Biology subjects

Chandra, G.

Publications and source records attributed to Chandra, G..

30 records · Page 2Linked to original sources

bioRxiv2022

Plasmid manipulation of bacterial behaviour through translational regulatory crosstalk

Beyond their role in horizontal gene transfer, conjugative plasmids commonly encode homologues of bacterial regulators. Known plasmid regulator homologues have highly targeted effects upon the transcription of specific bacterial traits. Here, we characterise a plasmid translational regulator, RsmQ, capable of taking global regulatory control in Pseudomonas fluorescens and causing a behavioural switch from motile to sessile lifestyle. RsmQ acts as a global regulator controlling the host proteome through direct interaction with host mRNAs and interference with the hosts translational regulatory network. This mRNA interference leads to largescale proteomic changes in metabolic genes, key regulators and genes involved in chemotaxis, thus controlling bacterial metabolism and motility. Moreover, comparative analyses found RsmQ on a large number of divergent plasmids isolated from multiple bacterial host taxa, suggesting the widespread importance of RsmQ for manipulating bacterial behaviour across clinical, environmental, and agricultural niches. RsmQ is a widespread plasmid global translational regulator primarily evolved for host chromosomal control to manipulate bacterial behaviour and lifestyle. Significance StatementPlasmids are recognised for their important role in bacterial evolution as drivers of horizontal gene transfer. Less well understood are the effects of plasmids upon bacterial behaviours by manipulating the expression of key bacterial phenotypes. Until now, examples of plasmid manipulation of their bacterial hosts were limited to highly targeted transcriptional control of a few related traits. In contrast, here we describe the first plasmid global translational regulator evolved to control the bacterial behavioural switch from a motile to a sessile lifestyle and bacterial metabolism, mediated through manipulation of the bacterial proteome. Moreover, this global translational regulator is common across divergent plasmids in a wide range of bacterial host taxa, suggesting that plasmids may commonly control bacterial lifestyle in the clinic, agricultural fields, and beyond.

microbiology↗

bioRxiv2022

Genome-wide identification of the LexA-mediated DNA damage response in Streptomyces venezuelae

DNA damage triggers a widely conserved stress response in bacteria called the SOS response that involves two key regulators, the activator RecA and the transcriptional repressor LexA. Despite the wide conservation of the SOS response, the number of genes controlled by LexA varies considerably between different organisms. The filamentous soil-dwelling bacteria of the genus Streptomyces contain LexA and RecA homologs but their roles in Streptomyces have not been systematically studied. Here, we demonstrate that RecA and LexA are required for the survival of Streptomyces venezuelae during DNA damaging conditions and for normal development during unperturbed growth. Monitoring the activity of a fluorescent recA promoter fusion and LexA protein levels revealed that the activation of the SOS response is delayed in S. venezuelae. By combining global transcriptional profiling and ChIP-seq analysis, we determined the LexA regulon and defined the core set of DNA damage repair genes that are expressed in response to treatment with the DNA alkylating agent mitomycin C. Our results show that DNA damage-induced degradation of LexA results in the differential regulation of LexA target genes. Using Surface Plasmon Resonance, we further confirm the LexA DNA binding motif (SOS box) and demonstrate that LexA displays tight but distinct binding affinities to its target promoters, indicating a graded response to DNA damage. IMPORTANCEThe transcriptional regulator LexA functions as a repressor of the bacterial SOS response, which is induced during DNA damaging conditions. This results in the expression of genes important for survival and adaptation. Here, we report the regulatory network controlled by LexA in the filamentous antibiotic producing Streptomyces bacteria and establish the existence of the SOS response in Streptomyces. Collectively, our work reveals significant insights into the DNA damage response in Streptomyces that will promote further studies to understand how these important bacteria adapt to their environment.

microbiology↗

bioRxiv2021

Multicellular growth and sporulation in filamentous actinobacteria require the conserved cell division protein SepX

Filamentous actinobacteria such as Streptomyces undergo two distinct modes of cell division, leading to partitioning of growing hyphae into multicellular compartments via cross-walls, and to septation and release of unicellular spores. Specific determinants for cross-wall formation and the importance of hyphal compartmentalization for Streptomyces development are largely unknown. Here we show that SepX, an actinobacterial-specific protein, is crucial for both cell division modes in Streptomyces venezuelae. Importantly, we find that sepX-deficient mutants grow without cross-walls and that this substantially impairs the fitness of colonies and the coordinated progression through the developmental life cycle. Protein interaction studies and live-cell imaging suggest that SepX contributes to the stabilization of the divisome, a mechanism that also requires the dynamin-like protein DynB. Thus, our work identifies an important determinant for cell division in Streptomyces that is required for cellular development and sporulation.

