AdpA Positively Regulates Morphological Differentiation and Chloramphenicol Biosynthesis in Streptomyces venezuelae.

In members of genus Streptomyces, AdpA is a master transcriptional regulator that controls the expression of hundreds of genes involved in morphological differentiation, secondary metabolite biosynthesis, chromosome replication, etc. However, the function of AdpASv, an AdpA ortholog of Streptomyces venezuelae, is unknown. This bacterial species is a natural producer of chloramphenicol and has recently become a model organism for studies on Streptomyces. Here, we demonstrate that AdpASv is essential for differentiation and antibiotic biosynthesis in S. venezuelae and provide evidence suggesting that AdpASv positively regulates its own gene expression. We speculate that the different modes of AdpA-dependent transcriptional autoregulation observed in S. venezuelae and other Streptomyces species reflect the arrangement of AdpA binding sites in relation to the transcription start site. Lastly, we present preliminary data suggesting that AdpA may undergo a proteolytic processing and we speculate that this may potentially constitute a novel regulatory mechanism controlling cellular abundance of AdpA in Streptomyces. IMPORTANCEStreptomyces are well-known producers of valuable secondary metabolites which include a large variety of antibiotics and important model organisms for developmental studies in multicellular bacteria. The conserved transcriptional regulator AdpA of Streptomyces exerts a pleiotropic effect on cellular processes, including the morphological differentiation and biosynthesis of secondary metabolites. Despite extensive studies, the function of AdpA in these processes remains elusive. This work provides insights into the role of a yet unstudied AdpA ortholog of Streptomyces venezuelae, now considered a novel model organism. We found that AdpA plays essential role in morphological differentiation and biosynthesis of chloramphenicol, a broad-spectrum antibiotic. We also propose that AdpA may undergo a proteolytic processing that presumably constitutes a novel mechanism regulating cellular abundance of this master regulator.

Genetic Engineering in Combination with Semi-Synthesis Leads to a New Route for Gram-Scale Production of the Immunosuppressive Natural Product Brasilicardin†A.

Brasilicardin A (1) consists of an unusual anti/syn/anti-perhydrophenanthrene skeleton with a carbohydrate side chain and an amino acid moiety. It exhibits potent immunosuppressive activity, yet its mode of action differs from standard drugs that are currently in use. Further pre-clinical evaluation of this promising, biologically active natural product is hampered by restricted access to the ready material, as its synthesis requires both a low-yielding fermentation process using a pathogenic organism and an elaborate, multi-step total synthesis. Our semi-synthetic approach included a) the heterologous expression of the brasilicardin A gene cluster in different non-pathogenic bacterial strains producing brasilicardin A aglycone (5) in excellent yield and b) the chemical transformation of the aglycone 5 into the trifluoroacetic acid salt of brasilicardin A (1 a) via a short and straightforward five-steps synthetic route. Additionally, we report the first preclinical data for brasilicardin A.

A novel LysR-type regulator negatively affects biosynthesis of the immunosuppressant brasilicardin.

Brasilicardin A (BraA) is a promising immunosuppressive compound produced naturally by the pathogenic bacterium Nocardia terpenica IFM 0406. Heterologous host expression of brasilicardin gene cluster showed to be efficient to bypass the safety issues, low production levels and lack of genetic tools related with the use of native producer. Further improvement of production yields requires better understanding of gene expression regulation within the BraA biosynthetic gene cluster (Bra-BGC); however, the only so far known regulator of this gene cluster is Bra12. In this study, we discovered the protein LysRNt, a novel member of the LysR-type transcriptional regulator family, as a regulator of the Bra-BGC. Using in vitro approaches, we identified the gene promoters which are controlled by LysRNt within the Bra-BGC. Corresponding genes encode enzymes involved in BraA biosynthesis as well as the key Bra-BGC regulator Bra12. Importantly, we provide in vivo evidence that LysRNt negatively affects production of brasilicardin congeners in the heterologous host Amycolatopsis japonicum. Finally, we demonstrate that some of the pathway related metabolites, and their chemical analogs, can interact with LysRNt which in turn affects its DNA-binding activity.

