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TRANSCRIPT
Salam et al. Bioresour. Bioprocess. (2020) 7:25 https://doi.org/10.1186/s40643-020-00314-w
RESEARCH
Effects of cadmium perturbation on the microbial community structure and heavy metal resistome of a tropical agricultural soilLateef B. Salam1* , Oluwafemi S. Obayori2, Mathew O. Ilori3 and Olukayode O. Amund3
Abstract
The effects of cadmium (Cd) contamination on the microbial community structure, soil physicochemistry and heavy metal resistome of a tropical agricultural soil were evaluated in field-moist soil microcosms. A Cd-contaminated agricultural soil (SL5) and an untreated control (SL4) were compared over a period of 5 weeks. Analysis of the physico-chemical properties and heavy metals content of the two microcosms revealed a statistically significant decrease in value of the soil physicochemical parameters (P < 0.05) and concentration of heavy metals (Cd, Pb, Cr, Zn, Fe, Cu, Se) content of the agricultural soil in SL5 microcosm. Illumina shotgun sequencing of the DNA extracted from the two microcosms showed the predominance of the phyla, classes, genera and species of Proteobacteria (37.38%), Actino-bacteria (35.02%), Prevotella (6.93%), and Conexibacter woesei (8.93%) in SL4, and Proteobacteria (50.50%), Alphaproteo-bacteria (22.28%), Methylobacterium (9.14%), and Methylobacterium radiotolerans (12,80%) in SL5, respectively. Statisti-cally significant (P < 0.05) difference between the metagenomes was observed at genus and species delineations. Functional annotation of the two metagenomes revealed diverse heavy metal resistome for the uptake, transport, efflux and detoxification of various heavy metals. It also revealed the exclusive detection in SL5 metagenome of members of RND (resistance nodulation division) protein czcCBA efflux system (czcA, czrA, czrB), CDF (cation diffusion facilitator) transporters (czcD), and genes for enzymes that protect the microbial cells against cadmium stress (sodA, sodB, ahpC). The results obtained in this study showed that Cd contamination significantly affects the soil microbial community structure and function, modifies the heavy metal resistome, alters the soil physicochemistry and results in massive loss of some autochthonous members of the community not adapted to the Cd stress.
Keywords: Cadmium, Agricultural soil, Heavy metals, Soil microcosm, Shotgun metagenomics, Microbial community structure and function, Heavy metal resistome
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IntroductionCadmium (Cd) is a highly toxic, carcinogenic heavy metal with an exceptionally high biological half-life (> 20 years) and propensity for accumulation in the food chain, drink-ing water and soil (Benavides et al. 2005; Khan et al. 2015; Fashola et al. 2016). Major sources of Cd in soil include
wet and dry atmospheric deposition (vehicular emission, incineration, burned fuel and tyre wear, residual ashes from wood, coal or other types of combustion) (Mielke et al. 1991; Steinnes and Friedland 2006); and geologi-cal weathering (Khan et al. 2010; Liu et al. 2013). Other primary anthropogenic sources of Cd in soil include min-ing, sewage sludge, composted municipal solid wastes, improper waste disposal practices, smelting, wastewater irrigation, manufacturing and agrochemicals (Alloway
Open Access
*Correspondence: [email protected] Department of Biological Sciences, Microbiology Unit, Summit University, Offa, Kwara, NigeriaFull list of author information is available at the end of the article
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and Steinnes 1999; Khan et al. 2016a, b; Nawab et al. 2016; Khan et al. 2017).
Elevated Cd concentration in soil poses significant threat to the quantity and diversity of soil microorgan-isms. Cd toxicity to microbial cells is believed to be due to depletion of glutathione and sulfhydryl groups in pro-teins, interaction with nucleic acids, oxidative damage by production of reactive oxygen species, and inactivation of metalloproteins due to displacement of Zn and Fe ions (Vallee and Ulmer 1972; Stohs and Bagchi 1995; Fortu-niak et al. 1996; Stohs et al. 2001; Banjerdkij et al. 2005). This result in protein denaturation, cell membrane and nucleic acid disruption, and inhibition of transcription, cell division and enzyme activities (Fashola et al. 2016). Several workers have also highlighted the debilitating effects of Cd toxicity on the lung, kidney, bones, and the nervous and immune systems of humans (Adriano 2001; Waisberg et al. 2003; Edwards and Prozialeck 2009; Yazdankhah et al. 2010; Satarug et al. 2001; Moynihan et al. 2017). Furthermore, Cd cytotoxicity has been impli-cated in destruction of plant mitochondria as well as dis-ruption of photosynthesis and transpiration (Imai and Siegel 1973; Toppi and Gabbrielli 1999; Lopez-Milla’n et al. 2009; Mohamed et al. 2012; Júnior et al. 2014; Khan et al. 2016a, b).
Bioremediation of Cd-inundated soil is predicated on the presence of highly efficient Cd uptake/transport/efflux/detoxification system within the soil microbial community well-adapted to Cd stress. Mechanisms such as intracellular or extracellular precipitation, active efflux, and transformation to less toxic species have been used by microorganisms to counteract heavy metal stress (Nies 1999, 2003; Hu et al. 2005). In Cd resistance, three families of efflux transporters are deployed by microor-ganisms. They are the P-type ATPases, which traverse the inner membrane and use ATP energy to pump metal ions from the cytoplasm (Nucifora et al. 1989; Rensing et al. 1997); the CBA (capsule biogenesis assembly) transport-ers, which act as cation–proton antiporters (Nies and Sil-ver 1989; Nies 1995; Hassan et al. 1999); and the cation diffusion facilitator (CDF) transporters, which act as che-miosmotic ion–proton exchanger (Xiong and Jayaswal 1998; Anton et al. 1999; Grass et al. 2001; Nies 2003).
Previous works have deployed culture-based and culture-independent methods to monitor the effects of heavy metal contamination on autochthonous soil microbial community. In most cases, where culture-inde-pendent approach was used, specific resistance genes are amplified via PCR techniques (Rhee et al. 2004; Bhadra et al. 2005; Altimira et al. 2012). Information obtained from such studies cannot be adapted to design effective bioremediation strategies as it does not reflect the true picture of heavy metal resistome in such environments.
The use of shotgun metagenomics allows deep metagen-omic sequencing providing unprecedented insight into the genetic potentials of microbial communities as well as underrepresented populations (Handelsman 2004; Oulas et al. 2015). It also reveals the communal nature of micro-bial existence and the interplay between diverse genes and processes produced and marshalled by members of the microbial community to counteract various environ-mental stressors. This exciting approach have been used to decipher the microbial community structure and func-tion of diverse polluted and pristine soils (Salam et al. 2017, 2018; Feng et al. 2018; Salam et al. 2019).
In recent time, attempts have been made to use next-generation shotgun metagenomics to characterize the microbial community structure and function of heavy metal-inundated soils. However, to the best of our knowledge, none of the reports have used the approach to extensively decipher the specific resistance systems deployed by members of the microbial community to counteract the stress imposed by the studied heavy metal. Here, we report the use of shotgun metagenomics to decipher the effects of Cd contamination on the micro-bial community structure and heavy metal resistome of a tropical agricultural soil.
Materials and methodsSampling site descriptionSoil samples were collected from an agricultural farm in Ilorin, Kwara State, Nigeria. The coordinates of the sam-pling site were latitude 8° 27′ 45.36ʺ N and longitude 4° 32′ 7.08ʺ E. Historically, farming at the sampling site dated back to 10–15 years and crops such as maize, cas-sava, cocoyam, beans and guinea corn were grown. In addition, livestock manures are routinely used to enhance soil nutrients while NIMBUS® Space Spray (5 g/kg soil pyrethrum + 40 g/kg soil piperonyl butoxide) is used on the farm to arrest grain weevil infestation.
Source of heavy metalCadmium chloride (CdCl2), the source of cadmium used in this study was purchased from Sigma Aldrich Corp (St Louis MO, USA).
Sampling, microcosm setup, physicochemical and heavy metal content analysisSoil samples were collected from upper 10–12 cm using a sterile hand trowel after removing the debris from the soil surface. The soil samples, collected via compos-ite sampling were passed through a 2-mm mesh sieve. Sieved soils were made homogenous by thorough mix-ing in a large plastic bag. Sieved soil (1 kg) weighed and placed in an open pan was designated SL4. The second soil microcosm designated SL5 contained 1 kg of sieved
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soil amended with 250 mg CdCl2, respectively. The two setups (in triplicates) were incubated at room tempera-ture for 5 weeks and flooded weekly with 50 ml distilled water to maintain a moisture content of 25%.
The pH of the soil samples was measured using a pH meter (model 3051, Jenway, UK) by dipping the glass electrode in a soil solution slurry that contains a fivefold volume of water containing 1 M KCl. Moisture and total organic matter contents were determined gravimetrically, while total nitrogen content was determined by macro-Kjeldahl digestion method. Potassium content was deter-mined by flame photometry (Flame photometer model PFP-7, Buck Scientific Inc, USA) method while phos-phorus content was determined spectrophotometrically. Heavy metals composition of the soils was determined using atomic absorption spectrophotometer (model Alpha 4, Chem Tech Analytical, UK) following mixed acid digestion and extraction of the soil samples.
Total DNA extraction and shotgun metagenomicsTotal DNA used for metagenomic analysis was extracted directly from the two soil microcosms, SL4 and SL5. To unravel the microbial community structure of the agri-cultural soil prior to Cd amendment, total DNA was extracted from the agricultural soil (SL4) immediately after sampling. For metagenomic evaluation of the effects of cadmium contamination (250 mg kg−1) on the micro-bial community of the agricultural soil, the total DNA was extracted from SL5 microcosm 5 weeks post-Cd amendment. Total DNA were extracted from the sieved soil samples (0.25 g) using ZYMO soil DNA extraction Kit (Model D 6001, Zymo Research, USA) following man-ufacturer’s instructions. The quality and concentration of the extracted total DNA was ascertained using Nan-oDrop spectrophotometer and electrophoresed on a 0.9% (w/v) agarose gel, respectively. Shotgun metagenomics of SL4 and SL5 microcosms was prepared using the Illu-mina Nextera XT sample processing kit and sequenced on a MiSeq. The protocols for total DNA preparation for Illumina shotgun sequencing were as described previ-ously (Salam 2018; Salam and Ishaq 2019).
Processing of fastq raw reads, quality control, assembly and taxonomic classificationProcessing and quality control of fastq raw reads, assem-bly and taxonomic classification were carried out using the analysis tools in EDGE Bioinformatics web server (Li et al. 2017). The pre-processing of the raw Illumina fastq file of the two metagenomes (SL4 and SL5) for quality control check, de novo assembly of the trimmed reads and assembly validation were carried out using FastQ Quality Control Software (FaQCs) (Lo and Chain 2014),
IDBA-UD (Peng et al. 2012), and Bowtie2 (Langmead and Salzberg 2012), respectively.
Read-based and contig-based classifications in the EDGE Bioinformatics web-server were deployed for tax-onomic classification of the SL4 and SL5 metagenomes. Although there are several read-based classification tools (GOTTCHA, Kraken, MetaPhlAN, BWA) in the EDGE, Kraken (Wood and Salzberg 2014) was selected for read-based taxonomic classification of the metagenomes due to the depth and accurateness of its database. Contig-based taxonomic classification is premised on alignment of the SL4 and SL5 contigs to NCBI’s RefSeq database using the BWA-mem aligner. Metagenomic data of SL4 and SL5 have been deposited and made public in EDGE Bioinformatics web server.
Functional annotation of metagenomics readsSequence reads generated from each of the metagen-ome were assembled individually using the make.contig command in the MOTHUR metagenomic analysis suite (Schloss et al. 2009). Gene calling was performed on the SL4 and SL5 sequence reads using MetaGene (Nogu-chi et al. 2006) to predict open reading frames (ORFs). The predicted genes were functionally annotated using the KEGG KofamOALA (Aramaki et al. 2019), which assigns K numbers to the predicted genes by HMMER/HMMSEARCH against KOfam (a customized HMM database of KEGG Orthologs). Other functional anno-tation tools used include the NCBI’s conserved domain database CDSEARCH/cdd v 3.15 (CDD; Marchler-Bauer et al. 2015), PANNZER2 (Protein Annotation with Z-score) designed to predict the functional description (DE) and GO (Gene Ontology) classes (Törönen et al. 2018), and BacMet (Pal et al. 2014), a function-specific bioinformatics resource for detection of antibacterial biocide and metal-resistance genes.
