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: babssalaam@yahoo.com1 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

Page 3 of 19Salam et al. Bioresour. Bioprocess. (2020) 7:25

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

ReferencesAdriano DC (2001) Trace elements in terrestrial environments: biogeochemis-

try, bioavailability, and risks of metals, 2nd edn. Springer, New York, p 867Aislabie J, Deslippe JR (2013) Soil microbes and their contribution to soil ser-

vices. In: Dymond JR (ed) Ecosystem services in New Zealand-conditions and trends. Manaaki Whenua Press, Lincoln, pp 143–161

Alloway BJ, Steinnes E (1999) Anthropogenic additions of cadmium to soils. In: McLaughlin MJ, Singh BR (eds) Cadmium in soils and plants Develop-ments in plants and soil sciences. Springer, Dordrecht, pp 97–123

Altimira F, Yáñez C, Bravo G, González M, Rojas LA, Seeger M (2012) Char-acterization of copper-resistant bacteria and bacterial communities from copper-polluted agricultural soils of central Chile. BMC Microbiol 12(1):193

Ammendola S, Cerasi M, Battistoni A (2014) Deregulation of transition metals homeostasis is a key feature of cadmium toxicity in Salmonella. Biometals 27:703–714

Anton A, Grosse C, Reissmann C, Pribyl T, Nies DH (1999) CzcD is a heavy metal ion transporter involved in regulation of heavy metal resistance in Ralsto-nia sp. strain CH34. J Bacteriol 181:6876–6881

Aramaki T, Blanc-Mathieu R, Endo H, Ohkubo K, Kanehisa M, Goto S, Ogata H (2019) KofamKOALA: KEGG ortholog assignment based on profile HMM and adaptive score threshold. Bioinformatics Btz859

Banjerdkij P, Vattanaviboon P, Mongkolsuk S (2005) Exposure to cadmium elevates expression of genes in the OxyR and OhrR regulons and induces cross-resistance to peroxide killing treatment in Xanthomonas campestris. Appl Environ Microbiol 71(4):1843–1849

Benavides MP, Gallego SM, Tomaro ML (2005) Cadmium toxicity in plants. Braz J Plant Physiol 17(1):21–34

Bhadra B, Nanda AK, Chakraborty R (2005) Inducible nickel resistance in a river isolate of india phylogenetically ascertained as a novel strain of Acineto-bacter junii. World J Microbiol Biotechnol 22(3):225–232

Braz VS, Marques MV (2005) Genes involved in cadmium resistance in Caulo-bacter crescentus. FEMS Microbiol Lett 251:289–295

Bruins MR, Kapil S, Oehme FW (2000) Microbial resistance to metals in the environment. Ecotoxicol Environ Saf 45:198–207

Cheema S, Lavania M, Lal B (2015) Impact of petroleum hydrocarbon contamination on the indigenous microbial community. Ann Microbiol 66:359–369

De Marco P, Pacheco CC, Figueiredo AR, Moradas-Ferreira P (2004) Novel pollutant-resistant methylotrophic bacteria for use in bioremediation. FEMS Microbiol Lett 234:75–80

Dourado MN, Andreote FD, Dini-Andreote F, Conti R, Araujo JM, Araujo WL (2012) Analysis of the 16S rRNA and mxaF genes revealing insights into Methylobacterium niche-specific plant association. Gen Mol Biol 35(1):142–148

Dourado MN, Neves AAC, Santos DS, Araujo WL (2015) Biotechnological and agronomic potential of endophytic pink-pigmented methylotrophic Methylobacterium spp. Biomed Res Int 2015:909016

Edwards JR, Prozialeck WC (2009) Cadmium, diabetes and chronic kidney disease. Toxicol Appl Pharmacol 238(3):289–293

Fashola MO, Ngole-Jeme VM, Babalola OO (2016) Heavy metal pollution from gold mines: environmental effects and bacterial strategies for resistance. Int J Environ Res Public Health 13:1047

Feng G, Xie T, Wang X, Bai J et al (2018) Metagenomic analysis of microbial community and function involved in cd-contaminated soil. BMC Micro-biol 18:1

Ferianc P, Farewell A, Nystrom T (1998) The cadmium-stress stimulon of Escheri-chia coli K-12. Microbiology 144:1045–1050

Fernandes VC, Albergaria JT, Oliva-Teles T, Delerue-Matos C, de Marco P (2009) Dual augmentation foe aerobic bioremediation of MTBE and TCE pollu-tion in heavy metal-contaminated soil. Biodegradation 20(3):375–382

Fortuniak A, Zadzinski R, Bilinski T, Bartosz G (1996) Glutathione depletion in the yeast Saccharomyces cerevisiae. Biochem Mol Biol Int 38:901–910

