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. 2021 Oct 25;11(11):473. doi: 10.1007/s13205-021-03020-2

Revealing the potential of Klebsiella pneumoniae PVN-1 for plant beneficial attributes by genome sequencing and analysis

Varsha Jha 1,2, Hemant Purohit 1, Nishant A Dafale 1,2,✉
PMCID: PMC8546017  PMID: 34777930

Abstract

Genome sequencing of Klebsiella pneumoniae PVN-1, isolated from effluent treatment plant (ETP), generates a 5.064 Mb draft genome with 57.6% GC content. The draft genome assembled into 19 contigs comprises 4783 proteins, 3 rRNA, 44 tRNA, 8 other RNA, 4911 genes, and 73 pseudogenes. Genome information revealed the presence of phosphate metabolism/solubilizing, potassium solubilizing, auxin production, and other plant benefiting attributes like enterobactin and pyrroloquinoline quinone biosynthesis genes. Presence of gcd and pqq genes in K. pneumoniae PVN-1 genome validates the inorganic phosphate solubilizing potential (528.5 mg/L). Pangenome analysis identified a unique 5ʹ-Nucleotidase that further assists in enhanced phosphate acquisition. Additionally, the genetic potential for complete benzoate, catechol, and phenylacetate degradation with stress response and heavy metal (Cu, Zn, Ni, Co) resistance was identified in K. pneumoniae PVN-1. Functioning of annotated plant benefiting genes validates by the metabolic activity of auxin production (7.40 µg/mL), nitrogen fixation, catalase activity, potassium solubilization (solubilization index—3.47), and protease activity (proteolytic index—2.27). In conclusion, the K. pneumoniae PVN-1 genome has numerous beneficial qualities that can be employed to enhance plant growth as well as for phytoremediation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-021-03020-2.

Keywords: Functional genes, Genome characterization, Pangenome, Phosphate solubilization, Plant-growth attributes

Klebsiella pneumoniae is a Gram-negative, non-motile bacterium, belongs to the family Enterobacteriaceae (class—Gammaproteobacteria; phylum—Proteobacteria) (Ashurst and Dawson 2018). K. pneumoniae occur ubiquitously in nature (soil, sewage, drinking water, industrial effluents, and vegetation), few strains can fix nitrogen anaerobically while others are (opportunistic) pathogens associated with community-acquired pneumonia, bloodstream infections, and urinary tract infections (UTIs) (Iniguez et al. 2004; Martin and Bachman 2018). The K. pneumoniae isolates from rhizospheric soil are of agricultural interest as they promote plant growth. Additionally, the ability to degrade toxic aromatic compounds and produce necessary chemicals such as 2,3-butanediol makes them ecologically and economically significant (Jung et al. 2013; Bhardwaj et al. 2017; Liu et al. 2018; Rajkumari et al. 2018; Mazumdar et al. 2018; Wang et al. 2020). Klebsiella spp. are also reported to stimulate Induced Systemic Resistance (ISR) phenomenon against pathogenic fungus and nematodes in rice and soybean, respectively (Ji et al., 2014; Liu et al. 2018). Though introducing "foreign" microbes into the environment may be concerning, strain optimization and metabolic engineering could support the design of desirable traits in strains (Kim et al. 2018; Bhattacharjee et al. 2020) that subsequently can be used in increasing plant productivity and phytoremediation.

Genome sequencing explores genes responsible for stress tolerance, biocontrol, bioremediation, and growth-enhancing properties in agriculturally effective microbes (Besset-Manzoni et al. 2018; Rajkumari et al. 2018; Khan and Bano 2018; Wu et al. 2019; Agrahari et al. 2020). Recently, genomes of Klebsiella variicola, Klebsiella pneumoniae, Pantoea agglomerans, and Pseudomonas koreensis isolated from plants, soil, and lake, respectively, have been reported and studied to understand these purposes (Reyna-Flores et al. 2018; Srivastava et al. 2019; Alkaabi et al. 2020).

The present work reports the draft genome sequence of K. pneumoniae PVN-1, which can solubilize inorganic phosphate. Genes for PGP traits, such as nitrogen fixation, potassium solubilization, auxin production, catalase, and protease, in addition to stress response, heavy metal resistance, aromatic compound degradation, and pesticide biotransformation were identified. Enterobactin and pyrroloquinoline quinone biosynthesis genes were also identified in the genome using antiSMASH and RAST server. Pangenome analysis of strain PVN-1 with other clinical and environmental K. pneumoniae isolates revealed the presence of a unique gene identified as 5ʹ Nucleotidase by Pfam 34.0 and Conserved Domains Database (CDD). In summary, K. pneumoniae PVN-1 has multiple beneficial properties that could be exploited for increased plant productivity and phytoremediation.

