 Concentration on Uptake and Assimilation of Inorganic Carbon in the Extreme Acidophile Acidithiobacillus ferrooxidans

“…PCC 7002 (Price et al, 2004). Additional SulP protein sequences were added to the analysis (Ward et al, 2004; Boden et al, 2011; Huntley et al, 2011; Svenning et al, 2011; Scott et al, 2019). A multiple sequence alignment was constructed using MAFFT with accurate option L-INS-i as alignment tools (Katoh and Standley, 2013; Nakamura et al, 2018) and ClustalW alignment program (Larkin et al, 2007).…”