Reeta Goel

Development of heavy metal-resistant mutants of phosphate solubilizing Pseudomonas sp. NBRI 4014 and their characterization.

Pseudomonas sp. NBRI 4014 is a potent phosphorus solubilizer (284 μg/ml). It also produced significant levels of siderophore (143.87 μg/ml) and IAA (5.6 μg/ml). Siderotyping indicated it was P. aeruginosa siderovar 1. Cadmium (180 μM), nickel (420 μM), and chromium (370 μM) resistant mutants were developed and characterized for their PGPR properties. Mutants were stable under non-selective pressure. In cases of nickel and cadmium, there were reductions of the siderophore levels. However, they were able to promote root and shoot elongation in soybeans (Glycine max PK 564) at a significant level (p < 0.05) in the presence of metals unfamiliar to the wild type. The persistence and stability of mutants were evident in rhizospheric soil, thus their exploitation for polluted/contaminated sites was supported.

Heavy Metal Pollution: Source, Impact, and Remedies

Although some heavy metals are essential trace elements, most of them can be toxic to all forms of life at high concentrations due to formation of complex compounds within the cell. Unlike organic pollutants, heavy metals once introduced into the environment cannot be biodegraded. They persist indefinitely and cause pollution of air, water, and soils. Thus, the main strategies of pollution control are to reduce the bioavailability, mobility, and toxicity of metals. Methods for remediation of heavy metal-contaminated environments include physical removal, detoxification, bioleaching, and phytoremediation. Because heavy metals are increasingly found in microbial habitats due to natural and industrial processes, microorganisms have evolved several mechanisms to tolerate their presence by adsorption, complexation, or chemical reduction of metal ions or to use them as terminal electron acceptors in anaerobic respiration. In heavy metals, pollution abatement, microbial sensors, and transformations are getting increased focus because of high efficiency and cost effectiveness. The sources and impacts of heavy metal pollution as well as various remediation techniques are described.

Siderophore mediated plant growth promotion at low temperature by mutant of fluorescent pseudomonad

A cold resistant mutant of Pseudomonas fluorescens ATCC 13525 was developed, which could grow equally well at 25 and 10 °C and its effect on plant growth promotion under in vitro and in situ conditions was observed. Siderophore estimation revealed it to be a siderophore-overproducing mutant (17-fold increase) when compared to its wild type counterpart. A gnotobiotic root elongation assay indicated that the mutant (CRPF9) promoted growth more than its wild type both at 25 and 10 °C, indicating its effectiveness at low temperature. Further, root colonization studies showed that CRPF9 was an efficient rhizosphere colonizer, inducing a significant increase in root (35%) and shoot length (28%) of mung bean plants in unsterilized soil system. The persistence and stability of the mutant was evident in rhizospheric soil. A sand culture experiment showed that ferric citrate was better than Fe(OH)3 as an iron source for plant growth, but in the presence of CRPF9 both salts were comparable. This study demonstrates the potential of chemical mutagenesis for improving the plant growth promoting properties of a P. fluorescens strain and its stimulating impact on plant growth promotion at low temperature both under in vitro and in situ conditions.

Differential Proteomics in Response to Low Temperature Diazotrophy of Himalayan Psychrophilic Nitrogen Fixing Pseudomonas migulae S10724 Strain

Himalayas are considered as a reservoir of diversified and dynamic gene pool. This study describes the response of a Himalayan psychrophilic diazotroph to low temperature diazotrophy. Seven cold adaptive N2 fixing bacteria were isolated and identified as Bacillus sp., Arthrobacter sp., Rhodococcus sp., Pseudomonas sp., etc. In order to examine the physiological response to low temperature diazotrophy, differential proteomic analysis of Pseudomonas migulae S10724 strain was carried out using two dimensional electrophoresis and MALDI–TOF–MS. Functional assessment of 66 differentially expressed proteins revealed several mechanisms thought to be involved in low temperature adaptation and nitrogen fixation, including general stress adaptation, protein and nucleic acid synthesis, energy metabolism, cell growth/maintenance, etc. Major fraction of the upregulated proteins was stress proteins, while majority of the downregulated proteins were related to cell division. Furthermore, MALDI–TOF–MS-based identification of randomly selected peptides encountered two exclusively expressed proteins: NifU family SUF system FeS assembly protein and membrane protein, suppressor for copper-sensitivity B precursor which might have a crucial role at low temperature nitrogen fixation. To the best of our knowledge, this is the first report of the isolation and differential proteomic analysis of psychrophilic diazotroph from Himalayan high altitude rhizospheric soil.

