Genome Sequencing Revealed Chromium and Other Heavy Metal Resistance Genes in E. cloacae B2-Dha
Received Date: Aug 28, 2017 / Accepted Date: Sep 18, 2017 / Published Date: Sep 25, 2017
The previously described chromium resistant bacterium, Enterobacter cloacae B2-DHA, was isolated from leather manufacturing tannery landfill in Bangladesh. Here we report the entire genome sequence of this bacterium containing chromium and other heavy metal resistance genes. The genome size and the number of genes, determined by massive parallel sequencing and comparative analysis with other known Enterobacter genomes, are predicted to be 4.22 Mb and 3958, respectively. Nearly 160 of these genes were found to be involved in binding, transport, and catabolism of ions as well as efflux of inorganic and organic compounds. Specifically, the presence of two chromium resistance genes, chrR and chrA was verified by polymerase chain reaction. The outcome of this research highlights the significance of this bacterium in bioremediation of chromium and other toxic metals from the contaminated sources.
Keywords: Bioremediation; Toxic metals; Enterobacter cloacae; Genome sequencing; De novo assembly; Gene annotation
The global urbanization and industrialization creates increasing levels of pollution including toxic heavy metal contamination . In particular, chromium toxicity is generated through widespread anthropogenic activity via leather processing, steel production, wood preservation, chromium/electroplating, metal processing, alloy formation, textiles, ceramics and thermonuclear weapons manufacturing, and together with agronomic practices such as the use of organic biomass (sewage sludge or fertilizers), which continues to be a major threat to the environment [2-6]. Furthermore, chromium exerts damage directly on human health through toxic and mutagenic effects causing severe DNA damage . However, chromium has multiple effects on bacteria including competitive inhibition of sulphate transport, DNA mutagenesis and protein damage . Microorganisms have developed various mechanisms to survive chromium toxicity: (i) transmembrane efflux of chromate (ii) the ChrR transport system (iii) the reduction of chromate (iv) protection against oxidative stress and (v) DNA repair systems [3,9-15]. In addition, chromate resistance is attributed to the functions of a series of chromosomal or plasmid encoded genes, including the chromium resistance (chr) operon comprising of either chrBAC or chrBACF in bacteria [9,16,17]. The ChrA protein, a member of the CHR superfamily of transporters appears to be active in chromate efflux driven by the membrane potential, whereas the chrB gene encodes for a membrane bound protein necessary for the regulation of chromate resistance [18-20]. The chrC gene encodes a protein almost similar to iron-containing superoxide dismutase, while the chrE gene encodes a protein resembling a rhodanese type enzyme in Orthrobacterium tritici 5bvI1 . The chrF gene likely encodes a repressor of chromatedependent induction, whereas the ChrR protein catalyzes one-electron shuttle followed by a two-electron transfer to Cr6+ .
Previously, we have characterized E. cloacae B2-DHA, a soilborne bacterium, that can survive and grow on medium containing up to 5.5 mM chromate. By using inductively coupled plasma atomic emission spectroscopy (ICP-AES) we have shown that after 120 h of exposure to 100 μg/mL chromium the B2-DHA cells can accumulate 320 μg of chromium per gram dry weight of bacterial biomass thus the concentration of chromium in the cell free growth medium is decreased from 100 μg/mL to 19 μg/mL (81%) . In addition, B2-DHA, can grow on medium containing sodium arsenate, ferric chloride, manganese chloride, zinc chloride, nickel chloride and silver nitrate. However, the mechanisms by which this chromium-adapted B2-DHA survives were not elucidated. Thus, the present study was aimed at demonstrating whether the strain B2-DHA harbored genes that were responsible for chromium and other metal resistance. In this study, we have performed massive parallel genome sequencing of E. cloacae B2-DHA to investigate the metal responsive genes. All the genes involved in metal binding activity and reduction of metal by the E. cloacae B2-DHA strain were predicted by Rapid Annotations using Subsystems Technology, RAST and/or Blast2GO [22,23]. Furthermore, we have conducted comparative genome analyses of E. cloacae B2-DHA with other known Enterobacter genome sequences and characterized the genetic rearrangement among the various lineages to understand the evolutionary processes involved in shaping the genomes.
