Browsing by Author "Vijay Shankar Singh"

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A comparative analysis of mycobacterial ribonucleases: Towards a therapeutic novel drug target
(Elsevier B.V., 2024) Lav Kumar Jaiswal; Rakesh Kumar Singh; Tanmayee Nayak; Anuja Kakkar; Garima Kandwal; Vijay Shankar Singh; Ankush Gupta
Bacterial responses to continuously changing environments are addressed through modulation of gene expression at the level of transcription initiation, RNA processing and/or decay. Ribonucleases (RNases) are hydrolytic or phosphorolytic enzymes involved in a majority of RNA metabolism reactions. RNases play a crucial role in RNA degradation, either independently or in collaboration with various trans-acting regulatory factors. The genus Mycobacterium consists of five subgenera: Mycobacteroides, Mycolicibacterium, Mycobacterium, Mycolicibacter and Mycolicibacillus, which include 63 fully sequenced species (pathogenic/non-pathogenic) to date. These include 13 different RNases, among which 5 are exonucleases (RNase PH, PNPase, RNase D, nano-RNases and RNase AS) and 8 are endonucleases (RNase J, RNase H, RNase P, RNase III, RNase BN, RNase Z, RNase G and RNase E), although RNase J and RNase BN were later identified to have exoribonuclease functions also. Here, we provide a detailed comparative insight into the Escherichia coli and mycobacterial RNases with respect to their types, phylogeny, structure, function, regulation and mechanism of action, with the main emphasis on RNase E. Among these 13 different mycobacterial RNases, 10 are essential for cell survival and have diverse structures hence, they are promising drug targets. RNase E is also an essential endonuclease that is abundant in many bacteria, forms an RNA degradosome complex that controls central RNA processing/degradation and has a conserved 5′ sensor domain/DNase-I like region in its RNase domain. The essential mycobacterial RNases especially RNase E provide a potential repertoire of drug targets that can be exploited for inhibitor/modulator screening against many deadly mycobacterial diseases. © 2024 The Authors
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.
Bacteriophytochrome controls carotenoid-independent response to photodynamic stress in a non-photosynthetic rhizobacterium, Azospirillum brasilense Sp7
(2012) Santosh Kumar; Suneel Kateriya; Vijay Shankar Singh; Meenakshi Tanwar; Shweta Agarwal; Hina Singh; Jitendra Paul Khurana; Devinder Vijay Amla; Anil Kumar Tripathi
Ever since the discovery of the role of bacteriophytochrome (BphP) in inducing carotenoid synthesis in Deinococcus radiodurans in response to light the role of BphPs in other non-photosynthetic bacteria is not clear yet. Azospirillum brasilense, a non-photosynthetic rhizobacterium, harbours a pair of BphPs out of which AbBphP1 is a homolog of AtBphP1 of Agrobacterium tumefaciens. By overexpression, purification, biochemical and spectral characterization we have shown that AbBphP1 is a photochromic bacteriophytochrome. Phenotypic study of the ΔAbBphP1 mutant showed that it is required for the survival of A. brasilense on minimal medium under red light. The mutant also showed reduced chemotaxis towards dicarboxylates and increased sensitivity to the photooxidative stress. Unlike D. radiodurans, AbBphP1 was not involved in controlling carotenoid synthesis. Proteome analysis of the ΔAbBphP1 indicated that AbBphP1 is involved in inducing a cellular response that enables A. brasilense in regenerating proteins that might be damaged due to photodynamic stress. © 2012 Macmillan Publishers Limited. All rights reserved.
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.
