The changing paradigm of rhizobial taxonomy and its systematic growth upto postgenomic technologies.

Rhizobia are a diazotrophic group of bacteria that are usually isolated form the nodules in roots, stem of leguminous plants and are able to form nodules in the host plant owing to the presence of symbiotic genes. The rhizobial community is highly diverse, and therefore, the taxonomy and genera-wise classification of rhizobia has been constantly changing since the last three decades. This is mainly due to technical advancements, and shifts in definitions, resulting in a changing paradigm of rhizobia taxonomy. Initially, the taxonomic definitions at the species and sub species level were based on phylogenetic analysis of 16S rRNA sequence, followed by polyphasic approach to have phenotypic, biochemical, and genetic analysis including multilocus sequence analysis. Rhizobia mainly belonging to α- and ß-proteobacteria, and recently new additions from γ-proteobacteria had been classified. Nowadays rhizobial taxonomy has been replaced by genome-based taxonomy that allows gaining more insights of genomic characteristics. These omics-technologies provide genome specific information that considers nodulation and symbiotic genes, along with molecular markers as taxonomic traits. Taxonomy based on complete genome sequence (genotaxonomy), average nucleotide identity, is now being considered as primary approach, resulting in an ongoing paradigm shift in rhizobial taxonomy. Also, pairwise whole-genome comparisons, phylogenomic analyses offer correlations between DNA and DNA re-association values that have delineated biologically important species. This review elaborates the present classification and taxonomy of rhizobia, vis-a-vis development of technical advancements, parameters and controversies associated with it, and describe the updated information on evolutionary lineages of rhizobia.

Rhizodegradation of Pyrene by a Non-pathogenic Klebsiella pneumoniae Isolate Applied With Tagetes erecta L. and Changes in the Rhizobacterial Community.

The non-clinical Klebsiella pneumoniae variants, isolated from different environments, are now well acknowledged for their role in plant-growth promotion and biodegradation of pollutants. In the present study, a non-clinical environmental isolate K. pneumoniae AWD5 is being described for rhizoremediation of pyrene, applied through the rhizosphere of an ornamental plant, Tagetes erecta L (marigold). The non-pathogenic nature of AWD5 was established using an in vivo mouse model experiment, where AWD5 was unable to cause lung infection in tested mice. Degradation of pyrene, in the presence of succinate as co-substrate, was observed to be 87.5% by AWD5, after 21 days of incubation in minimal (Bushnell-Hass) medium in vitro conditions. Consequently, the bacterial inoculation through the rhizosphere of T. erecta L. plants resulted in 68.61% degradation of pyrene, which was significantly higher than control soil. Inoculation of AWD5 also improved plant growth and exhibited an increase in root length (14.64%), dry root weight (80.56%), shoot length (3.26%), and dry shoot weight (45.35%) after 60 days of incubation. T. erecta L., an ornamental plant, was also found to be suitable for bioremediation of pyrene. The effect of AWD5 application, and rhizoremediation process, on rhizosphere bacterial diversity and community structure has been studied using the metagenomic analysis of the 16S (V3-V4) region of rRNA. 37 bacterial phyla constituted the core microbiome, which was dominated by Proteobacteria followed by Actinobacteria, Actinobacteria, and Planctomycetes for all the treatments. AWD5 inoculation enhanced the relative abundance of Firmicutes and Acidobacteria as compared with other treatments. Genus Kaistobacter and Verrucomicrobia were found to be an abundant indigenous population in pyrene-spiked soils. Bacterial richness and diversity were analyzed using the Shannon-Wiener (H) index. A lower diversity index was observed in pyrene-spiked soils. Canonical correspondence analysis (CCA) showed a possible linkage with plant growth attributes and available nitrogen content that influences diversity and abundance of the bacterial community.

The Endophytic Microbiome as a Hotspot of Synergistic Interactions, with Prospects of Plant Growth Promotion.

The plant root is the primary site of interaction between plants and associated microorganisms and constitutes the main components of plant microbiomes that impact crop production. The endophytic bacteria in the root zone have an important role in plant growth promotion. Diverse microbial communities inhabit plant root tissues, and they directly or indirectly promote plant growth by inhibiting the growth of plant pathogens, producing various secondary metabolites. Mechanisms of plant growth promotion and response of root endophytic microorganisms for their survival and colonization in the host plants are the result of complex plant-microbe interactions. Endophytic microorganisms also assist the host to sustain different biotic and abiotic stresses. Better insights are emerging for the endophyte, such as host plant interactions due to advancements in 'omic' technologies, which facilitate the exploration of genes that are responsible for plant tissue colonization. Consequently, this is informative to envisage putative functions and metabolic processes crucial for endophytic adaptations. Detection of cell signaling molecules between host plants and identification of compounds synthesized by root endophytes are effective means for their utilization in the agriculture sector as biofertilizers. In addition, it is interesting that the endophytic microorganism colonization impacts the relative abundance of indigenous microbial communities and suppresses the deleterious microorganisms in plant tissues. Natural products released by endophytes act as biocontrol agents and inhibit pathogen growth. The symbiosis of endophytic bacteria and arbuscular mycorrhizal fungi (AMF) affects plant symbiotic signaling pathways and root colonization patterns and phytohormone synthesis. In this review, the potential of the root endophytic community, colonization, and role in the improvement of plant growth has been explained in the light of intricate plant-microbe interactions.

