Genome and Transcriptome Sequencing of Novel Pseudomonas sp. NLX-4 Strain Involved in Bio-Restoration of Over Exploited Mining Sites


 Microbial weathering processes can significantly promote soil properties and enhance rock to soil ratio. Some soil-inhabiting bacteria exhibit efficient rock-dissolution abilities by releasing organic acids and other chemical elements from the silicate rocks. However, our understanding of the molecular mechanisms involved during bacterial rock-dissolution is still limited. In this study, we performed silicate rock-dissolution experiments on a novel Pseudomonas sp. NLX-4 strain isolated from an over-exploited mining site. The results revealed that Pseudomonas sp. NLX-4 strain efficiently accelerates the dissolution of silicate rocks by secreting amino acids, exopolysaccharides, and organic acids. Through employing genome and transcriptome sequencing (RNA-seq), we identified the major regulatory genes. Specifically, fifteen differentially expressed genes (DEGs) encoding for siderophore transport, EPS and amino acids synthesis, organic acids metabolism, and bacterial resistance to adverse environmental conditions were highly up-regulated in silicate rock cultures of NLX-4 strain. Our study proposes a novel bacterial based approach for improving the ecological restoration of over-exploited rock mining-sites.


Introduction
About 90% of the earth crust is composed of silicate minerals and these silicates are building blocks of most rock types (Prothero and Schwab, 2004). Human encroachments of earth surface in the form of mining have severely destroyed the ecology of the mining areas, causing soil erosion, exposure of bedrock and loss of productivity (Comino et al., 2016;Erkossa et al., 2015;Ochoa-Cueva et al., 2015). The area of soil and water loss in mining sites caused by overexploitation is increasing at a rate of 1.5×10 4 km 2 yr −1 , and more than 20% of these areas have been classi ed as rocky deserti cation (Balland-Bolou-Bi and Poszwa, 2012). Studies were being conducted to resolve the environmental destruction of these mining sites by developing e cient ecological restoration technologies (Jeffries et al., 2003). Spraying material on the surface of exposed rocks supplemented with plant seeds, soil and nutrients, which is known as external-soil spray seeding technology, is one of the most e cient and highly implemented approaches in the ecological restoration of mining areas (Russell, 2002). However, the current spraying material cannot break down rocks to continuously provide essential nutrients for plant growth. Therefore, soil formation for sustainable growth of plant is the key to solve the problem in long-term maintenance of external-soil spray seeding technology.
Microorganisms are widely present in natural environments. They break down the organic and inorganic compounds, and release them back into the environment as metabolic compounds (Bouwer and Zehnder, 1993). This process is known as bioweathering that promotes the rock-to-soil transition and improves the soil environment for plant growth (Gleeson et al., 2006;Vandevivere et al., 1994). Studies have reported that microorganisms accelerate the process of bioweathering through chemical dissolution, formation of rock-bio lm and chelating reactions (Finlay et al., 2009;Lian et al., 2008). Various microbial proteins have been proven that play vital roles in the bioweathering of rocks. For example, the bacterial membrane transport/channel proteins are crucial in increasing the absorption and transportation rate of mineral elements during the process of rock dissolution (Chen et al., 2008). Conversely, the unavailability of minerals and nutrients in rocky areas forces the microorganisms to regulate the expression of corresponding genes that control the synthesis of proteins involved in rock solubilization (Alahari and Apte, 2004).
Comprehensive understanding of the microbial gene regulation is of great concern in developing e cient microorganisms with rockdissolving abilities (Finlay et al., 2009). Xiao et al (2012a) have conducted a PCR-based suppression-subtractive hybridization (SSH) experiments for exploring the differential expression of cDNA fragments in the control and experimental transcriptomes of Aspergillus fumigatus, cultured on potassium supplemented rock-dissolution growth medium (Xiao et al., 2012c). Xiao et al (2012b) have also conducted a two-dimensional gel electrophoresis (2DGE) to understand the expression patterns of Bacillus mucilaginosus and its extracellular proteins secreted during the process of potassium rock dissolution (Xiao et al., 2012a). Nevertheless, it is di cult to draw convincing conclusions by analyzing the effect of individual factors on few candidate genes. Therefore, genome-wide transcriptome sequencing (RNA-seq) should be employed to capture the cellular snapshot of a rock-dissolving microorganisms at a given conditions (Kawahara et al., 2012).
Next generation sequencing technologies have been applied for analyzing and revealing the complete gene expression patterns of an organism for understanding the molecular mechanisms underlying its biological process (Kawahara et al., 2012;Wang et al., 2009). Wang et al (2015) have conducted a high-throughput RNA sequencing study to understand the molecular mechanisms of potassium feldspardissolving Aspergillus niger (Wang et al., 2015). The A. niger genes encoding for proteins involved in synthesis and transportation of organic acids, polysaccharides, cystathionine beta-synthase, cysteine synthase, and glutathione synthase were found to be highly upregulated (Wang et al., 2015). However, studies related to microbial weathering of silicate minerals were not conducted till date. Our present research is focused on understanding and exploring the effects and participating genes of an e cient silicate rock-dissolving bacterial strain. We have isolated 22 bacterial strains from a disturbed silicate rock mining site. The most e cient rock-dissolving bacterium was screened out to understand the molecular complexities of silicate rock solubilization. We have conducted a whole-genome sequencing followed by genome-wide transcriptome analysis and validated the results using real-time uorescent quantitative PCR (RT-qPCR) (Jiang et al., 2011). We propose that the experimental and sequencing data generated and analyzed in this study provide the e cient bacterial and genetic resources to improve the external-soil spray seeding technology for silicate rock mining areas.

