The complete genome sequences of sulfur-oxidizing Gammaproteobacteria Sulfurifustis variabilis skN76T and Sulfuricaulis limicola HA5T

Sulfurifustis variabilis and Sulfuricaulis limicola are autotrophic sulfur-oxidizing bacteria belonging to the family Acidiferrobacteraceae in the order Acidiferrobacterales. The type strains of these species, strain skN76T and strain HA5T, were isolated from lakes in Japan. Here we describe the complete genome sequences of Sulfurifustis variabilis skN76T and Sulfuricaulis limicola HA5T. The genome of Sulfurifustis variabilis skN76T consists of one circular chromosome with size of 4.0 Mbp including 3864 protein-coding sequences. The genome of Sulfuricaulis limicola HA5T is 2.9 Mbp chromosome with 2763 protein-coding sequences. In both genomes, 46 transfer RNA-coding genes and one ribosomal RNA operon were identified. In the genomes, redundancies of the genes involved in sulfur oxidation and inorganic carbon fixation pathways were observed. This is the first report to show the complete genome sequences of bacteria belonging to the order Acidiferrobacterales in the class Gammaproteobacteria.


Introduction
Sulfurifustis variabilis skN76 T and Sulfuricaulis limicola HA5 T are gammaproteobacterial sulfur-oxidizing bacteria isolated from sediments of Lake Mizugaki and Lake Harutori, respectively [1,2]. They both belong to the family Acidiferrobacteraceae in the order Acidiferrobacterales. In this order, only three species have been isolated in pure culture. They are all chemolithoautotrophs and can grow by oxidation of inorganic sulfur compounds. Sulfurifustis variabilis and Sulfuricaulis limicola are neutrophilic, whereas the other species, Acidiferrobacter thiooxydans, is acidophilic [3]. Taxonomy of Acidiferrobacter thiooxydans has been revised several times, and the family Acidiferrobacteraceae and order Acidiferrobacterales were recently established to accommodate the species [1,[3][4][5]. The members of the family Acidiferrobacteraceae have been frequently detected in various environments as gene sequences [2,3,6].
Here we show the complete genome sequences of Sulfurifustis variabilis skN76 T and Sulfuricaulis limicola HA5 T as the first genomes of the order Acidiferrobacterales.

Genome project history
Sulfurifustis variabillis skN76 T and Sulfuricaulis limicola HA5 T were selected for sequencing as representatives of sulfur-oxidizing bacteria belonging to the order Acidiferrobacterales, to reveal characteristics of their genomes.
A summary of the project information is shown in Table 2.

Growth conditions and genomic DNA preparation
Sulfurifustis variabilis skN76 T and Sulfuricaulis limicola HA5 T were grown with 20 mM thiosulfate as an energy source in a bicarbonate-buffered medium previously described [1], at 45 and 28°C, respectively. Genomic DNA samples were prepared by using Wizard® genomic DNA purification kit (Promega, Madison, WI, USA) from approximately 0.2 ml (skN76) or 0.1 ml (HA5) of and circular chromosome was manually constructed in the same manner.

Genome annotation
The genomes were annotated automatically using the Microbial Genome Annotation Pipeline [7]. Further manual annotation of the predicted protein-coding sequences was performed on the basis of BLASTP searches against the NCBI nonredundant database. CDSs were annotated as hypothetical protein-coding genes when they met any of the following four criteria in the top hit of the BLASTP analysis: (1) Evalue >1e-8, (2) length coverage <60 % against query sequence (3) sequence identity <30 % or (4) function of the hit was unidentified. The WebMGA server was used to assign the genes to Clusters of Ortholog Groups and Protein family domains [8][9][10][11]. The Phobius server was used to predict signal peptides and transmembrane helices [12]. Clustered Regularly Interspaced Short Palindromic Repeat loci were detected using CRISPRfinder [13].

Genome properties
The basic statistics of the genomes are shown in Table 3. Both genomes contained 46 tRNA genes and one rRNA operon. The genome size of Sulfurifustis variabillis skN76 T was approximately 1.4 times larger than that of Sulfuricaulis limicola HA5 T . CRISPR loci were found only in the genome of Sulfurifustis variabillis skN76 T ( Table 3). The distribution of genes into COGs functional categories is presented in Table 4.
Sulfurifustis variabillis skN76 T and Sulfuricaulis limicola HA5 T are autotrophic bacteria. They both have two  copies of the rbcL and rbcS genes, encoding large and small subunits of ribulose bisphosphate carboxylase/oxygenase (SVA_3460-3459, SVA_3471-3470; SCL_2417-2416, SCL_2425-2424), which is the key enzyme in the Calvin-Benson-Bassham cycle to catalyze inorganic carbon fixation. The two copies of RuBisCO in each genome are phylogenetically distinct, and belong to lineages referred to as green-like form IA and red-like form IC (Fig. 3) [21]. In the form IC RuBisCO coded by rbcL gene (SVA_3460, SCL_2417), Sulfurifustis variabillis skN76 T and Sulfuricaulis limicola HA5 T have six-aminoacid inserts at the same position where a similar insert was reported from Nitrosospira sp. 40KI [22]. There are two other RuBisCO sequences which have six-amino-acid inserts at the same position, and these sequences with inserts formed a monophyletic cluster in the tree of RuBisCO (Fig. 3). In general, RuBisCO of form IA and IC have different properties which are thought to be advantageous to fix inorganic carbon under different concentrations of carbon dioxide and/or oxygen [21]. Possession of the genes for these two distinct RuBisCO forms may be beneficial to cope with changing environmental conditions, or to thrive in various types of ecosystems.

Conclusion
This is the first report on complete genome sequences of bacteria belonging to the order Acidiferrobacterales.
The genome analysis of Sulfurifustis variabillis skN76 T and Sulfuricaulis limicola HA5 T revealed that they have similar sets of genes involved in sulfur oxidation pathways. In the both genomes, redundancies of the genes for sulfur oxidation and inorganic carbon fixation were observed, as represented by multiple copies of dsrAB, aprAB and rbcLS. Such redundancies may provide physiological flexibility to the chemolithotrophic sulfur oxidizers which are fully depending on these functions to obtain energy and carbon source for growth.