AfsK-Mediated Site-Specific Phosphorylation Regulates DnaA Initiator Protein Activity in Streptomyces coelicolor.

In all organisms, chromosome replication is regulated mainly at the initiation step. Most of the knowledge about the mechanisms that regulate replication initiation in bacteria has come from studies on rod-shaped bacteria, such as Escherichia coli and Bacillus subtilisStreptomyces is a bacterial genus that is characterized by distinctive features and a complex life cycle that shares some properties with the developmental cycle of filamentous fungi. The unusual lifestyle of streptomycetes suggests that these bacteria use various mechanisms to control key cellular processes. Here, we provide the first insights into the phosphorylation of the bacterial replication initiator protein, DnaA, from Streptomyces coelicolor We suggest that phosphorylation of DnaA triggers a conformational change that increases its ATPase activity and decreases its affinity for the replication origin, thereby blocking the formation of a functional orisome. We suggest that the phosphorylation of DnaA is catalyzed by Ser/Thr kinase AfsK, which was shown to regulate the polar growth of S. coelicolor Together, our results reveal that phosphorylation of the DnaA initiator protein functions as a negative regulatory mechanism to control the initiation of chromosome replication in a manner that presumably depends on the cellular localization of the protein.IMPORTANCE This work provides insights into the phosphorylation of the DnaA initiator protein in Streptomyces coelicolor and suggests a novel bacterial regulatory mechanism for initiation of chromosome replication. Although phosphorylation of DnaA has been reported earlier, its biological role was unknown. This work shows that upon phosphorylation, the cooperative binding of the replication origin by DnaA may be disturbed. We found that AfsK kinase is responsible for phosphorylation of DnaA. Upon upregulation of AfsK, chromosome replication occurred further from the hyphal tip. Orthologs of AfsK are exclusively found in mycelial actinomycetes that are related to Streptomyces and exhibit a complex life cycle. We propose that the AfsK-mediated regulatory pathway serves as a nonessential, energy-saving mechanism in S. coelicolor.

Correction to: Propionate represses the dnaA gene via the methylcitrate pathway-regulating transcription factor, PrpR, in Mycobacterium tuberculosis.

Subsequent to the publication of the above article, it has been noticed that data published in Figure 2A and Figure 2B of this article duplicate images previously published by this research group in the following paper.

Correction: A Novel Role of the PrpR as a Transcription Factor Involved in the Regulation of Methylcitrate Pathway in Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0043651.].

High-Quality Draft Genome Sequence of the Actinobacterium Nocardia terpenica IFM 0406, Producer of the Immunosuppressant Brasilicardins, Using Illumina and PacBio Technologies.

The bacterium Nocardia terpenica IFM 0406 is known as the producer of the immunosuppressant brasilicardin A. Here, we report the completely sequenced genome of strain IFM 0406, which facilitates the heterologous expression of the brasilicardin biosynthetic gene cluster but also unveils the intriguing biosynthetic capacity of the strain to produce secondary metabolites.

Two transcription factors, CabA and CabR, are independently involved in multilevel regulation of the biosynthetic gene cluster encoding the novel aminocoumarin, cacibiocin.

Aminocoumarins are potent antibiotics belonging to a relatively small group of secondary metabolites produced by actinomycetes. Genome mining of Catenulispora acidiphila has recently led to the discovery of a gene cluster responsible for biosynthesis of novel aminocoumarins, cacibiocins. However, regulation of the expression of this novel gene cluster has not yet been analyzed. In this study, we identify transcriptional regulators of the cacibiocin gene cluster. Using a heterologous expression system, we show that the CabA and CabR proteins encoded by cabA and cabR genes in the cacibiocin gene cluster control the expression of genes involved in the biosynthesis, modification, regulation, and potentially, efflux/resistance of cacibiocins. CabA positively regulates the expression of cabH (the first gene in the cabHIYJKL operon) and cabhal genes encoding key enzymes responsible for the biosynthesis and halogenation of the aminocoumarin moiety, respectively. We provide evidence that CabA is a direct inducer of cacibiocin production, whereas the second transcriptional factor, CabR, is involved in the negative regulation of its own gene and cabT-the latter of which encodes a putative cacibiocin transporter. We also demonstrate that CabR activity is negatively regulated in vitro by aminocoumarin compounds, suggesting the existence of analogous regulation in vivo. Finally, we propose a model of multilevel regulation of gene transcription in the cacibiocin gene cluster by CabA and CabR.

