Abstract
Bacteria must survive harsh environmental fluctuations at times and have evolved several strategies. “Collective” behaviors have been identified due to recent progress in single-cell analysis. Since most bacteria exist as single cells, bacterial populations are often considered clonal. However, accumulated evidence suggests this is not the case. Gene expression and protein expression are often not homogeneous, resulting in phenotypic heterogeneity. In extreme cases, this leads to bistability, the existence of two stable states. In many cases, expression of key master regulators is bimodal via positive feedback loops causing bimodal expression of the target genes. We observed bimodal expression of metabolic genes for alternative carbon sources. Expression profiles of the frlBONMD-yurJ operon driven by the frlB promoter (PfrlB), which encodes degradation enzymes and a transporter for amino sugars including fructoselysine, were investigated using transcriptional lacZ and gfp, and translational fluorescence reporter mCherry fusions. Disruption effects of genes encoding CodY, FrlR, RNaseY, and nucleoid-associated protein YlxR, four known regulatory factors for PfrlB, were examined for expression of each fusion construct. Expression of PfrlB-gfp and PfrlB-mCherry, which were located at amyE and its original locus, respectively, was bimodal; and disruption of ylxR resulted in the disappearance of the clear bimodal expression pattern in flow cytometric analyses. This suggested a role for YlxR on the bimodal expression of PfrlB. The data indicated that YlxR acted on the bimodal expression of PfrlB through both transcription and translation. YlxR regulates many genes, including those related to translation, supporting the above notion. Depletion of RNaseY abolished heterogenous expression of transcriptional PfrlB-gfp but not bimodal expression of translational PfrlB-mCherry, suggesting the role of RNaseY in regulation of the operon through mRNA stability control and regulatory mechanism for PfrlB-mCherry at the translational level. Based on these results, we discuss the meaning and possible cause of bimodal PfrlB expression.
Highlights
Bacterial “collective” behaviors of single cells have evolved to adapt to their harsh environments and have been identified as a result of recent progress in single-cell analysis (Veening et al, 2008; Bury-Moné and Sclavi, 2017)
Heterogeneous ylxR expression was subject to positive autoregulation of ylxR as disruption of ylxR resulted in no expression of PylxS-gfp (Figure 3)
All three of the promoter fusions showed homogeneous expression (Figure 5E). These results indicated that the observed bimodal expression of PfrlB-gfp was derived from regulated expression specific for PfrlB, not from gene and protein expression “noise.”
Summary
Bacterial “collective” behaviors of single cells have evolved to adapt to their harsh environments and have been identified as a result of recent progress in single-cell analysis (Veening et al, 2008; Bury-Moné and Sclavi, 2017). In Bacillus subtilis development of genetic competence for uptake of extracellular DNA, bimodal expression of the key master regulator ComK is observed, which leads to the differentiation of a fraction of cells among the cell population into the competent state (Maamar and Dubnau, 2005; Dubnau and Losick, 2006) When it comes to heterogeneous expression systems, bacteria sometime adopt a “bet-hedging strategy” where they differentiate into subpopulations in the same culture in order to facilitate adaptation to rapid environmental fluctuations (Veening et al, 2008; Norman et al, 2015). Highly heterogeneous phosphorylation of the master sporulation regulator Spo0A triggers the initiation of sporulation (Chastanet et al, 2010)
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