Genome-wide exploration of the molecular evolution and regulatory network of mitogen-activated protein kinase cascades upon multiple stresses in Brachypodium distachyon

Background

Brachypodium distachyon is emerging as a widely recognized model plant that has very close relations with several economically important Poaceae species. MAPK cascade is known to be an evolutionarily conserved signaling module involved in multiple stresses. Although the gene sequences of MAPK and MAPKK family have been fully identified in B. distachyon, the information related to the upstream MAPKKK gene family especially the regulatory network among MAPKs, MAPKKs and MAPKKKs upon multiple stresses remains to be understood.

Results

In this study, we have identified MAPKKKs which belong to the biggest gene family of MAPK cascade kinases. We have systematically investigated the evolution of whole MAPK cascade kinase gene family in terms of gene structures, protein structural organization, chromosomal localization, orthologs construction and gene duplication analysis. Our results showed that most BdMAPK cascade kinases were located at the low-CpG-density region, and the clustered members in each group shared similar structures of the genes and proteins. Synteny analysis showed that 62 or 21 pairs of duplicated orthologs were present between B. distachyon and Oryza sativa, or between B. distachyon and Arabidopsis thaliana respectively. Gene expression data revealed that BdMAPK cascade kinases were rapidly regulated by stresses and phytohormones. Importantly, we have constructed a regulation network based on co-expression patterns of the expression profiles upon multiple stresses performed in this study.

Conclusions

BdMAPK cascade kinases were involved in the signaling pathways of multiple stresses in B. distachyon. The network of co-expression regulation showed the most of duplicated BdMAPK cascade kinase gene orthologs demonstrated their convergent function, whereas few of them developed divergent function in the evolutionary process. The molecular evolution analysis of identified MAPK family genes and the constructed MAPK cascade regulation network under multiple stresses provide valuable information for further investigation of the functions of BdMAPK cascade kinase genes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1452-1) contains supplementary material, which is available to authorized users.     

Pivotal roles for the receiver domain in the mechanism of action of the response regulator RamR of Streptomyces coelicolor

The response regulator RamR activates expression of the ramCSAB operon, the source of the morphogenetic peptide SapB, and is therefore important for morphogenesis of the bacterium Streptomyces coelicolor. Like most response regulators, RamR consists of an amino-terminal receiver domain and a carboxy-terminal DNA binding domain. Four of five highly conserved active site residues known to be important in other response regulators are present in RamR: D12, D56 (the predicted site of phosphorylation), T84 and K105. Here, we show that in spite of this, RamR did not demonstrate an ability to autophosphorylate in vitro in the presence of small molecule phosphodonors. The unphosphorylated protein behaved as a dimer and bound cooperatively to three sites in the ramC promoter, one with very high affinity and two with lower affinity. On its own, the RamR DNA binding domain could not bind DNA but was able to interfere with the action of full length RamR in a manner suggesting direct protein-protein contact. Surprisingly, substitution of residues D12 or T84 had no effect on RamR function in vivo. In contrast, D56A and K105A substitutions caused defects in both dimer formation and DNA binding while the more conservative substitution, D56N permitted dimer formation but not DNA binding. L102 in RamR corresponds to a well-conserved tyrosine (or aromatic) residue that is important for function in the other response regulators. While a L102Y variant, which introduced the aromatic side-chain usually found at this position, functioned normally, L102A and L102W substitutions blocked RamR function in vivo. We show that these substitutions specifically impaired cooperative DNA binding by RamR at the lower affinity recognition sequences. The biochemical properties of RamR therefore differ markedly from those of other well-characterized response regulators.     

Streptomyces antibioticalis, a Novel Species from a Sanitary Landfill Soil

A novel isolate belonging to the genus Streptomyces, strain SL-4^T, was isolated from soil sample collected from a sanitary landfill, New Delhi, India. The taxonomic status of this isolate was studied by polyphasic approach including morphological, physiological and chemo-taxonomic characterization. Spore chains of SL-4^T were open loops, hooks or extended spirals of wide diameter (retinaculiperti). The cell wall peptidoglycan of the isolate SL-4^T contained L,L-diaminopimelic acid, suggesting that the strain has a cell wall of chemotype-I. The polar lipid profile of the isolate was of Type II, with phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. The 16SrRNA gene sequence similarity between SL-4^T and its phylogenetic relatives Streptomyces atrovirens NRRLB 16357^T ({"type":"entrez-nucleotide","attrs":{"text":"DQ026672","term_id":"68137786","term_text":"DQ026672"}}DQ026672), S. albogriseolus NRRLB 1305^T ({"type":"entrez-nucleotide","attrs":{"text":"AJ494865","term_id":"21742835","term_text":"AJ494865"}}AJ494865), S viridodiastaticus NBRC 13106^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184317","term_id":"90960133","term_text":"AB184317"}}AB184317), S. caelestis NRRL 2418^T ({"type":"entrez-nucleotide","attrs":{"text":"X80824","term_id":"587528","term_text":"X80824"}}X80824), S. flavoviridis NBRC 12772^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184842","term_id":"90960663","term_text":"AB184842"}}AB184842), S. pilosus NBRC 12807^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184161","term_id":"90959977","term_text":"AB184161"}}AB184161) and S. longispororuber NBRC 13488^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184440","term_id":"90960256","term_text":"AB184440"}}AB184440) was 99.65, 99.65, 99.64, 99.23, 99.15, 99.14 and 99.13 % respectively. Subsequent DNA–DNA hybridization experiments with the test strain and its clade members showed 55.27, 44.27, 36.86, and 15.65 % relatedness between SL-4^T and its relatives S. atrovirens,S. albogriseolus, S. viridodiastaticus and S. longispororuber respectively. The genotypic and phenotypic data was analyzed to verify possibility of the isolate SL-4^T representing novel member of the genus Streptomyces, for which the name S. antibioticalis is being proposed. The type strain is SL-4^T (=CCM 7434^T=MTCC 8588^T).     

