KNR4, a suppressor of Saccharomyces cerevisiae cwh mutants, is involved in the transcriptional control of chitin synthase genes

The KNR4 gene, originally isolated by complementation of a K9 killer-toxinresistant mutant displaying reduced levels of both 1,3-b-glucan and 1,3-bglucan synthase activity, was recloned from a YCp50 genomic library as a suppressor of Saccharomyces cerevisiae calcofluor-white-hypersensitive (cwh) mutants. In these mutants, which were characterized by increased chitin levels, the suppressor effect of KNR4 resulted, for some of them, in a lowering of polymer content to close to wild-type level, with no effect on the contents of b-glucan and mannan. In all cases, this effect was accompanied by a strong reduction in mRNA levels corresponding to CHS1, CHS2 and CHS3, encoding chitin synthases, without affecting expression of FKS1 and RHO1, two genes encoding the catalytic subunit and a regulatory component of 1,3-b-glucan synthase, respectively. Overexpression of KNR4 also inhibited expression of CHS genes in wild-type strains and in two other cwh mutants, whose sensitivity to calcofluor white was not suppressed by this gene. The physiological relevance of the KNR4 transcriptional effect was addressed in two different ways. In a wild-type strain exposed to a-factor, overexpression of this gene inhibited CHS1 induction and delayed shmoo formation, two events which are triggered in response to the pheromone, whereas it did not affect bud formation and cell growth in a chs1 chs2 double mutant. A chimeric protein made by fusing green fluorescent protein to the C terminus of Knr4p which fully complemented a knr4D mutation was found to localize in patches at presumptive bud sites in unbudded cells and at the incipient bud site during bud emergence. Taken together, these results demonstrate that KNR4 has a regulatory role in chitin deposition and in cell wall assembly. A mechanism by which this gene affects expression of CHS genes is proposed

Finding undetected protein associations in cell signaling by belief propagation

External information propagates in the cell mainly through signaling cascades and transcriptional activation, allowing it to react to a wide spectrum of environmental changes. High throughput experiments identify numerous molecular components of such cascades that may, however, interact through unknown partners. Some of them may be detected using data coming from the integration of a protein-protein interaction network and mRNA expression profiles. This inference problem can be mapped onto the problem of finding appropriate optimal connected subgraphs of a network defined by these datasets. The optimization procedure turns out to be computationally intractable in general. Here we present a new distributed algorithm for this task, inspired from statistical physics, and apply this scheme to alpha factor and drug perturbations data in yeast. We identify the role of the COS8 protein, a member of a gene family of previously unknown function, and validate the results by genetic experiments. The algorithm we present is specially suited for very large datasets, can run in parallel, and can be adapted to other problems in systems biology. On renowned benchmarks it outperforms other algorithms in the field.Comment: 6 pages, 3 figures, 1 table, Supporting Informatio

SMC is recruited to oriC by ParB and promotes chromosome segregation in Streptococcus pneumoniae

Segregation of replicated chromosomes is an essential process in all organisms. How bacteria, such as the oval-shaped human pathogen Streptococcus pneumoniae, efficiently segregate their chromosomes is poorly understood. Here we show that the pneumococcal homologue of the DNA-binding protein ParB recruits S. pneumoniae condensin (SMC) to centromere-like DNA sequences (parS) that are located near the origin of replication, in a similar fashion as was shown for the rod-shaped model bacterium Bacillus subtilis. In contrast to B. subtilis, smc is not essential in S. pneumoniae, and Δsmc cells do not show an increased sensitivity to gyrase inhibitors or high temperatures. However, deletion of smc and/or parB results in a mild chromosome segregation defect. Our results show that S. pneumoniae contains a functional chromosome segregation machine that promotes efficient chromosome segregation by recruitment of SMC via ParB. Intriguingly, the data indicate that other, as of yet unknown mechanisms, are at play to ensure proper chromosome segregation in this organism.