Strategies for Crop Improvement in Contaminated Soils Using Metal-Tolerant Bioinoculants

Heavy metal contamination due to natural and anthropogenic sources is a global environmental concern. Release of heavy metals without proper treatment poses a serious threat to public health because of its persistence, biomagnification and accumulation in food chain. Nonbiodegradability and sludge production are the two major constraints of metal treatment. The bioremediation of soil, sludge, sediments and wastes polluted with heavy metals generally involves the active microbiological processes of biosorption, bioaccumulation, sequestration and efflux. Bioremediation using microbes well adapted to diverse physiological conditions could be utilized for remediation of heavy metal-contaminated sites. The application of proteomics in environmental bioremediation program provides a global view of the protein compositions of the microbial cells and offers a promising approach to understand the molecular mechanisms of bioremediation. In this chapter, attention is paid to highlighting the strategies for crop improvement using metal-tolerant microbes in soils contaminated with heavy metals.

Bacterial diversity and community structure of Western Indian Himalayan red kidney bean (Phaseolus vulgaris) rhizosphere as revealed by 16S rRNA gene sequences

Abstract Agriculture is an important livelihood activity in the Himalayan regions. Our previous studies revealed the presence of diverse diazotrophic assemblage in indigenous red kidney bean (RKB) rhizospheric soil from two different locations of Western Indian Himalaya, namely S1 (Chhiplakot, 30.70◦ N/80.30° E) and S2 (Munsyari, 30.60◦ N/80.20° E), selected on the basis of real-time PCR analysis. In this study, two 16S rRNA gene clone libraries (SB1 and SB2, respectively) were constructed using the same rhizospheric soil samples for assessing the total bacterial diversity and their community structure. A total of 760 clones were obtained, with ∼54-59% of these sequences belonging to the phylum Proteobacteria. While sequences belonging to Bacteroidetes, Chloroflexi, Acidobactria, Planctomycetes, Firmicutes, Nitrospira, Gemmatimonadetes, Cyanobacteria, Verrucomicrobia, OD1, OP11 and Actinobacteria were encountered in both the soils, sequences belonging to bacteria from the classes Chlorobi and BRC1 were only detected in the S1 soil. Both the libraries showed similar bacterial community compositions, with Pseudomonas (∼33-34%) as predominant genus. Phylogenetic analysis revealed that all the clone sequences were clustered in different bacterial groups as per their resemblance with their respective phylogenetic neighbours. Major clusters were formed by Gammapreoteobacteria followed by Bacteroidetes and Alphaproteobacteria. A good fraction of the clone sequences has no resemblance with existing groups, thereby suggesting the need of culture-dependent studies from Himalayan regions. To the best of our knowledge, this study is the first major metagenomic effort on Himalayan RKBs rhizobacteria revealing fundamental information that needs to be explored for functional studies.

Phosphate solubilization by Chryseobacterium sp. and their combined effect with N and P fertilizers on plant growth promotion

The capabilities of soil microorganisms to solubilize phosphate have been known for many years, but their isolation and use as crop inoculants have met with little success. Thirty-five bacterial isolates were screened for their phosphate-solubilizing ability, and two of them, PSR10 and RGR13, identified through 16S rDNA sequencing as Chryseobacterium sp. PSR10 and Escherichia coli RGR13, respectively, screened for plant growth promotion in the greenhouse. Seed inoculation of Macrotyloma uniflorum (horsegram) by Chryseobacterium sp. PSR10 showed better plant growth promotion in sterilized and unsterilized soil under greenhouse conditions and was selected for a field experiment with 100, 50 and 30% of recommended doses of nitrogen and phosphorus fertilizer. Seed inoculation with 50% of the recommended dose of nitrogen and phosphorus increased plant growth (agronomical parameters, chlorophyll content, nitrate reductase activity, phosphorus content and crop yield). We conclude that effective plant growth-promoting bacterium Chryseobacterium sp. PSR10 broadens the spectrum of phosphate solubilizers available for field applications and might be used together with 50% dose of nitrogen and phosphate.