Fifty years after the replicon hypothesis: cell-specific master regulators as new players in chromosome replication control.

Numerous free-living bacteria undergo complex differentiation in response to unfavorable environmental conditions or as part of their natural cell cycle. Developmental programs require the de novo expression of several sets of genes responsible for morphological, physiological, and metabolic changes, such as spore/endospore formation, the generation of flagella, and the synthesis of antibiotics. Notably, the frequency of chromosomal replication initiation events must also be adjusted with respect to the developmental stage in order to ensure that each nascent cell receives a single copy of the chromosomal DNA. In this review, we focus on the master transcriptional factors, Spo0A, CtrA, and AdpA, which coordinate developmental program and which were recently demonstrated to control chromosome replication. We summarize the current state of knowledge on the role of these developmental regulators in synchronizing the replication with cell differentiation in Bacillus subtilis, Caulobacter crescentus, and Streptomyces coelicolor, respectively.

Propionate represses the dnaA gene via the methylcitrate pathway-regulating transcription factor, PrpR, in Mycobacterium tuberculosis.

During infection of macrophages, Mycobacterium tuberculosis, the pathogen that causes tuberculosis, utilizes fatty acids as a major carbon source. However, little is known about the coordination of the central carbon metabolism of M. tuberculosis with its chromosomal replication, particularly during infection. A recently characterized transcription factor called PrpR is known to directly regulate the genes involved in fatty acid catabolism by M. tuberculosis. Here, we report for the first time that PrpR also regulates the dnaA gene, which encodes the DnaA initiator protein responsible for initiating chromosomal replication. Using cell-free systems and intact cells, we demonstrated an interaction between PrpR and the dnaA promoter region. Moreover, real-time quantitative reverse-transcription PCR analysis revealed that PrpR acts as a transcriptional repressor of dnaA when propionate (a product of odd-chain-length fatty acid catabolism) was used as the sole carbon source. We hypothesize that PrpR may be an important element of the complex regulatory system(s) required for tubercle bacilli to survive within macrophages, presumably coordinating the catabolism of host-derived fatty acids with chromosomal replication.

oriC-encoded instructions for the initiation of bacterial chromosome replication.

Replication of the bacterial chromosome initiates at a single origin of replication that is called oriC. This occurs via the concerted action of numerous proteins, including DnaA, which acts as an initiator. The origin sequences vary across species, but all bacterial oriCs contain the information necessary to guide assembly of the DnaA protein complex at oriC, triggering the unwinding of DNA and the beginning of replication. The requisite information is encoded in the unique arrangement of specific sequences called DnaA boxes, which form a framework for DnaA binding and assembly. Other crucial sequences of bacterial origin include DNA unwinding element (DUE, which designates the site at which oriC melts under the influence of DnaA) and binding sites for additional proteins that positively or negatively regulate the initiation process. In this review, we summarize our current knowledge and understanding of the information encoded in bacterial origins of chromosomal replication, particularly in the context of replication initiation and its regulation. We show that oriC encoded instructions allow not only for initiation but also for precise regulation of replication initiation and coordination of chromosomal replication with the cell cycle (also in response to environmental signals). We focus on Escherichia coli, and then expand our discussion to include several other microorganisms in which additional regulatory proteins have been recently shown to be involved in coordinating replication initiation to other cellular processes (e.g., Bacillus, Caulobacter, Helicobacter, Mycobacterium, and Streptomyces). We discuss diversity of bacterial oriC regions with the main focus on roles of individual DNA recognition sequences at oriC in binding the initiator and regulatory proteins as well as the overall impact of these proteins on the formation of initiation complex.