Materials and Methods
Extraction of genomic DNA
Genomic DNA was extracted from E. cloacae B2-DHA using DNeasy Blood & Tissue Kit (Qiagen, Cat No 69506) according to manufacturer’s instructions with some modifications. The bacteria were cultured in Luria Bertani (LB) medium and pellets were collected from 1.0 ml of bacterial cultures by centrifugation at 8000 rpm for 10 min, the pellets were resuspended in TE buffer (10 mM Tris- HCl, 1 mM EDTA [pH 8.0]) containing RNase (50 mg/ml) and lysozyme (50 mg/ml) and incubated at 37°C for 2 h instead of using ATL (a tissue lysis buffer). The purity and concentration of the extracted DNA were measured using the Nanodrop® ND-1000 Spectrophotometer (Saveen Werner, USA). The DNA sample exhibiting a clear band in agarose gel electrophoresis was selected for sequencing of the whole genome.
The entire genome sequencing of E. cloacae B2-DHA was assisted by the Otogenetics Corporation (GA, USA) as follows: (i) Purified 0.5- 1.0 μg of genomic DNA sample was clipped into smaller fragments with a Covaris E210 ultrasonicator; (ii) the library of genomic DNA was prepared according to standard protocol of the NEB library preparation kit (New England Biolabs) for the Illumina sequencer with a single sequencing index; (iii) the sequencing was accomplished with the Illumina HiSeq2500 PE106 (106 bp paired-end) read format; (iv) properly paired reads (≥ 30 bp) were separated from the corrected read pool and the remaining singleton reads were combined as singleend reads; and (v) both of the single-end reads and corrected pairedend reads were used in the subsequent de novo assembly as described previously .
de novo assembly
The de novo assembly started with Illumina 106 bp paired-end reads of genomic DNA with an insert length of 300 bp and the read quality was measured with FastQC, version 1.10.1 . Adapter and quality trimming on raw reads were conducted with cutAdapt and K-mer error correction was performed on the adapter-free reads using Quake, version 0.3.5 [26,27]. The paired reads were extracted from the corrected read pool and the remaining singleton reads were listed as single-end reads. Both corrected paired-end and single-end reads were used in the k-mer-based de novo assembly. SOAPDenovo, version 2.04 was utilized to perform de novo assembly optimization with the error corrected reads . A wide range of K-mers (29-99) were used to identify the scaffold sequences with the largest N50. The optimal scaffold sequences were further subjected to gap closing by utilizing the corrected pairedend reads, and the resulting scaffolds of length ≥ 300 bp were chosen as the final assembly. The largest N50 of 492,970 bp was produced at the k-mer 97. All the scaffolds were ordered by finding the location of the best Blastn hit for each scaffold on the reference genome E. cloacae ECNIH2 [NCBI accession number CP008823]. A total of 13 scaffolds were used to order the contigs from a draft genome by comparison to a reference genome performed by following the Mauve Contigs Mover (http://darlinglab.org/mauve/user-guide/reordering.html).
Comparative analysis with other E-bacter genomes
The Whole Genome Shotgun project has been deposited at DDBJ/ EMBL/GenBank under the GenBank accession LFJA00000000 . The progressive MAUVE algorithm in the MAUVE genome alignment software, version 2.3.1was used to study genome rearrangements in E. cloacae B2-DHA and related bacteria. Furthermore, another nucleotidebased dot plot analysis was performed with the Gepard software to (i) compare the 4.21 Mbp chromosomal scaffolds of E. cloacae B2-DHA with that of 4.85 Mbp chromosomes in E. cloacae ECNIH2, and (ii) investigate the possible genome rearrangements in these strains.