Diversified diazotrophs associated with the rhizosphere of Western Indian Himalayan native red kidney beans (Phaseolus vulgaris L.)

Red kidney beans (RKBs) are one of the major components in the human diet of Western Indian Himalaya (WIH). Their cultivation in these habitats is strongly influenced by various biotic and abiotic stresses and therefore, there must be a selection of RKB associated microorganisms that are adapted to these harsh conditions. Seven cold adaptive diazotrophs from the same rhizosphere were isolated in our previous study to reveal the low-temperature associated proteins and mechanisms. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are still unknown. In this study, RKB rhizospheric soil from two different agro-ecosystems of WIH namely S1 (Chhiplakot, 30.70°N/80.30°E) and S2 (Munsyari, 30.60°N/80.20°E) were explored for the assessment of nitrogenase reductase gene (nifH) diversity by plating respective clone libraries SN1 and SN2. The RKB rhizosphere diazotroph assemblage was very diverse and apparently consists mainly of the genera Rhizobium, followed by unknown diazotrophic microorganisms. Deduced amino acid sequence analysis revealed the presence of diverse nifH sequences, affiliated with a wide range of taxa, encompassing members of the Proteobacteria, Actinobacteria and Firmicutes. Members of cyanobacteria, methanotrophs and archaea were also detected. To the best of our knowledge, this is the first major metagenomic effort that revealed the presence of diverse nitrogen-fixing microbial assemblages in indigenous RKB rhizospheric soil which can further be explored for improved crop yield/productivity.

Biodeterioration studies of thermoplastics in nature using indigenous bacterial consortium

Thermoplastics, poly vinyl chloride and low-density polyethylene were treated in the presence of indigenously developed bacterial consortium in laboratory and natural conditions. The consortium was developed using four bacteria, selected on the basis of utilization of PVC as primary carbon source, namely P. otitidis, B. aerius, B. cereus and A. pedis isolated from the plastic waste disposal sites in Northern India. The comparative in-vitro treatment studies as revealed by the spectral and thermal data, illustrated the relatively better biodegradation potential of developed consortium for PVC than the LDPE. Further, the progressive treatments of both the thermoplastics were conducted for three months under natural conditions. For this purpose, bioformulation of consortium was prepared and characterized for the viability up to 70 days of storage at 25±1oC. The consortium treated polymer samples were monitored through SEM and FT-IR spectroscopy. Analytical data revealed the biodeterioration potential of the developed consortium for PVC and LDPE, which could help in disposing the plastic waste.

Exploration of Csp Genes from Temperate and Glacier Soils of the Indian Himalayas and In Silico Analysis of Encoding Proteins

The metagenomic Csp library was constructed from the temperate and glacier soils of central Himalaya, India followed by polymerase chain reaction (PCR) amplification. The library was further screened for low-temperature adaptation, and the positive recombinants were sorted out by determining changes in the melting temperature (Tm). A homology search of cloned sequence showed their identity with the Csp genes of Pseudomonas fluorescens, Psychrobacter cryohalolentis K5, and Shewanella spp MR-4. Amino acid sequence analysis annotated the presence of conserved aromatic and basic amino acids as well as RNA binding motifs from the cold shock domain. Furthermore, a PROSITE scan showed a moderate identity of less than 60% with the known cold shock-inducible proteins (ribosomal proteins, rbfA, DEAD-box helicases), cold acclimation protein, and temperature-induced protein (SRP1/TIP1). This study highlighted the prevalence of Csp genes from cold Himalayan environments that can be explored for tailor-made crop constructions in future.