Deciphering the role of the two conserved motifs of the ECF41 family σ factor in the autoregulation of its own promoter in Azospirillum brasilense Sp245
(John Wiley and Sons Inc, 2022) Ekta Pathak; Ashutosh Prakash Dubey; Vijay Shankar Singh; Rajeev Mishra; Anil Kumar Tripathi
In Azospirillum brasilense, an extra-cytoplasmic function σ factor (RpoE10) shows the characteristic 119 amino acid long C-terminal extension found in ECF41-type σ factors, which possesses three conserved motifs (WLPEP, DGGGR, and NPDKV), one in the linker region between the σ2 and σ4, and the other two in the SnoaL_2 domain of the C-terminal extension. Here, we have described the role of the two conserved motifs in the SnoaL_2 domain of RpoE10 in the inhibition and activation of its activity, respectively. Truncation of the distal part of the C-terminal sequence of the RpoE10 (including NPDKV but excluding the DGGGR motif) results in its promoter's activation suggesting autoregulation. Further truncation of the C-terminal sequence up to its proximal part, including NPDKV and DGGGR motif, abolished promoter activation. Replacement of NPDKV motif with NAAAV in RpoE10 increased its ability to activate its promoter, whereas replacement of DGGGR motif led to reduced promoter activation. We have explored the dynamic modulation of σ2 -σ4 domains and the relevant molecular interactions mediated by the two conserved motifs of the SnoaL2 domain using molecular dynamics simulation. The analysis enabled us to explain that the NPDKV motif located distally in the C-terminus negatively impacts transcriptional activation. In contrast, the DGGGR motif found proximally of the C-terminal extension is required to activate RpoE10. © 2022 Wiley Periodicals LLC.
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 D-glucose utilization in Azospirillum brasilense Sp7 promotes rice root colonization
(Springer Science and Business Media Deutschland GmbH, 2022) Vijay Shankar Singh; Basant Kumar Dubey; Sushant Rai; Surendra Pratap Singh; Anil Kumar Tripathi
Abstract: Bacteria of the genus Azospirillum include several plant associated bacteria which often promote the growth of their host plants. Although the host range of Azospirillum brasilense Sp7 is much wider than its close relative Azospirillum lipoferum 4B, it lacks the ability to efficiently utilize D-glucose for its growth. By comparing the genomes of both the species, the genes of A. lipoferum 4B responsible for conferring D-glucose utilization ability in A. brasilese Sp7 were identified by cloning individual or a combination of genes in a broad host range expression vector, mobilizing them in A. brasilense Sp7 and examining the ability of exconjugants to use D-glucose as sole carbon source for growth. These genes also included the homologs of genes involved in N-acetyl glucosamine utilization in Pseudomonas aeruginosa PAO1. A transcriptional fusion of the 5 genes encoding glucose-6-phosphate dehydrogenase and 4 components of glucose phosphotransferase system were able to improve D-glucose utilization ability in A. brasilense Sp7. The A. brasilense Sp7 strain engineered with D-glucose utilization ability showed significantly improved root colonization of rice seedling. The improvement in the ability of A. brasilense Sp7 to colonize rice roots is expected to bring benefits to rice by promoting its growth. Key points: • Genes required for glucose utilization in Azospirillum lipoferum were identified. • A gene cassette encoding glucose utilization was constructed. • Transfer of gene cassette in A. brasilense improves glucose utilization and rice root colonization. © 2022, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
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.
Expression of alkyl hydroperoxide reductase is regulated negatively by OxyR1 and positively by RpoE2 sigma factor in Azospirillum brasilense Sp7
(Microbiology Society, 2016) Sudhir Singh; Susheel Kumar Dwivedi; Vijay Shankar Singh; Anil Kumar Tripathi
OxyR proteins are LysR-type transcriptional regulators, which play an important role in responding to oxidative stress in bacteria. Azospirillum brasilense Sp7 harbours two copies of OxyR. The inactivation of the oxyR1, the gene organized divergently to ahpC in A. brasilense Sp7, led to an increased tolerance to alkyl hydroperoxides, which was corroborated by an increase in alkyl hydroperoxide reductase (AhpC) activity, enhanced expression of ahpC:lacZ fusion and increased synthesis of AhpC protein in the oxyR1::km mutant. The upstream region of ahpC promoter harboured a putative OxyR binding site, T-N11-A. Mutation of T, A or both in the T-N11-Amotif caused derepression of ahpC in A. brasilense suggesting that T-N11-A might be the binding site for a negative regulator. Retardation of the electrophoretic mobility of the T-N11-A motif harbouring oxyR1-ahpC intergenic DNA by recombinant OxyR1, under reducing as well as oxidizing conditions, indicated that OxyR1 acts as a negative regulator of ahpC in A. brasilense. Sequence of the promoter of ahpC, predicted on the basis of transcriptional start site, and an enhanced expression of ahpC:lacZ fusion in chrR2::km mutant background suggested that ahpC promoter was RpoE2 dependent. Thus, this study shows that in A. brasilense Sp7, ahpC expression is regulated negatively by OxyR1 but is regulated positively by RpoE2, an oxidativestress- responsive sigma factor. It also shows that OxyR1 regulates the expression RpoE1, which is known to play an important role during photooxidative stress in A. brasilense. © 2016 The Authors.
