3.1 Enrichment of the most antimonite and arsenite resistant bacteria
The metal(loid) concentrations of isolation site were shown in Table 1, other physicochemical parameters of soil were as follows: pH: 7.95; total phosphorus 0.2663 g kg–1; total nitrogen 0.0221 mg kg–1; available phosphorus 16.6175 mg kg–1; available potassium 85.00 mg kg–1; organic matter 40.6704 g kg–1; electrical conductivity 98.60 uS cm–1. For bacterial isolation, enrichment cultures of a heavy metal contaminated chili peppers field (high concentration of contamination: 670 mg kg–1 Sb & 138 mg kg–1 As) near the biggest antimony mine in the world were performed to obtain the most antimonite and arsenite resistant microbes. For the isolates from this site, genus of Microbacterium and Pseudochrobactrum were obtained when performing As enrichment, while genus of Citrobacter, Pseudomonas and Enterobacter were obtained from Sb enrichment. Among them, C. portucalensis strain Sb-2 was one of the most antimonite resistant isolates and was further characterized here. The general features of Sb-2 are shown in Table 2.
C. portucalensis strain Sb-2 was highly resistant to arsenite (MIC 18 mM) and extremely resistant to antimonite (MIC 60 mM), with MICs much higher than the well-studied metal resistant bacteria Cupriavidus metallidurans AE104 & CH34 (MIC 2.5 mM for arsenite; 0.6 mM for antimonite) and well-studied strain Escherichia coli W3110 (MIC 3 mM for arsenite; 0.8 mM for antimonite) on the same solid minimal medium (Table 3).
3.2 Morphology, Growth and Physiology of Sb-2
The isolated strain C. portucalensis strain Sb-2 was shown to be a gram negative, motile Gammaproteobacterium with a morphology of short rods and forms creamy white colonies on minimal medium (Figure 1). 16S sequencing was performed to determine phylogeny and it could be determined to be most closely related to Citrobacter portucalensis (Figure 2). The genus Citrobacter belongs to the family of Enterobacteriaceae and is sorted in the “CESP” or “ESCPM” (Citrobacter, Enterobacter, Serratia and Providencia, and more recently, Morganella and Hafnia genres) group.
3.3 Genome and the phage driven arsenic and antimony resistance determinants of strain Sb-2
For the purpose of understanding the genetic mechanism for As(III) and Sb(III) resistance to Citrobacter portucalensis Sb-2, the whole genome of strain Sb-2 was sequenced and gene functions was annotated, the project of Sb-2 was shown in Table 4. The genome size of C. portucalensis Sb-2 is 4,794,853 bp, with a 51.9 mol% GC content, consists of 37 DNA Scaffolds (Table 5). The chromosome contains 4,625 Coding Sequences (CDS), 8 rRNAs, and 73 tRNAs (Table 5).
To better understand the genomic basis underlying this metalloid resistant phenotype, the draft genome sequence of C. portucalensis Sb-2 was analyzed using bioinformatics techniques for occurrence, homology and synteny in the genus Citrobacter. Furthermore, the clusters found were compared with the chromosomally encoded and well-studied reference determinant of the E. coli wild-type strain K12 to highlight similarities and uniqueness at the sequence level and synteny (Diorio et al. 1995; Oden et al. 1994; Silver et al. 1981). Two resistance gene clusters could be identified on the genome of strain Sb-2 (Figure 3). Both are higher in complexity of construction compared to the chromosomal encoded of E. coli K12. The operon encoding the arsenic resistance mediating components in E. coli K12 contains the arsR, arsB and arsC genes encoding the transcriptional repressor (ArsR), the transmembrane efflux protein (ArsB) and the arsenic reductase (ArsC) (Busenlehner et al. 2003; Meng et al. 2004; Zhu et al. 2014). A more complex operon structure occurs in E. coli R773 with two additional genes, arsD and arsA (Chen et al. 1986). Here, in addition to the efflux only mediated by ArsB, arsenite transporter exist that are composed of an ArsB pore plus an ArsA ATPase (Castillo and Saier 2010; Dey and Rosen 1995; Yang et al. 2012). The gene arsD encoding an arsenic metallochaperone that transfers trivalent metalloids to the ArsAB pump with an additional function as an inducer-independent, weak repressor of the ars operon. The ars1-cluster (locus tag: I9P40_RS1120 - I9P40_RS1120), one of the identified ars clusters, in strain