ShyA, a membrane protein for proper septation of hyphae in Streptomyces

The life cycle of Streptomyces involves the formation of filamentous substrate and aerial hyphae. Following cessation of growth of an aerial hypha, multiple septation occurs at the tip to produce a chain of unigenomic spores. A gene, shyA, which influences several aspects of this growth, was isolated and partially characterized in Streptomyces coelicolor. The gene product is a representative of a well-conserved family of small actinomycete proteins. The shyA mutant sporulates normally but displays hyper septum formation and altered spore-chain morphology. Biochemical separation experiments and immunofluorescence staining demonstrated that the shyA gene product locates at cell membranes. Moreover, yeast two-hybrid screen and GST-pull-down assay showed that ShyA can interact with itself. Altogether, ShyA belongs to a new family of membrane-associated proteins which plays a role in morphological differentiation in actinomycetes.     

High quality draft genome sequence of Streptomyces sp. strain AW19M42 isolated from a sea squirt in Northern Norway

Here we report the 8 Mb high quality draft genome of Streptomyces sp. strain AW19M42, together with specific properties of the organism and the generation, annotation and analysis of its genome sequence. The genome encodes 7,727 putative open reading frames, of which 6,400 could be assigned with COG categories. Also, 62 tRNA genes and 8 rRNA operons were identified. The genome harbors several gene clusters involved in the production of secondary metabolites. Functional screening of the isolate was positive for several enzymatic activities, and some candidate genes coding for those activities are listed in this report. We find that this isolate shows biotechnological potential and is an interesting target for bioprospecting.     

Pleiotropic regulatory genes bldA,adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin

Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production—bldA, adpA and absB—exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNA^Leu_UAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs—that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining.     

Morphological development and cytochrome c oxidase activity in Streptomyces lividans are dependent on the action of a copper bound Sco protein

Copper has an important role in the life cycle of many streptomycetes, stimulating the developmental switch between vegetative mycelium and aerial hyphae concomitant with the production of antibiotics. In streptomycetes, a gene encoding for a putative Sco-like protein has been identified and is part of an operon that contains two other genes predicted to handle cellular copper. We report on the Sco-like protein from Streptomyces lividans (Sco^Sl) and present a series of experiments that firmly establish a role for Sco^Sl as a copper metallochaperone as opposed to a role as a thiol-disulphide reductase that has been assigned to other bacterial Sco proteins. Under low copper concentrations, a Δsco mutant in S. lividans displays two phenotypes; the development switch between vegetative mycelium and aerial hyphae stalls and cytochrome c oxidase (CcO) activity is significantly decreased. At elevated copper levels, the development and CcO activity in the Δsco mutant are restored to wild-type levels and are thus independent of Sco^Sl. A CcO knockout reveals that morphological development is independent of CcO activity leading us to suggest that Sco^Sl has at least two targets in S. lividans. We establish that one Sco^Sl target is the dinuclear Cu_A domain of CcO and it is the cupric form of Sco^Sl that is functionally active. The mechanism of cupric ion capture by Sco^Sl has been investigated, and an important role for a conserved His residue is identified.     

The Arabidopsis thaliana flower organ specification gene regulatory network determines a robust differentiation process

The Arabidopsis thaliana flower organ specification gene regulatory network (FOS-GRN) has been modeled previously as a discrete dynamical system, recovering as steady states configurations that match the genetic profiles described in primordial cells of inflorescence, sepals, petals, stamens and carpels during early flower development. In this study, we first update the FOS-GRN by adding interactions and modifying some rules according to new experimental data. A discrete model of this updated version of the network has a dynamical behavior identical to previous versions, under both wild type and mutant conditions, thus confirming its robustness. Then, we develop a continuous version of the FOS-GRN using a new methodology that builds upon previous proposals. The fixed point attractors of the discrete system are all observed in the continuous model, but the latter also contains new steady states that might correspond to genetic activation states present briefly during the early phases of flower development. We show that both the discrete and the continuous models recover the observed stable gene configurations observed in the inflorescence meristem, as well as the primordial cells of sepals, petals, stamens and carpels. Additionally, both models are subjected to perturbations in order to establish the nature of additional signals that may suffice to determine the experimentally observed order of appearance of floral organs. Our results thus describe a possible mechanism by which the network canalizes molecular signals and/or noise, thus conferring robustness to the differentiation process.