Franke S, Grass G, Rensing C, Nies DH (2003) Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli. J Bacteriol 185:3804–3812

Grass G, Fan B, Rosen BP, Franke S, Nies DH, Rensing C (2001) ZitB (YbgR), a member of the cation diffusion facilitator family, is an additional zinc transporter in Escherichia coli. J Bacteriol 183:4664–4667

Gray CW, McLaren RG, Roberts AHC, Condron LM (1998) Sorption and desorp-tion of cadmium from some New Zealand soils: effect of pH and contact time. Australian J Soil Res 36:199–216

Gray CW, McLaren RG, Roberts AHC, Condron LM (1999) Effect of soil pH on cadmium phytoavailability in some New Zealand soils. N Z J Crop Hortic Sci 27:169–179

Handelsman J (2004) Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev 68:669–678

Hassan MT, van der Lelie D, Springael D, Romling U, Ahmed N, Mergeay M (1999) Identification of a gene cluster, czr, involved in cadmium and zinc resistance in Pseudomonas aeruginosa. Gene 238:417–425

Hu N, Zhao B (2007) Key genes involved in heavy-metal resistance in Pseu-domonas putida CD2. FEMS Microbiol Lett 267:17–22

Hu P, Brodie EL, Suzuki Y, McAdams HH, Andersen GL (2005) Whole-genome transcriptional analysis of heavy metal stresses in Caulobacter crescentus. J Bacteriol 187:8437–8449

Imai I, Siegel SM (1973) A specific response to toxic cadmium levels in red kidney bean embryos. Physiol Plant 29:118–120

Jones P, Kortenkamp A, O’Brien P, Wang G, Yang G (1991) Evidence for the gen-eration of hydroxyl radicals from a chromium(V) intermediate isolated

Page 18 of 19Salam et al. Bioresour. Bioprocess. (2020) 7:25

from the reaction of chromate with glutathione. Arch Biochem Biophys 286:652–655

Júnior CAL, Mazzafera P, Arruda MAZ (2014) A comparative ionomic approach focusing on cadmium effects in sunflowers (Helianthus annuus L). Environ Exp Bot 107:180–186

Kachur AV, Koch CJ, Biaglow JE (1998) Mechanism of copper-catalyzed oxida-tion of glutathione. Free Radic Res 28:259–269

Khan S, Rehman S, Khan A, Khan M, Shah M (2010) Soil and vegetables enrich-ment with heavy metals from geological sources in Gilgit, northern Pakistan. Ecotoxicol Environ Safety 73:1820–1827

Khan MIR, Nazir F, Asgher M, Per TS, Khan NA (2015) Selenium and sulfur influence ethylene formation and alleviate cadmium-induced oxidative stress by improving proline and glutathione production in wheat. J Plant Physiol 173:9–18

Khan A, Khan S, Alam M, Khan MA, Aamir M, Qamar Z, Rehman ZU, Perveen S (2016a) Toxic metal interactions affect the bioaccumulation and dietary intake of macro- and micro-nutrients. Chemosphere 146:121–128

Khan S, Munir S, Sajjad M, Li G (2016b) Urban park soil contamination by potentially harmful elements and human health risk in Peshawar City, Khyber Pakhtunkhwa, Pakistan. J Geochem Explor 165:102–110

Khan MA, Khan S, Khan A, Alam M (2017) Soil contamination with cadmium, consequences and remediation using organic amendments. Sci Total Environ 601–602:1591–1605

Kwak M-J, Jeong H, Madhaiyan M et al (2014) Genome information of Methy-lobacterium oryzae, a plant-probiotic methylotroph in the phyllosphere. PLoS ONE 9(9):e106704

Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie-2. Nat Methods 9(4):357–359

Larkin MJ, Kulakov LA, Allen CC (2005) Biodegradation and Rhodococcus—masters of catabolic versatility. Curr Opin Biotechnol 16:282–290

Lee SW, Glickmann E, Cooksey DA (2001) Chromosomal locus for cadmium resistance in Pseudomonas putida consisting of a cadmium-transporting ATPase and a MerR family response regulator. Appl Environ Microbiol 67:1437–1444

Legatzki A, Grass G, Anton A, Rensing C, Nies DH (2003) Interplay of the Czc system and two P-type ATPases in conferring metal resistance to Ralsto-nia metallidurans. J Bacteriol 185:4354–4361

Li P-E, Lo C-C, Anderson JJ et al (2017) Enabling the democratization of the genomics revolution with a fully integrated web-based bioinformatics platform. Nucleic Acids Res 45(1):67–80

Lo C-C, Chain PSG (2014) Rapid evaluation and quality control of next genera-tion sequencing data with FaQCs. BMC Bioinform 15:366