Rock samples
The silicate rock samples used in our study were obtained from Mount Lu (29°26'-29°41'N, 115°52'-116°08'E) in Jiangxi Province, southeast region in China (Fig. S1). This area was a typical overexploited silicate rock mining site with severely damaged ecological environment. The rock samples were fully rinsed in distilled water, dried and ground for 200 mesh sieve (Wu et al., 2017c). We have used Xray diffractometer (XRD, ARL EQUINOX 1000, Thermo Fisher Scienti c, USA) to analyze the composition of the rock samples. We have also conducted a rock mineral sheer analysis of the rock samples.

Screening of microbial strains
Bacterial strains were isolated from the soil around the weathered rocks in Mount Lu using continuous gradient dilution method (Wu et al., 2017b). The isolated bacteria were cultured on Alexander Rove agar plates (5.0 g sucrose, 2.0 g Na 2 HPO 4 , 0.5 g MgSO 4 ·7H 2 O, 0.005 g FeCl 3 , 0.l g CaCO 3 , l.0 g rock sample, 15.0 g agar, 1000 mL deionized water). Approximately, 200µL of each dilution (10 −4 , 10 −5 , 10 −6 ) was spread onto the surface of Alexander Rove agar plate. The inoculated plates were incubated at 28℃ for 24-72h to isolate individual colonies based on their size, color and morphology. Selected colonies were further puri ed using standard re-culturing techniques.
Each bacterial isolate was then tested for their capacity to produce indoleacetic acid (IAA) and siderophore, using the standard Salkowski colorimetric method and CAS assay respectively (Bric et al., 1991). The CAS assay was monitored using a spectrophotometer set at an absorbance of 630-nm, the treatment group (with bacteria) and control group (without bacteria) were represented as A and Ar respectively. The siderophore production was calculated as A/Ar ratio (represented as +/-), the A/Ar ratio is inversely proportional to the siderophore production thus, lower A/Ar ratio represents more siderophore production (Wu et al., 2019). Strains with more than 10 mg L −1 IAA and +++ siderophore productions were chosen for further experiments.
The above selected bacterial isolates were cultured in 10 mL Luria-Bertani broth (LB) medium incubated for 24 h at 180 rpm in a shaking incubator. After 24 h, 1.0 mL of each isolate was inoculated into a conical ask containing 20 mL of fermentation medium (10 g saccharose, 2.0 g Na 2 HPO 4 , 0.5 g MgSO 4 ·7H 2 O, 0.5 g (NH 4 ) 2 SO 4 , 0.1 g NaCl, 0.1 g CaCO 3 , and 1000 mL deionized water) supplemented with 0.2 g silicate rock sample. The inoculated samples were incubated at 30℃ with 180 rpm for 7 days in a shaking incubator, conical asks with 1.0mL of inactivated isolates are considered as control (Wu et al., 2017c). The amount of SiO 2 released from rock sample was determined using the standard silico-molybdenum blue spectrophotometric method (Meyer and Bloom, 1993). The NLX-4 strain which exhibited excellent IAA, siderophore and SiO 2 releasing abilities was selected for genomic analysis.

