Screening of the efficient strain
To confirm the type of rock used for the efficient strain screening, we analyzed the composition of the selected rock samples. The results revealed the percentages of potassium feldspar, quartz, and mica in isolated rock samples were 52%, 36%, and 10% by mass, respectively. The rock mineral sheers analysis showed that SiO2, Al2O3, and K2O accounted for 73.8%, 13.4%, and 9.9% by mass, respectively, which proved that the selected rock samples are silicate rocks.
Studies have proved the interaction between the IAA producing bacteria and plants has various applications to the plants ranging from pathogenesis to the phyto-stimulation (Spaepen et al. 2007). The siderophore secreted by bacteria has been proved to have a variety of vital capacities (Crosa and Walsh 2002). For example, it can inhibit the reproduction of pathogenic microorganisms causing plant diseases (Sahu and Sindhu 2011), chelate metal ions (Neilands 1995), and promote mineral decomposition (Neubauer et al. 2002). Therefore, the rock-dissolving abilities of 22 different microbial isolates were considered for further studies which were evaluated for their ability to produce IAA and siderophore (Additional file 1: Table S1). The highly efficient strains producing IAA more than 10 mg L−1 (Arancon et al. 2004) and exhibiting strong siderophore productivity (+ + +) (A/Ar: 0–0.6) were considered for further studies (Hesse et al. 2017). Based on the IAA and siderophore producing abilities, ten bacterial strains NLX-1, 3, 4, 7, 12, 14, 17, 18, 19 and 22 were further considered for the rock-dissolution experiments. The rock-dissolving capacity of a microbial strain is directly proportional to the concentration of silicon released from the rocks (in the form of SiO2 in the fermentation broth). The rock-dissolving ability was calculated by finding the difference between the experimental and control groups, respectively (Fig. 1a). The NLX-4 strain exhibited the significant advantageous effect in the release of Si (P < 0.001). It was reported that the Pseudomonas sp. strains were among these typical bacteria which produce both the IAA and siderophores (Bano and Musarrat 2003; Gupta et al. 2002; Rajkumar et al. 2005). It also showed that the release of Si from rock by NLX-4 increased by 7.51 mg L−1 compared to control after 7 days of cultivation, which was remarkably higher than the amount of Si-releasing induced by reported Pseudomonas strains (Maurice et al. 2001). Thus, we have selected NLX-4 strain for further genomic studies.
Identification of silicate rock-dissolving strain NLX-4
The standard morphological experiments of NLX-4 strain showed NLX-4 strain is a rod-shaped Gram-negative bacterium with single flagella (Additional file 1: Fig. S2a). To further identify the NLX-4 strain, sequenced 16S rRNA gene of NLX-4 strain was submitted to NCBI GenBank with an accession number KX379232 (Additional file 1: Fig. S2b). Results obtained from BLAST and the phylogenetic tree analysis endorsed that NLX-4 strain belonged to the Pseudomonas genus (Additional file 1: Fig. S2). Based on the results of the morphological and phylogenetic analysis, NLX-4 strain was identified as a Pseudomonas sp. strain.
Effects of Pseudomonas sp. NLX-4 on rock-dissolution
The final concentrations of K, Al, and Si released from silicate rock samples by NLX-4 strain showed an increasing trend, while the particle diameter variation of rock samples increased significantly in 0 to 15 days cultures followed by a slight decline in the PDV values in the later time periods, respectively (Fig. 1b). We have observed a gradual increase in release of elements from 2nd to 30th day cultures of NLX-4 strain, respectively (Fig. 1b). The concentrations of K, Al and Si (mg L−1) elements released by NLX-4 reached a maximum by the 30th day with 73.27 mg L−1, 62.43 mg L−1, and 26.50 mg L−1, respectively. The highest value of PDV was observed on 15th day cultures of NLX-4 (2.18 mm), which later reduced to 1.60 mm by 30th day, respectively (Fig. 1b).