microbiology↗

bioRxiv2020

Refactoring the formicamycin biosynthetic gene cluster to make high-level producing strains and new molecules

The formicamycins are promising antibiotics with potent activity against Gram-positive pathogens including VRE and MRSA and display a high barrier to selection of resistant isolates. They were first identified in Streptomyces formicae KY5, which produces the formicamycins at low levels on solid agar but not in liquid culture, thus hindering further investigation of these promising antibacterial compounds. We hypothesised that by understanding the organisation and regulation of the for biosynthetic gene cluster, we could rationally refactor the cluster to increase production levels. Here we report that the for biosynthetic gene cluster consists of 24 genes expressed on nine transcripts. Seven of these transcripts, including those containing all the major biosynthetic genes, are repressed by the MarR-regulator ForJ which also controls the expression of the ForGF two-component system that initiates biosynthesis. A third cluster-situated regulator, ForZ, autoregulates and controls production of the putative MFS transporter ForAA. Consistent with these findings, deletion of forJ increased formicamycin biosynthesis 5-fold, while over-expression of forGF in the {Delta}forJ background increased production 10-fold compared to the wild-type. De-repression by deleting forJ also switched on biosynthesis in liquid-culture and induced the production of two novel formicamycin congeners. By combining mutations in regulatory and biosynthetic genes, six new biosynthetic precursors with antibacterial activity were also isolated. This work demonstrates the power of synthetic biology for the rational redesign of antibiotic biosynthetic gene clusters both to engineer strains suitable for fermentation in large scale bioreactors and to generate new molecules. ImportanceAntimicrobial resistance is a growing threat as existing antibiotics become increasingly ineffective against drug resistant pathogens. Here we determine the transcriptional organisation and regulation of the gene cluster encoding biosynthesis of the formicamycins, promising new antibiotics with activity against drug resistant bacteria. By exploiting this knowledge, we construct stable mutant strains which over-produce these molecules in both liquid and solid culture whilst also making some new compound variants. This will facilitate large scale purification of these molecules for further study including in vivo experiments and the elucidation of their mechanism of action. Our work demonstrates that understanding the regulation of natural product biosynthetic pathways can enable rational improvement of the producing strains.

microbiology↗

bioRxiv2020

Second messenger control of mRNA translation by dynamic ribosome modification

Control of mRNA translation is a crucial regulatory mechanism used by bacteria to respond to their environment. In the soil bacterium Pseudomonas fluorescens, RimK modifies the C-terminus of ribosomal protein RpsF to influence important aspects of rhizosphere colonisation through proteome remodelling. In this study, we show that RimK activity is itself under complex, multifactorial control by the co-transcribed phosphodiesterase trigger enzyme (RimA) and a polyglutamate-specific protease (RimB). Furthermore, biochemical experimentation and mathematical modelling reveal a role for the nucleotide second messenger cyclic-di-GMP in coordinating these activities. Active ribosome regulation by RimK occurs by two main routes: indirectly, through changes in the abundance of the global translational regulator Hfq and directly, with translation of surface attachment factors, amino acid transporters and key secreted molecules linked specifically to RpsF modification. Our findings show that post-translational ribosomal modification functions as a rapid-response mechanism that tunes global gene translation in response to environmental signals.

microbiology↗

bioRxiv2019

Pan-genome analysis identifies intersecting roles for Pseudomonas specialized metabolites in potato pathogen inhibition

Agricultural soil harbors a diverse microbiome that can form beneficial relationships with plants, including the inhibition of plant pathogens. Pseudomonas spp. are one of the most abundant bacterial genera in the soil and rhizosphere and play important roles in promoting plant health. However, the genetic determinants of this beneficial activity are only partially understood. Here, we genetically and phenotypically characterize the Pseudomonas fluorescens population in a commercial potato field, where we identify strong correlations between specialized metabolite biosynthesis and antagonism of the potato pathogens Streptomyces scabies and Phytophthora infestans. Genetic and chemical analyses identified hydrogen cyanide and cyclic lipopeptides as key specialized metabolites associated with S. scabies inhibition, which was supported by in planta biocontrol experiments. We show that a single potato field contains a hugely diverse and dynamic population of Pseudomonas bacteria, whose capacity to produce specialized metabolites is shaped both by plant colonization and defined environmental inputs.

microbiology↗

bioRxiv2019

Self-immunity guided identification of threonyl-tRNA synthetase as the molecular target of obafluorin, a β-lactone antibiotic

