The transcriptional regulator Rok binds A+T-rich DNA and is involved in repression of a mobile genetic element in Bacillus subtilis

The rok gene of Bacillus subtilis was identified as a negative regulator of competence development. It also controls expression of several genes not related to competence. We found that Rok binds to extended regions of the B. subtilis genome. These regions are characterized by a high A+T content and are known or believed to have been acquired by horizontal gene transfer. Some of the Rok binding regions are in known mobile genetic elements. A deletion of rok resulted in higher excision of one such element, ICEBs1, a conjugative transposon found integrated in the B. subtilis genome. When expressed in the Gram negative E. coli, Rok also associated with A+T-rich DNA and a conserved C-terminal region of Rok contributed to this association. Together with previous work, our findings indicate that Rok is a nucleoid associated protein that serves to help repress expression of A+T-rich genes, many of which appear to have been acquired by horizontal gene transfer. In these ways, Rok appears to be functionally analogous to H-NS, a nucleoid associated protein found in Gram negative bacteria and Lsr2 of high G+C Mycobacteria.     

The ROK-family regulator Rok7B7 directly controls carbon catabolite repression,antibiotic biosynthesis,and morphological development in Streptomyces avermitilis

Carbon catabolite repression (CCR) is a common phenomenon in bacteria that modulates expression of genes involved in uptake of alternative carbon sources. In the filamentous streptomycetes, which produce half of all known antibiotics, the precise mechanism of CCR is yet unknown. We report here that the ROK-family regulator Rok7B7 pleiotropically controls xylose and glucose uptake, CCR, development, as well as production of the macrolide antibiotics avermectin and oligomycin A in Streptomyces avermitilis. Rok7B7 directly repressed structural genes for avermectin biosynthesis, whereas it activated olmRI, the cluster-situated activator gene for oligomycin A biosynthesis. Rok7B7 also directly repressed the xylose uptake operon xylFGH, whose expression was induced by xylose and repressed by glucose. Both xylose and glucose served as Rok7B7 ligands. rok7B7 deletion led to enhancement and reduction of avermectin and oligomycin A production, respectively, relieved CCR of xylFGH, and increased co-uptake efficiency of xylose and glucose. A consensus Rok7B7-binding site, 5′-TTKAMKHSTTSAV-3′, was identified within aveA1p, olmRIp, and xylFp, which allowed prediction of the Rok7B7 regulon and confirmation of 11 additional targets involved in development, secondary metabolism, glucose uptake, and primary metabolic processes. Our findings will facilitate methods for strain improvement, antibiotic overproduction, and co-uptake of xylose and glucose in Streptomyces species.     

Rho-kinase controls cell shape changes during cytokinesis

BACKGROUND: Animal cell cytokinesis is characterized by a sequence of dramatic cortical rearrangements. How these are coordinated and coupled with mitosis is largely unknown. To explore the initiation of cytokinesis, we focused on the earliest cell shape change, cell elongation, which occurs during anaphase B and prior to cytokinetic furrowing. RESULTS: Using RNAi and live video microscopy in Drosophila S2 cells, we implicate Rho-kinase (Rok) and myosin II in anaphase cell elongation. rok RNAi decreased equatorial myosin II recruitment, prevented cell elongation, and caused a remarkable spindle defect where the spindle poles collided with an unyielding cell cortex and the interpolar microtubules buckled outward as they continued to extend. Disruption of the actin cytoskeleton with Latrunculin A, which abolishes cortical rigidity, suppressed the spindle defect. rok RNAi also affected furrowing, which was delayed and slowed, sometimes distorted, and in severe cases blocked altogether. Codepletion of the myosin binding subunit (Mbs) of myosin phosphatase, an antagonist of myosin II activation, only partially suppressed the cell-elongation defect and the furrowing delay, but prevented cytokinesis failures induced by prolonged rok RNAi. The marked sensitivity of cell elongation to Rok depletion was highlighted by RNAi to other genes in the Rho pathway, such as pebble, racGAP50C, and diaphanous, which had profound effects on furrowing but lesser effects on elongation. CONCLUSIONS: We show that cortical changes underlying cell elongation are more sensitive to depletion of Rok and myosin II, in comparison to other regulators of cytokinesis, and suggest that a distinct regulatory pathway promotes cell elongation.     