In BacMet, the predicted genes (protein sequences of SL3 and SL4) were presented as query to the Bac-Met database (version 2.0) of predicted resistance genes (using default parameters) for identification of metal-resistance genes in the query sequences. A modified stand-alone version of the BLAST program (NCBI, ver-sion 2.2.2) implemented in the BacMet web server was used for similarity searches against the BacMet sequence databases.
Statistical analysisThe effects of Cd contamination on the soil physico-chemistry and the microbial community structure was statistically analysed using the t test tool in the Analysis ToolPak of Microsoft Excel 2013 software.
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ResultsPhysicochemical properties and heavy metals contentThe physicochemical properties and heavy metal con-tent of the agricultural soil (SL4) and cadmium-contam-inated agricultural soil (SL5) are shown in Table 1. The pH of the soil, which is close to neutral (6.87 ± 0.28) in SL4 became weakly acidic in SL5 (6.60 ± 0.06). The mois-ture content, which is less than 7% (6.75 ± 0.01) in SL4 dropped further to 4% in SL5 (4.32 ± 0.01). All the other physicochemical parameters also showed a declining trend in SL4 (Table 1). Statistical analysis of the physico-chemical parameters of the two metagenomes revealed that the difference is statistically significant (P < 0.05; P = 0.036). In addition, significant traces of heavy met-als were detected in the soil. While the concentrations of lead (0.02 ± 0.002 mg/kg), selenium (0.006 ± 0.001 mg/kg), and Cd (0.15 ± 0.001 mg/kg) detected in the agri-cultural soil are considerably low, high concentrations of zinc, iron, copper, and chromium were detected in the agricultural soil SL4. However, apart from Cd, the con-centrations of the heavy metals substantially decrease in SL5 (Table 1).
General characteristics of the metagenomesIllumina shotgun next-generation sequencing of the total DNA from the two soil microcosms revealed 73,402 and 46,294 sequence reads for SL4 and SL5, respectively. The SL4 and SL5 metagenomes consisted of 21,042,303 and 12,428,339 bp, mean sequence length of 286.67 ± 59.44 and 268.47 ± 86.22 bp, and mean GC contents of
55.08% ± 12.49 and 54.20% ± 10.61, respectively. After trimming, dereplication, and quality control, sequence reads in SL4 and SL5 reduced to 69,514 (94.70%) and 40,658 (87.83%) with 20,902,030 (99.33%) and 12,216,171 (98.29%) bp, mean sequence lengths of 300.69 ± 4.38 and 300.46 ± 7.23 bp, and mean GC contents of 57.49% ± 4.94 and 55.70% ± 4.49, respectively. Other general features of the soil metagenomes are indicated in Table 2.
Taxonomic characterization of the metagenomesTaxonomic characterization of the agricultural soil (SL4) revealed 29 phyla with the preponderance of the phyla Proteobacteria (37.38%), Actinobacteria (35.26%), Bacte-roidetes (13.45%), and Firmicutes (9.47%). In cadmium-contaminated SL5 microcosm, 25 phyla were recovered with the predominance of Proteobacteria (50.50%), Actinobacteria (17.17%), Firmicutes (16.42%), and Bac-teroidetes (10.70%). In SL5, 68.05% of members of Actino-bacteria were lost while there is massive reduction in the population of members of the phyla Candidatus Saccha-ribacteria, Chloroflexi, and Nitrospirae. In contrast, there is a massive upsurge in the population of members of the phyla Euryarchaeota (an archaeal phylum), Chlamy-diae, Spirochaetes, and Deferribacteres in SL5 microcosm (Fig. 1).
In class delineation, 42 and 38 classes were retrieved from SL4 and SL5 metagenomes with the dominance of Actinobacteria (35.02%), Alphaproteobacteria (12.31%), Betaproteobacteria (10.93%), and Gammaproteobacteria (8.99%) in SL4 and Alphaproteobacteria (22.28%), Act-inobacteria (18.36%), Gammaproteobacteria (15.54%), and Bacilli (11.34%) in SL5. In SL5, Massive decline was observed in the population of members of the classes Actinobacteria, Rubrobacteridae, Negativicutes, Acidimi-crobidae and Nitrospira while there is a huge upscale in the population of members of the classes Methanomicro-bia, Chlamydiia and Spirochaetia (Fig. 2).
In order classification where 94 and 78 orders were recovered in SL4 and SL5 metagenomes, there is pre-ponderance of Actinomycetales (25.81%), Burkholderiales (8.01%) and Bacteroidales (7.19%) in SL4 while Actinomy-cetales (17.18%), Rhizobiales (8.51%) and Burkholderiales (8.35%) dominates in SL5 (Additional file 1: Figure S1). In family delineation, 158 and 126 families were retrieved from SL4 and SL5 metagenomes. Caulobacteraceae (8.70%), Alcaligenaceae (7.10%), and Sphingobacteriaceae (6.12%) dominates in SL4 while Enterobacteriaceae (7.94%), Alcaligenaceae (7.45%) and Methyobacteriaceae (6.61%) were preponderant in SL5 (Additional file 1: Fig-ure S2).
In genus delineation, 270 and 205 genera were recov-ered in SL4 and SL5 metagenomes. The genera with the highest representation in SL4 include Prevotella (6.93%),
Table 1 Physicochemistry and heavy metals content of agricultural soil (SL4) and cadmium-contaminated agricultural soil (SL5)
ND not detected
SL4 SL5
Physicochemical parameters
pH 6.87 ± 0.28 6.60 ± 0.06
Moisture (%) 6.75 ± 0.01 4.32 ± 0.01
Total organic matter (%) 73.21 ± 0.21 64.79 ± 1.90
Total nitrogen (%) 53.48 ± 0.69 36.08 ± 2.13
Phosphorus (mg/kg) 29.41 ± 0.82 22.15 ± 1.39
Potassium (mg/kg) 17.880 ± 0.002 12.160 ± 0.003
Heavy metals content
Lead (mg/kg) 0.020 ± 0.001 ND
Chromium (mg/kg) 5.910 ± 0.003 3.580 ± 0.002
Cadmium (mg/kg) 0.150 ± 0.001 62.800 ± 0.002
Zinc (mg/kg) 14.080 ± 0.003 7.760 ± 0.004
Iron (mg/kg) 13.940 ± 0.003 7.230 ± 0.005
Copper (mg/kg) 12.580 ± 0.001 8.220 ± 0.004
Selenium (mg/kg) 0.006 ± 0.001 ND
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Conexibacter (5.91%), Brevundimonas (5.02%), and Bifi-dobacterium (4.46%). In Cd-contaminated SL5 metagen-ome, the predominant genera include Methylobacterium (9.14%), Streptococcus (4.29%), Paenibacillus (3.74%), and Prevotella (3.67%). Massive decline was observed in the population of Caulobacter, Acinetobacter, Megas-phaera, Conexibacter, Burkholderia, Prevotella and sev-eral others in SL5. In contrast, massive enrichment in the population of Methylobacterium, Paenibacillus, Modesto-bacter, Methanosaeta, Flexistipes, Desulfomicrobium, Arcobacter and few others were observed in the Cd-con-taminated SL5 metagenome (Fig. 3). Statistically signifi-cant (P < 0.05; P = 0.0016) difference in genus delineations was observed between SL4 and SL5 metagenome.
In species delineation, 310 and 230 species were retrieved from SL4 and SL5 metagenomes. The prepon-derant species in SL4 metagenome are Conexibacter woesei (8.93%), Brevundimonas subvibrioides (7.58%),
Sphingobacterium sp. 21 (6.47%), and Pedobacter sal-tans (4.59%). In Cd-amended SL5 metagenome, the dominant species are Methylobacterium radiotolerans (12.80%), Sphingobacterium sp. 21 (4.86%), Modesto-bacter marinus (4.60%) and Sphingomonas wittichii (3.60%), respectively. Population of C. woesei, Acine-tobacter baumannii, Megasphaera elsdenii, Acidimi-crobium ferrooxidans and several others massively nosedived in SL5 while species such as M. radiotoler-ans, M. marinus, Methanosaeta concilii, Flexistipes sinusarabici and many others were massively enriched (Fig. 4). Statistically significant (P < 0.05; P = 0.01) dif-ference in species delineations was observed between SL4 and SL5 metagenome.
Contig-based classification of the metagenomes (SL4 and SL5) conducted by aligning the SL4 and SL5 con-tigs to NCBI’s RefSeq database using the BWA-mem aligner is indicated in Additional file 1: Figs. S3 to S8.
Table 2 General characteristics of SL4 and SL5 metagenomes
SL4 SL5
1. Pre-processing
a. Raw reads
Reads 73,402 46,294
Total bases (bp) 21,042,303 12,428,339
Mean read length (bp) 286.67 ± 59.44 268.47 ± 86.22
Mean GC content (%) 55.08 ± 12.49 54.20 ± 10.61
b. Quality trimming
Trimmed reads
Reads 69,514 (94.70%) 40,658 (87.83%)
Total bases (bp) 20,902,030 (99.33%) 12,216,171 (98.29%)
Mean read length (bp) 300.69 ± 4.38 300.46 ± 7.23
Mean GC content (%) 57.49 ± 4.94 55.70 ± 4.49
Paired reads 69,494 (99.97%) 40,604 (99.87%)
Paired total bases 20,896,965 (99.98%) 12,200,818 (99.87%)
Unpaired reads 20 (0.03%) 54 (0.13%)
Unpaired total bases 5065 (0.02%) 15,353 (0.13%)
2. Assembly and annotation
a. De novo assembly by idba_ud
Number of contigs 117 76
N50 (bp) 420 424
Max contig size (bp) 458 462
Min contig size (bp) 255 270
Total assembly size (bp) 47,020 30,607
b. Assembly validation by read mapping
Number of mapped reads 40,629 23,795
% of total reads 58.45% 58.52%
Number of unmapped reads 28,885 16,863
% of total reads 41.55% 41.48%
Average fold coverage 214.34 X 204.18 X
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Functional annotation of the metagenomesFunctional characterization of the metagenomes revealed significant differences. In SL4 metagenome, putative genes for carbohydrate metabolism (fructose-6-phos-phate aldolase 2; arabinoxylan arabinofuranohydrolase; 2-dehydro-3-deoxygluconokinase/2-dehydro-3-deoxyg-alactonokinase), nitrogen metabolism (CFP/FNR family transcriptional regulator, nitrogen oxide reductase regu-lator), sulphur metabolism (sulphite oxidase), methane metabolism (Ni-sirohydrochlorin a,c-diamide reduc-tive cyclase, play a key role in methanogenesis and anaerobic methane oxidation), and autotrophic CO2 assimilation (energy-converting hydrogenase B) were detected. Other putative genes detected include genes responsible for biosynthesis of bioactive compounds and antibiotics (fumagillin biosynthesis methyltrans-ferase, nocardicin N-oxygenase, trigonelline monooxy-genase, oxygenase component), xenobiotic degradation (cyanamide hydratase, cytochrome P450 RapN, poly(3-hydroxyoctanoate) depolymerase), and stress response
(diacylglycerol diphosphate phosphatase/phosphatidate phosphatase).
In SL5 metagenome, putative genes and enzymes were detected for carbohydrate metabolism (2,3-bisphospho-glycerate-independent phosphoglycerate mutase, UDP-glucose-4 epimerase), amino acid metabolism (cysteine desulfurase, tryptophan synthase beta chain), xenobiotic degradation (carboxylesterase 1, alkene monooxygenase, effector subunit), polyketide synthases (nogalonic acid methyl ester cyclase/aklanonic acid methyl ester cyclase), and vitamin B12, porphyrin and chlorophyll metabolism (adenosylcobinamide-phosphate synthase).