To explore the effective components of NLX-4 strain promoting the rock dissolution, we have analyzed and compared the final concentrations of exopolysaccharides, amino acids, and organic acids secreted in fermentation broth (Additional file 1: Table S2). Pearson analysis was used to assess the correlation between PDV, element concentrations, EPS, amino acids, and organic acids, respectively (Fig. 1c). Apparently, PDV of rock samples has highly significant correlations (P < 0.001) with the release of K, Al, Si (Fig. 1c). Meanwhile, EPS, amino acids, and all organic acids exhibited highly significant correlations (P < 0.001) with PDV, with malic acid being the most relevant factor (Fig. 1c). In addition, EPS, amino acids, tartaric acid, and malic acid showed highly significant correlations (P < 0.001) with the release of three elements (Fig. 1c). In the study conducted by Welch and Vandevivere (1994), EPS did not affect mineral dissolution when it worked alone. However, EPS freshly secreted by bacteria appeared the capacity to accelerate the process of mineral dissolution by forming metal–organic complexes at mineral surface to weak the metal–oxygen bond in its structure (Tourney and Ngwenya 2014). During the process of rock dissolution, the EPS and amino acids produced by bacterial strain were proven to have the capacity to destroy the crystal structure of rocks, which attains strength in the acidic environments (Braissant et al. 2003). The organic acids secreted by a bacterial strain not only create favorable acidic environmental conditions for the dissolution of the rock, but also have advantages over inorganic acids to dissolve rock due to their complexation with the cations in the mineral crystal lattice of rocks (Wu et al. 2017b; c). In this study, the oxalic acid might show more complexation than acid solubilizing capacity to release K, Al, and Si, while the other three acids had different degrees of complexation with these elements. Effective and persistent microbial mineral bioweathering is dependent on bacterial acid production and tolerance of bacteria. A recent study conducted on Pseudomonas azotoformans F77 has revealed the silicate mineral weathering and acid tolerance capacities by culturing it on biotite (silicate mineral) (Li et al. 2021). Yuan-Li et al. also has revealed the genome, transcriptome and genetics of P. azotoformans F77 strain, explained the molecular mechanisms underlying the silicate mineral bioweathering and acid tolerance by deleting the genes involved in gluconic acid metabolism and acid tolerance mechanisms (Li et al. 2021). This study has reported that genes involved in acid production and tolerance were highly expressed when cultured on biotite.
Sequencing and assembly of NLX-4 strain
The genome of NLX-4 strain was sequenced and de novo assembled by implementing a hybrid approach involving PacBio RSII and Illumina HiSeq 4000 sequencing systems. The quality control analysis on the raw read sequences has resulted in 713,706, 632 base pairs of clean sequence data and total of 79,379 sequences with a mean read length of 8991 bp (Additional file 1: Table S4). Thus, obtained filtered subreads were further subjected to de novo assembly using RS_HGAP Assembly v3 in SMRT® analyser v2.3.0 software. The assembly of the PacBio sequence data was performed using the soapSNP and soapIndel by using the Illumina HiSeq 4000 sequence data.
The sheared genomic DNA of NLX-4 was also subjected to Illumina HiSeq 4000 sequencing system by constructing a 300-bp insert library with 2 × 100 bp read length. The Illumina sequencing has resulted in 904 Mb of raw data with an adapter and duplication percentages to be 0.23 and 8.03, respectively, and total reads were 9,049,948 (Additional file 1: Table S4 and S6b). The quality control analysis has resulted in clean data of 817 Mb of sequence data with filtered reads and low-quality filtered reads percentages were 9.68 and 1.39, respectively (Additional file 1: Table S4 and S6b). The final NLX-4 genome sequence assembly resulted in single chromosome with 0 gaps, single base quality of 1, structure base and reads usage percentages were 0.9975 and 0.9915, respectively, at a genome depth of 40.75 and genome size of 6.52 Mb (Additional file 1: Table S5a). The gene prediction of the NLX-4 genome sequence was performed using the Glimmer v3.0.2 and the whole-genome sequence was analyzed for the annotations using BLAST with non-redundant protein database (NR), Swiss-Prot, TrEMBL, COG, KEGG, InterPro and GO databases. The genome was also analyzed using the following servers RNAmmer, tRNAscan, Rfam, IslandPath-DIOM, SIGI-HMM, IslandPickerp and CRISPR Finder servers, respectively (Additional file 1: Fig. S3–S5).