Prediction and annotation of metal responsive genes
The prediction of all genes in B2-DHA genome was carried out using FGenesB and GeneMark. ARAGORN, version 1.2.36 employed to predict tRNA genes in B2-DHA genome. We have applied Blast2GO pipeline using all translated protein coding sequences resulting from the FGenesB to execute all functional annotation analyses. In Blast2GO, the BlastP option was chosen to find the closest homologs in the nonredundant protein databases (nr), followed by employment of Gene Ontology (GO) annotation terms to each gene . An InterPro scan was then performed through the Blast2GO interface with the InterPro IDs for obtaining integrated annotation results . Annotation of all putative metal responsive genes was manually curated. The assembled genome sequence was annotated with RAST which uses (i) the GLIMMER algorithm to predict protein-coding genes (ii) the tRNAscan-SE to predict tRNA genes , (iii) an internal script for identification of rRNA genes and (iv)the RNAmmer prediction server version 1.2, to identify rRNA genes . Furthermore, RAST (i) infers putative function(s) of the protein coding genes based on homology with known protein families in phylogenetic neighbor species, and (ii) detects subsystems represented in the genome, and helps to reconstruct the metabolic networks. RAST results obtained in prediction of protein coding genes were compared with the GeneMark and the FGenesB algorithms. Circular plot of ordered contigs of B2-DHA was generated with DNAPlotter to predict the graphical map of the genome .
PCR amplification of chromium-responsive genes
Primers for the gene chrR and chrA were designed by using the Primer3Plus web tool . The two primer pairs, chrR-F/chrR-R (5'-ATGTCTGATACGTTGAAAGTTGTTA- 3'/5'-CAGGCCTTCACCCGCTTA- 3') and chrA-F/chrA-R (5'-TGAAAAGCTGTTTACCCCACT- 3'/5'-TTACAGTGAAGGGTAGTCGGTATAA-3') were selected for the detection of chrR and chrA genes, respectively. PCR amplification of chromium-related marker genes was performed using bacterial genomic DNA as a template in a piko thermal cycler (Finzymes) under the following cycling conditions: 5 min of denaturation at 95°C, followed by 30 cycles of 1 min of denaturation at 95°C, 45 s of annealing at 54.5°C and primer extension at 72°C for 1 min of each Kb product size. All PCR reaction mixtures contained approximately 50 ng DNA templates, 0.2 mM of each deoxyribonucleoside triphosphate, 1X PCR buffer, 0.5 mM of each primer, and 1 U Taq DNA polymerase in a final volume of 50 μl. The final extension reaction was conducted at 72°C for 15 min. PCR products were purified with a QIAquick PCR Purification Kit (Qiagen, Cat No 28104).
Sequencing and de novo genome assembly
Illumina deep sequencing analysis revealed that the genome of B2-DHA consists of 1,756,877,072 bases containing 16,574,312 pairs of reads with an overall GC content of 55%. After quality trimming error correction followed by removal of the TruSeq adaptor sequence, 15,708,650 read pairs (94.78%) and 331,106 single end sequences remained for further analysis. Analysis of the raw reads with FastQC showed that the mean scores per base Phred and per sequence Phred were ≥ 36 and 36, respectively for all positions. The set of scaffold sequences with maximal N50 (492,970 bp) was detected at k-mer 97. The corresponding scaffold sequences were subjected to gap closure using the corrected paired-end reads and the resulting scaffolds (≥ 24300 bp) were defined as the final assembly. The genome summary including the nucleotide content and the gene count is posted in Table 1. The scaffolds were ordered by finding the location of the best Blastn hit for each scaffold on the reference genome Enterobacter cloacae ECNIH2. The final assembly of 4,218,945 bp was comprised of 13 scaffolds ranging from 72,208 to 777,700 bp.
|Attribute||Value||% of total|
|Genome size (bp)||4 218 945||100|
|DNA GC content (bp)||2 353 515||55|
|DNA coding region (bp)||3 768779||89,33|
|Number of replicons||1|
|Protein coding genes||3958||97,82|
|Genes assigned to RAST functional categories||3954||97,79|
|Genes assigned Gene Ontology terms by Blast2GO||3159||79,87|
Table 1: Summary of the genome of B2-DHA with nucleotide content and gene count.