Functional annotation of the predicted genes in SL4 and SL5 metagenomes for heavy metals resistance genes using the BacMet database revealed interesting find-ings. Diverse protein families responsible for transport, uptake and efflux of heavy metals were detected in the two metagenomes (Tables 3, 4). In agricultural soil SL4 metagenome, putative genes for transport, uptake, and efflux of copper (copA, copB, copC, copP, multicopper
0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000
SL4
SL5
SEQUENCE READS
PHYL
UM
Chlamydiae Euryarchaeota Verrucomicrobia
Cloacimonetes Fibrobacteres Aquificae
Synergistetes Spirochaetes Fusobacteria
Caldiserica Deinococcus-Thermus Thermodesulfobacteria
Chlorobi Elusimicrobia Chrysiogenetes
Ignavibacteriae Armatimonadetes Deferribacteres
Nitrospirae Thermotogae Tenericutes
Chloroflexi Candidatus Saccharibacteria Cyanobacteria
Acidobacteria Gemmatimonadetes Planctomycetes
Firmicutes Bacteroidetes Actinobacteria
L4
L5
ChCC lamydiaii e Euryayy rchaeota VerruVV comicrobia
ClCC oacimonetes Fibrobacteres Aquificae
Synergisii tetes Spirochaetes Fusuu obacteria
CaCC ldisii erica Deinococcusuu -ThTT ermus ThTT ermodesulfoff bacteria
ChCC lorobi Elusuu imicrobia ChCC rysyy iogenetes
IgII navibacteriaii e Armatimonadetes Deferribacteres
Nitrospirae ThTT ermotogae Tenericutes
ChCC loroflexi CaCC ndidadd tusuu Saccharibacteria CyCC ayy nobacteria
Acidobacteria Gemmatimonadetes Planctomycetes
Firmicutes Bacteroidetes Actinobacteria
Proteobacteria
Fig. 1 Comparative taxonomic profile of SL4 and SL5 metagenomes at phylum level, computed by EDGE Bioinformatics. Unclassified reads were not used for the analysis. All the phyla detected in SL4 and SL5 metagenomes were used
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oxidase type 2 and 3; CueO, cutC, cutE, etc.), chro-mium, cadmium, nickel, cobalt (chrA, chrB, nikA, nikB, nikR, cadmium-translocating P-type ATPase, nickel–cadmium–cobalt resistance protein nccC, etc.) were detected. Other putative genes detected include resist-ance genes for iron, zinc, magnesium, manganese (furA, BasS/PmrB, zinc/iron ZIP family permease, mgtB; mag-nesium-translocating P-type ATPase; NRAMP family Mn2+/Fe2+ transporter, etc.) and mercury, silver, molyb-denum, lead, arsenic, tungsten, tellurium and antimony (merA, merB, merR, merH, merP, pbrA, modA, modB, modC, TrgB, TehA, WtpA, arsenite oxidase, arsB, arsC, arsM, etc.) (Table 3).
In Cd-contaminated SL5 metagenome, putative genes were detected for cadmium, cobalt, nickel, zinc (heavy metal-translocating P-type ATPase, czcA, czcD, czrA,
czrB, zraR, zraP, znuA, cobalt–zinc–cadmium resist-ance protein, nikA, nikR, nikD, nikE, etc.), and copper, magnesium, and silver (copA, copB, copC, magnesium-transporting ATPase, corA, copper/silver efflux P-type ATPase, etc.). Also detected are putative resistance genes for iron, lead, chromium, manganese, tellurium, selenium (fpvA2 gene, fur, fbpC, ferroxidase, ctpC gene, tehB, chrA, chrC, trgB, etc.), and mercury, arsenic, molybdenum and tungsten (merA, merR, merT, merB1, arsB, arsC, arsH, arsenite oxidase, arsM, modB, wtpA, etc.) (Table 4).
It was observed that putative genes, responsible for cadmium homeostasis, transport, efflux and detoxifica-tion such as czcA, czcD, czrA, czrB, manganese trans-port protein, and manganese/iron superoxide dismutase (MnSOD, sodA; FeSOD, sodB) which were detected in Cd-amended SL5 metagenome were conspicuously
0 2000 4000 6000 8000 10000 12000 14000 16000 18000
SL4
SL5
SEQUENCE READS
CLA
SSErysipelotrichia Chlamydiia Methanomicrobia Fibrobacteria
Aquificae Verrucomicrobiae Dehalococcoidia Spirochaetia
Fusobacteriia Acidobacteriia Synergistia Caldisericia
Deinococci Thermodesulfobacteria Elusimicrobia Chlorobia
Chrysiogenetes Ignavibacteria Chthonomonadetes Chloroflexi
Anaerolineae Epsilonproteobacteria Deferribacteres Nitrospira
Thermotogae Cytophagia Thermomicrobia Mollicutes
Solibacteres Gemmatimonadetes Coriobacteridae Flavobacteriia
Acidimicrobidae Planctomycetia Negativicutes Clostridia
Deltaproteobacteria Rubrobacteridae Bacilli Sphingobacteriia
Bacteroidia Gammaproteobacteria Betaproteobacteria Alphaproteobacteria
SL4
SL5
Erysyy ipi elotrichia ChCC lamydiia Methanomicrobia Fibrobacteria
Aquificae VerruVV comicrobiaii e Dehalococcoidia Spirochaetia
Fusuu obacteriia Acidobacteriia Synergisii tia CaCC ldisii ericia
Deinococci ThTT ermodesulfoff bacteria Elusuu imicrobia ChCC lorobia
ChCC rysyy iogenetes IgII navibacteria ChCC thonomonadetes ChCC loroflexi
Anaerolineae Epsilonproteobacteria Deferribacteres Nitrospira
ThTT ermotogae CyCC tophagiaa ThTT ermomicrobia Mollicutes
Solibacteres Gemmatimonadetes CoCC riobacteridadd e FlFF avobacteriia
Acidimicrobidadd e Planctomycetia Negativicutes ClCC ostridia
Deltaproteobacteria Rubrobacteridadd e Bacilli Sphingn obacteriia
Bacteroidia Gammaproteobacteria Betaproteobacteria Alpl haproteobacteria
Actinobacteria
Fig. 2 Comparative taxonomic profile of SL4 and SL5 metagenomes at class level, computed by EDGE Bioinformatics. Unclassified reads were not used for the analysis. All the classes detected in SL4 and SL5 metagenomes were used
Page 8 of 19Salam et al. Bioresour. Bioprocess. (2020) 7:25
absent in SL4 metagenome. It was also observed based on functional annotation of protein sequences in Cd-amended SL5 metagenome using PANNZER2 that one thousand four hundred and forty (1440) of the sequences were annotated for alkyl hydroperoxide reductase (AhpC), an organic hydroperoxide detoxification enzyme.
However, the AhpC gene was not detected in the protein sequences of SL4 metagenome.
0 200 400 600 800 1000 1200 1400 1600 1800 2000
SL4
SL5
SEQUENCE READS
GEN
US
Leptothrix Capnocytophaga Proteus TreponemaSpirochaeta Renibacterium Klebsiella LactococcusFrateuria Acetobacter Delftia ArcobacterDesulfomicrobium Flexistipes Methanosaeta ModestobacterAcholeplasma Syntrophomonas Thermobacillus AchromobacterRiemerella Candidatus Symbiobacter Photorhabdus AcidobacteriumCatenulispora Wigglesworthia Sphingobium SalmonellaSulfobacillus Methylobacterium Paenibacillus LeuconostocChthonomonas Alkalilimnicola Rhodobacter LeifsoniaMethylocystis Aeromonas Spiroplasma BeutenbergiaVerrucosispora Actinosynnema Novosphingobium PsychrobacterThauera Rhodococcus Polaromonas BrevibacillusAnaerolinea Vibrio Nocardia GeobacillusCellulomonas Tannerella Rhodanobacter ParabacteroidesGordonia Helicobacter Sphaerobacter FrankiaThermotoga Candidatus Chloracidobacterium Clostridium SpirosomaThermomicrobium Actinoplanes Faecalibacterium RamlibacterCellvibrio Ureaplasma Tropheryma AnaeromyxobacterAnaplasma Acidovorax Singulisphaera ComamonasBlastococcus Chitinophaga Kytococcus BacteroidesMyxococcus Atopobium Cupriavidus MicrolunatusRhodospirillum Niastella Heliobacterium MicrococcusDesulfobacca Amycolatopsis Salinispora AnaerococcusSanguibacter Streptomyces Streptosporangium ParacoccusThermobispora Propionibacterium Symbiobacterium XylanimonasChelativorans Mesorhizobium Candidatus Liberibacter NitrosococcusAzospirillum Tepidanaerobacter Candidatus Koribacter HyphomicrobiumDesulfovibrio Sphingomonas Sorangium AgrobacteriumSaccharothrix Ilumatobacter Geodermatophilus RubrobacterKocuria Candidatus Solibacter Oscillibacter HalobacteroidesTaylorella Bacillus Kribbella CoriobacteriumMicromonospora Streptococcus Nakamurella AcidaminococcusGeobacter Xanthomonas Intrasporangium LactobacillusCandidatus Azobacteroides Gemmatimonas Acidimicrobium DesulfotomaculumBurkholderia Flavobacterium Staphylococcus MycobacteriumBrachybacterium Corynebacterium Pseudomonas StenotrophomonasMegasphaera Nocardioides Acinetobacter BordetellaPedobacter Caulobacter Sphingobacterium BifidobacteriumBrevundimonas Conexibacter
SL4
SL5
GEN
US
Leptothrixii CaCC pnocytophaga a Proteusuu TreponemaSpirochaeta Renibacterium KlKK ebsiella LactococcusuuFrFF ateuria Acetobacter Delftff iaii ArcobacterDesulfomicrobium Flexisii tipes Methanosaeta ModestobacterAcholeplasma Syntrophomonas ThTT ermobacillusuu AchromobacterRiemerella CaCC ndidadd tusuu Symyy biobacter Photorhabdusuu AcidobacteriumCaCC tenulisii pora WiWW gii ggg leswow rthiaii Sphingn obium SalmonellaSulfobacillusuu Methylobacterium Paenibacillusuu LeuconostocChCC thonomonas Alkalilimnicola Rhodobacter Leifsff oniaiiMethylocysyy tisii Aeromonas Spiroplasma Beutenbergr iaVeVV rrucosisii pora Actinosynnyy ema Novovv sphingn obium PsPP ychrobacterThaTT uera Rhodococcusuu Polaromonas BrevibacillusuuAnaerolinea ViVV brio Nocardiaii GeobacillusuuCeCC llulomonas Tannerella Rhodadd nobacter ParabacteroidesGordoniaii HeHH licobacter Sphaerobacter FrFF ankiaThTT ermotoga CaCC ndidadd tusuu ChCC loracidodd bacterium ClCC ostridium SpirosomaThTT ermomicrobium Actinoplanes Faecalibacterium RamlibacterCeCC llvibrio Ureaplasma Tropheryma AnaeromyxobacterAnaplasma Acidovovv rax Singn ulisii phaera ComamonasBlastococcusuu ChCC itinophaga a KyKK tococcusuu Bacteroidedd sMyxococcusuu Atopobium Cupu riaii vidusuu MicrolunatusuuRhodospirillum Niaii stella HeH liobacterium MicrococcusuuDesulfobacca Amycolatopsisii Salinisii pora AnaerococcusuuSangn uibacter Streptomyces Streptosporangn ium ParacoccusuuThTT ermobisii pora Propionibacterium Symbiobacterium XylXX animonasChCC elativovv rans Mesorhizobium CaCC ndidadd tusuu Liberibacter NitrosococcusuuAzospirillum TeTT pidadd naerobacter CaCC ndidadd tusuu KoKK ribacter Hypyy homicrobiumDesulfovibrio Sphingn omonas Sorangn ium AgrobacteriumSaccharothrixii Ilumatobacter Geodedd rmatophilusuu RubrobacterKoKK curiaii CaCC ndidadd tusuu Solibacter Oscillibacter HalHH obacteroidesTaylorella Bacillusuu KrKK ibbella CoriobacteriumMicromonospora Streptococcusuu NaNN kamurella Acidadd minococcusuuGeobacter XaXX nthomonas InII trasporangn ium LactobacillusuuCaCC ndidadd tusuu Azobacteroides Gemmatimonas Acidimicrobium DesulfotomaculumBurkholderiaii Flavovv bacterium Staphylococcusuu MycobacteriumBrachybacterium Corynyy ebacterium PsePP udomdd onas StenotrophomonasMege asphaera Nocardioides Acinetobacter BordetellaPePP dobacter CaCC ulobacter Sphingn obacterium BifidobacteriumBrevundimonas Conexibacter Prevotella