Functional metagenomics: A potential tool for mining of biomolecules from environmental samples
(Elsevier Inc., 2025) Vivek Kumar; Anjali Singh; Vijay Shankar Singh; Rajan Chaurasia; Mahesh Rao; Pramod Kumar Sahu; Durgesh Kumar Jaiswal
Functional metagenomics is emerging as a transformative tool for discovering biomolecules from environmental samples, offering unprecedented insights into microbial diversity and functionality. This review explores the potential of functional metagenomics to uncover novel enzymes, antibiotics, and therapeutic compounds by analyzing environmental DNA (eDNA) without the constraints of traditional culture-based methods. The vast microbial biomass in diverse ecosystems, from soil to extreme habitats, harbors a wealth of genetic resources that can be harnessed for biotechnological applications. Researchers can use modern sequencing techniques and functional screening approaches to find and characterize genes with advantageous features, such as bioremediation capacities and industrially relevant enzymes. Despite challenges in DNA extraction and host expression systems, integrating functional metagenomics with multi-omics approaches promises to enhance our understanding of microbial interactions and facilitate the development of eco-friendly bioprocesses. This review underscores the importance of functional metagenomics in expanding our repertoire of biomolecules and highlights its potential to significantly impact biotechnology and environmental sustainability. © 2025 Elsevier Inc.
Identification and characterization of a novel phosphodiesterase from the metagenome of an Indian coalbed
(Public Library of Science, 2015) Durgesh Narain Singh; Ankush Gupta; Vijay Shankar Singh; Rajeev Mishra; Suneel Kateriya; Anil Kumar Tripathi
Phosphoesterases are involved in the degradation of organophosphorus compounds. Although phosphomonoesterases and phosphotriesterases have been studied in detail, studies on phosphodiesterases are rather limited. In our search to find novel phosphodiesterases using metagenomic approach, we cloned a gene encoding a putative phosphodiesterase (PdeM) from the metagenome of the formation water collected from an Indian coal bed. Bioinformatic analysis showed that PdeM sequence possessed the characteristic signature motifs of the class III phosphodiesterases and phylogenetic study of PdeM enabled us to identify three distinct subclasses (A, B, and C) within class III phosphodiesterases, PdeM clustering in new subclass IIIB. Bioinformatic, biochemical and biophysical characterization of PdeM further revealed some of the characteristic features of the phosphodiesterases belonging to newly described subclass IIIB. PdeM is a monomer of 29.3 kDa, which exhibits optimum activity at 25° C and pH 8.5, but low affinity for bis(pNPP) as well as pNPPP. The recombinant PdeM possessed phosphodiesterase, phosphonate-ester hydrolase and nuclease activity. It lacked phosphomonoesterase, phosphotriesterase, and RNAse activities. Overexpression of PdeM in E.coli neither affected catabolite respression nor did the recombinant protein hydrolyzed cAMP in vitro, indicating its inability to hydrolyze cAMP. Although Mn 2+ was required for the activity of PdeM, but addition of metals (Mn2+ or Fe3+) did not induce oligomerization. Further increase in concentration of Mn 2+ upto 3 mM, increased α-helical content as well as the phosphodiesterase activity. Structural comparison of PdeM with its homologs showed that it lacked critical residues required for dimerization, cAMP hydrolysis, and for the high affinity binding of bis(pNPP). PdeM, thus, is a novel representative of new subclass of class III phosphodiesterases. © 2015 Singh et al.
