Kar4, the yeast homolog of METTL14, is required for mRNA m6A methylation and meiosis.

KAR4, the yeast homolog of the mammalian mRNA N6A-methyltransferase complex component METTL14, is required for two disparate developmental programs in Saccharomyces cerevisiae: mating and meiosis. To understand KAR4's role in yeast mating and meiosis, we used a genetic screen to isolate 25 function-specific mutant alleles, which map to non-overlapping surfaces on a predicted structure of the Kar4 protein (Kar4p). Most of the mating-specific alleles (Mat-) abolish Kar4p's interaction with the transcription factor Ste12p, indicating that Kar4p's mating function is through Ste12p. In yeast, the mRNA methyltransferase complex was previously defined as comprising Ime4p (Kar4p's paralog and the homolog of mammalian METTL3), Mum2p (homolog of mammalian WTAP), and Slz1p (MIS), but not Kar4p. During meiosis, Kar4p interacts with Ime4p, Mum2p, and Slz1p. Moreover, cells lacking Kar4p have highly reduced levels of mRNA methylation during meiosis indicating that Kar4p is a key member of the methyltransferase complex, as it is in humans. Analysis of kar4Δ/Δ and 7 meiosis-specific alleles (Mei-) revealed that Kar4p is required early in meiosis, before initiation of S-phase and meiotic recombination. High copy expression of the meiotic transcriptional activator IME1 rescued the defect of these Mei- alleles. Surprisingly, Kar4p was also found to be required at a second step for the completion of meiosis and sporulation. Over-expression of IME1 in kar4Δ/Δ permits pre-meiotic S-phase, but most cells remained arrested with a monopolar spindle. Analysis of the function-specific mutants revealed that roughly half became blocked after premeiotic DNA synthesis and did not sporulate (Spo-). Loss of Kar4p's Spo function was suppressed by overexpression of RIM4, a meiotic translational regulator. Overexpression of IME1 and RIM4 together allowed sporulation of kar4Δ/Δ cells. Taken together, these data suggest that Kar4p regulates meiosis at multiple steps, presumably reflecting requirements for methylation in different stages of meiotic gene expression.

Pseudomonas aeruginosa PumA acts on an endogenous phenazine to promote self-resistance.

The activities of critical metabolic and regulatory proteins can be altered by exposure to natural or synthetic redox-cycling compounds. Many bacteria, therefore, possess mechanisms to transport or transform these small molecules. The opportunistic pathogen Pseudomonas aeruginosa PA14 synthesizes phenazines, redox-active antibiotics that are toxic to other organisms but have beneficial effects for their producer. Phenazines activate the redox-sensing transcription factor SoxR and thereby induce the transcription of a small regulon, including the operon mexGHI-opmD, which encodes an efflux pump that transports phenazines, and PA14_35160 (pumA), which encodes a putative monooxygenase. Here, we provide evidence that PumA contributes to phenazine resistance and normal biofilm development, particularly during exposure to or production of strongly oxidizing N-methylated phenazines. We show that phenazine resistance depends on the presence of residues that are conserved in the active sites of other putative and characterized monooxygenases found in the antibiotic producer Streptomyces coelicolor. We also show that during biofilm growth, PumA is required for the conversion of phenazine methosulfate to unique phenazine metabolites. Finally, we compare ∆mexGHI-opmD and ∆pumA strains in assays for colony biofilm morphogenesis and SoxR activation, and find that these deletions have opposing phenotypic effects. Our results suggest that, while MexGHI-OpmD-mediated efflux has the effect of making the cellular phenazine pool more reducing, PumA acts on cellular phenazines to make the pool more oxidizing. We present a model in which these two SoxR targets function simultaneously to control the biological activity of the P. aeruginosa phenazine pool.

Redox-Based Regulation of Bacterial Development and Behavior.

Severe changes in the environmental redox potential, and resulting alterations in the oxidation states of intracellular metabolites and enzymes, have historically been considered negative stressors, requiring responses that are strictly defensive. However, recent work in diverse organisms has revealed that more subtle changes in the intracellular redox state can act as signals, eliciting responses with benefits beyond defense and detoxification. Changes in redox state have been shown to influence or trigger chromosome segregation, sporulation, aerotaxis, and social behaviors, including luminescence as well as biofilm establishment and dispersal. Connections between redox state and complex behavior allow bacteria to link developmental choices with metabolic state and coordinate appropriate responses. Promising future directions for this area of study include metabolomic analysis of species- and condition-dependent changes in metabolite oxidation states and elucidation of the mechanisms whereby the redox state influences circadian regulation.