The genome of Pseudomonas NLX-4 strain is a single circular chromosome with 6.7 Mb (6,771,445 bp) length and 63.21% of G + C content (Additional file 1: Table S6b). The genome contains 6,239 protein coding genes with an average length; internal length and internal GC content were 949 bp, 852,628 bp and 56.55%, respectively (Additional file 1: Table S5b). It also contains 72 tRNA genes, 19 rRNA genes and 17 sRNA genes. The NLX-4 genome has also showed sequencers for an intact prophage sequence with a length of 39,233 bp (start: 5,375,574, end: 5,414,806) with a GC% of 61.36. The results from Tandem Repeat Finder software shows that the genome contains a total of 192 tandem repeat fragments (TRF), 90 minisatellite and 36 microsatellite DNA sequences with total lengths of 35,083 bp, 4,597 bp and 1380 bp, respectively (Additional file 1: Table S5d). Total of four CRISPR (ID-1, 2, 3 and 4) spacer sequences with a sequence length ranging between 34, 35 40 and 48, respectively, and gene Island analysis has resulted in 54 gene islands (Additional file 1: Table S5e).
To benefit the further study on microbial weathering, a whole genomic sequencing was also conducted in our research. The whole genome was annotated using BLAST annotation pipeline by searching and retrieving the genome-wide annotations from different annotation databases such as prokaryotic orthologous groups (COG), gene ontology (GO), InterPro, KEGG, BLAST-NR, Swiss-Prot databases, respectively. The annotated genome of NLX-4 strain contains a total of 6041 (96.82%) protein coding gene sequences, with 5045 (80.86%) COG, 3996 (64.04%) GO, 5342 (85.62%) InterPro, 4386 (70.29%) KEGG, 6038 (96.77%) BLAST-NR and 3897 (62.46%) Swiss-Prot, respectively (Additional file 1: Table S5f). The COG has classified the protein encoding genes into 25%-cellular signaling and processing, 17%-information storage and processing, 42%-metabolism and 16%-poorly characterized, respectively (Fig. 2). The GO has classified protein coding genes into 55%-biological process, 16%-cellular process and 29%-molecular function, respectively (Fig. 3). The KEGG database has divided protein coding genes into 52%-metabolism, 6%-organismal systems, 5%-cellular processes, 14%-environmental information processing, 10%-genetic information and processing and 13%-human diseases, respectively (Fig. 3). Genes encoding for transcription regulator HTH (LysR), ABC transporter like, Signal transduction response regulator ABC transporter (Metl-like), GCN5-N-acetyltransferase (GNAT), PAS domain, major facilitator superfamily, homeodomain-like, histidine kinase, short-chain dehydrogenase, DNA-binding HTH, EamA domain, TonB-dependent receptor proteins and other genes were found to occur in multiple copies in the NLX-4 genome (Fig. 2).
Rock-dissolution molecular mechanisms and genes
In NLX-4 genome, 2124 genes exhibit catalytic activity, 29 genes exhibit antioxidant activity, 1595 genes exhibit binding activity (Fig. 3). Genome of NLX-4 encodes genes involved in important metabolic pathways with 819-amino acid metabolism pathways, 83-biosynthesis of secondary metabolites, 732-carbohydrate metabolism, 399-energy metabolism, 270-glycan biosynthesis and metabolism, 161-metabolism of other amino acids, 194-metabolism of terpenoids and polyketides, 480-xenobiotic biodegradation and metabolism, respectively. The prokaryotic orthologous group classification of NLX-4 genome contains 311 genes in energy production and conversion, 642-amino acid transport and metabolism, 315-carbohydrate transport and metabolism, 183-secondary metabolite biosynthesis, transport and metabolism, respectively (Fig. 3). Metabolically, some of the important genes include siderophore encoding genes (NZ1GL004767; NZ1GL000887; NZ1GL005349), polysaccharide biosynthesis protein (NZ1GL001889), nitric oxide dioxygenase (NZ1GL001246) and other proteins which are involved in the biosynthesis of organic acids, amino acids and secondary metabolites required for the dissolution of rocky material.