Comparative genome analysis
The chromosomal arrangement of E. cloacae B2-DHA was compared to E. cloacae ECNIH2 by employing progressive Mauve from the Mauve software  and Gepard dot plot software . While the alignment remained almost identical in chromosomal rearrangement, the progressive Mauve analysis found several inversions in scaffolds of E. cloacae B2-DHA compared to that in E. cloacae ECNIH2 (Figure 1A). The dot plot performed with E. cloacae B2-DHA and E. cloacae ECNIH2 depicted a similar observation of inversions in scaffolds of E. cloacae B2-DHA (Figure 1B). Furthermore, several large segments of high similarity were obtained when most parts of the chromosomes of E. cloacae B2-DHA and E. cloacae ECNIH2 were mapped onto each other (Figure 1B).
Figure 1: (A) Nucleotide-based alignment of a 4.21 Mbp chromosomal assembly of E. cloacae B2-DHA (upper) and 4.85 Mbp chromosomes of E. cloacae ECNIH2 (lower). A total of 12 homologous blocks are shown as identically colored regions and linked across the sequences. Regions that are inverted relative to E. cloacae B2-DHA are shifted to the right of center axis of the sequence. (B) Dot plot of nucleotide sequences of E. cloacae B2-DHA (X-axis) and E. cloacae ECNIH2 (Y-axis). Aligned segments are represented as dots, with regions of conservation appearing as lines.
The genome and the locations of all genes were predicted through RAST server and the results of this prediction are shown via a circular plot in Figure 2. The prediction of rRNA coding genes showed 22 rRNA genes including four LSU, four SSU, eight 16S and six 23S genes in E. cloacae B2-DHA (Figure 2). ARAGORN, version 1.2.36 , employed to predict tRNA genes, identified 66 tRNA genes with a GC content ranging from 48.0% to 67.5% in E. cloacae B2-DHA.
Figure 2: Circular plot of ordered contigs, generated with DNAPlotter. Tracks indicate (from outside inwards) protein coding genes in forward direction (blue) and protein coding genes in reverse direction (green), tRNA genes (red), rRNA genes (dark blue), metal responsive genes (black), GC ratio and GC skew.
RAST analysis using the GLIMMER algorithm predicted a total of 3958 protein coding genes of which 3401 could be annotated by RAST’s automated homology analysis procedure and assigned to functional categories (Figure 3) . For confirmation of the number of protein coding genes, the FGenesB and the GeneMark algorithms were also applied, yielding 3955 and 3764 genes, respectively [39,40]. By using RAST, we observed that the strain E. cloacae B2-DHA contained a large number of genes involved in the ion binding, transport, catabolism and efflux of inorganic as well as organic compounds. More specifically, B2-DHA strain contains many specific metal resistance genes, such as arsenic, chromium, cadmium, cobalt, lead and nickel (Table 2). The Blast2GO pipeline analysis also indicated that B2-DHA contains many genes that are directly responsive to toxic metal ions like arsenic, chromium, cadmium, cobalt, lead and nickel (Table 2). Moreover, these analyses revealed that B2-DHA strain also possesses many genes encoding binding and/or transport of calcium, copper, iron, magnesium, potassium and sodium ions as well as several trace elements like manganese, molybdenum and tellurite (Table 3). Also, a large number of zinc ion binding and/or transporter proteins are retained in this strain (Data not shown). Besides zinc the B2-DHA genome contains a total of 104 proteins involved in binding and transport of other metal ions (Data not shown).