Enrichment of antibiotic resistance genes (ARGs) in polyaromatic hydrocarbon-contaminated soils: a major challenge for environmental health.

Polyaromatic hydrocarbons (PAHs) are widely spread ecological contaminants. Antibiotic resistance genes (ARGs) are present with mobile genetic elements (MGE) in the bacteria. There are molecular evidences that PAHs may induce the development of ARGs in contaminated soils. Also, the abundance of ARGs related to tetracycline, sulfonamides, aminoglycosides, ampicillin, and fluoroquinolones is high in PAH-contaminated environments. Genes encoding the efflux pump are located in the MGE and, along with class 1 integrons, have a significant role as a connecting link between PAH contamination and enrichment of ARGs. The horizontal gene transfer mechanisms further make this interaction more dynamic. Therefore, necessary steps to control ARGs into the environment and risk management plan of PAHs should be enforced. In this review, influence of PAH on evolution of ARGs in the contaminated soil, and its spread in the environment, has been described. The co-occurrence of antibiotic resistance and PAH degradation abilities in bacterial isolates has raised the concerns. Also, presence of ARGs in the microbiome of PAH-contaminated soil has been discussed as environmental hotspots for ARG spread. In addition to this, the possible links of molecular interactions between ARGs and PAHs, and their effect on environmental health has been explored.

Development, spread and persistence of antibiotic resistance genes (ARGs) in the soil microbiomes through co-selection.

Bacterial pathogens resistant to multiple antibiotics are emergent threat to the public health which may evolve in the environment due to the co-selection of antibiotic resistance, driven by poly aromatic hydrocarbons (PAHs) and/or heavy metal contaminations. The co-selection of antibiotic resistance (AMR) evolves through the co-resistance or cross-resistance, or co-regulatory mechanisms, present in bacteria. The persistent toxic contaminants impose widespread pressure in both clinical and environmental setting, and may potentially cause the maintenance and spread of antibiotic resistance genes (ARGs). In the past few years, due to exponential increase of AMR, numerous drugs are now no longer effective to treat infectious diseases, especially in cases of bacterial infections. In this mini-review, we have described the role of co-resistance and cross-resistance as main sources for co-selection of ARGs; while other co-regulatory mechanisms are also involved with cross-resistance that regulates multiple ARGs. However, co-factors also support selections, which results in development and evolution of ARGs in absence of antibiotic pressure. Efflux pumps present on the same mobile genetic elements, possibly due to the function of Class 1 integrons (Int1), may increase the presence of ARGs into the environment, which further is promptly changed as per environmental conditions. This review also signifies that mutation plays important role in the expansion of ARGs due to presence of diverse types of anthropogenic pollutants, which results in overexpression of efflux pump with higher bacterial fitness cost; and these situations result in acquisition of resistant genes. The future aspects of co-selection with involvement of systems biology, synthetic biology and gene network approaches have also been discussed.

Distinctive features gleaned from the comparative genomes analysis of clinical and non-clinical isolates of Klebsiella pneumoniae.

It is of interest to describe the distinctive features gleaned from the comparative genome analysis of clinical and non-clinical isolates of Klebsiella pneumoniae. The core genome of K. pneumoinae consisted of 3568 genes. Comparative genome analysis shows that mdtABCD, toxin-antitoxin systems are unique to clinical isolates and catB, benA, and transporter genes for citrate utilization are exclusive to non-clinical isolates. We further noted aromatic compound degrading genes in non-clinical isolates unlike in the later isolates. We grouped 88 core genes into 3 groups linked to infections, drug-resistance or xenobiotic metabolism using codon usage variation analysis. It is inferred using the neutrality plot analysis of GC12 with GC3 that codon usage variation is dominant over mutation pressure. Thus, we document data to distinguish clinical and non-clinical isolates of K. pneumoniae using comparative genomes analysis for understanding of genome diversity during speciation.

The rhizosphere microbiome: Significance in rhizoremediation of polyaromatic hydrocarbon contaminated soil.