Lopez-Mill’a’n AF, Sagardoy R, Solanas M, Abadia A, Abadia J (2009) Cadmium toxicity in tomato (Lycopersicon esculentum) plants grown in hydroponics. Environ Exp Bot 65:376–385

Liu J, Zhang H, Zhang Y, Chai T (2013) Silicon attenuates cadmium toxicity in Solanum nigrum L. by reducing cadmium uptake and oxidative stress. Plant Physiol Biochem 68:1–7

Madhaiyan M, Poonguzhali S, Sa T (2007) Characterization of 1-aminocyclo-propane-1-carboxylate (ACC) deaminase containing Methylobacterium oryzae and interactions with auxins and ACC regulation of ethylene in canola (Brassica campestris). Planta 226(4):867–876

Marchler-Bauer A, Derbyshire MK, Gonzales NR et al (2015) CDD: NCBI’s con-served domain database. Nucleic Acids Res 43(D):222–226

Marschner P (2012) Marschner’s Mineral Nutrition of Higher Plants, 3rd edn. Academic Press, London

Mielke HW, Adams JL, Chaney RL, Mielke PW Jr, Ravikumar VC (1991) The pattern of cadmium in the environment of five Minnesota cities. Environ Geochem Health 13(1):29–34

Mohamed AA, Castagna A, Ranieri A, Sanita di Toppi L (2012) Cadmium toler-ance in Brassica juncea roots and shoots is affected by antioxidant status and phytochelatin biosynthesis. Plant Physiol Biochem 57:15–22

Mongkolsuk S, Helmann JD (2002) Regulation of inducible peroxide stress responses. Mol Microbiol 45:9–15

Montecchia MS, Tosi M, Soria MA, Vogrig JA, Sydorenko O, Correa OS (2015) Pyrosequencing reveals changes in soil bacterial communities after conversion of Yungas forests to agriculture. PLoS ONE 10:e0119426. https ://doi.org/10.1371/journ al.pone.01194 26

Moynihan M, Peterson KE, Cantoral A et al (2017) Dietary predictors of urinary cadmium among pregnant women and children. Sci Total Environ 575:1255–1262

Nawab J, Khan S, Aamir M, Shamshad I, Qamar Z, Din I, Huang Q (2016) Organic amendments impact the availability of heavy metal(loid)s in mine-impacted soil and their phytoremediation by Penisitum america-num and Sorghum bicolor. Environ Sci Pollut Res 23(3):2381–2390

Nies DH (1992) CzcR and CzcD, gene products affecting regulation of resist-ance to cobalt, zinc, and cadmium (czc system) in Alcaligenes eutrophus. J Bacteriol 174:8102–8110

Nies DH (1995) The cobalt, zinc, and cadmium efflux system CzcABC from Alcaligenes eutrophus functions as a cation-proton antiporter in Escheri-chia coli. J Bacteriol 177:2707–2712

Nies D (1999) Microbial heavy-metal resistance. Appl Microbiol Biotechnol 51:730–750

Nies DH (2003) Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev 27:313–339

Nies DH, Silver S (1989) Plasmid-determined inducible efflux is responsible for resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus. J Bacteriol 171:896–900

Nies DH, Nies A, Chu L, Silver S (1989) Expression and nucleotide sequence of a plasmid determined divalent cation efflux system from Alcaligenes eutrophus. Proc Natl Acad Sci USA 86:7351–7355

Noguchi H, Park J, Takagi T (2006) MetaGene: prokaryotic gene finding from environmental genome shotgun sequences. Nucleic Acids Res 34(19):5623–5630

Nucifora G, Chu L, Misra TK, Silver S (1989) Cadmium resistance from Staphy-lococcus aureus plasmid pI258 cadA gene results from a cadmium efflux ATPase. Proc Natl Acad Sci USA 86:3544–3548

Oulas A, Pavloudi G, Polymanakou P, Pavlopoulus GA, Papanikolaou N, Kotou-las G, Arvanitidis C, Iliopoulus I (2015) Metagenomics: tools and insights for analyzing next-generation sequencing data derived from biodiversity studies. Bioinform Biol Insights 9:75–88

Pal C, Bengtsson-Palme J, Rensing C, Kristiansson E, Larsson DG (2014) BacMet: antibacterial biocide and metal resistance genes database. Nucleic Acids Res 42(1):737–743

Peng Y, Leung HC, Yiu SM, Chin FY (2012) IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth. Bioinformatics 28(11):1420–1428

Rai PK, Lee SS, Zhang M, Tsang YF, Kim K-H (2019) Heavy metals in food crops: health risks, fate, mechanisms, and management. Environ Int 125:365–385

Rensing C, Mitra B, Rosen BP (1997) The zntA gene of Escherichia coli encodes a Zn(II)- translocating P-type ATPase. Proc Natl Acad Sci USA 94:14326–14331

