Effects of cadmium perturbation on the microbial community structure and heavy metal resistome of a tropical agricultural soil

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 physicochemical 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%), Actinobacteria (35.02%), Prevotella (6.93%), and Conexibacter woesei (8.93%) in SL4, and Proteobacteria (50.50%), Alphaproteobacteria (22.28%), Methylobacterium (9.14%), and Methylobacterium radiotolerans (12,80%) in SL5, respectively. Statistically 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.

Elevated Cd concentration in soil poses significant threat to the quantity and diversity of soil microorganisms. Cd toxicity to microbial cells is believed to be due to depletion of glutathione and sulfhydryl groups in proteins, 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;Fortuniak 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 implicated in destruction of plant mitochondria as well as disruption 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(Nies , 2003Hu et al. 2005). In Cd resistance, three families of efflux transporters are deployed by microorganisms. 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) transporters, which act as cation-proton antiporters Nies 1995;Hassan et al. 1999); and the cation diffusion facilitator (CDF) transporters, which act as chemiosmotic 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-independent 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 metagenomic 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 microbial existence and the interplay between diverse genes and processes produced and marshalled by members of the microbial community to counteract various environmental stressors. This exciting approach have been used to decipher the microbial community structure and function of diverse polluted and pristine soils (Salam et al. 2017Feng et al. 2018;).
In recent time, attempts have been made to use nextgeneration 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 microbial community structure and heavy metal resistome of a tropical agricultural soil.

Sampling site description
Soil samples were collected from an agricultural farm in Ilorin, Kwara State, Nigeria. The coordinates of the sampling 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, cassava, 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 metal
Cadmium chloride (CdCl 2 ), 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 analysis
Soil 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 composite sampling were passed through a 2-mm mesh sieve. Sieved soils were made homogenous by thorough mixing 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 soil amended with 250 mg CdCl 2 , respectively. The two setups (in triplicates) were incubated at room temperature 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 determined by flame photometry (Flame photometer model PFP-7, Buck Scientific Inc, USA) method while phosphorus 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 metagenomics
Total DNA used for metagenomic analysis was extracted directly from the two soil microcosms, SL4 and SL5. To unravel the microbial community structure of the agricultural 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 microbial 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 manufacturer'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 Illumina Nextera XT sample processing kit and sequenced on a MiSeq. The protocols for total DNA preparation for Illumina shotgun sequencing were as described previously (Salam 2018;Salam and Ishaq 2019).

Processing of fastq raw reads, quality control, assembly and taxonomic classification
Processing and quality control of fastq raw reads, assembly 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 taxonomic 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 readbased taxonomic classification of the metagenomes due to the depth and accurateness of its database. Contigbased 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 reads
Sequence reads generated from each of the metagenome 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 (Noguchi 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 annotation 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 metalresistance genes in the query sequences. A modified stand-alone version of the BLAST program (NCBI, version 2.2.2) implemented in the BacMet web server was used for similarity searches against the BacMet sequence databases.

Statistical analysis
The effects of Cd contamination on the soil physicochemistry and the microbial community structure was statistically analysed using the t test tool in the Analysis ToolPak of Microsoft Excel 2013 software.

Physicochemical properties and heavy metals content
The physicochemical properties and heavy metal content of the agricultural soil (SL4) and cadmium-contaminated 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 moisture 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 physicochemical parameters of the two metagenomes revealed that the difference is statistically significant (P < 0.05; P = 0.036). In addition, significant traces of heavy metals 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 agricultural soil are considerably low, high concentrations of zinc, iron, copper, and chromium were detected in the agricultural soil SL4. However, apart from Cd, the concentrations of the heavy metals substantially decrease in SL5 (Table 1).
Contig-based classification of the metagenomes (SL4 and SL5) conducted by aligning the SL4 and SL5 contigs to NCBI's RefSeq database using the BWA-mem aligner is indicated in Additional file 1: Figs. S3 to S8.
Functional annotation of the predicted genes in SL4 and SL5 metagenomes for heavy metals resistance genes using the BacMet database revealed interesting findings. 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  (Table 3).

Discussion
Point and non-point release of heavy metals and metalloids into soil environments via atmospheric deposition and diverse agricultural activities have negatively impacted soil ecological balance, alter soil physicochemistry and biogeochemistry, reduce soil microbial diversity and pose serious health risk to animals and humans (Feng et al. 2018;Rai et al. 2019;. In this study, all the physicochemical parameters considerably reduce in Cd-amended SL5 microcosm, though not as profound as those reported in our previous study on mercury ). 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(Gray et al. , 1999 ATCC Salam 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 agricultural 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 important heavy metals such as zinc, copper, iron and chromium are tightly linked to the metabolic functioning of soil biota as they are essential micronutrients required by most microorganisms, which possibly cause their reduction (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(Nies , 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 recalcitrant to various environmental stressors, and secretion of avalanche of enzymes, which degrade various macromolecules 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 morphological, physiological, and metabolic properties. These properties facilitate their preponderance in soils with various environmental conditions (Aislabie and Deslippe 2013; Montecchia et al. 2015;. 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 reactive oxygen species, and displacement of Zn and Fe ions from metalloproteins, resulting in their inactivation (Vallee 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, members of the genus Methylobacterium are reputed to be widely distributed in diverse environmental compartments 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 arsenic 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 resistance 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 community despite the heavy metals stress indicates the presence of resistance systems that tightly control intracellular concentrations of the heavy metal ions and their attendant toxicities (Nies 1999(Nies , 2003Hu et al. 2005).
One of the toxic effects of Cd is that it causes oxidative stress by depleting glutathione and protein-bound sulfhydryl 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 superoxide 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 previous reports have averred that the greatest induction of Mn superoxide dismutase (sodA) occurred under Cd and chromium stress, while induction of Fe superoxide dismutase (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

Conclusions
In summary, Illumina shotgun metagenomics and analysis 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 environmentally friendly methods for pest and herbage control and to improve crop yield is becoming more profound, due to the negative impacts of current agricultural practices on the general wellbeing of the soil ecosystem and its inhabitants.