|Start (bp)||End (bp)||Predicted function||RAST||Blast2GO|
|242454||243404||Magnesium and cobalt transport protein CorA||X||X|
|615926||616912||Cobalt, zinc, magnesium ion binding||X|
|964298||965137||Nickel, Cobalt cation transporter activity||X|
|997848||1000430||Copper, lead, cadmium, zinc, mercury transporting ATPase||X||X|
|1100748||1099984||Ferric enterobactin transport protein FepC||X||X|
|1101770||1100781||Ferric enterobactin transport protein FepG||X||X|
|1102774||1101770||Ferric enterobactin transport protein FepD||X||X|
|1105147||1104188||Ferric enterobactin transport protein FepB||X||X|
|1510555||1509272||Ferrous iron transport peroxidase EfeB||X||X|
|1511686||1510559||Ferrous iron transport periplasmic protein EfeO,||X|
|1512560||1511727||Ferrous iron transport permease EfeU||X||X|
|1703726||1704046||Arsenite resistance operon repressor||X||X|
|1704087||1705376||Arsenite efflux pump protein||X||X|
|1834407||1835345||Cobalt-zinc-cadmium resistance, Zinc transporter ZitB||X||X|
|1919484||1921043||Magnesium and cobalt efflux protein CorC||X|
|2058555||2059283||Ferric siderophore transport protein TonB||X|
|2216754||2216455||Transcriptional regulator, ArsR family||X|
|2592824||2595886||Cobalt-zinc-cadmium resistance protein CzcA||X|
|2735411||2736391||Nickel, Cobalt cation transporter activity||X|
|3168971||3169588||Nickel cation binding||X|
|3169598||3170242||Nickel cation binding||X|
|3170821||3171285||Nickel cation binding||X|
|3171295||3172998||Nickel cation binding||X|
|3173316||3173618||Nickel cation binding||X|
|3173629||3174456||Nickel cation binding||X|
|3170811||3170272||Transport of Nickel and Cobalt, Urea decomposition||X|
|3500732||3499869||Nickel incorporation-associated protein HypB||X||X|
|3505195||3506904||Nickel cation binding||X|
|3501086||3500736||Nickel incorporation protein HypA||X||X|
|3892272||3892499||Ferrous iron transport protein A||X||X|
|3892530||3894848||Ferrous iron transport protein B||X|
|3951655||3953826||Copper, lead, cadmium, zinc, mercury transporting ATPase||X||X|
|4172744||4173628||Cobalt-zinc-cadmium resistance protein||X||X|
|4176907||4178211||Arsenic efflux pump protein||X|
Table 2: Heavy metals responsive proteins in B2-DHA predicted by RAST and/or Blast2GO.
|Gene 802||858022||859119||manganese ion binding|
|Gene 196||202800||204344||manganese ion binding|
|Gene 209||215336||216379||manganese ion binding|
|Gene 271||277278||279605||molybdenum ion binding|
|Gene 597||628652||630154||manganese ion binding|
|Gene 626||664374||665294||manganese ion binding|
|Gene 720||771440||772036||manganese ion binding|
|Gene 861||919680||921458||manganese ion binding|
|Gene 1016||1088133||1089044||Manganese transporter protein SitA|
|Gene 1017||1089047||1089853||Manganese transporter protein SitB|
|Gene 1018||1089850||1090701||Manganese transporter protein SitC|
|Gene 1019||1090695||1091534||Manganese transporter protein SitD|
|Gene 1049||1125413||1124667||Molybdenum transport protein ModB|
|Gene 1071||1148010||1150448||molybdenum ion binding|
|Gene 1223||1294800||1296950||molybdenum ion binding|
|Gene 1504||1570809||1571876||molybdenum ion binding|
|Gene 1548||1623197||1625641||molybdenum ion binding|
|Gene 1610||1694840||1695742||manganese ion binding|
|Gene 1725||1822796||1821738||Molybdenum transport protein ModC|
|Gene 1726||1823488||1822796||Molybdenum transport protein ModB|
|Gene 1727||1824261||1823485||Molybdenum-binding protein ModA|
|Gene 1729||1824720||1825508||molybdate ion transport|
|Gene 1756||1853924||1855090||manganese ion binding|
|Gene 1818||1924327||1924905||manganese ion binding|
|Gene 1903||2021204||2023633||molybdenum ion binding|
|Gene 1908||2029753||2033496||molybdenum ion binding|