Rensing C, Ghosh M, Rosen BP (1999) Families of soft-metal-ion-transporting ATPases. J Bacteriol 181:5891–5897

Rhee SK, Liu X, Wu L, Chong SC, Wan X, Zhou J (2004) Detection of genes involved in biodegradation and biotransformation in microbial com-munities by using 50-mer oligonucleotide microarrays. Appl Environ Microbiol 70(7):4303–4317

Salam LB (2018) Detection of carbohydrate-active enzymes and genes in a spent engine oil-perturbed agricultural soil. Bull Natl Res Cent 42:10

Salam LB, Ishaq A (2019) Biostimulation potentials of corn steep liquor in enhanced hydrocarbon degradation in chronically polluted soil. Biotech 9:46

Salam LB, Obayori OS (2019) Structural and functional metagenomics analyses of a tropical agricultural soil. Spanish J Soil Sci 9(1):1–23

Salam LB, Obayori OS, Raji SA (2015) Biodegradation of used engine oil by a methylotrophic bacterium, Methylobacterium Mesophilicum isolated from tropical hydrocarbon-contaminated soil. Petrol Sci Technol 33(2):186–195

Salam LB, Obayori OS, Nwaokorie FO, Suleiman A, Mustapha R (2017) Metagenomic insights into effects of spent engine oil perturbation on the microbial community composition and function in a tropical agricul-tural soil. Environ Sci Pollut Res 24:7139–7159

Salam LB, Ilori MO, Amund OO, LiiMien Y, Nojiri H (2018) Characterization of bacterial community structure in a hydrocarbon-contaminated tropical African soil. Environ Technol 39(7):939–951

Salam LB, Shomope H, Ummi Z, Bukar F (2019) Mercury contamination imposes structural shift on the microbial community of an agricultural soil. Bull Natl Res Cent 43:163

Satarug S, Baker JR, Reilly PE, Moore MR, Williams DJ (2001) Changes in zinc and copper homeostasis in human livers and kidneys associated with exposure to environmental cadmium. Hum Exp Toxicol 20:205–213

Page 19 of 19Salam et al. Bioresour. Bioprocess. (2020) 7:25

Scherer J, Nies DH (2009) CzcP is a novel efflux system contributing to transi-tion metal resistance in Cupriavidus metallidurans CH34. Mol Microbiol 73:601–621

Schloss PD, Westcott SL, Ryabin T et al (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Micro-biol 75(23):7537–7541

Steinnes E, Friedland AJ (2006) Metal contamination of natural surface soils from long-range atmospheric transport: existing and missing knowledge. Environ Rev 14:169–186

Stohs SJ, Bagchi D (1995) Oxidative mechanisms in the toxicity of metal-ions. Free Radic Biol Med 18:321–336

Stohs SJ, Bagchi D, Hassoun E, Bagchi M (2001) Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 18:321–336

Toppi LSD, Gabbrielli R (1999) Response to cadmium in higher plants. Environ Exp Bot 41(2):105–130

Törönen P, Medlar A, Holm L (2018) PANNZER2: a rapid functional annotation webserver. Nucleic Acids Res 46(W1):W84–W88

Trivedi P, Delgado-Baquerizo M, Anderson IC, Singh BK (2016) Response of soil properties and microbial communities to agriculture: implications for primary productivity and health indicators. Front Plant Sci 7:990

Valencia EY, Braz VS, Guzzo C, Marques MV (2013) Two RND proteins involved in heavy metal efflux in Caulobacter crescentus belong to separate clus-ters within proteobacteria. BMC Microbiol 13:79

Vallee BL, Ulmer DD (1972) Biochemical effects of mercury, cadmium and lead. Annu Rev Biochem 41:91–128

Waisberg M, Joseph P, Hale B, Beyersmann D (2003) Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicol 192(2–3):95–117

WHO/FAO (2001). Codex alimentarius commission. Food additives and contaminants. Joint FAO/WHO Food Standards Programme, ALINORM 10/12A

Wood DE, Salzberg SL (2014) Kraken: ultrafast metagenomic sequence clas-sification using exact alignments. Genome Biol 15(3):R46

Xiong A, Jayaswal RK (1998) Molecular characterization of a chromosomal determinant conferring resistance to zinc and cobalt ions in Staphylococ-cus aureus. J Bacteriol 180:4024–4029

Yazdankhah A, Moradi S, Amirmahmoodi S, Abbasian M, Shoja SE (2010) Enhanced sorption of cadmium ion on highly ordered nanoporous car-bon by using different surfactant modification. Microporous Mesoporous Mater 133:45–53

Yin C, Mueth N, Hulbert S (2017) Bacterial community on wheat grown under long-term conventional tillage and no-till in the Pacific Northwest of the United States. Phytobiomes J 1(2):83–90

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