Phenotypic heterogeneity of microbial populations can facilitate survival in dynamic environments by generating sub-populations of cells that may have differential fitness in a future environment. Bacillus subtilis cultures experiencing nutrient limitation contain distinct sub-populations of cells exhibiting either comparatively high or low protein synthesis activity. This heterogeneity requires the production of phosphorylated guanosine nucleotides (pp)pGpp by three synthases: SasA, SasB, and RelA. Here we show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population of cells exhibiting low protein synthesis whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. Previously, it was reported that a RelA product allosterically activates SasB and we find that a SasA product competitively inhibits this activation. Finally, we provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This work therefore identifies the mechanism underlying phenotypic heterogeneity in protein synthesis.
Bacteriophage Cr30 has proven useful for the transduction of Caulobacter crescentus. Nucleotide sequencing of Cr30 DNA revealed that the Cr30 genome consists of 155,997 bp of DNA that codes for 287 proteins and five tRNAs. In contrast to the 67% GC content of the host genome, the GC content of the Cr30 genome is only 38%. This lower GC content causes both the codon usage pattern and the amino acid composition of the Cr30 proteins to be quite different from those of the host bacteria. As a consequence, the Cr30 mRNAs probably are translated at a rate that is slower than the normal rate for host mRNAs. A phylogenetic comparison of the genome indicates that Cr30 is a member of the T4-like family that is most closely related to a new group of T-like phages exemplified by ϕM12.
12Many bacteria exist in a state of metabolic quiescence where they must minimize energy 13 consumption so as to maximize available resources over a potentially extended period of time. 14 As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be 15 an appropriate target for downregulation during the transition from growth to quiescence. We find 16 that when Bacillus subtilis exits growth, a subpopulation of cells emerges with very low levels of 17 protein synthesis dependent on synthesis of the nucleotides (p)ppGpp. We show that (p)ppGpp 18 inhibits protein synthesis in vivo and in vitro by preventing the allosteric activation of the essential 19 GTPase Initiation Factor 2 (IF2) during translation initiation. Finally, we demonstrate that IF2 is 20 an authentic in vivo target of (p)ppGpp during the entry into quiescence, thus providing a 21 mechanistic basis for the observed attenuation of protein synthesis. 22 al., 2016), DNA primase (Wang et al., 2007) and the GTP biosynthetic enzymes HprT and Gmk 51 (Kriel et al., 2012), respectively. 52 Overexpression of a truncated RelA protein that synthesizes (p)ppGpp in the absence of 53 amino acid limitation results in a rapid decrease in 35 S-methionine incorporation (Svitil et al., 54 1993), consistent with an inhibitory effect of (p)ppGpp on translation. However, further 55investigation of a direct effect of (p)ppGpp on translation has been complicated by several factors. 56 First, (p)ppGpp affects synthesis of ribosomal proteins in E. coli (Lindahl et al., 1976). Whether 57 this effect is direct has been difficult to assess given the well-established effect of (p)ppGpp on 58 transcription by E. coli polymerase. Second, studies examining the effect of (p)ppGpp produced 59 by RelA use conditions that produce uncharged tRNAs in order to stimulate RelA (Arenz et al., 60 2016; Brown et al., 2016; Haseltine and Block, 1973; Loveland et al., 2016; O'Farrell, 1978). Since 61 uncharged tRNAs directly arrest translation, it has been difficult to differentiate this effect from a 62 direct effect of (p)ppGpp on translation. 63 In vitro, (p)ppGpp inhibits translation in a manner similar to that observed with non-64 hydrolyzable GTP analogs (Wagner and Kurland, 1980), suggesting that (p)ppGpp is targeting a 65 translational GTPase. Consistently, (p)ppGpp inhibits the GTPase activity of IF2 and EF-Tu 66 (Hamel and Cashel, 1974) as well as of GTPases that mediate other aspects of translation such 67 as ribosome assembly (Corrigan et al., 2016; Pausch et al., 2018), by acting as competitive 68 inhibitors. (p)ppGpp is capable of binding translational GTPases including EF-Tu, EF-G (Rojas et 69 al., 1984), IF2 (Milon et al., 2006; Mitkevich et al., 2010) and the ribosome assembly GTPase 70 ObgE (Persky et al., 2009) at affinities that are commensurate with the in vivo levels of (p)ppGpp 71 observed following stringent response induction, consistent with the enzymes being in vivo 72 targets. Of note, the affinity of IF2 f...
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