Browsing by Author "Parul Pandey"

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An ECF41 family σ factor controls motility and biogenesis of lateral flagella in azospirillum brasilense Sp245
(American Society for Microbiology, 2020) Ashutosh Prakash Dubey; Parul Pandey; Vijay Shankar Singh; Mukti Nath Mishra; Sudhir Singh; Rajeev Mishra; Anil Kumar Tripathi
ECF41 is a large family of bacterial extracytoplasmic function (ECF) σ factors. Their role in bacterial physiology or behavior, however, is not known. One of the 10 ECF σ factors encoded in the genome of Azospirillum brasilense Sp245, RpoE10, exhibits features characteristic of the typical ECF41-type σ factors. Inactivation of rpoE10 in A. brasilense Sp245 led to an increase in motility that could be complemented by the expression of rpoE10. By comparing the number of lateral flagella, transcriptome, and proteome of A. brasilense Sp245 with those of its rpoE10::km mutant, we show here that this ECF41-type σ factor is involved in the negative regulation of swimming motility and biogenesis of lateral flagella of A. brasilense Sp245. The genome of A. brasilense Sp245 also encodes two OmpR-type regulators (LafR1 and LafR2) and three flagellins, including Laf1, the major flagellin of lateral flagella. Elevated levels of laf1 transcripts and Laf1 protein in the rpoE10::km mutant indicated that RpoE10 negatively regulates the expression of Laf1. The elevated level of LafR1 in the rpoE10::km mutant indicated that LafR1 is also negatively regulated by RpoE10. The loss of motility and Laf1 in the lafR1::km mutant, complemented by lafR1 expression, showed that LafR1 is a positive regulator of Laf1 and motility in A. brasilense. In addition, upregulation of laf1:: lacZ and lafR1::lacZ fusions by RpoE10 and downregulation of the laf1::lacZ fusion by LafR1 suggest that RpoE10 negatively regulates swimming motility and the expression of LafR1 and Laf1. However, LafR1 positively regulates the swimming motility and Laf1 expression. IMPORTANCE Among extracytoplasmic function (ECF) σ factors, ECF41-type σ factors are unique due to the presence of a large C-terminal extension in place of a cognate anti-σ factor, which regulates their activity. Despite their wide distribution and abundance in bacterial genomes, their physiological or behavioral roles are not known. We show here an indirect negative role of an ECF41-type of σ factor in the expression of lateral flagellar genes and motility in A. brasilense. This study suggests that the motility of A. brasilense might be controlled by a regulatory cascade involving RpoE10, an unknown repressor, LafR1, and lateral flagellar genes, including that encoding Laf1. Copyright © 2020 American Society for Microbiology. All Rights Reserved.
Catalase expression in Azospirillum brasilense Sp7 is regulated by a network consisting of OxyR and two RpoH paralogs and including an RpoE1→RpoH5 regulatory cascade
(American Society for Microbiology, 2018) Ashutosh Kumar Rai; Sudhir Singh; Sushil Kumar Dwivedi; Amit Srivastava; Parul Pandey; Santosh Kumar; Bhupendra Narain Singh; Anil Kumar Tripathi
The genome of Azospirillum brasilense encodes five RpoH sigma factors: two OxyR transcription regulators and three catalases. The aim of this study was to understand the role they play during oxidative stress and their regulatory interconnection. Out of the 5 paralogs of RpoH present in A. brasilense, inactivation of only rpoH1 renders A. brasilense heat sensitive. While transcript levels of rpoH1 were elevated by heat stress, those of rpoH3 and rpoH5 were upregulated by H2O2. Catalase activity was upregulated in A. brasilense and its rpoH::km mutants in response to H2O2 except in the case of the rpoH5::km mutant, suggesting a role for RpoH5 in regulating inducible catalase. Transcriptional analysis of the katN, katAI, and katAII genes revealed that the expression of katN and katAII was severely compromised in the rpoH3::km and rpoH5::km mutants, respectively. Regulation of katN and katAII by RpoH3 and RpoH5, respectively, was further confirmed in an Escherichia coli twoplasmid system. Regulation of katAII by OxyR2 was evident by a drastic reduction in growth, KatAII activity, and katAII::lacZ expression in an oxyR2::km mutant. This study reports the involvement of RpoH3 and RpoH5 sigma factors in regulating oxidative stress response in alphaproteobacteria. We also report the regulation of an inducible catalase by a cascade of alternative sigma factors and an OxyR. Out of the three catalases in A. brasilense, those corresponding to katN and katAII are regulated by RpoH3 and RpoH5, respectively. The expression of katAII is regulated by a cascade of RpoE1→RpoH5 and OxyR2. © 2018 American Society for Microbiology.
