Sporulation in yeast consists of two highly coordinated processes. First, a diploid cell that is heterozygous at the mating-type locus undergoes meiosis, in which one round of DNA replication is followed by two rounds of nuclear division. Second, the meiotic products are packaged into spore cells that remain within the mother cell. A large number of genes are induced specifically during sporulation, and their products carry out different sporulation-specific events. Expression of these sporulation-specific genes is controlled by several regulators which function at different stages of the sporulation program, resulting in a cascade of gene expression following induction of meiosis. Here we describe one sporulation-specific gene, SSP2, which is induced midway through meiosis. Ssp2 shows significant homology to the predicted product of a hypothetical ORF in Candida albicans. Homozygous mutant ssp2 diploid cells fail to sporulate. In the mutant background, meiotic recombination and nuclear divisions remain normal; however, viability declines rapidly. Following meiosis, ssp2 cells form the prospore membrane, but fail to form the outer layer of the spore wall. The Ssp2 protein localizes to the spore wall after meiosis II. In addition, the ssp2 defect is also associated with delayed and reduced expression of late sporulation-specific genes. Our results suggest that SSP2 function is required after meiosis II and during spore wall formation.
Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis, must adapt to host-associated environments during infection by modulating gene expression. Small regulatory RNAs (sRNAs) are key regulators of bacterial gene expression, but their roles in Mtb are not well understood. Here, we address the expression and function of the Mtb sRNA Mcr11, which is associated with slow bacterial growth and latent infections in mice. We found, by using biochemical and genetic approaches, that the AbmR transcription factor and an extended region of native sequence 3’ to the mcr11 gene enhance production of mature Mcr11. Additionally, we found that expression of Mcr11 was unstable in the saprophyte Mycobacterium smegmatis, which lacks an mcr11 orthologue. Bioinformatic analyses used to predict regulatory targets of Mcr11 identified 9-11 nucleotide regions immediately upstream of Rv3282 and lipB with potential for direct base-pairing with Mcr11. mcr11-dependent regulation of Rv3282, lipB, Rv2216 and pknA was demonstrated using qRT-PCR in wild type versus mcr11-deleted Mtb and found to be responsive to the presence of fatty acids. These studies establish that Mcr11 has roles in regulating growth and central metabolism in Mtb that warrant further investigation. In addition, our finding that multiple factors are required for production of stable, mature Mcr11 emphasizes the need to study mechanisms of sRNA expression and stability in TB complex mycobacteria to understand their roles in TB pathogenesis.Author SummaryBacterial pathogens must continuously modulate their gene expression in response to changing conditions to successfully infect and survive within their hosts. Transcription factors are well known regulators of gene expression, but there is growing recognition that small RNAs (sRNAs) also have critically important roles in bacterial gene regulation. Many sRNAs have been identified in M. tuberculosis (Mtb), but little is known about their expression, regulatory targets or roles in Mtb biology. In this study, we found that the Mtb sRNA Mcr11, which is expressed at high levels in slowly replicating Mtb and during mouse infection, regulates expression of several target genes involved in central metabolism. Importantly, we also discovered that mcr11 has unexpected requirements for stable expression in mycobacteria. In particular, we identified RNA sequence elements immediately downstream of mcr11 that enhance transcription termination and production of mature Mcr11 RNA in TB-complex mycobacteria. Meanwhile, ectopic expression of Mcr11 was unstable in a non-pathogenic strain of mycobacteria, suggesting that factors specific to pathogenic mycobacteria are required for the stable production of Mcr11. These studies identify sRNA stability as a new frontier for understanding gene expression in Mtb.
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