Phosphate-solubilizing strain PVN-1 was isolated from activated sludge of effluent treatment plant (ETP) Mumbai, India (19.0690° N, 73.1331° E). The isolated bacterium, subjected to Gram staining and biochemical characterization like catalase, oxidase, motility test, and extracellular protease production using 30% hydrogen peroxide (H2O2), Kovac's oxidase, Luria agar and skim milk agar, respectively (O'May and Tufenkji 2011; Manzoor et al. 2017). The non-motile short-rod, Gram-negative, PVN-1 microbe showed catalase-positive and oxidase-negative properties. The Plant Growth-Promoting (PGP) attributes viz., phosphate solubilization, potassium solubilization, nitrogen fixation, HCN production, and auxin production were studied using Pikovskaya's (PKV) media, modified Aleksandrov’s media, Jensen’s media, King’s B, and Salkowski test, respectively, as per the protocol described by Manzoor et al. (2017) and Linu et al. (2019). The solubilization index (SI) was calculated as described by Pande et al. (2017). Findings of PGP attributes suggest that PVN-1 has nitrogen fixation, phosphate solubilization (SI—3.0), potassium solubilization (SI—3.47), auxin production (7.40 µg/mL), and protease activity (Proteolytic Index—2.27) (Table 1, Fig S1). Phosphate-solubilizing potential of PVN-1 strain was quantified using a phosphate test kit (Merck) in PKV broth supplemented with 5 g/L of tri-calcium phosphate. After 120 h incubation at 30 °C with 121 rpm shaking, the released phosphate in the PKV medium supernatant was estimated to be 528.5 mg/L using Spectroquant move 100 (Merck) (Fig S1).

Table 1.

Plant growth-promoting properties of Klebsiella pneumoniae PVN-1

Strain 16 s rRNA identification P-Solubilization Index P-Solubilization (mg/L) Potassium Solubilization Index Proteolytic Index Nitrogen Fixation Auxin production (µg/mL) HCN production Catalase
PVN-1 Klebsiella pneumoniae 3.0 528.5 3.47 2.27  +  7.40 −  + 
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The taxonomic status of isolated strain PVN-1 was determined using BLAST analysis of the sequenced 16S rRNA gene (NCBI GeneBank accession number MW440693.1) (Bohra et al. 2019; Singh et al. 2020; Srivastava et al. 2021a). The 16S rRNA identification showed the highest similarity to K. pneumoniae with 99% query coverage and 97.83% percent identity. Thus, to examine the molecular phylogenetic relationship between PVN-1 strain with its closest species, the 16S rRNA sequences with 97.83% and above sequence similarity were aligned by clustalW, and construct a phylogenetic tree by MEGA X using the Maximum Likelihood method based on the Jukes-Cantor model with 1000 bootstrap replications (Tamura et al. 2013). The clade of PVN-1 strain was dissimilar from the related strains, and the taxonomy was not conclusive (Fig. 1a). Thus, to elucidate the taxonomic classification, PVN-1 strain was compared with other Klebsiella sp. (Table S1) by a genome-based approach, using average nucleotide identity (ANI), using FastANI v0.1.2 and OrthoANI v0.93.1 algorithm (Lee et al. 2016; Jain et al. 2018) (Table 2). Based on the established cut-off values on species delimitation for ANI (> 95–96%) (Varghese et al. 2015), PVN-1 was strongly affiliated to Klebsiella pneumoniae (Fig. 1b).

Fig. 1.

Fig. 1

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a A phylogenetic tree representing the evolutionary relationship of Klebsiella pneumoniae PVN-1 with selected Klebsiella sp. was constructed by MEGA X software, using the Maximum Likelihood method (Jukes-Cantor model) with 1000 bootstrap replicates. Scale bar (0.10) at the bottom of the tree indicates the number of substitutions per site. b Heatmap represents the percentage Orthologous Average Nucleotide Identity (orthoANI) of Klebsiella pneumoniae PVN-1

Table 2.