Fig. 3 Comparative taxonomic profile of SL4 and SL5 metagenomes at genus level, computed by EDGE Bioinformatics. Unclassified reads were not used. Only genera with ≥ 10 sequence reads were used for the analysis
Page 9 of 19Salam et al. Bioresour. Bioprocess. (2020) 7:25
DiscussionPoint and non-point release of heavy metals and metal-loids into soil environments via atmospheric deposi-tion and diverse agricultural activities have negatively impacted soil ecological balance, alter soil physicochem-istry and biogeochemistry, reduce soil microbial diver-sity and pose serious health risk to animals and humans (Feng et al. 2018; Rai et al. 2019; Salam et al. 2019). In
this study, all the physicochemical parameters consider-ably reduce in Cd-amended SL5 microcosm, though not as profound as those reported in our previous study on mercury (Salam et al. 2019). This may be attributed to Cd contamination. Previous reports have indicated that increase in soil pH increases Cd sorption to soil organic matter (Gray et al. 1998, 1999). The decrease in soil pH observed in SL5 microcosm may thus be indicative of
0 200 400 600 800 1000 1200 1400 1600
SL4
SL5
SEQUENCE READS
SPEC
IES
Staphylococcus carnosus Streptococcus sanguinis Leptothrix cholodniiFlavobacteriaceae bacterium 3519-10 Capnocytophaga ochracea Bacillus infantisProteus mirabilis Treponema brennaborense Streptococcus thermophilusSpirochaeta smaragdinae Klebsiella oxytoca Lactococcus lactisClostridium pasteurianum Renibacterium salmoninarum Clostridium cellulovoransFrateuria aurantia Arcobacter butzleri Acetobacter pasteurianusDesulfomicrobium baculatum Flexistipes sinusarabici Methanosaeta conciliiModestobacter marinus Riemerella anatipestifer Bacteroides helcogenesThermobacillus composti Paenibacillus sp. Y412MC10 Achromobacter xylosoxidansAcholeplasma laidlawii Syntrophomonas wolfei Bacillus coagulansPhotorhabdus asymbiotica Acidobacterium capsulatum Candidatus Symbiobacter mobilisDesulfotomaculum acetoxidans Wigglesworthia glossinidia Pseudomonas stutzeriCatenulispora acidiphila Salmonella enterica Sulfobacillus acidophilusMethylobacterium radiotolerans Bordetella petrii Alkalilimnicola ehrlichiiChthonomonas calidirosea Azoarcus sp. BH72 Streptococcus suisRhodobacter capsulatus Leifsonia xyli Methylocystis sp. SC2[Cellvibrio] gilvus Prevotella ruminicola Actinoplanes missouriensisSpiroplasma diminutum Rhodococcus jostii Bacillus sp. 1NLA3EVerrucosispora maris Beutenbergia cavernae Pseudomonas putidaActinosynnema mirum Thauera sp. MZ1T Xanthomonas albilineansAnaerolinea thermophila Brevibacillus brevis Bacteroides fragilisVibrio cholerae Geobacter lovleyi Corynebacterium diphtheriaeTannerella forsythia Parabacteroides distasonis Rhodanobacter denitrificansPseudomonas fluorescens Sphaerobacter thermophilus Candidatus Chloracidobacterium thermophilumAmycolatopsis orientalis Thermomicrobium roseum Spirosoma lingualeCupriavidus necator Sphingomonas wittichii Faecalibacterium prausnitziiRamlibacter tataouinensis Azospirillum lipoferum Agrobacterium vitisCellvibrio japonicus Tropheryma whipplei Acidovorax sp. KKS102Sphingomonas sp. MM-1 [Clostridium] sticklandii Singulisphaera acidiphilaFlavobacterium branchiophilum Corynebacterium glutamicum Comamonas testosteroniBlastococcus saxobsidens Chitinophaga pinensis Kytococcus sedentariusAtopobium parvulum Rhodospirillum centenum Microlunatus phosphovorusCorynebacterium urealyticum Niastella koreensis Micrococcus luteusHeliobacterium modesticaldum Desulfobacca acetoxidans Agrobacterium sp. H13-3Anaerococcus prevotii Propionibacterium acnes Taylorella asinigenitalisSanguibacter keddieii Streptosporangium roseum Thermobispora bisporaSymbiobacterium thermophilum Flavobacterium columnare Xylanimonas cellulosilyticaChelativorans sp. BNC1 Desulfovibrio magneticus Candidatus Koribacter versatilisTepidanaerobacter acetatoxydans Pseudomonas aeruginosa Sorangium cellulosumSaccharothrix espanaensis Ilumatobacter coccineus Geodermatophilus obscurusRubrobacter xylanophilus Bacillus cereus Kocuria rhizophilaCandidatus Solibacter usitatus Oscillibacter valericigenes Burkholderia sp. YI23Halobacteroides halobius Lactobacillus ruminis Streptococcus agalactiaeKribbella flavida Bifidobacterium longum Coriobacterium glomeransNakamurella multipartita Geobacter sulfurreducens Prevotella melaninogenicaIntrasporangium calvum Candidatus Azobacteroides pseudotrichonymphae Gemmatimonas aurantiacaAcidimicrobium ferrooxidans Brachybacterium faecium Stenotrophomonas maltophiliaMegasphaera elsdenii Nocardioides sp. JS614 Acinetobacter baumanniiPedobacter saltans Sphingobacterium sp. 21 Brevundimonas subvibrioidesConexibacter woesei
Fig. 4 Comparative taxonomic profile of SL4 and SL5 metagenomes at species level, computed by EDGE Bioinformatics. Unclassified reads were not used. Only species with ≥ 10 sequence reads were used for the analysis
Page 10 of 19Salam et al. Bioresour. Bioprocess. (2020) 7:25
Tabl
e 3
Pred
icte
d he
avy
met
als
resi
stan
ce g
enes
det
ecte
d in
SL4
met
agen
ome
and
thei
r tax
onom
ic a
ffilia
tion
s
Hea
vy m
etal
sEn
zym
e/ge
nes
Taxo
nom
ic a
ffilia
tion
Copp
erCo
pper
resi
stan
ce p
rote
in C
opC
; mul
ticop
per o
xida
se ty
pe 3
; cop
per e
xpor
ting
ATPa
se; p
utat
ive
mul
ticop
per o
xida
se (l
acca
se-li
ke);
copp
er re
sist
ance
pro
tein
B
prec
urso
r; co
pper
hom
eost
asis
pro
tein
, cut
C; c
oppe
r res
ista
nce
prot
ein
A,
copA
; tw
in-a
rgin
ine
tran
sloc
atio
n pa
thw
ay s
igna
l; pu
tativ
e co
pper
bin
ding
pr
otei
n; c
oppe
r-tr
ansl
ocat
ing
P-ty
pe A
TPas
e, c
opB;
mul
ticop
per o
xida
se ty
pe
2; a
polip
opro
tein
N-a
cyltr
ansf
eras
e; tw
o-co
mpo
nent
hea
vy m
etal
tran
-sc
riptio
nal r
egul
ator
; apo
lipop
rote
in N
-acy
ltran
sfer
ase/
copp
er h
omeo
stas
is
prot
ein,
cut
E; h
eavy
-met
al tr
ansp
ortin
g P-
type
ATP
ase;
P-A
TPas
e su
perf
amily
P-
type
ATP
ase
copp
er tr
ansp
orte
r; pe
nici
llina
se re
pres
sor/
tran
scrip
tion
regu
la-
tor,
copY
/tcr
Y; tw
o-co
mpo
nent
sen
sor,
copS
; lip
opro
tein
invo
lved
with
cop
per
hom
eost
asis
and
adh
esio
n, c
utF;
blu
e co
pper
oxi
dase
Cue
O; c
oppe
r tol
eran
ce
prot
ein;
hea
vy m
etal
tran
spor
t/de
toxi
ficat
ion
prot
ein,
cop
P; p
utat
ive
lacc
ase
Fran
kia
sp. C
cI3;
Shi
gella
dys
ente
riae
1012
; Hal
oalk
alic
occu
s jeo
tgal
i B3;
Intr
aspo
-ra
ngiu
m c
alvu
m D
SM 4
3,04
3; M
etha
nosa
eta
haru
ndin
acea
6A
c; R
hodo
cocc
us
pyrid
iniv
oran
s AK3
7; H
alor
ubru
m la
cusp
rofu
ndi A
TCC
492
39; A
zosp
irillu
m b
rasi-
lens
e Sp
245;
Geo
baci
llus t
herm
oden
itrifi
cans
NG
80-2
; Pse
udom
onas
aer
ugin
osa
2192
; Citr
obac
ter r
oden
tium
ICC
168;
Citr
obac
ter k
oser
i ATC
C B
AA
-895
; Ace
toba
cter
po
mor
um D
M00
1; K
ytoc
occu
s sed
enta
rius D
SM 2
0547
; Azo
rhiz
obiu
m c
aulin
odan
s O
RS 5
71; N
itros
omon
as e
utro
pha
C91
; Cor
yneb
acte
rium
am
mon
iage
nes D
SM
2030
6; S
alm
onel
la e
nter
ica
subs
p. e
nter
ica
sero
var W
elte
vred
en s
tr. H
I_N
05-
537;
Oce
anic
ola
sp. S
124;
Pol
ymor
phum
gilv
um S
L-00
3B-2
6A1;
Xan
thob
acte
r au
totr
ophi
cus P
y2; B
rady
rhiz
obiu
m s
p. O
RS 3
75; D
elfti
a ac
idov
oran
s SPH
-1;
Achr
omob
acte
r sp.
AO
22; V
ibrio
sp.
RC
586;
Oxa
loba
cter
form
igen
es O
XCC
13;
Myc
obac
teriu
m p
aras
crof
ulac
eum
ATC
C B
AA
-614
; Lac
toba
cillu
s pen
tosu
s MP-
10;
Lact
obac
illus
pen
tosu
s IG
1; L
acto
baci
llus p
lant
arum
JDM
1; P
seud
omon
as
aeru
gino
sa P
A01
; Pse
udom
onas
aer
ugin
osa
M18
; Pec
toba
cter
ium
car
otov
orum
su
bsp.
bra
silie
nsis
PBR
1692
; Rah
nella
sp.
Y96
02; P
rovi
denc
ia ru
stig
iani
i DSM
454
1;
Rhod
obac
tera
les
bact
eriu
m Y
4I; T
herm
omon
ospo
ra c
urva
ta D
SM 4
3183
; Ver
-ru
com
icro
biac
eae
bact
eriu
m C
NC
16
Chr
omiu
m, n
icke
l, co
balt
NA
DPH
-dep
ende
nt F
MN
redu
ctas
e, tr
ansc
riptio
nal r
egul
ator
Nik
R, C
opG
fam
ily
(Ni);
inte
gral
mem
bran
e se
nsor
sig
nal t
rans
duct
ion
hist
idin
e ki
nase
nrs
S; n
icke
l A
BC tr
ansp
orte
r, ni
ckel
/met
allo
phor
e pe
ripla
smic
bin
ding
pro
tein
; chr
omat
e re
sist
ance
pro
tein
, chr
B; b
indi
ng-p
rote
in-d
epen
dent
tran
spor
t sys
tem
inne
r m
embr
ane;
nic
kel A
BC tr
ansp
orte
r, pe
ripla
smic
nic
kel-b
indi
ng p
rote
in, n
ikA
; iro
n-di
citr
ate
tran
spor
ter s
ubun
it, m
embr
ane
com
pone
nt o
f ABC
sup
erfa
mily
(N
i/Co)
; chr
omat
e tr
ansp
orte
r, ch
rA; m
ajor
faci
litat
or s
uper
fam
ily p
rote
in (N
i/Co
); A
rsR
fam
ily tr
ansc
riptio
nal r
egul
ator
(Co/
Ni);
pep
tide/
nick
el tr
ansp
ort
syst
em s
ubst
rate
-bin
ding
pro
tein
; reg
ulat
ory
prot
ein,
chr
B1; c
atio
n di
ffusi
on
faci
litat
or fa
mily
tran
spor
ter (
Ni/C
o); p
erm
ease
s of
the
maj
or fa
cilit
ator
sup
er-
fam
ily (N
i/Co)
; bin
ding
-pro
tein
-dep
ende
nt tr
ansp
ort s
yste
m in
ner m
embr
ane
prot
ein,
nik
B; c
adm
ium
-tra
nslo
catin
g P-
type
ATP
ase
(Co/
Ni)
Baci
llus c
oagu
lans
2-6
; Met
hano
cocc
us v
olta
e A
3; A
cary
ochl
oris
sp. C
CM
EE 5
410;
Pe
pton
iphi
lus s
p. o
ral t
axon
375
str.
