Biology subjects

Chandra, G.

Publications and source records attributed to Chandra, G..

4 recordsLinked to original sources

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↗