The genome of deep-sea vent chemolithoautotroph Thiomicrospira crunogena XCL-2

Abstract

Presented here is the complete genome sequence of Thiomicrospira crunogena XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 base pairs), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of coding sequences (CDSs) encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety of options for acquiring these substrates from the environment. Thiom. crunogena XCL-2 is unusual among obligate sulfur-oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome.

Figure 1. Circular Map of the Thiom. crunogena XCL-2 Genome

The outer two rings (rings 1 and 2) are protein-encoding genes, which are color-coded according to COG category. Rings 3 and 4 are tRNA and rRNA genes. Ring 5 indicates the location of a prophage (magenta), phosphonate/heavy metal resistance island (cyan), and four insertion sequences (red; two insertion sequences at 2028543 and 2035034 are superimposed on this figure). The black circle indicates the deviation from the average %GC, and the purple and green circle is the GC skew (= [G − C]/[G + C]). Both the %GC and GC skew were calculated using a sliding window of 10,000 bp with a window step of 100.

Figure 2. Cell Model for Thiom. crunogena XCL-2, with an Emphasis on Ultrastructure, Transport, Energy, Carbon Metabolism, and Chemotaxis

Genes encoding virtually all of the steps for the synthesis of nucleotides and amino acids by canonical pathways are present in the bacterium, but are omitted here for simplicity. Electron transport components are yellow, and abbreviations are as follows: bc₁, bc₁ complex; cbb₃, cbb₃-type cytochrome C oxidase; cytC, cytochrome C; NDH, NADH dehydrogenase; Sox, Sox system; UQ, ubiquinone. MCPs are fuchsia, as are MCPs with PAS domains or PAS folds. Influx and efflux transporter families with representatives in this genome are indicated on the figure, with the number of each type of transporter in parentheses. ATP-dependent transporters are red, secondary transporters are sky blue, ion channels are light green, and unclassified transporters are purple. Abbreviations for transporter families are as follows: ABC, ATP-binding cassette superfamily; AGCS, alanine or glycine:cation symporter family; AMT, ammonium transporter family; APC, amino acid-polyamine-organocation family; ATP syn, ATP synthetase; BASS, bile acid:Na⁺ symporter family; BCCT, betaine/carnitine/choline transporter family; CaCA, Ca²⁺:cation antiporter family; CDF, cation diffusion facilitator family; CHR, chromate ion transporter family; CPA, monovalent cation:proton antiporter-1, −2, and −3 families; DAACS, dicarboxylate/amino acid:cation symporter family; DASS, divalent anion:Na⁺ symporter family; DMT, drug/metabolite transporter superfamily; FeoB, ferrous iron uptake family; IRT, iron/lead transporter superfamily; MATE, multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) flippase superfamily, MATE family; McsS, small conductance mechanosensitive ion channel family; MFS, major facilitator superfamily; MgtE, Mg²⁺ transporter-E family; MIT, CorA metal ion transporter family; NCS2, nucleobase:cation symporter-2 family; NRAMP, metal ion transporter family; NSS, neurotransmitter:sodium symporter family; P-ATP, P-type ATPase superfamily; Pit, inorganic phosphate transporter family; PNaS, phosphate:Na⁺ symporter family; PnuC, nicotamide mononucleotide uptake permease family; RhtB, resistance to homoserine/threonine family; RND, resistance-nodulation-cell division superfamily; SSS, solute:sodium symporter family; SulP, sulfate permease family; TRAP, tripartite ATP-independent periplasmic transporter family; TRK, K⁺ transporter family; VIC, voltage-gated ion channel superfamily.

Figure 3. Prophage Genome within the Thiom. crunogena XCL-2 Genome

Lysogenic and lytic genes are delineated, as are predicted gene functions.

Figure 4. Phylogenetic Relationship of Thiom. crunogena XCL-2 SoxB to Sequences of Selected Bacteria

Sequences were aligned using the program package MacVector. Neighbor-joining and parsimony trees based on the predicted amino acid sequences were calculated using PAUP 4.0b10 [113]. Bootstrap values (1,000 replicates) are given for the neighbor-joining (first value) and parsimony analyses (second value).

