Genomic Analysis of Citrobacter Portucalensis Sb-2 Identifies a Phage Predation Driven Metalloid Resistance


 Bacterial adaptation to extreme environments is often mediated by horizontal gene transfer (HGT). At the same time, phage mediated HGT for conferring bacterial arsenite and antimonite resistance has not been documented before. In this study, a highly arsenite and antimonite resistant bacterium, C. portucalensis strain Sb-2, was isolated and subsequent genome analysis showed that putative arsenite and antimonite resistance determinants were flanked or embedded by prophages. We predict these phage-mediated resistances play a significant role in maintaining genetic diversity within the genus of Citrobacter and are responsible for endowing the corresponding resistances to C. portucalensis strain Sb-2.

2012). Other metal(loids)s (e.g., As, Sb, Ag, Al, Au, Cd, Hg, and Pb) have no or limited biological roles and are only cytotoxic (Bruins et al. 2000). Regions of toxic metal(loid) pollutants are widespread around the world (Romaniuk et al. 2018). This is a consequence of both natural processes (e.g. weathering of metal-containing minerals, volcanic emissions, forest res, deep sea springs and geysers) and anthropogenic activities (e.g. agriculture, animal husbandry, large-scale burning of fossil fuels, fracking, mining and metallurgical production (Charlesworth et  Arsenic (As) and antimony (Sb) are toxic metalloids that often coexist in the environment (Lu et al. 2018; Shtangeeva et al. 2011) and share chemical and toxicological properties (Nies 1999). The toxicity of As and Sb depends upon their chemical species and oxidation state. In many bacterial species Sb(III) and As(III) are taken up by aquaglyceroporins such as the glycerol facilitator GlpF of Escherichia coli, producing toxicity (Meng et al. 2004;Sanders et al. 1997). Detoxi cation is frequently conferred by e ux via As(III)/Sb(III) e ux permeases such as ArsB and ACR3 permeases (Meng et al. 2004). Pentavalent inorganic arsenate (As(V)) enters cells of E. coli by phosphate uptake systems such as Pit and/or Pst. As(V) is reduced by ArsC arsenate reductases to the trivalent arsenite (As(III)), which is removed from the cells by the e ux permeases. Alternatively, As(III) is methylated by the ArsM As(III) S-adenosylmethionine methyltransferase, producing highly toxic methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)), as well as volatile trimethylarsenite (TMAs(III)). These are oxidized in air to the relatively nontoxic pentavalent species (Yang and Rosen 2016). Sb(III) is also detoxi ed by e ux via ArsB or methylated by ArsM, but, in general, the comparable reactions with antimony are not well characterized (Butcher et al. 2000;Silver et al. 1981). Here we report isolation, of a highly antimony-resistant bacterium, Citrobacter portucalensis Sb-2, from an agricultural eld near the biggest antimony mine in the world. From the draft sequence of its genome, we identi ed putative arsenic/antimony resistance genes and characterized its resistance to antimony and arsenic.

Culture enrichment and strain isolation
For bacterial enrichment, 1.0 g sample was mixed thoroughly with 20 mL of 0.85% saline, 50 ul suspension was inoculated into 5 mL minimal (Mergeay et al. 1985) / R2A (Zhang et al. 2020) and TY media (Mergeay et al. 1985) respectively containing 0.5 mM potassium antimonyl tartrate (C 8 H 4 K 2 O 12 Sb 2 ·3H 2 O) or sodium arsenite (NaAsO 2 ). The cultures were incubated at 28°C with 180 rpm shaking for about 1 day until the medium became turbid. The culture was transferred to fresh medium containing twice the concentrations of Sb(III) or As(III). The concentrations of Sb(III) or As(III) were gradually increased until the cultures stopped growing. The microbial suspension was subsequently serially diluted onto agar plates with 4 mM Sb(III) or As(III). Single colonies were picked and re-streaked at least three times to obtain pure isolates. Metal(loid) concentrations from the isolation site were determined by inductively coupled plasma mass spectroscopy (ICP-MS) (NexIon 300 X, PerkinElmer, US).

Phylogenetic analysis and genome sequencing
Total genomic DNA of Citrobacter portucalensis Sb-2 was extracted using a TIANamp Bacteria DNA kit (TIANGEN, China) following the suggestions of manufacturer and used as a template to amplify 16S rRNA sequence by PCR. Close relative and phylogenetic a liation of the obtained 16S rRNA sequences were determined by using the BLAST search program at the NCBI website (www.ncbi.nlm.nih.gov). The 16S rRNA gene sequences were submitted for comparison and identi cation to the GenBank databases using the NCBI Blastn algorithm and to the EMBL databases using the Fasta algorithm. The phylogenetic tree of 16S rRNA gene sequences and the amino acid sequences of compared resistance determinants were inferred by Geneious prime

Determination of the Minimal Inhibitory Concentration (MIC)
The single colonies were later used to examine the minimal inhibitory concentration (MIC) on C 8 H 4

Scanning electron microscopy
Overnight cultures were diluted 1:100 in fresh mineral salts medium and incubated at 28°C. Samples were harvested at a logarithmic phase by centrifuging for 5 min, 2500 rpm at 4°C and were immobilized using glutaraldehyde (5%, v/v) for 4 h. Samples were washed three times in 0.

Genome annotation and characterization
The shot gun sequence of Citrobacter portucalensis Sb-2 was submitted to NCBI Prokaryotic Genome Annotation Pipeline (PGAP; Annotation Software revision 4.6) for gene annotation following the standard operating procedures and was released on 26rd of December, 2020 with GenBank assembly accession number GCA_016406035.1, under BioProject: PRJNA684029 and BioSample: SAMN017050479.

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 eld (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).

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.

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 identi ed 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 e ux 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 e ux 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 ars 1 -cluster (locus tag: I9P40_RS1120 -I9P40_RS1120), one of the identi ed ars clusters, in strain Sb-2 contains all these ve components, but also a second arsR gene (arsR 1 , 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 signi cant 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 e ciency in conferring roxarsone and MMA(III) resistance to Sb-2. The second ars 2 -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 ars 1 -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 cluster 1 with an interrupted arsD´ can be identi ed ( Figure 3).
Interestingly, both arsenic resistance determinants of strain Sb-2 are anked by DNA region belonging to prophage Klebsi_phiKO2 (NC_005857) (ars 2 -cluster) or embedded in prophage Entero_mEp237 (N C_019704) DNA region (ars 1 -cluster) respectively (Table 6). Both phages anking 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 <-> arsR 1 <-> 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 anking and enclosing prophages in the genomes of these members of the Citrobacter genus. This nding 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  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 ve questionable.
The presence of these putative arsenite and antimonite resistance determinants anked 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).

Conclusion
The contamination of heavy metals at high concentrations in different areas around the world is predicted to signi cantly in uence the taxonomic and functional diversities of soil microbial communities. Here we showed evidence that phage predation plays a major role in maintaining genetic diversity within population of a single species or genus. These results support a better understanding of diversity dynamics, in which the diversity of prokaryotic populations is driven by phage predation. In addition, the role of HGT in adaptation of bacteria to extreme environments is highlighted.

Declarations Data availability
This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JAEKIJ000000000. The data used to support the ndings of this study are included within the article. Please check https://www.ncbi.nlm.nih.gov/assembly/ GCF_016406035.1 for more details.

Declaration of competing interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.