A novel role of the PrpR as a transcription factor involved in the regulation of methylcitrate pathway in Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, presumably utilizes fatty acids as a major carbon source during infection within the host. Metabolism of even-chain-length fatty acids yields acetyl-CoA, whereas metabolism of odd-chain-length fatty acids additionally yields propionyl-CoA. Utilization of these compounds by tubercle bacilli requires functional glyoxylate and methylcitrate cycles, respectively. Enzymes involved in both pathways are essential for M. tuberculosis viability and persistence during growth on fatty acids. However, little is known about regulatory factors responsible for adjusting the expression of genes encoding these enzymes to particular growth conditions. Here, we characterized the novel role of PrpR as a transcription factor that is directly involved in regulating genes encoding the key enzymes of methylcitrate (methylcitrate dehydratase [PrpD] and methylcitrate synthase [PrpC]) and glyoxylate (isocitrate lyase [Icl1]) cycles. Using cell-free systems and intact cells, we demonstrated an interaction of PrpR protein with prpDC and icl1 promoter regions and identified a consensus sequence recognized by PrpR. Moreover, we showed that an M. tuberculosis prpR-deletion strain exhibits impaired growth in vitro on propionate as the sole carbon source. Real-time quantitative reverse transcription-polymerase chain reaction confirmed that PrpR acts as a transcriptional activator of prpDC and icl1 genes when propionate is the main carbon source. Similar results were also obtained for a non-pathogenic Mycobacterium smegmatis strain. Additionally, we found that ramB, a prpR paralog that controls the glyoxylate cycle, is negatively regulated by PrpR. Our data demonstrate that PrpR is essential for the utilization of odd-chain-length fatty acids by tubercle bacilli. Since PrpR also acts as a ramB repressor, our findings suggest that it plays a key role in regulating expression of enzymes involved in both glyoxylate and methylcitrate pathways.

AdpA, key regulator for morphological differentiation regulates bacterial chromosome replication.

AdpA, one of the most pleiotropic transcription regulators in bacteria, controls expression of several dozen genes during Streptomyces differentiation. Here, we report a novel function for the AdpA protein: inhibitor of chromosome replication at the initiation stage. AdpA specifically recognizes the 5' region of the Streptomyces coelicolor replication origin (oriC). Our in vitro results show that binding of AdpA protein decreased access of initiator protein (DnaA) to the oriC region. We also found that mutation of AdpA-binding sequences increased the accessibility of oriC to DnaA, which led to more frequent replication and acceleration of Streptomyces differentiation (at the stage of aerial hyphae formation). Moreover, we also provide evidence that AdpA and DnaA proteins compete for oriC binding in an ATP-dependent manner, with low ATP levels causing preferential binding of AdpA, and high ATP levels causing dissociation of AdpA and association of DnaA. This would be consistent with a role for ATP levels in determining when aerial hyphae emerge.

The level of AdpA directly affects expression of developmental genes in Streptomyces coelicolor.

AdpA is a key regulator of morphological differentiation in Streptomyces. In contrast to Streptomyces griseus, relatively little is known about AdpA protein functions in Streptomyces coelicolor. Here, we report for the first time the translation accumulation profile of the S. coelicolor adpA (adpA(Sc)) gene; the level of S. coelicolor AdpA (AdpA(Sc)) increased, reaching a maximum in the early stage of aerial mycelium formation (after 36 h), and remained relatively stable for the next several hours (48 to 60 h), and then the signal intensity decreased considerably. AdpA(Sc) specifically binds the adpA(Sc) promoter region in vitro and in vivo, suggesting that its expression is autoregulated; surprisingly, in contrast to S. griseus, the protein presumably acts as a transcriptional activator. We also demonstrate a direct influence of AdpA(Sc) on the expression of several genes whose products play key roles in the differentiation of S. coelicolor: STI, a protease inhibitor; RamR, an atypical response regulator that itself activates expression of the genes for a small modified peptide that is required for aerial growth; and ClpP1, an ATP-dependent protease. The diverse influence of AdpA(Sc) protein on the expression of the analyzed genes presumably results mainly from different affinities of AdpA(Sc) protein to individual promoters.

Replisome trafficking in growing vegetative hyphae of Streptomyces coelicolor A3(2).

We observed movies of replisome trafficking during Streptomyces coelicolor growth. A replisome(s) in the spore served as a replication center(s) until hyphae reached a certain length, when a tip-proximal replisome formed and moved at a fixed distance behind the tip at a speed equivalent to the extension rate of the tip.