Rock-dissolution experiments
The culture medium supplemented with 5.0 g L −1 silicate rock sample (sole K + source) was inoculated with NLX-4 strain and incubated at 30℃ with 180 rpm in a shaking incubator for different time periods ranging from 0, 2, 5, 9, 12, 15, 22, and 30 days respectively. The culture medium was regularly monitored (the above mentioned time periods) for determining the concentration of K + and Al 3+ ions, using inductively coupled plasma-atomic emission spectrometry (ICP-AES, Vista MPX, Varian, USA) as per the protocol reported by Wu et al. (2017c) and Kanicky and Mermet (1997). The concentration of Si ion released from the rock cultures was measured using the silicomolybdenum blue spectrophotometric method as mentioned above. The exopolysaccharides (EPS) secreted by NLX-4 strain was determined using the standard ethanol precipitation method (Sambrook and Russell, 2006). Amino acids and organic acids were analyzed using high performance liquid chromatography (HPLC, 1260 In nity, Agilent Technologies, USA) using the well-established protocols (Wu et al., 2017b). The rock culture samples inoculated with inactivated NLX-4 strain was considered as control. We have also determined the particle diameter of the rock samples by retrieving the bacteria-rock mixtures and the rocks were separated based on the median particle diameter (D 50 ) and analyzed using laser particle size analyser (LDSA, S3500 SI, Microtrac, USA) (Wu et al., 2017c). The particle diameter variations (PDV) were calculated as the differences between the experimental group and the control group.
Whole genome sequencing and assembly of NLX-4 The genomic DNA of NLX-4 strain was extracted using the standard SDS-based method (Zhou et al., 1996) and sequenced using the Illumina Hiseq 4000 (Illumina, Inc., San Diego, CA) and PacBio RSII sequencing platform (BGI, Beijing, China). The raw data produced by Illumina Hiseq 4000 were initially processed to obtain clean data by discarding the low-quality reads (reads containing more than 36 bases, reads with ≤ 20 base-qualities, reads with ploy-N≥9bp, reads with adapter or duplication bases). Similarly, the raw data of NLX-4 genome obtained from PacBio RSII were ltered by removing the polymerase reads shorter than 100bp or the read mass less than 0.80, then the subreads with length ≥1000bp and reads without adapters were extracted from ltered polymerase reads respectively. The clean reads obtained after ltering were assembled using RS_HGAP Assembly3 (SMRT Analysis v2.3.0). The correct K-mers were observed for the reads with deep sequencing (high frequency), while random sequencing error-containing K-mers were observed for reads with low read frequency . The error correction method used in this study was based on K-mer frequency information. The K-mer size was determined by the genome size, read length, and supercomputer memory , thus K = 15 bp was chosen here. The corrections of single-base error in contig were performed using two-round analysis methods to process the data from Illumina Hiseq 4000. The rstround error correction method was carried out by soap SNP and soap Indel software, while the second-round of error correction was conducted by GATK analysis pipeline. The circle form by contigs was vili ed by checking the overlap using SSPACE-LongRead to determine the subreads or corrected reads based on the data obtained from PacBio RSII (Boetzer and Pirovano, 2014). The assembly of the bacterial genome was inspected by: 1) comparing 100X clean data to the assembly result (reads utilization >95%, the depth of backward and forward bunches >3X, the insert size of backward and forward bunches was distributed within the proper range, satis ed randomness, and the depth of single bunches = 0); 2) carrying out the statistics of repetitive sequence (parameters were set as -e 1e-10); 3) performing GC-depth assessment. Gene annotation was mainly based on amino acid sequence alignment (Wyman et al., 2004). The amino acid sequence of the gene was compared with the BLAST database, and the corresponding functional annotation information was obtained. Since each of these sequence comparisons had more than one result, the optimal comparison was retained as the annotation of gene, so as to ensure its biological signi cance (Slater and Birney, 2005). All annotations were completed by using BLAST software to combine various databases, including Gene Ontology (GO), Clusters of Orthologous Group (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-Prot, and NCBI non-redundant protein (nr).