Transcriptome of NLX-4
The sequencing has resulted in a total of 143 million reads with each sample the number of reads varied between 23 to 24 million reads, respectively (Additional file 1: Table S7a and b). The quality control analysis was performed to discard the low-quality reads and obtain clean reads; these clean reads were further mapped both to the gene and genome of NLX-4 strain obtained above. The RNA sequence data of six samples (control and treatment (NLX-4) in triplicates) were aligned to the reference genome using SOAPv2.0. The sequence reads were aligned to the NLX-4 genome and non-ribosomal reads which did not align uniquely with the NLX-4 genomic sequences were removed. Total number of rRNA reads mapped to the NLX-4 reference genome varied from 0.10% to 1.51%, respectively. The alignment results obtained from gene and genome level are reported in Additional file 1: Table S7a and b, respectively. On average 90.53% of transcripts were found to be mapped to the genome of NLX-4, which demonstrates the suitability of using RNA sequencing data for further studies.
We have observed that in control samples the total number of rRNA reads count were slightly higher than the treatment samples, which might be due to the insignificantly higher rRNA to mRNA ratios in control samples. The present genome-wide transcriptome study of NLX-4 completely covered the majority of all the genes with more than one read. Higher reproducibility was observed among the biological replicates, they have similar number of total and the mapped reads (Additional file 1: Fig. S11–S13). The mapped reads were further subjected to a series of analysis including sequence assessment, biological contextualization, gene structure refinement, alternative splicing, novel transcript detection and SNP analysis (Additional file 1: Fig. S14). Transcriptome analysis conducted by edgeR, limma and Glimma packages showed that the strain NLX-4 cultured with/without silicate rocks resulted in 539 (288-up and 251-down) DEGs. Those DEGs were further analyzed for its biological contextualization using GO and KEGG pathway enrichment analysis (Additional file 1: Fig. S15).
Differentially expressed genes
As the bacteria were previously reported to release nutrients from rocks for their growth (Alahari and Apte 2004), the rocks using as a kind of nutritional source were considered to have its influence on the metabolic process of bacteria (Xiao et al. 2012a). Compared to the samples composed of soluble K (KCl) as potassium source, the NLX-4 strain using K-bearing silicate rock as potassium source has resulted in higher EPS, amino acids, and organic acids. These differences illustrated that the interaction of NLX-4 strain and rock to release nutrients for bacterial growth significantly accelerated the process of rock dissolution (Wu et al. 2017c). The RPKM values obtained from the quantification of the control and treatment samples were used for finding the significant DEG. We have followed two different approaches for finding the DEGs among the samples (a) using the Audic Claverie method, (b) using a customized pipeline implementing edgeR, limma and Glimma packages. The results obtained from both the approaches have given us almost the same list of DEGs. Genes encoding for nitric oxide dioxygenase, flagellin, glutathione-S-transferase (GST), chaperonin GroES, heat shock protein HSP10, elongation factor prokaryotic, bacterioferritin, polysaccharide biosynthesis protein Epsc, DNA-methyl transferase, flagellar FliS, FlaG, FlgN protein, diguanylate cyclase, large ribosomal protein L7 and two-component system-NARXL were highly expressed with a fold change values log > 2.5 (Fig. 4 and Additional file 1: Fig. S15). While the genes encoding for fatty acyl-CoA synthase, sulfur dioxygenase, DNA polymerase III, acetyltransferase, cation symporter, sulfide: quinone oxidoreductase, alcohol dehydrogenase, pyrroloquinoline quinone biosynthesis protein B, hydrogen cyanide synthase (HcnB), CBS domain, cytochrome c peroxidase, polar amino acid transport system, cytochrome c oxidase and nitrate reductases were down-regulated in the treatment samples with a fold change value of log < 2.5, respectively (Fig. 4 and Additional file 1: Fig. S15). We have also analyzed the enrichment of the DEGs in biological pathways using KEGG pathways and GO analysis, respectively (Fig. 4 and Additional file 1: Fig. S15). RT-qPCR was used to further validate the expression level of genes identified in Illumina sequencing analysis. A total of 5 DEGs, encoding for basic amino acid, nitric oxide dioxygenase, GST, bacterioferritin, and polysaccharide biosynthesis protein, with relatively higher gene expressions were chosen as candidates for the RT-qPCR study (Additional file 1: Table S6a). These 5 DEGs showed a consistent pattern between Illumina sequencing results and RT-qPCR (Additional file 1: Fig. S17), which indicated that the transcriptome data are reliable and accurate.