To meet the ever-growing demand of antibiotic discovery, new chemical matter and antibiotic targets are urgently needed. Many potent natural product antibiotics which were previously discarded can also provide lead molecules and drug targets. One such example is the structurally unique {beta}-lactone obafluorin, produced by Pseudomonas fluorescens ATCC 39502. Obafluorin is active against both Grampositive and -negative pathogens, however the biological target was unknown. We now report that obafluorin targets threonyl-tRNA-synthetase and we identify a homologue, ObaO, which confers self-immunity to the obafluorin producer. Disruption of obaO in P. fluorescens ATCC 39502 results in obafluorin sensitivity, whereas expression in sensitive E. coli strains confers resistance. Enzyme assays demonstrate that E. coli threonyl-tRNA synthetase is fully inhibited by obafluorin, whereas ObaO is only partly susceptible, exhibiting a very unusual partial inhibition mechanism. Altogether, our data highlight the utility of a self-immunity guided approach for the identification of an antibiotic target de novo and will ultimately enable the generation of improved obafluorin variants.

microbiology↗

bioRxiv2018

Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool

The rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are particularly refractory to reliable bioinformatic predictions due to the absence of a common biosynthetic feature across all pathways. Here, we describe RiPPER, a new tool for the family-independent identification of RiPP precursor peptides and apply this methodology to search for novel thioamidated RiPPs in Actinobacteria. Until now, thioamidation was believed to be a rare post-translational modification, which is catalyzed by a pair of proteins (YcaO and TfuA) in Archaea. In Actinobacteria, the thioviridamide-like molecules are a family of cytotoxic RiPPs that feature multiple thioamides, and it has been proposed that a YcaO-TfuA pair of proteins also catalyzes their formation. Potential biosynthetic gene clusters encoding YcaO and TfuA protein pairs are common in Actinobacteria but the chemical diversity generated by these pathways is almost completely unexplored. A RiPPER analysis reveals a highly diverse landscape of precursor peptides encoded in previously undescribed gene clusters that are predicted to make thioamidated RiPPs. To illustrate this strategy, we describe the first rational discovery of a new family of thioamidated natural products, the thiovarsolins from Streptomyces varsoviensis.

microbiology↗

bioRxiv2017

Discovery and biosynthesis of the antibiotic bicyclomycin in distant bacterial classes

Bicyclomycin (BCM) is a clinically promising antibiotic that is biosynthesised by Streptomyces cinnamoneus DSM 41675. BCM is structurally characterized by a core cyclo(L-Ile-L-Leu) 2,5-diketopiperazine (DKP) that is extensively oxidized. Here, we identify the BCM biosynthetic gene cluster, which shows that the core of BCM is biosynthesised by a cyclodipeptide synthase and the oxidative modifications are introduced by five 2-oxoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase. The discovery of the gene cluster enabled the identification of BCM pathways encoded in the genomes of hundreds of Pseudomonas aeruginosa isolates distributed globally, and heterologous expression of the pathway from P. aeruginosa SCV20265 demonstrated that the product is chemically identical to BCM produced by S. cinnamoneus. Overall, putative BCM gene clusters have been found in at least seven genera spanning Actinobacteria and Proteobacteria (Alpha-, Beta- and Gamma-). This represents a rare example of horizontal gene transfer of an intact biosynthetic gene cluster across such distantly related bacteria, and we show that these gene clusters are almost always associated with mobile genetic elements.\n\nIMPORTANCEBicyclomycin is the only natural product antibiotic that selectively inhibits the transcription termination factor Rho. This mechanism of action, combined with its proven biological safety and its activity against clinically relevant Gram-negative bacterial pathogens, makes it a very promising antibiotic candidate. Here, we report the identification of the bicyclomycin biosynthetic gene cluster in the known producing organism Streptomyces cinnamoneus, which will enable the engineered production of new bicyclomycin derivatives. The identification of this gene cluster also led to the discovery of hundreds of bicyclomycin pathways encoded in highly diverse bacteria, including the opportunistic pathogen Pseudomonas aeruginosa. This wide distribution of a complex biosynthetic pathway is very unusual, and provides an insight into how a pathway for an antibiotic can be transferred between diverse bacteria.

microbiology↗

bioRxiv2017

BldC delays entry into development to produce a sustained period of vegetative growth in Streptomyces venezuelae