Use of genomic DNA control features and predicted operon structure in microarray data analysis: ArrayLeaRNA – a Bayesian approach

<p>Abstract</p> <p>Background</p> <p>Microarrays are widely used for the study of gene expression; however deciding on whether observed differences in expression are significant remains a challenge.</p> <p>Results</p> <p>A computing tool (ArrayLeaRNA) has been developed for gene expression analysis. It implements a Bayesian approach which is based on the Gumbel distribution and uses printed genomic DNA control features for normalization and for estimation of the parameters of the Bayesian model and prior knowledge from predicted operon structure. The method is compared with two other approaches: the classical LOWESS normalization followed by a two fold cut-off criterion and the OpWise method (Price, et al. 2006. BMC Bioinformatics. 7, 19), a published Bayesian approach also using predicted operon structure. The three methods were compared on experimental datasets with prior knowledge of gene expression. With ArrayLeaRNA, data normalization is carried out according to the genomic features which reflect the results of equally transcribed genes; also the statistical significance of the difference in expression is based on the variability of the equally transcribed genes. The operon information helps the classification of genes with low confidence measurements.</p> <p>ArrayLeaRNA is implemented in Visual Basic and freely available as an Excel add-in at <url>http://www.ifr.ac.uk/safety/ArrayLeaRNA/</url></p> <p>Conclusion</p> <p>We have introduced a novel Bayesian model and demonstrated that it is a robust method for analysing microarray expression profiles. ArrayLeaRNA showed a considerable improvement in data normalization, in the estimation of the experimental variability intrinsic to each hybridization and in the establishment of a clear boundary between non-changing and differentially expressed genes. The method is applicable to data derived from hybridizations of labelled cDNA samples as well as from hybridizations of labelled cDNA with genomic DNA and can be used for the analysis of datasets where differentially regulated genes predominate.</p

A novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and DNA viral systems

The use of nucleases as toxins for defense, offense or addiction of selfish elements is widely encountered across all life forms. Using sensitive sequence profile analysis methods, we characterize a novel superfamily (the SUKH superfamily) that unites a diverse group of proteins including Smi1/Knr4, PGs2, FBXO3, SKIP16, Syd, herpesviral US22, IRS1 and TRS1, and their bacterial homologs. Using contextual analysis we present evidence that the bacterial members of this superfamily are potential immunity proteins for a variety of toxin systems that also include the recently characterized contact-dependent inhibition (CDI) systems of proteobacteria. By analyzing the toxin proteins encoded in the neighborhood of the SUKH superfamily we predict that they possess domains belonging to diverse nuclease and nucleic acid deaminase families. These include at least eight distinct types of DNases belonging to HNH/EndoVII- and restriction endonuclease-fold, and RNases of the EndoU-like and colicin E3-like cytotoxic RNases-folds. The N-terminal domains of these toxins indicate that they are extruded by several distinct secretory mechanisms such as the two-partner system (shared with the CDI systems) in proteobacteria, ESAT-6/WXG-like ATP-dependent secretory systems in Gram-positive bacteria and the conventional Sec-dependent system in several bacterial lineages. The hedgehog-intein domain might also release a subset of toxic nuclease domains through auto-proteolytic action. Unlike classical colicin-like nuclease toxins, the overwhelming majority of toxin systems with the SUKH superfamily is chromosomally encoded and appears to have diversified through a recombination process combining different C-terminal nuclease domains to N-terminal secretion-related domains. Across the bacterial superkingdom these systems might participate in discriminating `self’ or kin from `non-self’ or non-kin strains. Using structural analysis we demonstrate that the SUKH domain possesses a versatile scaffold that can be used to bind a wide range of protein partners. In eukaryotes it appears to have been recruited as an adaptor to regulate modification of proteins by ubiquitination or polyglutamylation. Similarly, another widespread immunity protein from these toxin systems, namely the suppressor of fused (SuFu) superfamily has been recruited for comparable roles in eukaryotes. In animal DNA viruses, such as herpesviruses, poxviruses, iridoviruses and adenoviruses, the ability of the SUKH domain to bind diverse targets has been deployed to counter diverse anti-viral responses by interacting with specific host proteins