Genome-based comparison of Klebsiella pneumoniae PVN-1 with other Klebsiella species by computing average nucleotide identity (ANI)

Query Reference FastANI OrthoANI (%)
ANI Estimate (%) Matches Total
Klebsiella pneumoniae strain PVN-1 Klebsiella huaxiensis strain WCHKl090001T 83.1914 1110 1678 82.11
Klebsiella pneumoniae strain PVN-1 Klebsiella oxytoca NBRC 105695T 83.871 1162 1678 82.92
Klebsiella pneumoniae strain PVN-1 Klebsiella grimontii strain 06D021T 84.2524 1164 1678 83.24
Klebsiella pneumoniae strain PVN-1 Klebsiella michiganensis strain DSM 25444T 84.328 1166 1678 83.14
Klebsiella pneumoniae strain PVN-1 Klebsiella aerogenes KCTC 2190T 85.8586 1092 1678 84.38
Klebsiella pneumoniae strain PVN-1 Klebsiella quasipneumoniae subsp. Quasipneumoniae strain 01A030T 93.7032 1373 1678 93.53
Klebsiella pneumoniae strain PVN-1 Klebsiella quasivariicola strain KPN1705T 94.0214 1380 1678 93.82
Klebsiella pneumoniae strain PVN-1 Klebsiella variicola strain DSM 15968T 94.7613 1409 1678 94.53
Klebsiella pneumoniae strain PVN-1 Klebsiella pneumoniae strain ATCC 13883T 99.0913* 1435 1678 99.19*
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*ANI values around 95% (≥ 95% ANI) represent the same species as the reference isolate; Ttype cultures

Several K. pneumoniae strains have been identified as opportunistic pathogens, and antibiotic resistance is a serious concern with this bacterium. Thus, the strain PVN-1 was checked for virulent phenotype, such as hypermucoviscosity, hemolytic, and phospholipase/lecithinase activity, using blood agar and yolk agar, respectively (Pereira and Vanetti 2015), and no such pathogenic phenotype was observed in PVN-1 (Fig S2). The antibiotic susceptibility assay was performed in four sets using 20 different antibiotics to investigate the antibiotic sensitivity of the isolated strain PVN-1 by disk diffusion method. The maximum antibiotics utilized in the antibiotic susceptibility test showed a zone of inhibition with diameters ranging from 10 to 20 mm. PVN-1 was resistant against Fosfomycin, Levofloxacin, Ofloxacin (fluoroquinolones class) and Colistin (polymyxin class) while it is highly vulnerable toward Streptomycin, Doxycycline Hydrochloride, Trimethoprim and Chloramphenicol (Fig. S3, Table S2).

Genomic DNA for sequencing was extracted from 24 h grown (at 30 °C with 120 rpm) pure culture in Luria Broth (LB broth), using FastDNA Spin kit for soil as directed in the manufacturer’s protocol (Srivastava et al. 2020). DNA quality and quantity/purity were checked on 0.8% agarose gel and Nanodrop DS-11 + (DeNovix) spectrophotometer, respectively. A paired-end sequencing library was prepared by Illumina TruSeq Nano DNA Library Prep Kit. The quality and quantity of the PCR enriched library were checked on 4200 Tape Station System (Agilent technologies) using high-sensitivity D1000 Screen tape as per the manufacturer’s instructions. The library was sequenced on the NextSeq500 platform using 2 × 150 bp chemistry. The high-quality de novo genome assembly was performed by SPAdes v. 3.13.0. The high-purity extracted DNA with 70.5 ng/µL concentration (A260/280 ratio—1.87; A260/A230 ratio—1.03) was sequenced and assembled to generate a 5.064 Mb draft genome with 19 contigs and scaffold N50 value of 349,756 bp (Table 3).

Table 3.

Genome assembly statics of Klebsiella pneumoniae strain PVN-1and other Klebsiella sp