F04
36; B
urkh
olde
ria m
ultiv
oran
s CG
D1;
Bu
rkho
lder
ia m
ultiv
oran
s CG
D2M
; Can
dida
tus D
esul
foru
dis a
udax
viat
or M
P104
C;
Syne
rgist
etes
bac
teriu
m S
GP1
; Esc
heric
hia
coli
S88;
Lep
toth
rix c
holo
dnii
SP-6
; Cel
-lu
lom
onas
fim
i ATC
C 4
84; J
ones
ia d
enitr
ifica
ns D
SM 2
0603
; Azo
spiri
llum
sp.
B51
0;
Cupr
iavi
dus
met
allid
uran
s C
H34
(pla
smid
); M
ethy
loba
cter
ium
nod
ulan
s ORS
20
60; M
ethy
loba
cter
ium
ext
orqu
ens D
M4;
Sin
orhi
zobi
um m
elilo
ti CC
NW
SX00
20;
Cand
idat
us D
esul
foru
dis a
udax
viat
or M
P104
C; S
trep
tom
yces
gris
eofla
vus T
u400
0
Mer
cury
, silv
er, c
adm
ium
, lea
dCo
pper
-tra
nspo
rtin
g AT
Pase
RA
N1,
mer
P; m
ercu
ric io
n re
duct
ase,
mer
A; m
ercu
-ric
redu
ctas
e m
erB1
; rig
ht o
rigin
-bin
ding
pro
tein
robA
(Ag/
Hg/
Cd);
mer
curic
tr
ansp
ort p
rote
in p
erip
lasm
ic p
rote
in, m
erP;
Pb-
efflux
ATP
ase
pbrA
(Pb)
; or
gano
mer
curia
l lya
se, m
erB;
Mer
R fa
mily
tran
scrip
tiona
l reg
ulat
or; d
isul
fide
bond
form
atio
n pr
otei
n B
(Cd/
Hg)
; Pb/
Cd/Z
n/H
g tr
ansp
ortin
g AT
Pase
; sen
sor
prot
ein
ZraS
(Pb,
Zn)
; mer
curic
tran
spor
ter,
mer
H
Vert
icill
ium
dah
liae
VdLs
. 17;
Ver
ticill
ium
alb
o-at
rum
VaM
s. 10
2; P
seud
omon
as
fluor
esce
ns; X
anth
obac
ter a
utot
roph
icus
Py2
; Yer
sinia
ruck
eri A
TCC
294
73; D
elfti
a ac
idov
oran
s SPH
-1; R
alst
onia
met
allid
uran
s CH
34 (p
lasm
id);
Jant
hino
bact
eriu
m
sp. M
arse
ille
(pla
smid
); Ps
eudo
mon
as st
utze
ri (p
lasm
id);
Hal
iang
ium
och
race
um
DSM
143
65; V
ibrio
car
ibbe
nthi
cus A
TCC
BA
A-2
122;
Tol
umon
as a
uens
is D
SM 9
187;
M
esor
hizo
bium
am
orph
ae C
CN
WG
S012
3; C
itrob
acte
r you
ngae
ATC
C 2
9220
; M
ycob
acte
rium
sp.
Zinc
, man
gane
se, c
adm
ium
Zinc
resi
stan
ce p
rote
in, z
raP;
Fis
fam
ily tr
ansc
riptio
nal r
egul
ator
; per
ipla
smic
sol
-ut
e-bi
ndin
g pr
otei
n (M
n/Cd
); RN
D fa
mily
effl
ux tr
ansp
orte
r, M
FP s
ubun
it (Z
n);
DSB
A O
xido
redu
ctas
e (C
d); m
embr
ane
fusi
on p
rote
in (M
FP-R
ND
) hea
vy m
etal
ca
tion
tric
ompo
nent
effl
ux H
mxB
(Zn)
; cat
ion
tran
spor
ting
ATPa
se, P
-typ
e;
high
-affi
nity
zin
c tr
ansp
orte
r per
ipla
smic
pro
tein
; DSB
A g
ene
prod
uct (
Cd/Z
n/H
g); c
adm
ium
-tra
nslo
catin
g P-
type
ATP
ase
(Zn)
; dith
iol-d
isul
fide
isom
eras
e (C
d); h
eavy
met
al-t
rans
loca
ting
P-ty
pe A
TPas
e (Z
n); z
inc/
man
gane
se/ir
on A
BC
tran
spor
ter,
perip
lasm
ic z
inc/
man
gane
se/ir
on-b
indi
ng p
rote
in
Esch
eric
hia
sp. T
W09
308;
Citr
obac
ter f
reun
di 4
_7_4
7CFA
A; C
itrob
acte
r you
ngae
AT
CC 2
9220
; Sal
mon
ella
ent
eric
a su
bsp.
ent
eric
a se
rova
r Had
ar s
tr. R
I_05
P066
; D
esul
fovi
brio
vul
garis
str.
‘Miy
azak
i F’;
Ther
mob
acill
us c
ompo
sti K
WC
4; P
seu-
dom
onas
put
ida
S16;
Pae
niba
cillu
s muc
ilagi
nosu
s KN
P414
; Cup
riavi
dus
met
allid
uran
s CH
34 (p
lasm
id);
Pyro
cocc
us a
byss
i GE5
; Dic
keya
dad
antii
Ech
703;
Ca
ndid
atus
Blo
chm
anni
a pe
nnsy
lvan
icus
str.
BPE
N; T
herm
aero
bact
er su
bter
rane
us
DSM
139
65; R
hodo
cocc
us e
qui 1
035;
Bac
illus
cyt
otox
icus
NVH
391
-98;
Rho
doba
c-te
rale
s ba
cter
ium
HTC
C20
83
Page 11 of 19Salam et al. Bioresour. Bioprocess. (2020) 7:25
Tabl
e 3
(con
tinu
ed)
Hea
vy m
etal
sEn
zym
e/ge
nes
Taxo
nom
ic a
ffilia
tion
Iron,
cob
alt,
nick
el, g
alliu
m, c
ad-
miu
m, m
agne
sium
, man
gane
se,
zinc
Fe(II
I)-py
oche
lin o
uter
mem
bran
e re
cept
or p
recu
rsor
; pep
tide/
nick
el tr
ansp
ort
syst
em s
ubst
rate
-bin
ding
pro
tein
; fer
richr
ome
ABC
tran
spor
ter p
erm
ease
(Ni/
Co);
mgt
B ge
ne p
rodu
ct (M
g/Co
); m
agne
sium
-tra
nslo
catin
g P-
type
ATP
ase
(Mg/
Co);
iron-
depe
nden
t rep
ress
or, i
deR;
hea
vy m
etal
-tra
nslo
catin
g P-
type
AT
Pase
(Co/
Ni);
aco
nita
se A
(Fe)
; ABC
tran
spor
ter,
ATP-
bind
ing
prot
ein
YbbL
(F
e); T
onB-
depe
nden
t sid
erop
hore
rece
ptor
; zin
c/iro
n ZI
P fa
mily
per
mea
se
(Zn/
Fe/C
o/N
i/Cu/
Cd);
nick
el–c
obal
t–ca
dmiu
m re
sist
ance
pro
tein
ncc
C
(pla
smid
); ac
onita
te h
ydra
tase
(Fe)
; fer
ritin
(Fe/
Cu/M
n); f
pvA
2 ge
ne p
rodu
ct;
pred
icte
d di
vale
nt h
eavy
-met
al c
atio
ns tr
ansp
orte
r (Zn
/Fe/
Co/N
i/Cu/
Cd);
ferr
ic u
ptak
e re
gula
tor f
amily
pro
tein
, fur
A (F
e); D
NA
-bin
ding
tran
scrip
tiona
l re
gula
tor B
asR
(Fe)
; iro
n-di
citr
ate
tran
spor
ter A
TP-b
indi
ng s
ubun
it; N
RAM
P fa
mily
Mn2+
/Fe2+
tran
spor
ter (
Mn/
Fe/C
d/Co
/Zn)
; sen
sor p
rote
in B
asS/
PmrB
(F
e); f
errip
yove
rdin
e re
cept
or 2
Pseu
dom
onas
aer
ugin
osa
C37
19; P
seud
omon
as a
erug
inos
a M
18; P
seud
omon
as
aeru
gino
sa P
A01
; Azo
spiri
llum
sp.
B51
0; V
ibrio
par
ahae
mol
ytic
us R
IMD
221
0633
; Pe
ctob
acte
rium
atr
osep
ticum
SC
RI10
43; K
lebs
iella
pne
umon
iae
342;
Kle
bsie
lla
varii
cola
At-
22; P
seud
omon
as a
erug
inos
a 13
8244
; Kin
eoco
ccus
radi
otol
eran
s SR
S302
16; A
ctin
osyn
nem
a m
irum
DSM
438
27; B
ifido
bact
eriu
m b
ifidu
m N
CIM
B 41
171;
Bifi
doba
cter
ium
bifi
dum
S17
; Esc
heric
hia
ferg
uson
ii AT
CC 3
5469
; Pse
u-do
mon
as fu
lva
12-X
; Com
amon
as te
stos
tero
ni S
44; V
ario
vora
x pa
rado
xus E
PS;
Met
hano
saet
aH
arun
dina
cea
6Ac;
Cor
yneb
acte
rium
glu
curo
noly
ticum
ATC
C 5
1867
; Str
epto
myc
es
viol
aceu
snig
er T
u 41
13; P
rote
us m
irabi
lis H
I432
0; A
zoar
cus s
p. B
H72
; Sol
ibac
il-lu
s silv
estr
is St
LB04
6; S
egni
lipar
us ri
gosu
s ATC
C B
AA
-974
; Sod
alis
glos
sinid
ius s
tr.
‘mor
sita
ns’;
Citr
obac
ter k
oser
i ATC
C B
AA
-895
; The
rmob
acul
um te
rren
um A
TCC
BA
A-7
98; E
nter
obac
ter c
loac
ae s
ubsp
. clo
acae
ATC
C 1
3047
; Ach
rom
obac
ter
xylo
soxi
dans
A8
Mol
ybde
num
, tun
gste
n; te
lluriu
mm
odE
gene
pro
duct
; tel
lurit
e re
sist
ance
pro
tein
Trg
B; K
laB
prot
ein
(Te)
; mol
yb-
date
ABC
sup
erfa
mily
ATP
-bin
ding
cas
sett
e tr
ansp
orte
r, A
BC p
rote
in, m
odC
; m
olyb
date
ABC
tran
spor
ter p
erip
lasm
ic m
olyb
date
-bin
ding
pro
tein
mod
A;
tung
stat
e A
BC tr
ansp
orte
r bin
ding
pro
tein
Wtp
A; m
olyb
denu
m A
BC tr
ans-
port
er p
erm
ease
pro
tein
mod
B; te
llurit
e re
sist
ance
pro
tein
Teh
A
Xeno
rhab
dus b
ovie
nii S
S-20
04; R
oseo
bact
er s
p. G
AI1
01; u
ncul
ture
d ba
cter
ium
(p
lasm
id);
Serra
tia o
dorif
era
DSM
458
2; S
inor
hizo
bium
mel
iloti
1021
; Des
ulfu
ro-
bact
eriu
m th
erm
olith
otro
phum
DSM
116
99; G
emm
atim
onas
aur
antia
ca T
-27;
N
eiss
eria
muc
osa
ATCC
259
96
Ars
enic
, ant
imon
yA
BC tr
ansp
orte
r; m
ultid
rug
resi
stan
ce p
rote
in, p
-gly
copr
otei
n; a
rsen
ite o
xida
se,
larg
e su
buni
t, A
oxB;
pho
spha
te A
BC tr
ansp
orte
r per
mea
se (A
s); p
hosp
hate
A
BC tr
ansp
orte
r sub
stra
te-b
indi
ng p
rote
in; p
rote
in-t
yros
ine
phos
phat
ase,
low
m
olec
ular
wei
ght,
arsC
; ars
enat
e re
duct
ase;
ars
enat
e re
duct
ase,
glu
tath
ione
/gl
utar
edox
in ty
pe; a
rsen
ic re
sist
ance
pro
tein
ars
B; a
rsen
ite o
xida
se s
mal
l sub
u-ni
t, A
oxA
; ABC
thio
l tra
nspo
rter
; met
hyltr
ansf
eras
e, a
rsM
; met
hyltr
ansf
eras
e ty
pe II
; ars
enite
S-a
deno
sylm
ethy
ltran
sfer
ase;
ars
enat
e re
duct
ase
(azu
rin);
arse
nica
l res
ista
nce
prot
ein
Ars
H; a
rsen
ical
resi
stan
ce p
rote
in a
rsB
Leish
man
ia m
ajor
str
ain
Frie
dlin
; Ros
eoba
cter
lito
ralis
och
149
; Nitr
osom
onas
sp.