Sb-2 contains all these five components, but also a second arsR gene (arsR1, I9P40_RS1120) and a gene encoding an uncharacterized gene product YraQ (yraQ, I9P40_RS1125) in a divergon orientation (Figure 3). This gene product is generally annotated as a permease and predicted to be transporter with eight transmembrane helices (Aziz et al. 2008; Kelley et al. 2015; Krogh et al. 2001). YraQ from Sb-2 is no ortholog to the yraQ (locus tag: b13151) encoded gene product of E. coli K12 and shares only 18.5 % of amino acid (AA) identity with different topology (data not shown). Furthermore, this gene is encoded independently of an arsenic cluster on the chromosome of E. coli. In contrast, YraQ from Sb-2 displayed significant homologies to ArsP ( ~ 90% identity on AA level) from Campylobacter jejunii and is therefore predicted to confer resistance to roxarsone and MMA(III) (Shen et al. 2014), suggesting YraQ could also have a high efficiency in conferring roxarsone and MMA(III) resistance to Sb-2. The second ars2-cluster (locus tag: I9P40_RS10540 - I9P40_RS10555) of strain Sb-2 is homologous in synteny with simple cluster of E. coli K12 but with the additional gene arsH (I9P40_RS10540) in a divergon orientation to arsR (I9P40_RS10545). The encoded gene products show a higher degree of AA identity to the E. coliars operon when compared to the gene products of ars1-cluster (Figure 3). Both clusters are found independently and frequently on the genomes of other C. portucalensis strains and Citrobacter species. Sometimes only the simple cluster 2 is present, as in C. braakii FDAARGOS_253, or only the more complex cluster 1 in C. portucalensis P10159 and C. freundii RHB12-C20. In the genome of C. freundii R17, an intact cluster and a cluster1 with an interrupted arsD´ can be identified (Figure 3).
Interestingly, both arsenic resistance determinants of strain Sb-2 are flanked by DNA region belonging to prophage Klebsi_phiKO2 (NC_005857) (ars2 - cluster) or embedded in prophage Entero_mEp237 (N C_019704) DNA region (ars1 - cluster) respectively (Table 6). Both phages flanking arsH <-> arsRBC clusters in the genomes of C. braaki strain FDAARGOS_253 and C. portucalensis Sb-2 strains show the same closest relative, PHAGE_Klebsi_phiKO2, according to PHASTER annotation (Arndt et al. 2019). In comparison, the more complex arsRDABC <-> arsR1 <-> yraQ clusters in C. portucalensis strain Sb-2 and P10159 as well as in C. freundii RHB12-C20 are embedded in the homologous annotated phage, Entero_mEp237. This arrangement displays a corresponding degree of similarity within these two ars clusters and the respective flanking and enclosing prophages in the genomes of these members of the Citrobacter genus. This finding gives a direct indication for phage-driven HGT metalloid resistance spread. These phages are widespread in the Enterobacteriacea family and, due to their broad host range, enable not only horizontal gene spread and transmission of metal resistance determinants, but also of antibiotic resistance islands or virulence factors (Ekundayo and Okoh 2018; Magaziner et al. 2019). The results of comparative phageomics of the five Citrobacter species shown in Table 6 indicate that strain Sb-2 harbors, in addition to two mentioned intact phages (size of 59.3 and 25.5 kb), another intact phage of size 34.4 kb, an incomplete and a questionable phage, most similar to PHAGE_Salmon_SP_004 (NC_021774;), PHAGE_Pectob_CBB (NC_041878) and PHAGE_Escher_500465_1 (NC049342). Comparable in number of harbored prophages is C. braaki strain 253 with 4 intact and one incomplete and C. freudii strain R17 with two intact, two incomplete and two questionable. In contrast, the genome of C. portucalensis P10159 followed by C. freudii RHB12-20 displayed a higher number of phage DNA regions with up to 5 intact, 4-10 incomplete and also up to five questionable.
The presence of these putative arsenite and antimonite resistance determinants flanked and embedded by prophages and present in different Citrobacter species from different environments indicates widespread transduction of this phage. It is possible that this determinant also protects Citrobacter species from protist predation that are known to use both arsenite in addition to copper and zinc to poison the prey (Hao et al. 2017).