Functional analysis of the type 3 effector nodulation outer protein L (NopL) from Rhizobium sp. NGR234: symbiotic effects, phosphorylation, and interference with mitogen-activated protein kinase signaling

Pathogenic bacteria use type 3 secretion systems to deliver virulence factors (type 3 effector proteins) directly into eukaryotic host cells. Similarly, type 3 effectors of certain nitrogen-fixing rhizobial strains affect nodule formation in the symbiosis with host legumes. Nodulation outer protein L (NopL) of Rhizobium sp. strain NGR234 is a Rhizobium-specific type 3 effector. Nodulation tests and microscopic analysis showed that distinct necrotic areas were rapidly formed in ineffective nodules of Phaseolus vulgaris (cv. Tendergreen) induced by strain NGRΩnopL (NGR234 mutated in nopL), indicating that NopL antagonized nodule senescence. Further experiments revealed that NopL interfered with mitogen-activated protein kinase (MAPK) signaling in yeast and plant cells (Nicotiana tabacum). Expression of nopL in yeast disrupted the mating pheromone (α-factor) response pathway, whereas nopL expression in N. tabacum suppressed cell death induced either by overexpression of the MAPK gene SIPK (salicylic acid-induced protein kinase) or by SIPK(DD) (mutation in the TXY motif resulting in constitutive MAPK activity). These data indicate that NopL impaired function of MAPK proteins or MAPK substrates. Furthermore, we demonstrate that NopL was multiply phosphorylated either in yeast or N. tabacum cells that expressed nopL. Four phosphorylated serines were confirmed by mass spectrometry. All four phosphorylation sites exhibit a Ser-Pro pattern, a typical motif in MAPK substrates. Taken together, data suggest that NopL mimics a MAPK substrate and that NopL suppresses premature nodule senescence by impairing MAPK signaling in host cells.     

AtMPK4 is required for male-specific meiotic cytokinesis in Arabidopsis

Mitogen-activated protein kinase (MAPK) cascades have been implicated in regulating various aspects of plant development, including somatic cytokinesis. The evolution of expanded plant MAPK gene families has enabled the diversification of potential MAPK cascades, but functionally overlapping components are also well documented. Here we report that Arabidopsis MPK4, an MAPK that was previously described as a regulator of disease resistance, can interact with and be phosphorylated by the cytokinesis-related MAP kinase kinase, AtMKK6. In mpk4 mutant plants, anthers can develop normal microspore mother cells (MMCs) and peripheral supporting tissues, but the MMCs fail to form a normal intersporal callose wall after male meiosis, and thus cannot complete meiotic cytokinesis. Nevertheless, the multinucleate mpk4 microspores subsequently proceed through mitotic cytokinesis, resulting in enlarged mature pollen grains that possess increased sets of the tricellular structure. This pollen development phenotype is reminiscent of those observed in both atnack2/tes/stud and anq1/mkk6 mutants, and protein-protein interaction analysis defines a putative signalling module linking AtNACK2/TES/STUD, AtANP3, AtMKK6 and AtMPK4 together as a cascade that facilitates male-specific meiotic cytokinesis in Arabidopsis.     

Oscillatory phosphorylation of yeast Fus3 MAP kinase controls periodic gene expression and morphogenesis

Signal-transduction networks can display complex dynamic behavior such as oscillations in the activity of key components [1-6], but it is often unclear whether such dynamic complexity is actually important for the network's regulatory functions [7, 8]. Here, we found that the mitogen-activated protein kinase (MAPK) Fus3, a key regulator of the yeast mating-pheromone response, undergoes sustained oscillations in its phosphorylation and activation state during continuous pheromone exposure. These MAPK activity oscillations led to corresponding oscillations in mating-gene expression. Oscillations in MAPK activity and gene expression required the negative regulator of G protein signaling Sst2 and partially required the MAPK phosphatase Msg5. Peaks in Fus3 activation correlated with periodic rounds of cell morphogenesis, with each peak preceding the formation of an additional mating projection. Preventing projection formation did not eliminate MAPK oscillation, but preventing MAPK oscillation blocked the formation of additional projections. A mathematical model was developed that reproduced several features of the observed oscillatory dynamics. These observations demonstrate a role for MAPK activity oscillation in driving a periodic downstream response and explain how the pheromone signaling pathway, previously thought to desensitize after 1-3 hr, controls morphology changes that continue for a much longer time.     