Identification and functional characterization of a fructose-inducible phosphotransferase system in Azospirillum brasilense Sp7
(American Society for Microbiology, 2025) Sushant Rai; Vijay Shankar Singh; Parikshit Gupta; Anil Kumar Tripathi
Plant growth-promoting rhizobacterium Azospirillum brasilense Sp7 utilizes fructose efficiently via a fructose phosphotransferase system (Fru-PTS). Its genome encodes two putative Fru-PTS, each consisting of FruB (EIIA), FruK (Pfk), and FruA (EIIBC) proteins. We compared the proteomes of A. brasilense Sp7 grown with malate or fructose as sole carbon source, and noticed upregulation of the constituent proteins of Fru-PTS1 only on fructose. Inactivation of fruA gene of both the Fru-PTS showed that Fru-PTS1 is the main PTS involved in fructose utilization. Overexpression of fruA1 in A. brasilense Sp7 enhanced its growth on fructose showing improved consumption of fructose. This suggested that fructose utilization in A. brasilense Sp7 is limited due to the limitation of EIIBC component. A FruR-type regulator, encoded divergently to the Fru-PTS1 operon, was required for chemotaxis toward fructose. Although not an absolute necessity for the growth of fructose, FruR was required for the optimal growth of fructose. The fruB1 promoter was activated by fructose and repressed by malate, but FruR does not seem to regulate its expression. A 27-nucleotide stem-loop structure located between the −125 and −99 promoter proximal region of fruB1 was involved in fructose inducibility and malate repression. Fructose also upregulated several proteins involved in the biogenesis of a Type 6 secretion system. Here, we have shown that A. brasilense Sp7 was able to inhibit the growth of Escherichia coli and Agrobacterium tumefaciens in the presence of fructose, and that an intact T6SS was required for contact-dependent growth inhibition of the two Gram-negative bacteria. © © 2025 Rai et al.
Management of Agriculture Waste: Bioconversion of Agro-Waste into Valued Products
(Bentham Science Publishers, 2022) Durgesh Narain Singh; Manikant Tripathi; Vijay Shankar Singh; Rakshpal Singh; Rajeeva Gaur; Neelam Pathak
Agriculture wastes or agro-wastes are byproducts obtained after the processing of crops and other agriculture products. The worldwide production of a huge quantity of agro-wastes presents different challenges in the environment. Agriculture wastes are potentially toxic to plants, humans, animals, as well as different components of the environment. The burning of agricultural waste causes serious environmental pollution, while dumping causes leaching and soil deterioration. Despite several drawbacks, the valorization of agriculture waste has been a promising approach for their sustainable management. Agriculture wastes are rich in lignocellulosic material that include cellulose, hemicellulose, and lignin and also contain pectin, proteins, lipids, and polyphenols. About 50% agro-wastes are obtained from wheat, rice, and oilseed crops that contain 0.5% N, 0.2% P2O5and 1.5% K2O. The rich nutrient and mineral content of agro-wastes presents them as a good raw material for the production of different valued products. Production of valued products such as enzyme, ethanol, compost, biogas, mushroom, and animal feed using agriculture wastes as a substrate has been discussed. The present chapter converses the utilization of agrowaste for the production of different value-added products and also describes the challenges and advancements during the fermentation of wastes into products. © 2022, Bentham Science Publishers.
Regulation of a glycerol-induced quinoprotein alcohol dehydrogenase by σ54 and a LuxR-type regulator in Azospirillum brasilense Sp7
(American Society for Microbiology, 2017) Vijay Shankar Singh; Ashutosh Prakash Dubey; Ankush Gupta; Sudhir Singh; Bhupendra Narain Singh; Anil Kumar Tripathi
Azospirillum brasilense Sp7 uses glycerol as a carbon source for growth and nitrogen fixation. When grown in medium containing glycerol as a source of carbon, it upregulates the expression of a protein which was identified as quinoprotein alcohol dehydrogenase (ExaA). Inactivation of exaA adversely affects the growth of A. brasilense on glycerol. A determination of the transcription start site of exaA revealed an RpoN-dependent -12/-24 promoter consensus. The expression of an exaA::lacZ fusion was induced maximally by glycerol and was dependent on σ54. Bioinformatic analysis of the sequence flanking the -12/-24 promoter revealed a 17-bp sequence motif with a dyad symmetry of 6 nucleotides upstream of the promoter, the disruption of which caused a drastic reduction in promoter activity. The electrophoretic mobility of a DNA fragment containing the 17-bp sequence motif was retarded by purified EraR, a LuxR-type transcription regulator that is transcribed divergently from exaA. EraR also showed a positive interaction with RpoN in twohybrid and pulldown assays. © 2017 American Society for Microbiology.
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.