As has been already known, a total of 15 genes involved in the regulations of
ionophore transportation, EPS and amino acids synthesis, and organic acids metabolism were highly expressed with a fold change values log > 2.5. The bacterioferritin encoding gene (involved in ionophore and transportation of siderophore) (Carrondo 2003), was highly up-regulated in the tested conditions.
The gene ontology analysis of the DEGs showed that majority of these gene products are mainly involved in the process of protein translation, metabolic, and cellular macromolecule biosynthesis (Fig. 4). This study also proposes the involvement of EPS, amino acids and organic acid are associated with various factors playing crucial roles in protein translation, metabolic, and cellular macromolecule biosynthetic, such as elongation factors and large ribosomal protein L7 processes. In addition, the oxidoreductases such as nitric oxide dioxygenase were also found to regulate the amino acids and organic acid metabolism reactions (Litvinova et al. 2015). The up-regulation of genes involved in macromolecular assembly of extracellular proteins, polysaccharides, and glycoproteins, might be involved in creating a better weathering microenvironment during (a) bacterial attachment to rock particles; (b) formation of the bacterial–rock aggregates. Genes encoding for flagellin, polysaccharide biosynthesis protein Epsc, flagellar FliS, FlaG, FlgN protein, diguanylate cyclase (Chen and Schaap 2012) endorses the above functions. The gene encoding for DNA-methyl transferase with a function inducing the lipopolysaccharide, was found to be highly up-regulated (Zhang et al. 2013) and these results also confirms the existence of polysaccharides in the environment (Fig. 4). Recent study conducted by Picard et al. 2021, have reported about a novel pyrroloquinoline quinone (PQQ) cofactor independent glucose dehydrogenase enzyme involved in production of gluconic acid which is involved in mineral bioweathering of biotite (Picard et al. 2021). Picard et al. have conducted a genome analysis study on Collimonas pratensis strain PMB3 strain and developed three mutant strains which did not produce gluconic acid which has revealed that the mutant strains exhibited low mineral weathering abilities compared to wild type (Picard et al. 2021). Genes encoding for glucose dehydrogenase (GDH) and glucose/methanol/choline oxidoreductases were highly expressed in the NLX4-strain samples cultured on rock samples. Similarly, quinoprotein glucose dehydrogenase (gcd) genes were found to be differentially expressed among the datasets. Recent study conducted by Wang et al. (2020) on Pseudomonas azotoformans F77 strain and its bioweathering capacity of biotite (abundant mineral identified in acidic soil) (Wang et.al. 2020). This study has revealed the importance and involvement of gcd gene (involved in gluconic acid metabolism) and adh gene (involved in pilus formation) in biotite mineral weathering.
Glutathione (GSH) is essential for the cells to adapt to the deficiency of K+ deficiency by acting as an important antioxidant and detoxifying agent in cells (Wang et al. 2015). GST has the function to catalyze the conjugation of glutathione with a wide variety of electrophiles (Beckett and Hayes 1993). In the process of rock dissolving, various elements in the crystal lattice of rocks are released. Apart from cell biogenic elements, some heavy metal elements and impurities also enter into the bacterial cells as exogenous substances, which generate toxic effects such as inducing oxidative stress (Sun et al. 2013). Therefore, Pseudomonas sp. NLX-4 performs cell detoxification by up-regulating the expression of GST genes, and homeostasis in its cells can be maintained to overcome the harsh environment. GST has been found up-regulated in the bio-dissolving of K-bearing mineral affected by Aspergillus fumigatus (Xiao et al. 2012b), which is consistent with the results of this study. Furthermore, two-component system-NARXL in bacteria, which referred to numerous sensory-response circuits operate by making use of a phosphorylation control mechanism (Kobayashi et al. 2016), is also helpful for bacteria to be masters at adapting and coordinating cellular events to accommodate adverse conditions.
Moreover, as K+ is an essential element for the metabolism of bacteria because of its indispensability for synthesis of proteins and enzymes, Pseudomonas sp. NLX-4 may first cause incorrect folding of proteins and enzymes without K+ (Fresht et al. 1992). Therefore, amounts of mis-/un-folded proteins will accumulate in the cell. In the case of K+ deficiency, Pseudomonas sp. NLX-4 launched a stress response to up-regulate expression of chaperonin (GroES) and heat shock protein (HSP10), which played an important role in protein folding (Maleki et al. 2016).