Cometabolism of ethanol in azospirillum brasilense Sp7 Is mediated by fructose and glycerol and regulated negatively by an alternative sigma factor RpoH2
(American Society for Microbiology, 2021) Vijay Shankar Singh; Basant Kumar Dubey; Parul Pandey; Sushant Rai; Anil Kumar Tripathi
Azospirillum brasilense is a plant growth-promoting rhizobacterium that is not known to utilize ethanol as a sole source of carbon for growth. This study shows that A. brasilense can cometabolize ethanol in medium containing fructose or glycerol as a carbon source and contribute to its growth. In minimal medium containing fructose or glycerol as a carbon source, supplementation of ethanol caused enhanced production of an alcohol dehydrogenase (ExaA) and an aldehyde dehydrogenase (AldA) in A. brasilense. However, this was not the case when malate was used as a carbon source. Inactivation of aldA in A. brasilense resulted in the loss of the AldA protein and its ethanol utilizing ability in fructose- or glycerol-supplemented medium. Furthermore, ethanol inhibited the growth of the aldA::Km mutant. The exaA::Km mutant also lost its ability to utilize ethanol in fructose-supplemented medium. However, in glycerol-supplemented medium, A. brasilense utilized ethanol due to the synthesis of a new paralog of alcohol dehydrogenase (ExaA1). The expression of exaA1 was induced by glycerol but not by fructose. Unlike exaA, expression of aldA and exaA1 were not dependent on s54. Instead, they were negatively regulated by the RpoH2 sigma factor. Inactivation of rpoH2 in A. brasilense conferred the ability to use ethanol as a carbon source without or with malate, overcoming catabolite repression caused by malate. This is the first study showing the role of glycerol and fructose in facilitating cometabolism of ethanol by inducing the expression of ethanol-oxidizing enzymes and the role of RpoH2 in repressing them. IMPORTANCE This study unraveled a hidden ability of Azospirillum brasilense to utilize ethanol as a secondary source of carbon when fructose or glycerol were used as a primary growth substrate. It opens the possibility of studying the regulation of expression of the ethanol oxidation pathway for generating high yielding strains that can efficiently utilize ethanol. Such strains would be useful for economical production of secondary metabolites by A. brasilense in fermenters. The ability of A. brasilense to utilize ethanol might be beneficial to the host plant under the submerged growth conditions. Copyright © 2021 American Society for Microbiology. All Rights Reserved.
Dicarboxylate transporters of Azospirillum brasilense Sp7 play an important role in the colonization of finger millet (Eleusine coracana) roots
(American Phytopathological Society, 2019) Vijay Shankar Singh; Prajna Tripathi; Parul Pandey; Durgesh Narain Singh; Basant Kumar Dubey; Chhaya Singh; Surendra Pratap Singh; Rachana Pandey; Anil Kumar Tripathi
Azospirillum brasilense is a plant growth-promoting bacterium that colonizes the roots of a large number of plants, including C3 and C4 grasses. Malate has been used as a preferred source of carbon for the enrichment and isolation Azospirillum spp., but the genes involved in their transport and utilization are not yet characterized. In this study, we investigated the role of the two types of dicarboxylate transporters (DctP and DctA) of A. brasilense in their ability to colonize and promote growth of the roots of a C4 grass. We found that DctP protein was distinctly upregulated in A. brasilense grown with malate as sole carbon source. Inactivation of dctP in A. brasilense led to a drastic reduction in its ability to grow on dicarboxylates and form cell aggregates. Inactivation of dctA, however, showed a marginal reduction in growth and flocculation. The growth and nitrogen fixation of a dctP and dctA double mutant of A. brasilense were severely compromised. We have shown here that DctPQM and DctA transporters play a major and a minor role in the transport of C4-dicarboxylates in A. brasilense, respectively. Studies on inoculation of the seedlings of a C4 grass, Eleusine corcana, with A. brasilense and its dicarboxylate transport mutants revealed that dicarboxylate transporters are required by A. brasilense for an efficient colonization of plant roots and their growth. © 2019 The American Phytopathological Society.