Is
79A
3; A
cido
vora
x av
enae
sub
sp. a
vena
e AT
CC 1
9860
; Sph
aero
bact
er th
erm
ophi
-lu
s DSM
207
45; S
taph
yloc
occu
s sap
roph
ytic
us s
ubsp
. sap
roph
ytic
us; S
taph
yloc
oc-
cus a
ureu
s; Ba
cillu
s sub
tilis
subs
p. s
ubtil
is R
O-N
N-1
; Mic
roco
leus
cht
hono
plas
tes
PCC
742
0; T
haue
ra s
p. M
ZIT;
Rho
dom
icro
bium
van
ielii
ATC
C 1
7100
; Bur
khol
deria
m
ultiv
oran
s ATC
C 1
7616
; Lei
shm
ania
infa
ntum
JPC
MS;
Mic
rolu
natu
s pho
spho
-vo
rus N
M-1
; Lep
tone
ma
illin
i DSM
215
28; D
esul
foha
lobi
um re
tbae
nse
DSM
569
2;
Rhod
ofer
ax fe
rrire
duce
ns T
118;
Glu
cona
ceto
bact
er s
p. S
KCC
-1; P
epto
niph
ilus h
arei
A
CS-
146-
V-Sc
h2b;
Ace
tivib
rio c
ellu
loly
ticus
CD
2
Page 12 of 19Salam et al. Bioresour. Bioprocess. (2020) 7:25
Tabl
e 4
Pred
icte
d he
avy
met
als
resi
stan
ce g
enes
det
ecte
d in
cad
miu
m-a
men
ded
SL5
met
agen
ome
and
thei
r tax
onom
ic a
ffilia
tion
s
Hea
vy m
etal
sEn
zym
e/ge
nes
Taxo
nom
ic a
ffilia
tion
Cadm
ium
, cob
alt,
mer
cury
, lea
d, z
inc
Hea
vy m
etal
-tra
nslo
catin
g P-
type
ATP
ase,
Cd/
Co/H
g/Pb
/Zn-
tran
spor
ting;
Apa
G p
rote
in; p
rote
in d
isul
phid
e is
omer
ase
I, ds
bA g
ene
prod
uct (
Cd/Z
n/H
g); D
SBA
oxi
dore
duct
ase
(Cd)
; RN
D d
ival
ent m
etal
cat
ion
efflux
tran
spor
ter C
zcA
(Zn/
Cd);
mem
bran
e-bo
und
catio
n-pr
oton
-ant
ipor
ter C
zrA
gen
e (Z
n/Cd
); co
balt-
zinc
-cad
miu
m re
sist
ance
pro
tein
; cat
ion
diffu
sion
fa
cilit
ator
fam
ily tr
ansp
orte
r; C
zrB
gene
Myc
obac
teriu
m s
p. S
pyr1
; Sac
char
omon
ospo
ra v
iridi
s DSM
430
17;
Myc
obac
teriu
m v
anba
alen
ii PY
R-1;
Myc
obac
teriu
m g
ilvum
PYR
-G
CK;
She
wan
ella
sedi
min
is H
AW-E
B3; S
erra
tia sy
mbi
otic
a st
r. ‘C
inar
a ce
dri’;
Cand
idat
us B
loch
man
nia
penn
sylv
anic
us s
tr. B
PEN
; Pa
enib
acill
us m
ucila
gino
sus K
NP4
14; P
seud
omon
as a
erug
inos
a PA
7; P
seud
omon
as a
erug
inos
a 39
016,
Pse
udom
onas
aer
ugin
osa
1382
44; P
seud
omon
as p
utid
a; P
seud
omon
as s
p. 2
_1_2
6; X
an-
thom
onas
gar
dner
i ATC
C 1
9865
; Pse
udom
onas
aer
ugin
osa
Copp
er, m
agne
sium
, silv
erCo
pper
-res
ista
nce
prot
ein
A (c
opA
); m
ultic
oppe
r oxi
dase
type
3
(cut
O);
copp
er re
sist
ance
pro
tein
C (c
opC
); pu
tativ
e co
pper
bi
ndin
g pr
otei
n; m
ultic
oppe
r oxi
dase
; mul
ticop
per o
xida
se
type
2; m
olec
ular
cha
pero
ne D
naK;
cop
per-
tran
sloc
atin
g P-
type
AT
Pase
; mag
nesi
um-t
rans
port
ing
ATPa
se; c
oppe
r exp
ortin
g AT
Pase
; hea
vy m
etal
-tra
nslo
catin
g P-
type
ATP
ase;
blu
e co
pper
ox
idas
e cu
eO p
recu
rsor
; P-A
TPas
e su
perf
amily
P-t
ype
ATPa
se
copp
er tr
ansp
orte
r, ct
pV; a
polip
opro
tein
N-a
cyltr
ansf
eras
e;
Apa
G p
rote
in; c
atio
n-tr
ansp
ortin
g AT
Pase
; tw
in-a
rgin
ine
tran
sloc
atio
n pa
thw
ay s
igna
l: co
pper
resi
stan
ce p
rote
in; c
atio
n effl
ux s
yste
m p
rote
in C
usB
(Cu/
Ag)
; hea
vy m
etal
-tra
nslo
catin
g P-
type
ATP
ase,
cop
B; ln
t gen
e pr
oduc
t (Cu
); Cu
(I)-r
espo
nsiv
e tr
ansc
riptio
nal r
egul
ator
; Mer
E fa
mily
tran
scrip
tiona
l reg
ula-
tor (
Cu);
Mer
R fa
mily
tran
scrip
tiona
l reg
ulat
or (C
u); l
ipop
rote
in
invo
lved
with
cop
per h
omeo
stas
is a
nd a
dhes
ion,
cut
F/nl
pE;
heav
y m
etal
tran
spor
t/de
toxi
ficat
ion
prot
ein,
cop
P; c
oppe
r/si
lver
effl
ux P
-typ
e AT
Pase
Acid
obac
teriu
m c
apsu
latu
m A
TCC
511
96; A
ceto
bact
er p
omor
um
DM
001;
Ach
rom
obac
ter x
ylos
oxid
ans A
8; A
naer
omyx
obac
ter s
p.
Fw10
9-5;
Hal
alka
licoc
cus j
eotg
ali B
3; S
teno
trop
hom
onas
mal
t-op
hila
R55
13; F
rank
ia s
p. C
cI3;
Hal
orub
rum
Iacu
spro
fund
i ATC
C
4923
9; C
oryn
ebac
teriu
m a
mm
onia
gene
s DSM
203
06; C
oryn
ebac
-te
rium
lipo
philo
flavu
m D
SM 4
4291
; Xan
thob
acte
r aut
otro
phic
us
Py2;
Hal
adap
tatu
s pau
ciha
loph
ilus D
X253
; Rho
doco
ccus
jost
ii RH
A1;
Intr
aspo
rang
ium
cal
vum
DSM
430
43; S
trep
tom
yces
sp.
S4;
St
rept
omyc
es a
lbus
J107
4; H
alob
acte
rium
sp.
DL1
; Ach
rom
obac
ter
xylo
soxi
dans
C54
; Cup
riavi
dus m
etal
lidur
ans C
H34
; Met
hano
saet
a ha
rund
inac
ea 6
Ac;
Rho
dofe
rax
ferr
iredu
cens
; Erw
inia
sp.
Ejp
617;
M
icro
cocc
us lu
teus
NC
TC 2
665;
Kyt
ococ
cus s
eden
tariu
s DSM
205
47;
Myc
obac
teriu
m p
aras
crof
ulac
eum
ATC
C B
AA
-614
; Pro
teus
mira
bilis
AT
CC 2
9906
; She
wan
ella
sedi
min
is H
AW-E
B3; B
acill
us a
myl
oliq
uefa
-ci
ens T
A20
8; P
seud
omon
as sy
ringa
e pv
. gly
cine
a st
r. B0
76; K
lebs
iella
pn
eum
onia
e 34
2; G
ordo
nia
poly
isopr
eniv
oran
s NBR
C 1
6320
; M
ethy
loba
cter
ium
chl
orom
etha
nicu
m C
M4;
Shi
gella
dys
ente
riae
1012
; Aca
ryoc
hlor
is sp
. CC
MEE
541
0; H
ypho
mon
as n
eptu
nium
AT
CC 1
5444
; Cau
loba
cter
sp.
K31
; Pse
udom
onas
syrin
gae
pv. a
ceris
st
r. M
3022
73PT
; Pse
udom
onas
ent
omop
hila
L48
; Pse
udom
onas
pu
tida
S16;
Rah
nella
sp.
Y96
02; P
seud
onoc
ardi
a sp
. P1;
Citr
omic
ro-
bium
bat
hyom
arin
um JL
354
Nic
kel
Pept
ide/
nick
el tr
ansp
ort s
yste
m, s
ubst
rate
-bin
ding
pro
tein
, ni
kA; t
rans
crip
tiona
l reg
ulat
or N
ikR,
Cop
G fa
mily
(nik
R); n
icke
l tr
ansp
orte
r ATP
-bin
ding
pro
tein
, nik
D; n
icke
l ABC
tran
spor
ter,
perip
lasm
ic n
icke
l-bin
ding
pro
tein
; nic
kel t
rans
port
er A
TP-
bind
ing
prot
ein,
nik
E
Azo
spiri
llum
sp.
B51
0; S
trep
toco
ccus
sang
uini
s SK1
056;
Met
hano
coc-
cus v
olta
e A
3; M
etah
nosp
irillu
m h
unga
tei J
F-1;
Pse
udom
onas
sp.
TJ
I-51;
Syn
ergi
stet
es b
acte
rium
SG
P1; R
hodo
spiri
llum
rubr
um A
TCC
11
170
Page 13 of 19Salam et al. Bioresour. Bioprocess. (2020) 7:25
Tabl
e 4
(con
tinu
ed)
Hea
vy m
etal
sEn
zym
e/ge
nes
Taxo
nom
ic a
ffilia
tion
Zinc
, tel
luriu
mRN
D fa
mily
effl
ux tr
ansp
orte
r MFP
sub
unit;
zin
c re
sist
ance
-ass
oci-
ated
pro
tein
, zra
P; lo
w-a
ffini
ty in
orga
nic
phos
phat
e tr
ansp
ort
Prot
ein;
dru
g effl
ux p
ump
tran
smem
bran
e pr
otei
n, m
dtB;
tellu
rite
resi
stan
ce g
ene,
trgB
; tra
nscr
iptio
nal r
egul
ator
y pr
otei
n, z
raR
(Zn)
; sig
nal t
rans
duct
ion
hist
idin
e-pr
otei
n ki
nase
, bae
S; ln
t ge
ne p
rodu
ct (Z
n); s
enso
ry h
istid
ine
kina
se in
two-
com
pone
nt
regu
lato
ry s
yste
m w
ith B
aeR;
zin
c-re
spon
sive
tran
scrip
tiona
l re
gula
tor;
high
-affi
nity
zin
c tr
ansp
orte
r, pe
ripla
smic
com
pone
nt
znuA
; low
-affi
nity
inor
gani
c ph
osph
ate
tran
spor
ter 1
(Zn/
Te)
Pseu
dom
onas
put
ida
KT24
40; C
itrob
acte
r sp.
30_
2; C
itrob
acte
r fre
undi
i 4_7
_47C
FAA
; Citr
obac
ter y
oung
ae A
TCC
292
20; S
alm
o-ne
lla e
nter
ica
subs
p. e
nter
ica
sero
var J
ohan
nesb
urg
str.
S5-7
03;
Salm
onel
la e
nter
ica
subs
p. e
nter
ica
sero
var T
yphi
str.
CT1
8; S
odal
is gl
ossin
idiu
s str.