|Gene 2091||2224133||2227873||molybdenum ion binding|
|Gene 2098||2235200||2237611||molybdenum ion binding|
|Gene 2209||2348836||2349429||Tellurite resistance protein TehB|
|Gene 2210||2349429||2350424||Tellurite resistance protein TehA|
|Gene 2610||2758489||2760867||molybdenum ion binding|
|Gene 2650||2805061||2806479||manganese ion binding|
|Gene 2682||2845329||2847608||manganese ion binding|
|Gene 2713||2876204||2877379||Manganese transport protein MntH|
|Gene 2785||2950966||2953689||molybdenum ion binding|
|Gene 3085||3280624||3281571||manganese ion binding|
|Gene 3130||3326886||3328205||manganese ion binding|
|Gene 3151||3347474||3349207||manganese ion binding|
|Gene 3540||3756054||3757565||manganese ion binding|
|Gene 3885||4139417||4141831||molybdenum ion binding|
|Gene 3886||4141880||4142467||molybdenum ion binding|
Table 3: Manganese, molybdenum and tellurite resistant proteins in B2-DHA predicted by RAST and/or Blast2GO.
Detection of putative chromium resistance genes
The Blast2GO and RAST analyses detected two chromium reductase genes in B2-DHA. These gene were named as chrR and chrA (not to be confused with chromate transporter gene). Presence of these genes in this bacterium was verified by PCR amplification (Figure 4). In addition, a number of other chromate responsive genes were confirmed in B2-DHA. Most of these genes have NAD (P) H dependent oxidoreductase activity (Table 4).
Figure 4: Molecular analysis of chromium responsive genes of B2-DHA and gel electrophoresis. PCR amplification of chrR and chrA genes. L represents 2 log DNA marker, lane 1 and 2 are the amplified fragments of chrR gene in two replicates whereas lane 3 and 4 are the amplified fragments of chrA gene in two replicates.
|Seq. Name||Start||End||Predicted function|
|Gene- 343||355848||356153||Cytochrome-c oxidase activity|
|Gene- 488||512342||513904||Oxidoreductase activity, reduced flavin or flavoprotein|
|Gene- 650||692191||693615||Dihydrolipoamide dehydrogenase|
|Gene- 1057||1135310||1134879||Universal stress protein G|
|Gene- 1121||1198337||1199287||Universal stress protein E|
|Gene- 1150||1228221||1230242||NAD(P)H dependent oxidoreductase activity|
|Gene- 1554||1634190||1635158||Thioredoxin reductase|
|Gene- 2184||2325505||2324840||Multiple antibiotic resistance protein MarC|
|Gene- 2185||2325818||2326195||Multiple antibiotic resistance protein MarR|
|Gene- 2186||2326216||2326596||Multiple antibiotic resistance protein MarA|
|Gene- 2187||2326629||2326844||Multiple antibiotic resistance protein MarB|
|Gene- 2364||2487111||2487539||Universal stress protein C|
|Gene- 2436||2553300||2556080||NADH: flavin oxidoreductase|
|Gene- 2463||2591265||2590834||Universal stress protein G|
|Gene- 2531||2669613||2672735||Multidrug resistance MdtB|
|Gene- 2533||2672736||2675813||Multidrug resistance MdtC|
|Gene- 2534||2675814||2677229||Multidrug resistance MdtD|
|Gene- 3334||3544616||3543069||Multidrug resistance MdtB|
|Gene- 3335||3545805||3544633||Multidrug resistance MdtA|
|Gene- 3582||3809416||3810390||Quinone oxidoreductase|
|Gene- 3638||3860171||3862714||Nitrite reductases|
|Gene- 3746||3974324||3973941||Polymyxin resistance protein PmrM|
|Gene- 3747||3974644||3974321||Polymyxin resistance protein PmrL,|
|Gene- 3749||3977186||3976284||Polymyxin resistance protein PmrJ|
|Gene- 3751||3980145||3979162||Polymyxin resistance protein ArnC|
|Gene- 3760||3989137||3988850||Universal stress protein B|
|Gene- 3761||3989469||3989906||Universal stress protein A|
Table 4: Universal stress proteins, multiple antibiotic resistant proteins, multidrug resistance proteins and polymyxin resistance protein in B2-DHA as predicted by RAST and/or Blast2GO.