Streptomycetes are filamentous bacteria that differentiate by producing spore-bearing reproductive structures called aerial hyphae. The transition from vegetative to reproductive growth is controlled by the bld (bald) loci, and mutations in bld genes prevent the formation of aerial hyphae, either by blocking entry into development (typically mutations in activators) or by inducing precocious sporulation in the vegetative mycelium (typically mutations in repressors). One of the bld genes, bldC, encodes a 68-residue DNA-binding protein related to the DNA-binding domain of MerR-family transcription factors. Recent work has shown that BldC binds DNA by a novel mechanism, but there is less insight into its impact on Streptomyces development. Here we used ChIP-seq coupled with RNA-seq to define the BldC regulon in the model species Streptomyces venezuelae, showing that BldC can function both as a repressor and as an activator of transcription. Using electron microscopy and time-lapse imaging, we show that bldC mutants are bald because they initiate development prematurely, bypassing the formation of aerial hyphae. This is consistent with the premature expression of BldC target genes encoding proteins with key roles in development (e.g. whiD, whiI, sigF), chromosome condensation and segregation (e.g. smeA-sffA, hupS), and sporulation-specific cell division (e.g. dynAB), suggesting that BldC-mediated repression is critical to maintain a sustained period of vegetative growth prior to sporulation. We discuss the possible significance of BldC as an evolutionary link between MerR family transcription factors and DNA architectural proteins.\n\nIMPORTANCEUnderstanding the mechanisms that drive bacterial morphogenesis depends on the dissection of the regulatory networks that underpin the cell biological processes involved. Recently, Streptomyces venezuelae has emerged as an attractive new model system for the study of morphological differentiation in Streptomyces. This has led to significant progress in identifying the genes controlled by the transcription factors that regulate aerial mycelium formation (Bld regulators) and sporulation (Whi regulators). Taking advantage of S. venezuelae, we used ChIP-seq coupled with RNA-seq to identify the genes directly under the control of BldC. Because S. venezuelae sporulates in liquid culture, the complete spore-to-spore life cycle can be examined using time-lapse microscopy, and we applied this technique to the bldC mutant. These combined approaches reveal BldC to be a member of an emerging class of Bld regulators that function principally to repress key sporulation genes, thereby extending vegetative growth and blocking the onset of morphological differentiation.

microbiology↗

bioRxiv2017

A unifying mechanism for the biogenesis of prokaryotic membrane proteins co-operatively integrated by the Sec and Tat pathways

The vast majority of polytopic membrane proteins are inserted into the cytoplasmic membrane of prokaryotes by the general secretory (Sec) pathway. However, a subset of monotopic proteins that contain non-covalently-bound redox cofactors depend on the twin-arginine translocase (Tat) machinery for membrane integration. Recently actinobacterial Rieske iron-sulfur cluster-containing proteins were identified as an unusual class of membrane proteins that require both the Sec and Tat pathways for the insertion of their three transmembrane domains (TMDs). The Sec pathway inserts the first two TMDs of these proteins co-translationally, but releases the polypeptide prior to the integration of TMD3 to allow folding of the cofactor-containing domain and its translocation by Tat. Here we have investigated features of the Streptomyces coelicolor Rieske polypeptide that modulate its interaction with the Sec and Tat machineries. Mutagenesis of a highly conserved loop region between Sec-dependent TMD2 and Tat-dependent TMD3 shows that it plays no significant role in coordinating the activities of the two translocases, but that a minimum loop length of approximately eight amino acids is required for the Tat machinery to recognise TMD3. Instead we show that a combination of relatively low hydrophobicity of TMD3, coupled with the presence of C-terminal positively-charged amino acids, results in abortive insertion of TMD3 by the Sec pathway and its release at the cytoplasmic side of the membrane. Bioinformatic analysis identified two further families of polytopic membrane proteins that share features of dual Sec-Tat-targeted membrane proteins. A predicted heme-molybdenum cofactor-containing protein with five TMDs, and a polyferredoxin also with five predicted TMDs, are encoded across bacterial and archaeal genomes. We demonstrate that membrane insertion of representatives of each of these newly-identified protein families is dependent on more than one protein translocase, with the Tat machinery recognising TMD5. Importantly, the combination of low hydrophobicity of the final TMD and the presence of multiple C-terminal positive charges that serve as critical Sec-release features for the actinobacterial Rieske protein also dictate Sec release in these further protein families. Therefore we conclude that a simple unifying mechanism governs the assembly of dual targeted membrane proteins.

microbiology↗

bioRxiv2016

MtrA is an essential regulator that coordinates antibiotic production and development in Streptomyces species

Streptomyces bacteria make numerous secondary metabolites, including half of all known antibiotics. Understanding the global regulation of secondary metabolism is important because most Streptomyces natural products are not made under laboratory conditions and unlocking cryptic biosynthetic gene clusters (BGCs) is a major focus for natural product discovery. Production is coordinated with sporulation but the regulators that coordinate development with antibiotic biosynthesis are largely unknown. Here we characterise a highly conserved actinobacterial response regulator called MtrA in antibiotic-producing Streptomyces species. We show that MtrA is an essential global regulator of secondary metabolism that directly activates antibiotic production in in S. coelicolor and S. venezuelae. MtrA also controls key developmental genes required for DNA replication and cell division and we propose that MtrA is the missing link that coordinates secondary metabolism with development in Streptomyces species.

microbiology↗