Characterization and genomics identification of key genes involved in denitrification-DNRA-nitrification pathway of plant growth-promoting rhizobacteria (Serratia marcescens OK482790)

Abstract

Background: A wide variety of microorganisms, including bacteria, live in the rhizosphere zone of plants and have an impact on plant development both favorably and adversely. The beneficial outcome is due to the presence of rhizobacteria that promote plant growth (PGPR).

Results: In this study, a bacterial strain was isolated from lupin rhizosphere and identified genetically as Serratia marcescens (OK482790). Several biochemically and genetically characteristics were confirmed in vitro and in vivo to determine the OK482790 strain ability to be PGPR. The in vitro results revealed production of different lytic enzymes (protease, lipase, cellulase, and catalase), antimicrobial compounds (hydrogen cyanide, and siderophores), ammonia, nitrite, and nitrate and its ability to reduce nitrate to nitrite. In silico and in vitro screening proposed possible denitrification-DNRA-nitrification pathway for OK482790 strain. The genome screening indicated the presence of nitrite and nitrate genes encoding Nar membrane bound sensor proteins (NarK, NarQ and NarX). Nitrate and nitrite reductase encoding genes (NarI, NarJ, NarH, NarG and NapC/NirT) and (NirB, NirC, and NirD) are also found in addition to nitroreductases (NTR) and several oxidoreductases. In vivo results on wheat seedlings confirmed that seedlings growth was significantly improved by soil inoculation of OK482790 strain.

Conclusions: This study provides evidence for participation of S. marcescens OK482790 in nitrogen cycling via the denitrification-DNRA-nitrification pathway and for its ability to produce several enzymes and compounds that support the beneficial role of plant-microbe interactions to sustain plant growth and development for a safer environment.

Keywords: 16S rDNA; Ammonia production; HCN production; Lytic enzymes; Nar genes; NirBD gene; Nitrate/nitrite reduction; Siderophore production.

Fig. 1

Morphological and molecular identification of Serratia marcescens isolated from Lupinus rhizosphere. A The growth of S. marcescens strain on TS agar, showing the typical morphological feature for S. marcescens. B Gel electrophoresis image for 1500 bp of 16 S rRNA encoding gene PCR fragment. Sma refers to the amplified product, -ve refers to negative control, no amplifications confirm no PCR contamination. 1 kb + refers to DNA ladder (BioLabs). C Phylogenetic tree of the 16S rRNA encoding gene amplified from the current strain. The phylogeny indicated that the sequence of the current strain; labelled in bold purple font color, belongs to Serratia marcescens

Fig. 2

Agarose gel electrophoresis for PCR products of six nitrogen cycling related genes amplified from Serratia marcescens (OK482790); lanes 1 to 6 refers to the amplified products of NarK, NarQ, NarX, NTR, NapC/NirT and oxyred genes, respectively, 1KB refers to DNA ladder. The expected product sizes were indicated

Fig. 3

Reduction of nitrate and production of ammonia, nitrite, and nitrate by Serratia marcescens OK482790 strain. Where A- Deep red colour with sulfanilic acid and alpha-naphthylamine mixture (1:1) indicated nitrite production. B- Golden yellow colour with Nessler’s reagent indicated ammonia production. C- Deep blue colour with Trommsdorf^,s reagent indicated nitrite production. D- Deep blue colour with Diphenyl amine reagent indicated nitrate production

Fig. 4

Production of hydrogen cyanide in A and siderophores in B by Serratia marcescens OK482790 strain

Fig. 5

Different enzymatic activities of Serratia marcescens OK482790 strain. A) Protease B) Cellulase C) Lipase D) Catalase

Fig. 6

Effect of PGPR; Serratia marcescens (OK482790) by soil inoculation or seed coating on wheat’s shoots and roots’ heights, fresh and dry weights in comparison to uninoculated plants (the control) of two-week-old seedlings. A- Pot experiment of two weeks old seedlings showing the growth promoting effect of S. marcescens inoculation. B- Effect of S. marcescens on the length of wheat shoot and root stock

Fig. 7

Possible denitrification-DNRA-nitrification pathway of Serratia marcescens (OK482790) strain based on genome and biochemical screening analysis