Global expression of novel transcripts and sRNA
The novel transcripts expressed differentially among the control and treatment samples were also analyzed using the customized pipeline using edgeR, limma and Glimma packages. The RNA sequencing has resulted in total of 9681(CK-1), 9931(CK-2), 9424 (CK-3), 10,010 (Treatment-1), 11,470 (Treatment-2) and 11,695 (Treatment-3) transcripts, respectively. Out of which, 208, 229, 215, 186, 222 and 230 novel transcripts encoded for the coding regions and 9473, 9702, 9209, 9824, 11,248 and 11,465 novel transcripts encoded for the non-coding regions, respectively (Additional file 1: Fig. S18). Results obtained from the differential expression results have showed that a total of 847 novel transcript encoding genes were highly up-regulated with ≥ 2.0-fold change values and 169 novel transcript encoding genes were down regulated with ≤ 2.0-fold change values, respectively. Novel transcripts encoding for succinate dehydrogenase/fumarate reductase (TU1181), uncharacterized MFS-type transporter YbfB (TU1209), DNAJ domain (TU1447) were highly up-regulated and transcripts encoding for domain of unknown function (TU263), GGDEF domain (TU882), glucose-6-phosphate dehydrogenase (TU798), aldo/keto reductase (TU209), P-loop nucleoside triphosphate hydrolase (TU427), polyhydroxyalkanoic acid protein system (TU1525), HTH ArsR-type DNA-binding domain (TU955), thioesterase (TU1556), cytidine and deoxycytidylate deaminase (TU331), ABC-type transporter (TU1129), metal-sensitive transcriptional repressor (TU957), DNA polymerase (TU967), acyl-CoA dehydrogenase (TU1534), GNAT domain (TU315), alcohol dehydrogenase (TU1632), MarR-type HTH domain (TU710) and WD40 repeat (TU879) transcripts were down-regulated.
In the analysis of novel transcripts, succinate dehydrogenase is found out as one of the key enzymes in the pathway tricarboxylic acid cycle, which is part of carbohydrate metabolism (Araujo et al. 2012), and catalyzes the reaction of succinic acid converting to tartaric acid (Pan et al. 2010). Uncharacterized MFS-type transporter YbfB is involved in the transmembrane transport (Aminov et al. 2002), which is related to the secretion of bacterial secondary metabolites (ionophore, EPS, amino acids, and organic acids, et al.). DNAJ domain is involved in protein folding in the cell by acting as a co-molecular chaperone under environmental stresses (Bascos 2008). Therefore, combining with the analysis results of novel transcripts indicating the genes encoding succinate dehydrogenase/fumarate reductase (TU1181), uncharacterized MFS-type transporter YbfB (TU1209), DNAJ domain (TU1447) were highly up up-regulated, our opinion is that the essential elements bacteria need caused rock microbial-dissolving and the EPS, amino acids, organic acids secreted by bacterial strain accelerate rock dissolving were further confirmed.
Overall, a major disadvantage in current external-soil spray seeding technology is that the soil substrates used in these eco-restoration technologies showed poor rock interface fusion. Our present study displays a great potential to overcome these disadvantages by employing multifunctional bacteria such as Pseudomonas sp. NLX-4 strain to improve the interactions between the soil substrates and rock samples. Moreover, our study lays a firm foundation to further explore the deeper molecular mechanisms of rock dissolution by identifying the DEGs involved in this process.
Limitations of the study
Although we have isolated and screened out the efficient bacterium Pseudomonas sp. NLX-4 that can be applied for improving the current external-soil spray seeding technology, all experiments were completed under the optimal experimental conditions. Therefore, a field experiment combining Pseudomonas sp. NLX-4 with external-soil spray seeding technology should be conducted to verify the effect of this strain in the natural environment of the rock mining site. Furthermore, proteome sequencing will be a valuable tool to further research and explain the relationship between genes, pathways, proteins and metabolites, so that the molecular mechanisms could be fully revealed. In any case, the role of Pseudomonas sp. NLX-4 and its selected genetic resources in the improvement of external-soil spray seeding technology is worth exploring.