RNA extraction, library preparation and RNA-seq
The NLX-4 strain samples cultured with rock sample (5.0 g L − 1 ) growth medium was incubated at 30℃ with 180 rpm for 15 d, while strain NLX-4 cultured in fermentation medium added with 0.3 g L − 1 KCl was set as control. To further investigate the molecular mechanisms resulting involved in rock-dissolution we have performed a genome-wide RNA sequencing. The total RNA extraction was performed using TRIzol® Invitrogen reagent (Invitrogen, Carlsbad, CA). The total RNA extraction of NLX-4 strain cultures were performed separately for the control and treated group cultures (Gómez-Lozano et al., 2012). RNA quality was analyzed using Agilent® bioanalyzer (Agilent 2100, Agilent Technologies, USA). Ribo-Zero rRNA removal kit (Illumina, Inc. San Diego, CA) was used for extracting total RNA devoid of rRNA. After extracting sequencing quality total RNA we have added fragmentation buffer was for breaking down the mRNA to short fragments. Taking these short fragments as templates, random hexamer-primers were used to synthesize the rst-strand cDNA. The second-strand cDNA was synthesized using buffer supplemented with dATPs, dGTPs, dCTPs, dUTPs, RNase H and DNA polymerase I respectively after removing dNTPs. Short fragments were puri ed using the QiaQuick® PCR extraction kit (QIAGEN, Hilden, Germany) and later resolved using the elution buffer for repairing the ends of the strands and adding poly (A) tails. After that, the short fragments were connected with sequencing adapters. Then, the UNG enzyme was used to degrade the second-strand cDNA, followed by purifying the product using the MinElute® PCR Puri cation Kit (QIAGEN, Hilden, Germany) before the PCR ampli cation process. Thus, obtained mRNA library was sequenced using Illumina HiSeq2000 (Illumina, Inc., San Diego, CA).

Bioinformatic analysis of NLX-4 transcriptome data
Raw data produced by Illumina Hiseq 2000 were initially processed to obtain clean data by discarding the low-quality reads (reads containing adapter sequences, ploy-N, or low-quality bases). Clean reads obtained after ltering were mapped to the NLX-4 reference genome (fully assembled genome of strain NLX-4) using SOAP2 (Li et al., 2009). Mismatches with no more than 5 bases were allowed during the process of alignment. Reads per kb per million reads (RPKM) method was used to calculate individual gene expression of the test and control samples (Mortazavi et al., 2008). The RPKM level data of control and treatment samples were also analyzed using edgeR, limma, and Glimma statistical analysis packages to obtain statistically signi cant DEGs (Audic and Claverie, 1997). DEGs with false discovery rate (FDR) ≤ 0.001 and fold change ratio larger than 2 were chosen for gene ontology (GO) functional enrichment analysis and KEGG pathway analysis. Only the genes exhibiting the P-value < 0.05 and Q-value < 0.05 were considered as differentially expressed (Tribelli et al., 2015).

Quantitative real-time PCR (RT-qPCR) validation
We have speci cally performed the RT-qPCR analysis using the signi cant list of differentially expressed RNA transcript sequences encoding for 5 metabolically important genes. The forward and reverse primers were designed using the Primer Premier 5.0 software (Table 1). RT-qPCR procedures were performed as previously suggested (Tribelli et al., 2015). The relative quanti cation of the control and test samples were performed by including three biological replications and the obtained gene expression results were normalized to the reference gene (16S rRNA). The differential expression of the subjected genes were calculated according to the standard 2 −ΔΔCt method (Livak and Schmittgen, 2001).

Statistical analysis
All experiments were conducted in triplicates to obtain statistically signi cant results. One-way analysis of variance (ANOVA) and two independent samples t-test were carried out to quantify the signi cant differences between different conditions. Pearson correlation analysis was performed to assess the effects of different factors on rock dissolution and release of elements. Statistical analysis was accomplished using the IBM SPSS 22.0 and R 3.3.0 software.