Prediction other proteins
Several polymyxin resistant proteins such as PmrM, PmrL, PmrJ and ArnC were aslo predicted by RAST and Blast2GO (Table 4). RAST analysis enabled us to detect several multidrug transporter proteins like MdtA, MdtB, MdtC and MdtD in B2-DHA strain (Table 4). This strain also contains universal stress proteins A, B, C, E and G, as well as several multiple antibiotic resistance proteins such as MarA, MarB, MarC and MarR (Table 4). Other proteins that catalyze binding and transport of the metal ions are metalloendopeptidase, metalloexopeptidase, metallopeptidase, metallocarboxypeptidase and metallochaperone. Some metallocenter assembly proteins such as HypA, HypB, HypC, HypD, HypE and HypF are also present in this strain.
Previously we have reported chromium-resistant bacterial strain E. cloacae B2-DHA isolated from the Hazaribagh tannery areas in Bangladesh . In this paper we report the results of sequencing of the whole-genome of this bacterium. After quality trimming, error correction, and removal of the TruSeq adaptor sequence the genome was de novo assembled resulting an approximate genome length of 4.22 Mbp. Several other Enterobacter strains have been sequenced previously. For example, E. cloacae UW5 had a genome size of 4.9-Mbp and E. cloacae ENHKU01 had 4.72-Mbp [41,42]. Our strain E. cloacae B2-DHA contained a total of 3958 protein coding genes, whereas in E. cloacae ENHKU01 the total number of these genes was 4338. The results we obtained in B2-DHA are in agreement with those reported by other researchers, although the genome size and number of protein coding genes in B2-DHA are slightly smaller than those in E. cloacae ENHKU0. The difference in number of protein coding genes in the bacterial strains can be attributed to a common phenomenon. Even in a Gram-positive bacterium, Lysinibacillus sphaericus B1-CDA, the number of protein coding genes analyzed by different web tools was found to be different [43,44]. The goal of gene prediction in B2- DHA was to catalogue all the genes encoded within its genome. This prediction facilitates understanding of the mechanisms that might be involved in resistance of this bacterium to chromium and other toxic metals. The annotation of the assembled genome, number of tRNA and rRNA in B2-DHA varied from those found in the reference genome of E. cloacae ECNIH2. B2-DHA genome contained 22 rRNA and 66 tRNA genes, whereas in the reference genome ECNIH2 these were 25 and 87, respectively (http://www.ncbi.nlm.nih.gov/nuccore/CP008823). The difference in the number of tRNA genes is a common feature of bacterial and archaeal genomes . These differences could likely be due to the draft status of their B2-DHA genome compared to the reference. However, sometimes annotation systems miss some RNA genes. Furthermore, the bacteria which have the highest number of 16S rRNA genes also have the highest number of tRNA genes .
Results obtained from RAST and Blast2GO analyses showed that the bacterium contains many metal resistance genes and there is no significant difference in the obtained results between these two methods (Table 2). Genome sequencing also revealed that B2-DHA harbors many other genes conferring resistance of this bacterium to polymyxins, multiple drugs and antibiotics. These type genes or their homologues have been identified previously in both Gram-positive and Gram-negative bacteria as well as archaea [47,48]. The proteins encoded by these genes contain many metal-binding residues, which may bind to several metal ions, primarily nickel ions [49,50]. Polymyxin resistance proteins are polycationic antimicrobial peptides that serve as antibiotics for the treatment of infectious diseases caused by multidrug-resistant Gram-negative bacteria. Several bacteria such as Serratia sp., Burkholderia sp. and Proteus sp. are naturally resistant to these antibiotics, whereas other bacteria like Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae develop resistance to polymyxins through acquired resistance . The B2-DHA strain contains many metalloproteinase or metalloprotease enzymes. The possible explanation for this is that the bacteria often need to protect themselves from adverse environmental stimuli, including exposure to stress factor, cationic antimicrobial peptides, and toxic metals . To survive in these stress conditions bacteria develop various strategies mainly based on alterations of the lipopolysaccharides (LPSs) in their cell walls, which have overall negative charges and are the initial targets of polymyxins . Other strategies may include efflux pumps and capsule formation [54,55]. Thus, the strain B2-DHA, isolated from highly chromium contaminated tannery industry area may have developed similar mechanisms to survive under adverse conditions.