Genome Accession no Size (Mb) Contigs GC Content Protein rRNA tRNA Other RNA Gene Pseudogene No. of subsystem
PVN-1 GCA_014297455.1 5.06 19 57.57 4783 3 44 8 4911 73 349
AWD5 GCA_001876675.1 4.8 137 58.17 4528 25 81 10 4695 51 375
KpMx1 GCA_900087895.1 5.36 50 57.46 5089 12 82 14 5299 102 385
2483 GCA_011044745.1 5.84 132 56.5 5583 39 84 14 5918 198 390
ATCC 13883T GCA_000742135.1 5.55 16 57 5180 21 77 7 5517 233 395
342 GCA_000019565.1 5.92 3 56.87 5249 25 88 13 5460 85 398
DSM 15968T GCA_000828055.2 5.52 1 57.6 5117 25 86 14 5309 67 390
KCTC 2190T GCA_000215745.1 5.28 1 54.8 4791 25 84 10 4999 89 390
01A030T GCA_000751755.1 5.47 65 58 5091 10 70 12 5267 84 393
KPN1705T GCA_002269255.1 5.54 4 56.9 5181 25 87 12 5414 109 393
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*Genome stats given as per RAST and NCBI prokaryotic genomes automatic annotation pipeline (PGAAP) annotation

The assembled genome was submitted to NCBI under the accession number NZ_JACORD010000001.1. Rapid Annotations using Subsystems Technology (RAST v2.0) and NCBI prokaryotic genomes automatic annotation pipeline (PGAAP) were used to perform the genome annotation (Srivastava et al. 2021b). K. pneumoniae PVN-1 genome was screened for the presence of prophage and plasmid using PHASTER and PlasmidFinder v2.0.1 (minimum percent coverage—60%; threshold percent identity—95%), respectively (Carattoli et al. 2014; Arndt et al. 2016). PVN-1 draft genome annotation and functional characterization identify 4783 protein count, 73 pseudogenes, 3 rRNA genes, 44 tRNAs, and 349 subsystems. Similar genome assembly reports on K. pneumoniae strains isolated from automobile workshop and sugarcane stem have been documented (Rajkumari et al. 2018; Reyna-Flores et al. 2018). One plasmid in scaffold 18 and three intact prophage regions with 50.5 kb, 49.7 kb, and 107.6 kb in scaffold 1, 14, and 18 of the genome were identified, respectively.

A circular genome map of K. pneumoniae PVN-1 was constructed using CG viewer beta version with default parameters and compared with other Klebsiella species/sub-species (listed in Table 3) by BLAST plugin provided in CG view server (Stothard and Wishart 2005; Bohra et al. 2018). Comparative genomics among different Klebsiella strains and species using NCBI identified around 4500–5590 coding sequences (CDS) where strain 2483 has the highest CDS and strain AWD5 has the lowest CDS (Table 3). Graphical representation of genome comparison by CG viewer (Fig. 2) indicated gaps among the sequences due to genome dissimilarity, thus reflects the adaptation of Klebsiella species to the particular climatic conditions of different inhabiting geographical location. Genome analysis identifies genes for inorganic and organic phosphate solubilization, phosphorous metabolism, phosphonate metabolism, aromatic compound degradation, heavy metal tolerance/resistance, auxin, and pyrroloquinoline quinone production in PVN-1 genome (Fig. 3, Fig S4, Table 4). Hence, this bacterium can be considered a potential genomic resource for sustainable agriculture and remediation with synthetic biology and metabolic engineering intervention.

Fig. 2.

Fig. 2

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Graphical representation of genome comparison between Klebsiella pneumoniae PVN-1 and other Klebsiella species/sub-species using CG viewer beta version with default parameters

Fig. 3.

Fig. 3

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Differential arrangement of phosphate metabolism and solubilizing gene cluster in Klebsiella pneumoniae PVN-1 and selected Klebsiella sp. genome

Table 4.

Functional genes involved in the PGP activities annotated in the genome of Klebsiella pneumoniae PVN-1