Microbial communities are an essential part of plant rhizosphere and participate in the functioning of plants, including rhizoremediation of petroleum contaminants. Rhizoremediation is a promising technology for removal of polyaromatic hydrocarbons based on interactions between plants and microbiome in the rhizosphere. Root exudation in the rhizosphere provides better nutrient uptake for rhizosphere microbiome, and therefore it is considered to be one of the major factors of microbial community function in the rhizosphere that plays a key role in the enhanced PAH biodegradation. Although the importance of the rhizosphere microbiome for plant growth has been widely recognized, the interactions between microbiome and plant roots in the process of rhizosphere mediated remediation of PAH still needs attention. Most of the current researches target PAH degradation by plant or single microorganism, separately, whereas the interactions between plants and whole microbiome are overlooked and its role has been ignored. This review summarizes recent knowledge of PAH degradation in the rhizosphere in the process of plant-microbiome interactions based on emerging omics approaches such as metagenomics, metatranscriptomics, metabolomics and metaproteomics. These omics approaches with combinations to bioinformatics tools provide us a better understanding in integrated activity patterns between plants and rhizosphere microbes, and insight into the biochemical and molecular modification of the meta-organisms (plant-microbiome) to maximize rhizoremediation activity. Moreover, a better understanding of the interactions could lead to the development of techniques to engineer rhizosphere microbiome for better hydrocarbon degradation.

Genomic insights of aromatic hydrocarbon degrading Klebsiella pneumoniae AWD5 with plant growth promoting attributes: a paradigm of soil isolate with elements of biodegradation.

This research employs draft genome sequence data of Klebsiella pneumoniae AWD5 to explore genes that contribute to the degradation of polyaromatic hydrocarbon (PAH) and stimulate plant growth, for rhizosphere-mediated bioremediation. Annotation analysis suggests that the strain AWD5 not only possess gene clusters for PAH utilization, but also for utilization of benzoate, fluorobenzoate, phenylacetate (paa), hydroxyphenylacetic acid (hpa), 3-hydroxyphenyl propionate (mhp). A comparative genome analysis revealed that the genome of AWD5 was highly similar with genomes of environmental as well as clinical K. pneumoniae isolates. The artemis output confirmed that there are 139 different genes present in AWD5 which were absent in genome of clinical strain K. pneumoniae ATCC BAA-2146, and 25 genes were identified to be present in AWD5 genome but absent in genome of environmental strain K. pneumoniae KP-1. Pathway analyzed using Kyoto Encyclopedia of Genes and Genomes enzyme database revealed the presence of gene clusters that code for enzymes to initiate the opening of aromatic rings. The polyaromatic hydrocarbon and benzoate degradation were found to be metabolized through ortho-cleavage pathway, mineralizing the compounds to TCA cycle intermediates. Genes for plant growth promoting attributes such as Indole acetic acid (IAA) synthesis, siderophore production, and phosphate solubilization were detected in the genome. These attributes were verified in vitro, including IAA (14.75 µg/ml), siderophore production (13.56%), phosphate solubilization (198.28 ng/ml), and ACC deaminase (0.118 mM α-ketobutyrate/mg) in the presence of pyrene, and also compared with results obtained in glucose amended medium. K. pneumoniae AWD5 enhanced the growth of Jatropha curcas in the presence of pyrene-contaminated soil. Moreover, AWD5 harbors heavy metal resistance genes indicating adaptation to contaminants. The study revealed the genomic attributes of K. pneumoniae AWD5 for its catabolic characteristics for different aromatic compounds, which makes it suitable for rhizoremediation of PAH-contaminated soil.

Shape dependent physical mutilation and lethal effects of silver nanoparticles on bacteria.

In this report, spherical silver nanoparticle (AgNP-sp) and rod-shaped silver nanoparticle (AgNR) were prepared by chemical reduction method and their antibacterial activity against various Gram-positive and Gram-negative bacteria had been evaluated for their efficiency. Minimal inhibitory concentration (MIC) tests were conducted to study the antibacterial properties, and substantiated with killing kinetics of silver nanoparticles (AgNPs). The study revealed that both AgNP-sp and AgNRs are good antibacterial candidates. Bacterial sensitivity to nanoparticles (NPs) was found to vary depending on microbial species. Disc diffusion studies revealed the greater effectiveness of AgNP-sp and AgNR against Klebsiella pneumoniae AWD5 at the doses of 249 and 392 µg. The dose dependent activities of prepared NPs were also observed on the batch studies of disc diffusion and MIC with various strains. The optical and morphological structures of NPs were analyzed by UV-visible, XRD, FE-SEM and TEM. Further, FESEM of bacterial culture treated with AgNPs confirmed antibacterial activity of NPs by showing rupture of bacterial cell wall. Also, the genome of test organism was found to have CusCFBA and CusRS operons. The killing kinetics confirmed that the death rate of K. pneumoniae was higher against AgNP-sp as compared to AgNR.

Draft Genome Sequence of Klebsiella pneumoniae AWD5.

Here, we report the draft genome sequence of Klebsiella pneumoniae strain AWD5, isolated from an automobile workshop in India. The de novo assembly resulted in a 4,807,409 bp genome containing 25 rRNA genes, 81 tRNAs, and 4,636 coding sequences (CDS). It carries important genes for polyaromatic hydrocarbon degradation and benzoate degradation.