‘mor
sita
ns’;
Oxa
loba
cter
form
igen
es O
XCC
13;
Rose
obac
ter l
itora
lis O
ch 1
49; R
oseo
bact
er d
enitr
ifica
ns O
ch 1
14;
Des
ulfo
vibr
io m
agne
ticus
RS-
1; E
sche
richi
a co
li 83
972;
Hyp
ho-
mon
as n
eptu
nium
ATC
C 1
5444
; Ral
ston
ia so
lana
cear
um C
MR1
5;
Phot
orha
bdus
lum
ines
cens
sub
sp. l
aum
ondi
i TTO
1; H
aem
ophi
lus
pitt
man
iae
HK
85; Y
ersin
ia ru
cker
i ATC
C 2
9473
; Dic
keya
dad
an-
tii E
ch70
3; E
rwin
ia p
yrifo
liae
DSM
121
63; E
rwin
ia a
myl
ovor
a C
FBP1
430
Ars
enic
Ars
enite
oxi
dase
, lar
ge s
ubun
it (a
oxB)
; ars
enite
S-a
deno
sylm
ethy
l-tr
ansf
eras
e; a
rsen
ite m
ethy
ltran
sfer
ase
arsM
; ars
enic
al re
sist
ance
pr
otei
n, a
rsB;
pro
tein
-tyr
osin
e ph
osph
atas
e, lo
w m
olec
ular
w
eigh
t, ar
sC; a
rsen
ite o
xida
se, s
mal
l sub
unit
(aox
A);
arse
nica
l re
sist
ance
pro
tein
Ars
H; p
hosp
hate
ABC
sup
erfa
mily
ATP
-bin
d-in
g ca
sset
te tr
ansp
orte
r, bi
ndin
g pr
otei
n
Burk
hold
eria
okl
ahom
ensis
C67
86; R
oseo
bact
er li
tora
lis o
ch 1
49;
Pseu
dovi
brio
sp.
JE06
2; D
esul
foha
lobi
um re
tbae
nse
DSM
569
2;
Des
ulfo
vibr
io a
lkal
iphi
lus A
HT2
; Des
ulfo
vibr
io sa
lexi
gens
DSM
263
8;
Mag
neto
spiri
llum
gry
phisw
alde
nse
MSR
-1; R
ubro
bact
er x
ylan
ophi
-lu
s D
SM 9
941;
Myc
obac
teriu
m p
aras
crof
ulac
eum
ATC
C B
AA
-614
; Sp
haer
obac
ter t
herm
ophi
lus D
SM 2
0745
; Des
ulfo
bacc
a ac
etoo
x-id
ans D
SM 1
1109
; Bur
khol
deria
mul
tivor
ans A
TCC
176
16; A
cidi
phi-
lium
mul
tivor
um A
IU30
1; M
annh
eim
ia h
aem
olyt
ica
PHL2
13
Cadm
ium
, cob
alt,
nick
el, m
anga
nese
, mag
nesi
umCa
dmiu
m-t
rans
loca
ting
P-ty
pe A
TPas
e; p
erip
lasm
ic s
olut
e-bi
ndin
g pr
otei
n; c
zcD
gen
e pr
oduc
t; he
avy
met
al-t
rans
loca
ting
P-ty
pe A
TPas
e, C
o/N
i; pu
tativ
e nr
bE-li
ke p
rote
in (N
i/Co)
; maj
or
faci
litat
or tr
ansp
orte
r, nr
sD/n
reB
(Ni/C
o); m
agne
sium
and
co
balt
tran
spor
t pro
tein
, cor
A (M
g/Co
/Ni/M
n); p
roba
ble
Nre
B pr
otei
n (N
i/Co)
; man
gane
se tr
ansp
ort p
rote
in (M
n/Fe
/Cd/
Co/
Zn);
mag
nesi
um-t
rans
loca
ting
P-ty
pe A
TPas
e (C
o/M
g); A
paG
ge
ne p
rodu
ct (C
o/M
g)
Stre
ptom
yces
flav
ogris
eus A
TCC
333
31; T
herm
obac
illus
com
post
i KW
C4;
Alk
aniv
orax
bor
kum
ensis
SK2
; Act
inos
ynne
ma
miru
m
DSM
438
27; H
oefle
a ph
otot
roph
ica
DFL
-43;
Ros
eifle
xus s
p. R
S-1;
M
icro
mon
ospo
ra s
p. A
TCC
391
49; P
aeni
baci
llus v
orte
x V4
53;
Phot
obac
teriu
m le
iogn
athi
sub
sp. m
anda
pam
ensi
s sv
ers.1
.1.;
Pho-
toba
cter
ium
sp.
SKA
34; V
ibrio
ang
ustu
m S
14; O
cean
ospi
rillu
m s
p.
MED
92; P
anto
ea st
ewar
tii s
ubsp
. ste
war
tii; S
erra
tia s
p. A
S12;
Vib
rio
carib
bent
hicu
s ATC
C B
AA
-212
2; S
acch
arop
hagu
s deg
rada
ns 2
-40
Chr
omiu
m, t
ellu
rium
, sel
eniu
mAT
P-de
pend
ent D
NA
hel
icas
e Re
cG; t
ellu
rite
resi
stan
ce p
rote
in
TehB
; chr
omat
e tr
ansp
orte
r; ch
rom
ate
ion
tran
spor
ter f
amily
pr
otei
n; c
hrom
ate
tran
spor
t pro
tein
Chr
A; t
ellu
rite
resi
stan
ce
gene
, trg
B; m
anga
nese
/iron
sup
erox
ide
dism
utas
e, c
hrC
Tolu
mon
as a
uens
is D
SM 9
187;
Yer
sinia
pes
tis K
IM10
+ ; M
ethy
loba
c-te
rium
sp.
4-4
6; M
ethy
loba
cter
ium
nod
ulan
s ORS
206
0; D
esul
fovi
-br
io s
p. A
2; A
lpha
-pro
teob
acte
rium
BA
L199
; Ros
eoba
cter
lito
ralis
O
ch 1
49; R
oseo
bact
er d
enitr
ifica
ns O
ch 1
14; B
eije
rinck
ia in
dica
su
bsp.
indi
ca A
TCC
903
9; C
upria
vidu
s bas
ilens
is O
R16;
Ent
erob
acte
r cl
oace
ae s
ubsp
. clo
acea
e N
CTC
939
4; A
licyc
liphi
lus
deni
trifi
cans
; Cu
pria
vidu
s m
etal
lidur
ans
CH
34 (p
lasm
id);
Zuno
ngw
angi
a pr
ofun
da S
M-A
B7
Page 14 of 19Salam et al. Bioresour. Bioprocess. (2020) 7:25
Tabl
e 4
(con
tinu
ed)
Hea
vy m
etal
sEn
zym
e/ge
nes
Taxo
nom
ic a
ffilia
tion
Man
gane
se, i
ron,
cob
alt,
zinc
, nic
kel,
copp
er, c
adm
ium
, gal
lium
TonB
-dep
ende
nt s
ider
opho
re re
cept
or; f
errip
yove
rdin
e re
cept
or;
ABC
tran
spor
ter-
like
prot
ein;
zin
c/iro
n pe
rmea
se; i
ron-
depe
nd-
ent r
egul
ator
y pr
otei
n, id
eR; D
NA
pro
tect
ion
prot
ein,
dps
A;
fpvA
2 ge
ne p
rodu
ct; a
coni
tate
hyd
rata
se 1
; ABC
tran
spor
ter
tran
smem
bran
e re
gion
fam
ily p
rote
in (F
e); l
ipop
rote
in in
ner
mem
bran
e A
BC tr
ansp
orte
r, Irp
6; p
erm
ease
and
ATP
-bin
ding
pr
otei
n of
yer
sini
abac
tin-ir
on A
BC tr
ansp
orte
r Ybt
P; A
BC tr
ans-
port
er p
recu
rsor
of t
he in
ner m
embr
ane
lipop
rote
in; i
ron(
III)-
tran
spor
t ATP
-bin
ding
pro
tein
, fbp
C; f
erro
xida
se; z
inc/
iron
ZIP
fam
ily p
erm
ease
(Zn/
Fe/C
o/N
i/Cu/
Cd);
czcD
gen
e pr
oduc
t (Fe
/Zn
/Co/
Cd/N
i); z
inc
tran
spor
ter z
upT
(Zn/
Fe/C
o/N
i/Cu/
Cd);
ferr
ic
upta
ke re
gula
tor,
Fur f
amily
; ctp
C g
ene
prod
uct (
Mn/
Zn)
Sten
otro
phom
onas
mal
toph
ila R
5513
; Ste
notr
opho
mon
as s
p. S
KA14
; Ve
rmin
ephr
obac
ter e
iseni
ae E
F01-
2; E
than
olig
enes
her
bine
nse
YUA
N-3
; Pse
udom
onas
fulv
a 12
-X; K
ocur
ia rh
izop
hila
DC
2201
; Sy
nech
ococ
cus s
p. C
C93
11; P
roch
loro
cocc
us m
arin
us s
tr. M
IT 9
303;
A
zoar
cus s
p. B
H72
; Pse
udom
onas
fluo
resc
ens P
F-5;
Str
epto
my-
ces h
ygro
scop
icus
ATC
C 5
3653
; Str
epto
myc
es v
iola
ceus
nige
r Tu
4113
; Esc
heric
hia
coli
DEC
BC; Y
ersin
ia e
nter
ocol
itica
; Esc
heric
hia
coli
5598
9; E
sche
richi
a co
li O
104:
H4
str.
01-0
9591
; Esc
heric
hia
coli
NA
114;
Yer
sinia
pse
udot
uber
culo
sis IP
329
53; Y
ersi
nia
pest
is
KIM
10 +
; Nei
sser
ia m
enin
gitid
is AT
CC 1
3091
; Nei
sser
ia m
enin
gitid
is M
C58
; Nei
sser
ia m
enin
gitid
is M
01-2
4035
5; N
eiss
eria
men
ingi
tidis
alph
a153
; Rho
doco
ccus
equ
i 103
S; M
etha
nosa
eta
haru
ndin
acea
6A
c; A
erom
onas
salm
onic
ida
subs
p. s
alm
onic
ida
A44
9; E
rwin
ia
billi
ngia
e Eb
661;
Ser
ratia
sp.
AS1
2; S
erra
tia o
dorif
era
4Rx1
3; S
erra
tia
prot
eam
acul
ans 5
68; S
erra
tia o
dorif
era
DSM
458
2; P
revo
tella
den
-ta
lis D
SM 3
688;
Myc
obac
teriu
m u
lcer
ans A
gy99
; Myc
obac
teriu
m
mar
inum
M
Mer
cury
Mer
curic
(Hg(
II)) r
educ
tase
, mer
A; m
ercu
ric tr
ansp
ort p
rote
in,
mer
T; M
erR
fam
ily tr
ansc
riptio
nal r
egul
ator
; Hg(
II)-r
espo
nsiv
e tr
ansc
riptio
nal r
egul
ator
; mer
curic
tran
spor
ter,
mer
H; m
erR
gene
pro
duct
; mer
curic
redu
ctas
e, m
erB1
Thio
mon
as s
p. 3
As;
Endo
riftia
Per
seph
one ‘
Hot
96_1
+ H
ot96
_2′;
Baci
llus c
ellu
losil
ytic
us D
SM 2
522;
Agr
obac
teriu
m tu
mef
acie
ns F
2;
Lept
ospi
rillu
m fe
rrod
iazo
trop
hum
; Mei
othe
rmus
silv
anus
DSM
994
6;
Ther
mus
ther
mop
hilu
s HB2
7; T
herm
us th
erm
ophi
lus H
B8; O
ce-
anith
erm
us p
rofu
ndus
DSM
149
77; E
nter
obac
ter c
loac
eae
subs
p.
cloa
ceae
ATC
C 1
3047
; Sph
ingo
pyxi
s ala
sken
sis R
B225
6; M
ethy
l-ov
ersa
tilis
univ
ersa
lis F
AM
5; N
ovos
phin
gobi
um n
itrog
enifi
gens
DSM
19
370;
Bur
khol
deria
glu
mae
BG
R1; A
cido
vora
x sp
. JS4
2; M
ycob
acte
-riu
m s
p.; S
phin
gobi
um s
p. S
YK-6
; Xan
thob
acte
r aut
otro
phic
us P
y2
Mol
ybde
num
, tun
gste
nM
olyd
benu
m A
BC tr
ansp
orte
r ATP
-bin
ding
; ext
race
llula
r sol
ute-
bind
ing
prot
ein,
wtp
A; s
igna
l tra
nsdu
ctio
n hi
stid
ine-
prot
ein
kina
se, b
aeS;
sen
sory
his
tidin
e ki
nase
in tw
o-co
mpo
nent
re
gula
tory
sys
tem
with
Bae
R; A
BC tr
ansp
orte
r fam
ily p
rote
in;
mol
ybda
te A
BC tr
ansp
orte
r, pe
rmea
se p
rote
in m
odB
Met
hano
sarc
ina
acet
ivor
ans C
2A; M
etha
noha
lobi
um e
vest
igat
um
Z-73
03; E
sche
richi
a co
li 83
972;
Ral
ston
ia so
lana
cear
um C
MR1
5;
Des
ulfo
vibr
io s
p. A
2; R
aphi
diop
sis b
rook
ii D
9
Page 15 of 19Salam et al. Bioresour. Bioprocess. (2020) 7:25
solubility of cadmium in the soil and its availability in soil solution.
