309 metagenome assembled microbial genomes from deep sediment samples in the Gulfs of Kathiawar Peninsula

Marine microbiome is considered as the largest environment on earth which has many secrets concealed into it^1,2. Many marine microbes play a key role in biogeochemical cycles. However, high proportions of microbes remain uncultured in vitro³ and so instead of analysing the microbes individually, cultivation-independent genome-level characterization methods notably single-cell genomics and metagenomics are frequently being applied for microbiome analysis⁴. Amplicon sequencing based cultivation-independent studies are enriching the microbial diversity knowledge of various hitherto less studied environmental niche, specifically within the marine resources. However, amplicon analysis is just a preliminary step in metagenomics as it focuses only on one gene for the community diversity assessment.

With the view of studying the marine microbial community for determination of its composition in terms of diversity as well as function, whole metagenomics has become the preferred approach. Recently, it has been realized that the actual understanding of metagenomics data can be obtained by individual genome binning, which eventually also enhances the microbial genome database⁵. This requires use of various complex computational algorithms including those relying on previous data findings viz., the supervised classifiers and the unsupervised classifiers that rely on sequence specific features like the GC content, k-mer frequency and coverage estimation for binning the genomes. Most of the recently developed tools for binning include a combined approach of both the algorithms⁶. Binning aids in revealing the link between the potential functional genes in a given microbiome to its taxonomy.

The unique properties of the Gulfs of Kathiawar Peninsula like extreme tidal variations, different sediment texture and physicochemical variations make them an ideal place for studying the microbial diversity. Varied onshore anthropogenic activities may have imparted unique features to the microflora of the Gulfs. Study of microbial diversity and functions in the mentioned Gulfs have largely been focused on cultivation based approaches and very few molecular studies have been conducted on the shore sediments. Additionally, the presence of several on-shore industries like fertilizer, chemicals, oil refineries, power plants and ASSBRY (Alang Ship Breaking Yard) may have also influenced the deeper sediment microbiome leading to their variable gene profile⁷. Our previous insights into the pelagic sediment resistome profile by metagenomics approach have shown that the deeper sediments, earlier thought to be primeval are actually hosting microbes with a concerning number of resistance genes^7,8. This acted as a propeller to the present study wherein we tried to look deeper into the metagenomics data of the samples collected from the Gulfs of Kathiawar Peninsula and a sample from the Arabian Sea by sorting individual prokaryoplankton genomes from the data using the binning approach.

We successfully reconstructed 309 Metagenome Assembled Genomes (MAGs) from the nine sediment metagenomics sequences (Table 1) from Gulf of Khambhat (GOC), Gulf of Kutch (GOK) and Arabian Sea (A) by differential coverage approach and considering the GC percent and tetranucleotide frequencies. Out of the 309 MAGs, 39 were archaeal genomes (Online-only Table 1) and 270 were bacterial genomes (Online-only Table 2). Seventy-one were high quality drafts with a completeness of ≥90% and contamination <10%, 120 were medium quality (completeness: 70–90%, contamination: <10%) and the remaining 118 were draft genomes with a final completeness of >50%. The distribution of the bins as per the MIMAG quality standards⁹ is described in Table 2. To the best of our knowledge, this is the first report of multiple MAGs from the studied sites.

Single nucleotide polymorphisms were correlated to quality of bins to understand the influence of strain heterogeneity on the fragmentation of the MAGs (Fig. 1). Phylogenomic analysis revealed that the archaeal populations were quite different in two Gulfs, with GOC bins (n = 15) encompassing 3 major phyla: Thaumarchaeota and Aenigmarchaeota from the DPANN superphylum andBathyarchaeota. The GOK genomes (n = 24) were falling under the Bathyarchaeota, Thaumarchaeota, Euryarchaeota and the Korarchaeota phyla (Figs. 2 and 3). Based on the community profile assessment of the samples by considering all the reads, the above mentioned archaeal phyla represented <3% of the total microbial population at each sample site. Majority of the phyla were those reported earlier in the marine and estuarine environments, with most having few or no cultured representatives^10,11. The observed genomes like Thaumarchaeota have been reported to be nitrifiers in the sediment niche, thus, the insights into their gene content will provide details on the functional significance of the archaea in the respective sample site. Genomes from Thaumarchaeota were recovered from both the sites (Fig. 2). Nevertheless, the difference in the populations observed in two Gulfs can also be studied based on the predicted roles of the genomes and correlation with the niche properties.

Among the bacterial members, five phyla were commonly observed between the Gulfs viz., Proteobacteria, Zixibacteria, Gemmatimonadetes, Dadabacteria and Planctomycetes (Figs. 2 and 4). Among the common bacterial phyla, Proteobacteria majorly comprised of Gammaproteobacteria members which are the most abundant reported bacteria in the marine sediments and have been reported to perform versatile roles including metabolite production, hydrocarbon degradation, acetate assimilation and many more^12,13. Zixibacteria and Dadabacteria MAGs have been reported from marine environments as an evolutionary phyla and these have been observed to play role in the nutrient cycling of the niche^14,15. Apart from these, few genomes in GOC encompassed Bacteroidetes, FCB superphylum, Armatimonadates, Acidobacteria, Chloroflexi and Aminicenantes phyla; while those in GOK were falling under Actinobacteria, KSB1, Saccharibacteria (TM7), Nitrospinae, Caldithrix, Verrucomicrobia and Balneolaeota. Species belonging to Nitrospinae are reported to be exclusively abundant in marine niche, where they play a role in nitrite oxidization, as well as these are ubiquitously observed in sites demanding thermoprotection^16,17. Community profiling of the samples by considering all the reads revealed that the MAGs identified within Proteobacteria (>40%) and Chloroflexi (~15%) phyla represented a substantial population, while rest of the MAGs corresponded to 0.01% to 5% of the total microbial community at each sample site (details in Supplementary Table 1a and b).

The genomic bins described here would prominently enhance the repertoire of microbial genomic information from the Gulfs of Kathiawar Peninsula. It will also provide the insights for better understanding the effects of on-shore activities on the microbiome of deeper sediment in the Gulfs. In the long term, the data will fortify further applications of the genomic information for 1) understanding the microbes involved in the marine nutrient cycling, 2) open gates for bioprospection of novel thermophilic and halophilic enzymes and 3) allow understanding of microbial-host and microbial-niche interactions as the phylum distribution reflects the variability across the 2 Gulfs under study.

Source: Ecology - nature.com