We also report that B2-DHA contains 219 genes which are responsive to cell wall and capsule development as well as 164 genes which are involved in stress response (Figure 3). Presence of these genes in this bacterium might be accounted for its morphological changes when exposed to chromium. This type of changes is an advantageous trait for this bacterium and it facilitates accumulation of chromium inside the cells . As described in the results, B2-DHA contains a number of chromate reductase genes and most of these have NAD(P) H-dependent oxidoreductase activity (Table 4). Similar kind of results has been reported previously by . B2-DHA also harbors soluble quinone oxidoreductases that are expected to reduce Fe3+ and Cr6+ and counter oxidative stress . In addition, B2-DHA possesses many other functional genes such as thioredoxin and thioredoxin reductase, dihydrolipoamide dehydrogenase, nitrite reductases, NADH: flavin oxidoreductase, quinones, cytochromes, flavoproteins and proteins with iron sulphur centers (Table 4). These genes are believed to be involved in metal oxidoreductase exhibiting Cr6+ reduction as reported previously [58-60]. The proteins encoded by these genes initially catalyze one-electron shuttle followed by a two-electron transfer to Cr6+ with the formation of intermediate(s) Cr5+ and/or Cr4+ before further reduction to Cr3+ which is a critical process involved in detoxification of chromium inside the cells . Thus bacteria can survive and grow in a chromium contaminated environment. One of many possible ways to increase the effectiveness of chromium bioremediation by using bacteria is to alter the expression of these genes to minimize oxidative stress during chromate reduction. This approach has been proposed by several other researchers [12,48]. Previously, we have postulated that the B2-DHA is resistant to chromium and it can decrease chromium content significantly in the contaminated source by accumulating it in the cells . Furthermore, we report that the bacterium can reduce Cr6+ to Cr3+, corroborating the presence of chromium resistance genes chrR and chrA as described in this paper.
In this paper we report the genome sequence and annotation of a chromium resistant bacterium, E. cloacae B2-DHA. Furthermore, bioinformatics analyses revealed that this bacterium harbours two chromium resistance genes, chrA and chrR among many metal resistance and other genes. Our previous findings of chromium accumulation and the recent data on genomics and functionality of the genes in B2-DHA (which is under investigation) will provide insights to establish the mechanism of chromium resistance in this strain. Altogether, our findings can be employed in bioremediation of these toxic metals in polluted environments especially industry effluents. In a long-term perspective, millions of people worldwide, in turn, can avoid many lethal diseases caused by chronic exposure of toxic metal poisoning. Therefore, our discoveries have a great potential through further investigations in contributing to a significant positive impact on the socioeconomic status of the people particularly in the developing world.
This research was supported by a major grant (AKT-2010-018) from the Swedish International Development Cooperation Agency (SIDA), and partly by a small grant from the Nilsson-Ehle (The Royal Physiographic Society in Lund) foundation in Sweden.
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Citation: Aminur R, Björn O, Jana J, Neelu NN, Sibdas G, et al. (2017) Genome Sequencing Revealed Chromium and Other Heavy Metal Resistance Genes in E. cloacae B2-Dha. J Microb Biochem Technol 09:191-199. DOI: 10.4172/1948-5948.1000365
Copyright: © 2017 Aminur R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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