The detection of various heavy metals in SL4 agricul-tural soil as revealed in the heavy metal content analysis, though at thresholds permitted for soils (WHO/FAO 2001) may be attributed to atmospheric deposition and various agricultural practices, which introduce the heavy metals into the soil. The significant reduction of these metals in Cd-amended SL5 microcosm may be due to several reasons. First, utilization of biologically impor-tant heavy metals such as zinc, copper, iron and chro-mium are tightly linked to the metabolic functioning of soil biota as they are essential micronutrients required by most microorganisms, which possibly cause their reduc-tion (Bruins et al. 2000; Marschner 2012; Rai et al. 2019). Also, addition of Cd to the agricultural soil induces the activation of Cd resistance systems, which are also used by microorganisms for uptake, transport, efflux, and detoxification of other heavy metals detected in this study (Nies 1999, 2003).
The predominance of the phyla Proteobacteria and Actinobacteria in the agricultural soil is not surprising as the two phyla comprise members that are well adapted to agricultural soils (Cheema et al. 2015; Trivedi et al. 2016; Salam et al. 2017; Yin et al. 2017). The exhibition of filamentous growth, possession of spores that are recal-citrant to various environmental stressors, and secretion of avalanche of enzymes, which degrade various macro-molecules that abound in soil provide distinctive edge for members of Actinobacteria phylum in soil environments (Larkin et al. 2005; Salam and Obayori 2019). Members of the phylum Proteobacteria have diverse morphologi-cal, physiological, and metabolic properties. These prop-erties facilitate their preponderance in soils with various environmental conditions (Aislabie and Deslippe 2013; Montecchia et al. 2015; Salam et al. 2019).
While about 11% of proteobacterial members were lost due to Cd contamination in SL5, it still constitutes the most abundant phylum (50.50%). In contrast, though the second most abundant phylum in SL5 (17.17%), the phylum Actinobacteria loses 68.05% of its members. This may be due to Cd toxicity to majority of its members, which results in oxidative damage via production of reac-tive oxygen species, and displacement of Zn and Fe ions from metalloproteins, resulting in their inactivation (Val-lee and Ulmer 1972; Stohs and Bagchi 1995; Fortuniak et al. 1996; Stohs et al. 2001; Banjerdkij et al. 2005).
Structural analysis of the SL5 metagenome revealed the dominance of the class Alphaproteobacteria and the genus Methylobacterium. The preponderance of members of the class and the genus may be attributed to several factors. The preponderance of czrCBA efflux system and other Cd uptake/transport/efflux systems
among members of the class Alphaproteobacteria may have contributed immensely to their abundance in SL5 system. The czrCBA efflux system is involved mainly in response to Cd and zinc showing significant induction in their presence (Nies 2003; Braz and Marques 2005; Hu et al. 2005; Valencia et al. 2013). In addition, mem-bers of the genus Methylobacterium are reputed to be widely distributed in diverse environmental compart-ments with propensity for detoxification of heavy metals (De Marco et al. 2004; Fernandes et al. 2009; Salam et al. 2015). They are renowned for possession of heavy metal resistance genes such as cation efflux system protein czcA gene, ABC transporters involved in metal uptake, copper-translocating P-type and genes encoding arse-nic resistance and chromate transport (Madhaiyan et al. 2007; Dourado et al. 2012; Kwak et al. 2014; Dourado et al. 2015).
Functional characterization of the two metagenomes (SL4, SL5) revealed the presence of heavy metal resist-ance genes (Tables 3, 4). Detection of resistance genes in SL4 agricultural soil metagenome is not surprising as traces of various heavy metals were detected in the soil (Table 1). The survival of some members of the com-munity despite the heavy metals stress indicates the presence of resistance systems that tightly control intra-cellular concentrations of the heavy metal ions and their attendant toxicities (Nies 1999, 2003; Hu et al. 2005).
One of the toxic effects of Cd is that it causes oxidative stress by depleting glutathione and protein-bound sulf-hydryl groups resulting in formation of reactive oxygen species (ROS). The resultant ROS causes enhanced lipid peroxidation, DNA damage and distorted calcium and sulfhydryl homeostasis (Kachur et al. 1998). In this study, thioredoxin-based thiol disulfide oxidoreductase (dsbA, dsbB) and dithiol disulfide isomerase, which protect microbial cells against oxidative stress were detected in the two metagenomes. However, manganese/iron super-oxide dismutase, two superoxide dismutases known to remove superoxide radicals that may be generated upon exposure to heavy metals (Jones et al. 1991; Stohs and Bagchi 1995; Kachur et al. 1998; Nies 1999) were only detected in SL5 metagenome. This is interesting as pre-vious reports have averred that the greatest induction of Mn superoxide dismutase (sodA) occurred under Cd and chromium stress, while induction of Fe superoxide dis-mutase (sodB) occurred only under Cd stress (Hu et al. 2005; Ammendola et al. 2014). Thus, the induction of these two intracellular superoxide dismutases required to control Cd-mediated oxidative stress in SL5 metagenome could only be attributed to elevated concentration of Cd in SL5 microcosm.
Another interesting finding is the detection of alkyl hydroperoxide reductase (ahpC) gene in 1440 protein
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sequences of SL5 metagenome, which is not detected in the protein sequences of SL4 metagenome. The detec-tion of this gene in SL5 metagenome may be attributed to Cd contamination. Previous works have reported cad-mium-induced cross-protection against H2O2 in E. coli cells pre-treated with CdCl2 while others have reported increase in induction of AhpC gene by tenfold after cells were exposed to Cd (Ferianc et al. 1998; Mongkolsuk and Helmann 2002; Banjerdkij et al. 2005).
The three major families of efflux transporters involved in Cd2+/Zn2+ resistance namely the P-type ATPases (Nucifora et al. 1989; Rensing et al. 1997), the CBA trans-porters (Nies and Silver 1989; Nies 1995; Hassan et al. 1999), and the cation diffusion facilitator (CDF) trans-porters (Xiong and Jayaswal 1998; Anton et al. 1999; Grass et al. 2001; Nies 2003) were detected in this study. Several P-type ATPases were detected in SL4 (cadmium-translocating P-type ATPase; Pb/Cd/Zn/Hg transport-ing ATPase; cation transporting P-type ATPase) and SL5 (cadmium-translocating P-type ATPase; heavy metal-translocating P-type ATPase Cd/Co/Hg/Pb/Zn-trans-porting) metagenomes. The functional features of these pumps include maintenance of homeostasis of essential metals (Cu+, Co2+, Zn2+) and mediating resistance to toxic metals (Cd2+, Pb2+, Ag+) (Rensing et al. 1997, 1999; Lee et al. 2001; Hu and Zhao 2007; Scherer and Nies 2009).
It is instructive to note that while CBA transport-ers (czcA, czrA, czrB) were detected in Cd-amended SL5 metagenome (Table 4), only the nccC (nickel–cad-mium–cobalt) protein, which confers resistance to nickel, cadmium and cobalt was detected in the SL4 metagen-ome (Table 3). The RND protein CzcA component of the three-component CzcCBA (cadmium–zinc–cobalt) efflux system detected in SL5 metagenome mediates the active part of the transport process, determines the sub-strate specificity and is involved in the assembly of the trans-envelope protein complex. Its presence in a heavy metal-polluted system is exceptional and indicates high-level resistance to heavy metal ions (Nies et al. 1989; Franke et al. 2003; Nies 2003). Another RND efflux sys-tem detected in SL5 metagenome is the czrCBA efflux system, a prototype of the czcCBA efflux system (Hassan et al. 1999; Valencia et al. 2013). It is an efflux system that showed significant induction in the presence of cadmium and zinc. The detection of czrA and czrB in SL5 metage-nome could only be attributed to Cd amendment, which upregulate the czr regulon in the metagenome. CBA transporters mainly carried out outer membrane efflux by removing periplasmic metal ions transported there by ATPases or CDF transporters or expelling the ions before they entered the cytoplasm (Scherer and Nies 2009).
The cation diffusion facilitator (CDF) transporters are represented in Cd-amended SL5 metagenome with the czcD gene, the archetype of the family. The gene, first described as a regulator of expression of the CzcCBA high-resistance system in Ralstonia (now Cupriavidus) metallidurans strain CH34 can also mediate resistance to small degree of Zn2+/Co2+/Cd2+ in the absence of Czc-CBA system (Nies 1992; Anton et al. 1999; Nies 2003).
The interplay of different transporters in Cd and zinc resistance clearly indicated, as shown in several stud-ies that full resistance to Cd2+ requires both the activity of CBA transporter and P-type ATPase (Legatzki et al. 2003; Scherer and Nies 2009). This is because some Cd2+ can escape the CBA transporter and enter the cytoplasm. In such instance, they will be exported by the P-type ATPases (Scherer and Nies 2009). This per-haps explains the reason why both P-type ATPases and CBA transporters were upregulated in Cd-perturbed SL5 metagenome.
A cursory look at the taxonomic affiliation of the heavy metal genes detected in SL4 and SL5 metagenome revealed they belong exclusively to the two dominant phyla, Proteobacteria and Actinobacteria, with Proteo-bacteria members largely dominating. This is in tan-dem with the structural analysis results, which shows the dominance of Proteobacteria and Actinobacteria in the two metagenomes. This is interesting as it revealed that the two phyla not only dominate the ‘who is there?’ part of the two microbial community, but were equally responsible for the detoxification of Cd (SL5) and other heavy metals in the communities.
ConclusionsIn summary, Illumina shotgun metagenomics and analy-sis of soil physicochemistry and heavy metals content has revealed the presence of several heavy metals and the effects of Cd contamination on soil physicochemistry and microbial community structure of SL4 agricultural soil. Detection of various heavy metals in the agricultural soil, though at low threshold is concerning as heavy metals are not biodegradable and can bioaccumulate in the food chain over time. Possession of diverse resistance genes by members of the microbial community may be exploited for depuration of agricultural soils inundated with Cd and other heavy metals. The need to embrace environ-mentally friendly methods for pest and herbage control and to improve crop yield is becoming more profound, due to the negative impacts of current agricultural prac-tices on the general wellbeing of the soil ecosystem and its inhabitants.
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Supplementary informationSupplementary information accompanies this paper at https ://doi.org/10.1186/s4064 3-020-00314 -w.
Additional file 1. Additional figures.
AbbreviationsCd: Cadmium; CDF: Cation diffusion facilitator; RND: Resistance nodulation division; CBA: Capsule biogenesis/assembly; NRAMP: Natural resistance-associ-ated macrophage protein.
AcknowledgementsNot applicable.
Authors’ contributionsLBS conceived the study and performed the experiments. OSO coordinated the study and in consultation with LBS wrote the Materials and Methods and Results. MOI and OOA contributed to the Discussion section. All authors read and approved the final manuscript.
FundingNo external funding was received to conduct this study.
Availability of data and materialsAll data generated or analysed during this study are included in this published article and its additional files
Ethics approval and consent to participateNot applicable.
Consent for publicationNot applicable.
Competing interestThe authors declare that they have no competing interest.
Author details1 Department of Biological Sciences, Microbiology Unit, Summit University, Offa, Kwara, Nigeria. 2 Department of Microbiology, Lagos State University, Ojo, Lagos, Nigeria. 3 Department of Microbiology, University of Lagos, Akoka, Lagos, Nigeria.
Received: 3 March 2020 Accepted: 5 May 2020
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