Subsystem Description Accession number Locus tag Gene name Protein product
Pyrroloquinoline Quinone biosynthesis pyrroloquinoline quinone precursor peptide PqqA NZ_JACORD010000001.1 H8S94_RS01265 pqqA WP_002905689.1
pyrroloquinoline quinone biosynthesis protein PqqB NZ_JACORD010000001.1 H8S94_RS01270 pqqB WP_032434592.1
pyrroloquinoline quinone synthase PqqC NZ_JACORD010000001.1 H8S94_RS01275 pqqC WP_032434594.1
pyrroloquinoline quinone biosynthesis peptide chaperone PqqD NZ_JACORD010000001.1 H8S94_RS01280 pqqD WP_004143686.1
pyrroloquinoline quinone biosynthesis protein PqqE NZ_JACORD010000001.1 H8S94_RS01285 pqqE WP_004184158.1
pyrroloquinoline quinone biosynthesis protein PqqF NZ_JACORD010000001.1 H8S94_RS01290 pqqF WP_032434596.1
d-gluconate and ketogluconates metabolism Glucose dehydrogenase, PQQ-dependent (EC 1.1.5.2) NZ_JACORD010000009.1 H8S94_RS16250 gcd WP_002888816.1
Phosphate metabolism Alkaline phosphatase (EC 3.1.3.1) NZ_JACORD010000005.1 H8S94_RS11870 phoA WP_004147286.1
Auxin biosynthesis Tryptophan synthase alpha chain (EC 4.2.1.20) NZ_JACORD010000003.1 H8S94_RS09675 trpA WP_002901728.1
Anthranilate phosphoribosyltransferase (EC 2.4.2.18) NZ_JACORD010000003.1 H8S94_RS09690 trpD WP_004148109.1
Tryptophan synthase beta chain (EC 4.2.1.20) NZ_JACORD010000003.1 H8S94_RS09680 trpB WP_117061140.1
Monoamine oxidase (1.4.3.4) NZ_JACORD010000003.1 H8S94_RS10765 tynA WP_117061214.1
Phosphoribosylanthranilate isomerase (EC 5.3.1.24) NZ_JACORD010000003.1 H8S94_RS09685 trpCF WP_014907770.1
Phosphonate metabolism 2-aminoethylphosphonate ABC transporter permease protein I (EC 3.A.1.9.1) NZ_JACORD010000013.1 H8S94_RS19975 phnU WP_004150257.1
2-aminoethylphosphonate:pyruvate aminotransferase (EC 2.6.1.37) NZ_JACORD010000013.1 H8S94_RS19995 phnW WP_004181579.1
2-aminoethylphosphonate uptake and metabolism regulator NZ_JACORD010000013.1 H8S94_RS19990 phnR WP_002923196.1
2-aminoethylphosphonate ABC transporter permease protein II (EC 3.A.1.9.1) NZ_JACORD010000013.1 H8S94_RS19970 phnV WP_004145044.1
Phosphonoacetaldehyde hydrolase (EC 3.11.1.1) NZ_JACORD010000013.1 H8S94_RS20000 phnX WP_040189763.1
2-aminoethylphosphonate ABC transporter periplasmic-binding component (EC 3.A.1.9.1) NZ_JACORD010000013.1 H8S94_RS19985 phnS WP_040189761.1
2-aminoethylphosphonate ABC transporter ATP-binding protein (EC 3.A.1.9.1) NZ_JACORD010000013.1 H8S94_RS19980 phnT WP_117061251.1
Siderophore Enterobactin Enterobactin esterase NZ_JACORD010000017.1 H8S94_RS23820 fes WP_023284837.1
2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (EC 1.3.1.28) [enterobactin] siderophore NZ_JACORD010000017.1 H8S94_RS23770 entB WP_020324582.1
Ferric enterobactin transport system permease protein FepG (EC 3.A.1.14.2) NZ_JACORD010000017.1 H8S94_RS23800 fepG WP_038992512.1
Ferric enterobactin-binding periplasmic protein FepB (EC 3.A.1.14.2) NZ_JACORD010000017.1 H8S94_RS23785 fepB WP_004147526.1
Ferric enterobactin transport ATP-binding protein FepC (EC 3.A.1.14.2) NZ_JACORD010000017.1 H8S94_RS23805 fepC WP_002893737.1
2,3-dihydroxybenzoate-AMP ligase (EC 2.7.7.58) [enterobactin] siderophore NZ_JACORD010000017.1 H8S94_RS23775 entE WP_019705365.1
Outer membrane receptor for ferric enterobactin and colicins B, D NZ_JACORD010000017.1 H8S94_RS23825 fepA WP_004210903.1
Enterobactin exporter EntS NZ_JACORD010000017.1 H8S94_RS23790 entS WP_004147525.1
Enterobactin synthetase component F, serine activating enzyme (EC 2.7.7.-) NZ_JACORD010000017.1 H8S94_RS23810 entF WP_038992511.1
Isochorismate synthase (EC 5.4.4.2) [enterobactin] siderophore NZ_JACORD010000017.1 H8S94_RS23780 entC WP_004176916.1
4'-phosphopantetheinyl transferase (EC 2.7.8.-) [enterobactin] siderophore NZ_JACORD010000017.1 H8S94_RS23830 entD WP_032434174.1
Proofreading thioesterase in enterobactin biosynthesis EntH NZ_JACORD010000017.1 H8S94_RS23760 entH WP_002893892.1
