AURTHO: Autoregulation of transcription factors as facilitator of cis-acting element discovery.

Transcriptional regulation is key in bacteria for providing an adequate response in time and space to changing environmental conditions. However, despite decades of research, the binding sites and therefore the target genes and the function of most transcription factors (TFs) remain unknown. Filling this gap in knowledge through conventional methods represents a colossal task which we demonstrate here can be significantly facilitated by a widespread feature in transcriptional control: the autoregulation of TFs implying that the yet unknown transcription factor binding site (TFBS) is neighboring the TF itself. In this work, we describe the "AURTHO" methodology (AUtoregulation of oRTHOlogous transcription factors), consisting of analyzing upstream regions of orthologous TFs in order to uncover their associated TFBSs. AURTHO enabled the de novo identification of novel TFBSs with an unprecedented improvement in terms of quantity and reliability. DNA-protein interaction studies on a selection of candidate cis-acting elements yielded an >90 % success rate, demonstrating the efficacy of AURTHO at highlighting true TF-TFBS couples and confirming the identification in a near future of a plethora of TFBSs across all bacterial species.

The virulome of Streptomyces scabiei in response to cello-oligosaccharide elicitors.

The development of spots or lesions symptomatic of common scab on root and tuber crops is caused by few pathogenic Streptomyces with Streptomyces scabiei 87-22 as the model species. Thaxtomin phytotoxins are the primary virulence determinants, mainly acting by impairing cellulose synthesis, and their production in S. scabiei is in turn boosted by cello-oligosaccharides released from host plants. In this work we aimed to determine which molecules and which biosynthetic gene clusters (BGCs) of the specialized metabolism of S. scabiei 87-22 show a production and/or a transcriptional response to cello-oligosaccharides. Comparative metabolomic analyses revealed that molecules of the virulome of S. scabiei induced by cellobiose and cellotriose include (i) thaxtomin and concanamycin phytotoxins, (ii) desferrioxamines, scabichelin and turgichelin siderophores in order to acquire iron essential for housekeeping functions, (iii) ectoine for protection against osmotic shock once inside the host, and (iv) bottromycin and concanamycin antimicrobials possibly to prevent other microorganisms from colonizing the same niche. Importantly, both cello-oligosaccharides reduced the production of the spore germination inhibitors germicidins thereby giving the 'green light' to escape dormancy and trigger the onset of the pathogenic lifestyle. For most metabolites - either with induced or reduced production - cellotriose was revealed to be a slightly stronger elicitor compared to cellobiose, supporting an earlier hypothesis which suggested the trisaccharide was the real trigger for virulence released from the plant cell wall through the action of thaxtomins. Interestingly, except for thaxtomins, none of these BGCs' expression seems to be under direct control of the cellulose utilization repressor CebR suggesting the existence of a yet unknown mechanism for switching on the virulome. Finally, a transcriptomic analysis revealed nine additional cryptic BGCs that have their expression awakened by cello-oligosaccharides, suggesting that other and yet to be discovered metabolites could be part of the virulome of S. scabiei.

Deletion of bglC triggers a genetic compensation response by awakening the expression of alternative beta-glucosidase.

In the plant pathogen Streptomyces scabies, the gene bglC encodes a GH1 family cellobiose beta-glucosidase that is both required for primary metabolism and for inducing virulence of the bacterium. Deletion of bglC (strain ΔbglC) surprisingly resulted in the augmentation of the global beta-glucosidase activity of S. scabies. This paradoxical phenotype is highly robust as it has been observed in all bglC deletion mutants independently generated, thereby highlighting a phenomenon of genetic compensation. Comparative proteomics allowed to identify two glycosyl hydrolases - named BcpE1 and BcpE2 - of which peptide levels were significantly increased in strain ΔbglC. Quantitative RT-PCR revealed that the higher abundance of BcpE1 and BcpE2 is triggered at the transcriptional level, the expression of their respective gene being 100 and 15 times upregulated. Enzymatic studies with pure BcpE proteins showed that they both possess beta-glucosidase activity thereby explaining the genotypic-phenotypic discrepancy of the bglC deletion mutant. The GH1 family BcpE1 could hydrolyze cellobiose and generate glucose similarly to BglC itself thereby mainly contributing to the survival of strain ΔbglC when cellobiose is provided as sole nutrient source. The low affinity of BcpE2 for cellobiose suggests that this GH3 family beta-glucosidase would instead primarily target another and yet unknown glucose-beta-1,4-linked substrate. These results make S. scabies a new model system to study genetic compensation. Discovering how, either the bglC DNA locus, its mRNA, the BglC protein, or either its enzymatic activity controls bcpE genes' expression, will unveil new mechanisms directing transcriptional repression.

Complete Genome Sequence of Streptomyces lunaelactis MM109T, Isolated from Cave Moonmilk Deposits.

Streptomyces lunaelactis MM109T is a ferroverdin A (anticholesterol) producer isolated from cave moonmilk deposits. The complete genome sequence of MM109T was obtained by combining Oxford Nanopore MinION and Illumina HiSeq and MiSeq technologies, revealing an 8.4-Mb linear chromosome and two plasmids, pSLUN1 (127,264 bp, linear) and pSLUN2 (46,827 bp, circular).

Cracking the regulatory code of biosynthetic gene clusters as a strategy for natural product discovery.

The World Health Organization (WHO) describes antibiotic resistance as "one of the biggest threats to global health, food security, and development today", as the number of multi- and pan-resistant bacteria is rising dangerously. Acquired resistance phenomena also impair antifungals, antivirals, anti-cancer drug therapy, while herbicide resistance in weeds threatens the crop industry. On the positive side, it is likely that the chemical space of natural products goes far beyond what has currently been discovered. This idea is fueled by genome sequencing of microorganisms which unveiled numerous so-called cryptic biosynthetic gene clusters (BGCs), many of which are transcriptionally silent under laboratory culture conditions, and by the fact that most bacteria cannot yet be cultivated in the laboratory. However, brute force antibiotic discovery does not yield the same results as it did in the past, and researchers have had to develop creative strategies in order to unravel the hidden potential of microorganisms such as Streptomyces and other antibiotic-producing microorganisms. Identifying the cis elements and their corresponding transcription factors(s) involved in the control of BGCs through bioinformatic approaches is a promising strategy. Theoretically, we are a few 'clicks' away from unveiling the culturing conditions or genetic changes needed to activate the production of cryptic metabolites or increase the production yield of known compounds to make them economically viable. In this opinion article, we describe and illustrate the idea beyond 'cracking' the regulatory code for natural product discovery, by presenting a series of proofs of concept, and discuss what still should be achieved to increase the rate of success of this strategy.