In this study, the complete genome of Citrobacter sp. XT1-2-2 was sequenced and comparative genomics analysis was also conducted with the other relevant reference sequenced genomes. In our previous study, the strain XT1-2-2 was isolated from high Cd-contaminated soils, and demonstrated an excellent ability to decrease the bioavailability of Cd in the soil and inhibit Cd uptake in rice. In addition, the strain XT1-2-2 could significantly promote rice growth and increase rice biomass. However, the genome sequence of this organism has not been reported so far.
The antigenic system of the Bethesna-Ballerup group bacteria was established by West and Edwards in 1954 . This group of bacteria is now called Citrobacter freundii . So far, Citrobacter genus contains eleven species: Citrobacter freundii, Citrobacter koseri, Citrobacter amalonaticus, Citrobacter farmeri, Citrobacter youngae, Citrobacter braakii, Citrobacter werkmanii, Citrobacter sedlakii, Citrobacter rodentium, Citrobacter genomospecies 10, Citrobacter genomospecies 11 [12, 13]. According to the results of phylogenetic analysis, the strain XT1-2-2 was most closely related to Citrobacter werkmanii strain BF-6 and Citrobacter youngae ATCC 29220 (Fig. S1). According to the physicochemical properties of these strains, some Citrobacter species immobilized biofilms were used to bioremediate heavy metal contaminated soils through an acid-type phosphatase enzymatic activity or their ability to accumulate heavy metals [14-16]. In this study, genome analysis of the strain XT1-2-2 revealed all genes of a complete set of sulfate reduction pathway according to the KEGG analysis (Fig.4). The occurrence of metabolic pathways involves the following steps: (1) Sulfate (SO42−) from outside is taken up into cells by putative sulfate transporter CysPUWA; (2) Sulfate (SO42−) entering the cell is first acetylated to adenylylsulphate (APS) by sulfate adenylyltransferases CysN and CysD; (3) The resulting APS is then phosphorylated to phosphoadenylyl-sulphate (PAPS) by the APS kinase CysC; (4) The resulting PAPS is further reduced to sulfite (SO32−) by PAPS reductase CysH; (5) The resulting sulfite (SO32−) is finally reduced to sulfide (S2-) by sulfite reductase CysIJ . The reason why the strain XT1-2-2 has a significant effect of removing cadmium is mainly because the strain generates sulfide (S2-) via the sulfur metabolism pathway, which can combine with Cd2+ in the soil to form the precipitated CdS, thereby reducing the uptake and transport of cadmium in the soil by rice plant.
Meanwhile, the strain XT1-2-2 also revealed various genes responsible for multiple heavy metal resistance (Fig. 5), which provided the genomic basis for the strain to adapt to the external complex harmful environment. CzcD is involved in resistance to the heavy metals Cd2+, Zn2+ and Co2+ . The membrane transporter ChrA is responsible for the efflux of intracellular Cr(VI) from the cell . Heavy metal-transporting ATPase (ZntA) is responsible for the efflux of Pb2+, Zn2+, Cd2+ and Hg2+ . The metal ABC transport system (ZnuABC) are involved in Zn2+ uptake . ArsB, ArsC, and ArsH proteins are involved in the functions of arsenical pump membrane protein, arsenate reductase and arsenical resistance protein, respectively . Cus copper resistance system consists of CusCBA efflux pump, CusF periplasmic protein and CusS regulatory protein . Mercury transport system (mer operon) encodes a group of proteins consisting of MerR mercury regulatory proteins, MerT, MerC, MerP mercury transport proteins and MerA, MerD, MerE mercury resistance proteins . The Co2+ ECF transporter complex is involved in Co2+ resistance and transmembrane transport .
The analysis of the core and pan genomes showed an uneven distribution among functional categories (Fig. 3). There were several notable differences in the numbers of genes, such as amino acid transport and metabolism (category E), transport and metabolism of carbohydrates (category G), translation (category K) and inorganic ion transport and metabolism (category P). In particular, this difference in the number of genes belonging to the same COG category was mainly reflected in transport and metabolism . For KEGG annotations, two gene functional categories were enriched in core gene families including metabolism and environmental information processing (Fig. 8). It may be due to the fact that the signal transduction system faces the complex and changeable external environment, and the metabolic pathway needs to respond quickly to adapt to the environmental changes.