Ferric enterobactin transport system permease protein FepD (EC 3.A.1.14.2) NZ_JACORD010000017.1 H8S94_RS23795 fepD WP_004176917.1
Heavy metal resistance Cytochrome c heme lyase subunit CcmH NZ_JACORD010000001.1 H8S94_RS02595 ccmH WP_072196948.1
Copper-resistance protein CopC NZ_JACORD010000001.1 H8S94_RS04075 yobA WP_023285314.1
Copper-resistance protein CopD NZ_JACORD010000001.1 H8S94_RS04070 copD WP_023285313.1
Blue copper oxidase CueO precursor NZ_JACORD010000009.1 H8S94_RS16245 cueO WP_040189974.1
Copper sensory histidine kinase CusS NZ_JACORD010000007.1 H8S94_RS13345 cusS WP_065801221.1
Copper-sensing two-component system response regulator CusR NZ_JACORD010000007.1 H8S94_RS13350 cusR WP_004214143.1
Cytochrome c heme lyase subunit CcmF NZ_JACORD010000001.1 H8S94_RS02605 ccmF WP_032432488.1
Copper-translocating P-type ATPase (EC 3.6.3.4) NZ_JACORD010000005.1 H8S94_RS12645 copA WP_032421659.1
Cytoplasmic copper homeostasis protein CutC NZ_JACORD010000001.1 H8S94_RS04575 cutC WP_009307530.1
Cu(I)-responsive transcriptional regulator NZ_JACORD010000005.1 H8S94_RS12650 cueR WP_002892208.1
Copper homeostasis protein CutE NZ_JACORD010000017.1 H8S94_RS23415 int/cutE WP_004147589.1
Periplasmic divalent cation tolerance protein CutA NZ_JACORD010000008.1 H8S94_RS14405 cutA WP_002885422.1
Magnesium and cobalt efflux protein CorC NZ_JACORD010000017.1 H8S94_RS23410 corC WP_002894719.1
Copper homeostasis protein CutF precursor NZ_JACORD010000009.1 H8S94_RS16635 nlpE WP_117061116.1
Cobalt–zinc–cadmium resistance protein NZ_JACORD010000001.1 H8S94_RS01360 czcB WP_004151892.1
Zinc transporter ZitB NZ_JACORD010000016.1 H8S94_RS23355 zitB WP_004147641.1
Nickel/Cobalt transporter NZ_JACORD010000002.1 H8S94_RS07715 WP_021440688.1
Nickel ABC transporter, nickel/metallophore periplasmic-binding protein NZ_JACORD010000011.1 H8S94_RS18885 nikA WP_040189497.1
Nickel ABC transporter permease subunit NikB NZ_JACORD010000011.1 H8S94_RS18890 nikB WP_004150102.1
Nickel ABC transporter permease subunit NikC NZ_JACORD010000011.1 H8S94_RS18895 nikC WP_004151422.1
Nickel import ATP-binding protein NikD NZ_JACORD010000011.1 H8S94_RS18900 nikD WP_040189498.1
Nickel import ATP-binding protein NikE NZ_JACORD010000011.1 H8S94_RS18905 nikE WP_032445328.1
Nickel-responsive transcriptional regulator NikR NZ_JACORD010000011.1 H8S94_RS18910 nikR WP_002921188.1
Nickel/cobalt efflux protein RcnA NZ_JACORD010000005.1 H8S94_RS12435 rcnA WP_117061068.1
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The antiSMASH 6 beta features server was used to screen gene clusters encoding secondary metabolite production in PNV-1 genomes with the following parameters: Detection strictness—relaxed, known cluster blast, sub-cluster blast, MIBiG cluster comparison, cluster Pfam analysis, active site finder, Pfam based GO term annotation, RRE finder, TIGR fam analysis checked (Blin et al. 2019). To characterize the genome and KO assignment, the BlastKOALA KEGG Tool was employed (Kanehisa et al. 2016), and the resulting file was then utilized to map metabolic pathways using KEGG Mapper. Secondary metabolite screening identified the enterobactin siderophore biosynthesis gene, which was confirmed by the KEGG mapper (Fig. 4, Fig S4, Table 4). Enterobactin is a high-affinity siderophore (catecholate-based) produced by bacteria that inhibits fungal phytopathogens by reducing ferric ion levels in the rhizosphere (Schippers et al. 1987). KEGG mapper also discovered metabolic pathways for other siderophore production, such as bacillibactin, vibriobactin, and Myxochelin (Fig. 4). Thus, in addition to direct plant growth-promoting features, the isolate possesses disease suppression properties and could be used as a potential biocontrol agent.