Engineering a carotenoid-overproducing strain of azospirillum brasilense for heterologous production of geraniol and amorphadiene
(American Society for Microbiology, 2020) Shivangi Mishra; Parul Pandey; Ashutosh Prakash Dubey; Aafreen Zehra; Chandan Singh Chanotiya; Anil Kumar Tripathi; Mukti Nath Mishra
Escherichia coli and Saccharomyces cerevisiae have been used extensively for heterologous production of a variety of secondary metabolites. Neither has an endogenous high-flux isoprenoid pathway, required for the production of terpenoids. Azospirillum brasilense, a nonphotosynthetic GRAS (generally recognized as safe) bacterium, produces carotenoids in the presence of light. The carotenoid production increases multifold upon inactivating a gene encoding an anti-sigma factor (ChrR1). We used this A. brasilense mutant (Car-1) as a host for the heterologous production of two high-value phytochemicals, geraniol and amorphadiene. Cloned genes (crtE1 and crtE2) of A. brasilense encoding native geranylgeranyl pyrophosphate synthases (GGPPS), when overexpressed and purified, did not produce geranyl pyrophosphate (GPP) in vitro. Therefore, we cloned codon-optimized copies of the Catharanthus roseus genes encoding GPP synthase (GPPS) and geraniol synthase (GES) to show the endogenous intermediates of the carotenoid biosynthetic pathway in the Car-1 strain were utilized for the heterologous production of geraniol in A. brasilense. Similarly, cloning and expression of a codon-optimized copy of the amorphadiene synthase (ads) gene from Artemisia annua also led to the heterologous production of amorphadiene in Car-1. Geraniol or amorphadiene content was estimated using gas chromatography-mass spectrometry (GC-MS) and GC. These results demonstrate that Car-1 is a promising host for metabolic engineering, as the naturally available endogenous pool of the intermediates of the carotenoid biosynthetic pathway of A. brasilense can be effectively utilized for the heterologous production of high-value phytochemicals. © 2020 American Society for Microbiology.
Evidence for high-risk pollutants and emerging microbial contaminants at two major bathing ghats of the river Ganga using high-resolution mass spectrometry and metagenomics
(Elsevier B.V., 2025) Durgesh Narain Singh; Parul Pandey; Vijay Shankar Singh; Anil Kumar Tripathi
An efficient wastewater treatment plant is imperative to limit the entry of emerging pollutants (EPs) and emerging microbial contaminants (EMCs) in the river ecosystem. The detection of emerging EPs in aquatic environments is challenging due to complex sample preparation methods, and the need for sophisticated accurate analytical tools. In Varanasi (India), the river Ganga holds immense significance as a holy river but is consistently polluted with municipal (MWW) and hospital wastewater (HWW). We developed an efficient method for untargeted detection of EPs in the water samples using High-resolution mass spectrometry (HRMS), and identified 577 and 670 chemicals (or chemical components) in the water samples from two major bathing ghats, Assi Ghat (AG) and Dashashwamedh Ghat (DG), respectively. The presence of EPs of different categories viz chemicals from research labs, diagnostic labs, lifestyle and industrial chemicals, toxins, flavor and food additives indicated the unsafe disposal of MWW and HWW or inefficient wastewater treatment plants (WWTPs). Besides, shotgun metagenomic analysis depicted the presence of bacteria associated with MWW viz Cloacibacterium normanse, Sphaerotilus natans (sewage fungi), E. coli, and Prevotella. Also, the presence of human pathogens Arcobacter, Polynucleobacter, Pseudomonas, Klebsiella, Aeromonas, Acinetobacter, Vibrio, and Campylobacter suggests the discharge of HWW. EPs are linked to the development, and transmission of antimicrobial resistance (AMR). Occurrence of antibiotic resistance genes (ARGs), plasmid-borne β-lactamases, aminoglycoside transferases, and ARGs associated with integrons, transposons and plasmids viz mcr-3 gene that confer resistance to colistin, the last resort of antibiotics confirmed the presence of emerging microbial contaminants. Subsequent genome reconstruction studies showed the presence of uncultivable ARB and transmission of ARGs through horizontal gene transfer. This study can be used to monitor the health of aquatic bodies as well as the efficiency of WWTPs and raise an urgent need for efficient WWTPs to safeguard the river, Ganga. © 2024 Elsevier B.V.