Upstream open reading frames regulate the cell cycle-dependent expression of the RNA helicase Rok1 in Saccharomyces cerevisiae

The RNA helicase Rok1 plays a role in rRNA processing and in control of cell cycle progression in Saccharomyces cerevisiae. We identified two upstream open reading frames (uORFs) within the ROK1 5′ untranslated region, which inhibited Rok1 translation. Mutating uATG to uAAG or generation of a premature stop codon in the uORFs resulted in increased Rok1p levels. Rok1 protein levels oscillated during the cell cycle, declining at G1/S and increasing at G2. The uAAG1 mutation caused a constitutive level of Rok1 proteins throughout the cell cycle, resulting in significant delays in mitotic bud emergence and recovery from pheromone arrest. Our study reveals that the Rok1 protein level is regulated by uORFs, which is critical in cell cycle progression.     

Sho1 and Msb2-Related Proteins Regulate Appressorium Development in the Smut Fungus Ustilago maydis

The dimorphic fungus Ustilago maydis switches from budding to hyphal growth on the plant surface. In response to hydrophobicity and hydroxy fatty acids, U. maydis develops infection structures called appressoria. Here, we report that, unlike in Saccharomyces cerevisiae and other fungi where Sho1 (synthetic high osmolarity sensitive) and Msb2 (multicopy suppressor of a budding defect) regulate stress responses and pseudohyphal growth, Sho1 and Msb2-like proteins play a key role during appressorium differentiation in U. maydis. Sho1 was identified through a two-hybrid screen as an interaction partner of the mitogen-activated protein (MAP) kinase Kpp6. Epistasis analysis revealed that sho1 and msb2 act upstream of the MAP kinases kpp2 and kpp6. Furthermore, Sho1 was shown to destabilize Kpp6 through direct interaction with the unique N-terminal domain in Kpp6, indicating a role of Sho1 in fine-tuning Kpp6 activity. Morphological differentiation in response to a hydrophobic surface was strongly attenuated in sho1 msb2 mutants, while hydroxy fatty acid–induced differentiation was unaffected. These data suggest that Sho1 and the transmembrane mucin Msb2 are involved in plant surface sensing in U. maydis.     

Ste50 adaptor protein governs sexual differentiation of Cryptococcus neoformans via the pheromone-response MAPK signaling pathway

The mitogen-activated protein kinase (MAPK) pathways control diverse cellular functions in pathogenic fungi, including sexual differentiation, stress response, and maintenance of cell wall integrity. Here we characterized a Cryptococcus neoformans gene, which is homologous to the yeast Ste50 that is known to play an important role in mating pheromone response and stress response as an adaptor protein to the Ste11 MAPK kinase kinase in Saccharomyces cerevisiae. The C. neoformans Ste50 was not involved in any of the stress responses or virulence factor production (capsule and melanin) that are controlled by the HOG and Ras/cAMP signaling pathways. However, Ste50 was required for mating in both serotype A and serotype D C. neoformans strains. The ste50Δ mutant was completely defective in cell-cell fusion and mating pheromone production. Double mutation of the STE50 gene blocked increased production of pheromone and the hyper-filamentation phenotype of cells deleted of the CRG1 gene, which encodes the RGS protein that negatively regulates pheromone responsive G-protein signaling via the MAPK pathway. Regardless of the presence of the basidiomycota-specific SH3 domains of Ste50 that are known to be required for full virulence of Ustilago maydis, Ste50 was dispensable for virulence of C. neoformans in a murine model of cryptococcosis. In conclusion, the Ste50 adaptor protein controls sexual differentiation of C. neoformans via the pheromone-responsive MAPK pathway but is not required for virulence.     

Regulation of ykrL (htpX) by Rok and YkrK, a novel type of regulator in Bacillus subtilis

Expression of ykrL of Bacillus subtilis, encoding a close homologue of the Escherichia coli membrane protein quality control protease HtpX, was shown to be upregulated under membrane protein overproduction stress. Using DNA affinity chromatography, two proteins were found to bind to the promoter region of ykrL: Rok, known as a repressor of competence and genes for extracytoplasmic functions, and YkrK, a novel type of regulator encoded by the gene adjacent to ykrL but divergently transcribed. Electrophoretic mobility shift assays showed Rok and YkrK binding to the ykrL promoter region as well as YkrK binding to the ykrK promoter region. Comparative bioinformatic analysis of the ykrL promoter regions in related Bacillus species revealed a consensus motif, which was demonstrated to be the binding site of YkrK. Deletion of rok and ykrK in a PykrL-gfp reporter strain showed that both proteins are repressors of ykrL expression. In addition, conditions which activated PykrL (membrane protein overproduction, dissipation of the membrane potential, and salt and phenol stress) point to the involvement of YkrL in membrane protein quality control.