Figure 5. Transporter Gene Frequencies within the Genomes of Thiom. crunogena XCL-2 (Arrow) and Other Proteobacteria

Nitrob. winogradskyi (Nitrobacter winogradskyi) is an alphaproteobacterium, Nitros. europaea (Nitrosomonas europaea) is a betaproteobacterium, and Nitrosoc. oceani (Nitrosococcus oceani) and Methylo. capsulatus (Methylococcus capsulatus) are gammaproteobacteria. Bars for intracellular pathogens are lighter red than the other heterotrophic gammaproteobacteria.

Figure 6. Calvin-Benson-Bassham Cycle Gene Organization in Proteobacteria

RubisCO genes (cbbLS and cbbM) are green, phosphoribulokinase genes (cbbP) are red, other genes encoding Calvin-Benson-Bassham cycle enzymes are black, and carboxysome structural genes are grey. For species in which cbbP is not near cbbLS or cbbM, the distance from the RubisCO gene to cbbP in kbp is indicated in parentheses. Thiob. denitrificans has two cbbP genes, so two distances are indicated for this species. Names of organisms that are unable to grow well as organoheterotrophs are boxed. Abbreviations and accession numbers for the 16S sequences used to construct the cladogram are as follows: A. ehrlichei, Alkalilimnicola ehrlichei, AF406554; Brady. sp., Bradyrhizobium sp., AF338169;B. japonicum, Bradyrhizobium japonicum, D13430; B. xenovorans, Burkholderia xenovorans, U86373; D. aromatica, Dechloromonas aromatica, AY032610; M. magneticum, Magnetospirillum magneticum, D17514; M. capsulatus, Methylococcus capsulatus BATH, AF331869; N. hamburgensis, Nitrobacter hamburgensis, L11663; N. winogradskyi, Nitrobacter winogradskyi, L11661; N. oceani, Nitrosococcus oceani, AF363287; N. europaea, Nitrosomonas europaea, BX321856; N. multiformis, Nitrosospira multiformis, L35509; P. denitrificans, Paracoccus denitrificans, X69159; R. sphaeroides, Rhodobacter sphaeroides, CP000144; R. ferrireducens, Rhodoferax ferrireducens, AF435948; R. palustris, Rhodopseudomonas palustris, NC 005296; R. rubrum, Rhodospirillum rubrum, D30778; R. gelatinosus, Rubrivivax gelatinosus, M60682; S. meliloti, Sinorhizobium meliloti, D14509; T. denitrificans, Thiobacillus denitrificans, AJ43144; T. crunogena, Thiomicrospira crunogena, AF064545. The cladogram was based on an alignment of 1,622 bp of the 16S rRNA genes, and is the most parsimonious tree (length 2,735) resulting from a heuristic search with 100 replicate random step-wise addition and TBR branch swapping (PAUP*4.0b10 [113]). Sequences were aligned using ClustalW [114], as implemented in BioEdit. Percent similarities and identities for cbbL, cbbM, and cbbP gene products, as well as gene locus tags, are provided as supporting information (Table S4).

Figure 7. Models for Glycolysis, Gluconeogenesis, and the CAC in Thiom. crunogena XCL-2

Models for central carbon metabolism for cells under environmental conditions with (A) sufficient reduced sulfur and oxygen; (B) sulfide scarcity; and (C) oxygen scarcity; green arrows represent the two “non-canonical” CAC enzymes, 2-oxoglutarate oxidoreductase (2-OG OR) and malate: quinone oxidoreductase (MQO).

Figure 8. Thiom. crunogena XCL-2 Phosphonate Operon

The DBR-1 genes are identical to each other, as are the DBR-2 genes. Gene abbreviations are: chp, conserved hypothetical protein; DBR-1 and -2, DNA breaking-rejoining enzymes; hyp, hypothetical protein; phnFDCEGHIJKLMNP, phosphonate operon. An asterisk (*) marks the location of a region (within and upstream of tRNA-phe) with a high level of similarity to the 5′ ends of the two direct repeat sequences noted in the figure. The transposase and integrase are actually a single CDS separated by a frameshift.

Figure 9. Numbers of MCP Genes in Thiom. crunogena XCL-2 and Other Proteobacteria

Thiom. crunogena is marked with an arrow.