Role of the Single-Stranded DNA–Binding Protein SsbB in Pneumococcal Transformation: Maintenance of a Reservoir for Genetic Plasticity

Bacteria encode a single-stranded DNA (ssDNA) binding protein (SSB) crucial for genome maintenance. In Bacillus subtilis and Streptococcus pneumoniae, an alternative SSB, SsbB, is expressed uniquely during competence for genetic transformation, but its precise role has been disappointingly obscure. Here, we report our investigations involving comparison of a null mutant (ssbB−) and a C-ter truncation (ssbBΔ7) of SsbB of S. pneumoniae, the latter constructed because SSBs' acidic tail has emerged as a key site for interactions with partner proteins. We provide evidence that SsbB directly protects internalized ssDNA. We show that SsbB is highly abundant, potentially allowing the binding of ∼1.15 Mb ssDNA (half a genome equivalent); that it participates in the processing of ssDNA into recombinants; and that, at high DNA concentration, it is of crucial importance for chromosomal transformation whilst antagonizing plasmid transformation. While the latter observation explains a long-standing observation that plasmid transformation is very inefficient in S. pneumoniae (compared to chromosomal transformation), the former supports our previous suggestion that SsbB creates a reservoir of ssDNA, allowing successive recombination cycles. SsbBΔ7 fulfils the reservoir function, suggesting that SsbB C-ter is not necessary for processing protein(s) to access stored ssDNA. We propose that the evolutionary raison d'être of SsbB and its abundance is maintenance of this reservoir, which contributes to the genetic plasticity of S. pneumoniae by increasing the likelihood of multiple transformation events in the same cell

KNR4 is a member of the PKC1 signalling pathway and genetically interacts with BCK2, a gene involved in cell progression in Saccharomyces cerevisiae

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The interaction of Slt2 MAP kinase with Knr4 is necessary for signalling through the cell wall integrity pathway in Saccharomyces cerevisiae

In budding yeast, PKC1 plays an essential role in cell integrity and proliferation through a linear MAP (Mitogen Activated Protein) kinase phosphorylation cascade, which ends up with the activation of the Slt2‐MAP kinase by dual phosphorylation on two conserved threonine and tyrosine residues. In this phosphorylated form, Slt2p kinase activates by phosphorylation at least two known downstream targets: Rlm1p, which is implicated in the expression of cell wall‐related genes, and SBF, required for transcription activation of cell cycle‐regulated genes at the G1 to S transition. In this paper, we demonstrate by two‐hybrid, in vitro immunoprecipitation and tandem affinity purification (TAP) methods that Knr4p physically interacts with Slt2p. Moreover, we show that the absence of Knr4p alters proper signalling of Slt2p to its two known downstream targets. In a knr4 null mutant, the SLT2‐dependent activation of Rlm1p is strongly reduced and the transcriptional activity of Rlm1p is decreased, although the phosphorylated form of Slt2p is more abundant than in wild‐type cells. On the contrary, SBF is abnormally activated in this mutant, as shown by a more abundant phosphorylated form of Swi6p, by higher β‐galactosidase levels from a SCB‐lacZ gene fusion, and by deregulation of the cyclic behaviour of several cell cycle‐regulated genes. These results, taken together with our recent finding that Bck2p requires Knr4p to activate additively with Cln3‐Cdc28p SBF target genes, lead to a model in which Knr4p is involved in co‐ordinating the Slt2p‐mediated cell wall integrity pathway with progression of the cell cycle

The interactome of Knr4/Smi1, a protein involved in coordinating cell wall assembly with cellular growth in yeast

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A disordered protein domain involved in morphogenesis and cell cycle progression.

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