Results And Discussion
Screening of the e cient strain To con rm the type of rock used for the e cient 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 SiO 2 , Al 2 O 3 , and K 2 O 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 has 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 (Table S1). The highly e cient 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 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 SiO 2 in the fermentation broth). The rock-dissolving ability was calculated by nding the difference between the experimental and control groups respectively (Fig. 1a). The NLX-4 strain exhibited the signi cant 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 d 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.
Identi cation 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 agella (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 (Fig. S2b). Results obtained from BLAST and the phylogenetic tree analysis endorsed that NLX-4 strain belonged to the Pseudomonas genus (Fig. S2). Based on the results of the morphological and phylogenetic analysis, NLX-4 strain was identi ed as a Pseudomonas sp. strain.

Effects of Pseudomonas sp. NLX-4 on rock-dissolution
The nal 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 signi cantly 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 nal concentrations of exo-polysaccharides, amino acids, and organic acids secreted in fermentation broth (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 signi cant correlations (P<0.001) with the release of K, Al, Si (Fig. 1c). Meanwhile, EPS, amino acids, and all organic acids exhibited highly signi cant 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 signi cant correlations (P<0.001) with the release of three elements (Fig. 1c). 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;Wu et al., 2017c). 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.

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 8,991bp (Table S4). Thus, obtained ltered 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 x 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 (Table 2 and S4). The quality control analysis has resulted in clean data of 817 Mb of sequence data with ltered reads and low-quality ltered reads percentages were 9.68 and 1.39 respectively (Table 2 and S4).
The nal 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 ( 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 (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 ( Table 2). The genome contains 6,239 protein coding genes with an average length; internal length and internal GC content were 949bp, 852,628bp and 56.55% respectively (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 39233 bp (start: 5375574, end: 5414806) 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 35,083bp, 4,597bp and 1380bp respectively ( 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 (Table S5e).
To bene t 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 ( Table S5f). The COG has classi ed 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 classi ed 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, 161metabolism of other amino acids, 194-metabolism of terpenoids and polyketides, 480-xenobiotic biodegradation and metabolism respectively. The prokaryotic orthologous group classi cation 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 (Table 3a 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 0.10-1.51% respectively. The alignment results obtained from gene and genome level were reported in Table 3a 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 insigni cantly 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 ( Fig. S11-S13). The mapped reads were further subjected to a series of analysis including sequence assessment, biological contextualization, gene structure re nement, alternative splicing, novel transcript detection and SNP analysis (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 (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 in uence 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 signi cantly accelerated the process of rock dissolution (Wu et al., 2017c). The RPKM values obtained from the quanti cation of the control and treatment samples were used for nding the signi cant DEG. We have followed two different approaches for nding 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, agellin, glutathione-S-transferase (GST), chaperonin GroES, heat shock protein HSP10, elongation factor prokaryotic, bacterioferritin, polysaccharide biosynthesis protein Epsc, DNA-methyl transferase, agellar 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 S15). While the genes encoding for fatty acyl-CoA synthase, sulfur dioxygenase, DNA polymerase III, acetyltransferase, cation symporter, sul de: 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 S15). We have also analyzed the enrichment of the DEGs in biological pathways using KEGG pathways and GO analysis respectively ( Fig. 4 and S15). RT-qPCR was used to further validate the expression level of genes identi ed 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 (Table 1). These 5 DEGs showed a consistent pattern between Illumina sequencing results and RT-qPCR (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 ironophore 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 ironophore 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 agellin, polysaccharide biosynthesis protein Epsc, agellar 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 con rms the existence of polysaccharides in the environment (Fig. 4).
Glutathione (GSH) is essential for the cells to adapt to the de ciency of K + de ciency 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 detoxi cation 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 rst cause incorrect folding of proteins and enzymes without K + . Therefore, amounts of mis-/un-folded proteins will accumulate in the cell. In the case of K + de ciency, 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.

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), 10010 (Treatment-1), 11470 (Treatment-2) and 11695 (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, 11248 and 11465 novel transcripts encoded for the non-coding regions respectively (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. 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 (ironophore, 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 con rmed.
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 rm 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 e cient 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 eld 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 expecting.

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