Fig. 4.

Fig. 4

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Comparative analysis of siderophore biosynthesis mapped in Klebsiella pneumoniae PVN-1 and other Klebsiella sp. genome using KEGG Mapper

In K. pneumoniae PVN-1 genome, cytochrome P450-based biotransformation/detoxification of xenobiotic chemical intermediates involved in pesticide production was mapped (Fig. 5, Fig S5). Toxic intermediates, such as 1-nitronaphthalene, naphthalene, bromobenzene, and 1,2-dibromoethane, are xenobiotics that are detoxified by glutathione S-transferase via glutathione (GSH) conjugation (Allocati et al. 2018). As a result, the PVN-1 strain could be effective in contaminated soil bioremediation.

Fig. 5.

Fig. 5

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Cytochrome P450-based biotransformation of xenobiotic chemical intermediates used in pesticides a 1-Nitronaphthalene; b Naphthalene; c bromobenzene; d 1,2-Dibromoethane

Furthermore, pangenome analysis with clinical and environmental K. pneumoniae was performed using Bacterial Pan Genome Analysis (BPGA) pipeline with default clustering tool and parameters to detect the unique features of PVN-1 (Chaudhari et al. 2016). Interestingly, a unique gene with annotated 5ʹNucleotidase domain was identified by Pfam and NCBI-CDD web-based tools (Fig. 6). Presence of 5ʹ Nucleotidase further supports the ability to scavenge phosphate from nucleotide degradation. Also, no genotypic information for pathogenic characters, such as colibactin, yersiniabactin, salmochelin, rmpA gene (regulator of mucoid phenotype A), or lecithinase, was observed in the accessory and unique genome of PVN-1. Screening of genome for antibiotic-resistance genes (ARGs) showed the presence of beta-lactamase, fosfomycin, and fluoroquinolones-resistance genes (Table S3). Presence of fosfomycin, and fluoroquinolones-resistance genes in PVN-1 geneome validates the antibitioc susceptibility findings.

Fig. 6.

Fig. 6

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Venn diagram showing core and unique genes in the pangenome of Klebsiella pneumoniae analyzed by BPGA

In conclusion, the assembled PVN-1 draft genome contains the genetic potential for plant growth-promoting properties, such as nitrogen-fixing, auxin production, phosphate, and potassium solubilizing, which were confirmed by metabolic estimations. Gene for enterobactin siderophore biosynthesis, involved in biocontrol activity was detected in PVN-1 genome, exhibits disease suppression ability in addition to plant growth. Complete metabolic pathways of benzoate, catechol, and phenylacetate degradation and heavy metal (Cu, Zn, Ni, Co)-resistance genes were also identified PVN-1 genome. Thus, these findings confer K. pneumoniae PVN-1 has ecologically significant properties that could be exploited for sustainably growing plants and phytoremediation.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOC 4052 KB) (4MB, doc)
Supplementary file2 (XLSX 44 KB) (44.5KB, xlsx)

Acknowledgements

Varsha Jha is grateful to the University Grant Commission (UGC) for the award of Senior Research Fellow (SRF). The authors highly acknowledge the Director, CSIR-NEERI, for providing facilities for this work. The manuscript has been checked for plagiarism by Knowledge Resource Centre, CSIR-NEERI, Nagpur, India, and assigned KRC number [KRC No.: CSIR-NEERI/KRC/2021/APRIL/EBGD/3].

Author contributions

VJ performed lab experiments, bioinformatics analysis and manuscript writing. NAD conceptualized the study and improved the manuscript. HJP conceptualized study, review and editing.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Accession numbers: The 16S rRNA sequence and the whole genome shotgun (WGS) sequence of K. pneumoniae PVN-1 were submitted in the GenBank database with accession number MW440693.1 and NCBI-WGS database under accession number JACORD000000000, respectively.

Contributor Information

Varsha Jha, Email: va.jha@neeri.res.in.

Hemant Purohit, Email: hj_purohit@neeri.res.in.

Nishant A. Dafale, Email: na_dafale@neeri.res.in

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