RelQ mediates the expression of β-Lactam resistance in methicillin-resistant staphylococcus aureus
(Frontiers Media S.A., 2019) Ajita Bhawini; Parul Pandey; Ashutosh Prakash Dubey; Aafreen Zehra; Gopal Nath; Mukti Nath Mishra
An induced stringent response, which is established by an increased level of (p)ppGpp, is required for the expression of β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). However, it is not clear whether RSH (enzyme mediating stringent response to amino acid starvation) or small alarmone synthetases (SASs) are involved in the maintenance of (p)ppGpp level in response to β-lactams. Since the S. aureus genome encodes two active SASs (RelP and RelQ), their contribution to the expression of β-lactam resistance in MRSA was investigated. It was determined that relQ deletion renders community-associated MRSA (CA-MRSA) sensitive to β-lactams by negatively affecting the expression of mecA, and induction of (p)ppGpp synthesis by mupirocin bypasses the requirement of relQ for the expression of high-level β-lactam resistance. Surprisingly, relP deletion increased the level of β-lactam resistance. Such contradictory observations could be attributed to the fact that relQ promoter is ~5-fold stronger than the relP and is induced by oxacillin as well as deletion of either of the SASs, while relP promoter responds only to oxacillin. The stronger promoter activity of relQ, coupled with the inducibility of the relQ promoter in response to the lack of relP, results in efficient expression of relQ in the relP-deleted background. This positively affects mecA expression and renders the ΔrelP strain highly resistant. These findings indicate an important role for RelQ in the expression of high-level β-lactam resistance in MRSA. Copyright © 2019 Bhawini, Pandey, Dubey, Zehra, Nath and Mishra. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Role of a fasciclin domain protein in photooxidative stress and flocculation in Azospirillum brasilense Sp7
(Elsevier Masson s.r.l., 2021) Ashutosh Prakash Dubey; Parul Pandey; Shivangi Mishra; Parikshit Gupta; Anil Kumar Tripathi
Fasciclin domain proteins (FDP) are found in all domains of life, but their biological role and regulation are not clearly understood. While studying the proteome of a mutant (Car1) of Azospirillum brasilense Sp7 with a Tn5 insertion in the gene encoding an anti-sigma factor (ChrR1), we found that FDP was maximally expressed. To study the biological role of this FDP, we inactivated fdp in A. brasilense Sp7 and in its Car1 mutant, which rendered them sensitive to methylene blue (MB) and toluidine blue (TB) in the presence of light. The transcription of fdp was also strongly upregulated by an ECF sigma factor (RpoE1) and photooxidative stress. The fdp null mutants of A. brasilense Sp7 and its Car1 mutant produced relatively fewer carotenoids and showed reduced flocculation. The reduced ability of fdp null mutants to flocculate was partly due to their reduced ability to produce carotenoids as inhibition of carotenoid synthesis by diphenylamine reduced their flocculation ability by 15–20%. Hence, FDP plays an important role in protecting A. brasilense Sp7 against photo-oxidative stress by supporting carotenoid accumulation and cell aggregation. © 2021 Institut Pasteur
Whole-genome sequences of four Indian isolates of azospirillum brasilense
(American Society for Microbiology, 2019) Chhaya Singh; Parul Pandey; Durgesh Narain Singh; Rachna Pandey; Ajit Kumar Shasany; Anil Kumar Tripathi
Azospirillum brasilense is used worldwide as a plant growth-promoting inoculant for agricultural crops. To understand how the genomes of Indian strains of A. brasilense compare with their South American counterparts, we determined the whole-genome sequences of four strains of A. brasilense isolated from the rhizosphere of grasses from India. Copyright © 2019 Singh et al.
β-lactam Resistance in Azospirillum baldaniorum Sp245 Is Mediated by Lytic Transglycosylase and β-lactamase and Regulated by a Cascade of RpoE7!RpoH3 Sigma Factors
(American Society for Microbiology, 2022) Parul Pandey; Ashutosh P. Dubey; Shivangi Mishra; Vijay Shankar Singh; Chhaya Singh; Anil K. Tripathi
Bacterial resistance to β-lactam antibiotics is often mediated by β-lactamases and lytic transglycosylases. Azospirillum baldaniorum Sp245 is a plant-growth-promoting rhizobacterium that shows high levels of resistance to ampicillin. Investigating the molecular basis of ampicillin resistance and its regulation in A. baldaniorum Sp245, we found that a gene encoding lytic transglycosylase (Ltg1) is organized divergently from a gene encoding an extracytoplasmic function (ECF) s factor (RpoE7) in its genome. Inactivation of rpoE7 in A. baldaniorum Sp245 led to increased ability to form cell-cell aggregates and produce exopolysaccharides and biofilm, suggesting that rpoE7 might contribute to antibiotic resistance. Inactivation of ltg1 in A. baldaniorum Sp245, however, adversely affected its growth, indicating a requirement of Ltg1 for optimal growth. The expression of rpoE7, as well that of as ltg1, was positively regulated by RpoE7, and overexpression of RpoE7 conferred ampicillin sensitivity to both the rpoE7:: km mutant and its parent. In addition, RpoE7 negatively regulated the expression of a gene encoding a β-lactamase (bla1). Out of the 5 paralogs of RpoH encoded in the genome of A. baldaniorum Sp245, RpoH3 played major roles in conferring ampicillin sensitivity and in the downregulation of bla1. The expression of rpoH3 was positively regulated by RpoE7. Collectively, these observations reveal a novel regulatory cascade of RpoE7-RpoH3 s factors that negatively regulates ampicillin resistance in A. baldaniorum Sp245 by controlling the expression of a β-lactamase and a lytic transglycosylase. In the absence of a cognate anti-sigma factor, addressing how the activity of RpoE7 is regulated by β-lactams will unravel new mechanisms of regulation of β-lactam resistance in bacteria. © 2022 American Society for Microbiology. All rights reserved.