No abstract

Random cell-to-cell variations in gene expression within an isogenic population can lead to transitions between alternative states of gene expression. Little is known about how these variations (noise) in natural systems affect such transitions. In Bacillus subtilis, noise in ComK, the protein that regulates competence for DNA uptake, is thought to cause cells to transition to the competent state in which genes encoding DNA uptake proteins are expressed. We demonstrate that noise in comK expression selects cells for competence and that experimental reduction of this noise decreases the number of competent cells. We also show that transitions are limited temporally by a reduction in comK transcription. These results illustrate how such stochastic transitions are regulated in a natural system and suggest that noise characteristics are subject to evolutionary forces.Variability in gene expression within a population of genetically identical cells enables those cells to maintain a diversity of phenotypes, potentially enhancing fitness (1, 2). When the underlying gene network contains regulatory positive feedback loops, individual cells can exist in different states; some cells may, for example, live in the "off" expression state of a particular gene, whereas others are in the "on" expression state (this is an example of bistable gene expression). These stochastic fluctuations in gene expression, commonly referred to as noise, have been proposed to cause transitions between these states (3-7). We apply recently developed theories of noise (8, 9) to examine how noise influences these transitions in a natural system. An example of bistable expression with associated stochastic transitions (10-16) involves the ability of the soil bacterium Bacillus subtilis to develop "competence" for DNA uptake as it enters stationary growth phase, potentially allowing bacteria to increase their fitness by incorporating new genetic material. The genes needed for competence are transcribed only in the presence of ComK, the master regulator of competence. comK expression is subject to positive autoregulation effected by the cooperative binding of ComK to its own promoter ( Fig. 1A) (21), effectively lowering the rate of ComK degradation and allowing random fluctuations in the level of ComK to occasionally cause transitions to the competent state. Cells continue to randomly transition to competence for 2 hours, by which time (T 2 ) transitions have ceased to occur (16) and the 15% of the cells that have become competent remain so until diluted into fresh growth medium ( Fig. 1C and movie S1). In this report, we ask why cells only transition to competence for a limited duration of time and investigate the source of the fluctuations that actuate the ComK feedback loop in a minority of cells.To understand why cells only transition to the competent state for ~2 hours during stationary phase, we examined the dynamics of comK expression in noncompetent cells. Because the level of ComK in noncompetent cells is very low, we used fluoresce...

SummaryGene expression in bacteria is traditionally studied from the average behaviour of cells in a population, which has led to the assumption that under a particular set of conditions all cells express genes in an approximately uniform manner. The advent of methods for visualizing gene expression in individual cells reveals, however, that populations of genetically identical bacteria are sometimes heterogeneous, with certain genes being expressed in a non-uniform manner across the population. In some cases, heterogeneity is manifested by the bifurcation into distinct subpopulations, and we adopt the common usage, referring to this phenomenon as bistability. Here we consider four cases of bistability, three from Bacillus subtilis and one from Escherichia coli, with an emphasis on random switching mechanisms that generate alternative cell states and the biological significance of phenotypic heterogeneity. A review describing additional examples of bistability in bacteria has been published recently.

Natural competence is widespread among bacterial species. The mechanism of DNA uptake in both gram-positive and gram-negative bacteria is reviewed. The transformation pathways are discussed, with attention to the fate of donor DNA as it is processed by the competent cell. The proteins involved in mediating various steps in these pathways are described, and models for the transformation mechanisms are presented. Uptake of DNA across the inner membrane is probably similar in gram-positive and gram-negative bacteria, and at least some of the required proteins are orthologs. The initial transformation steps differ, as expected, from the presence of an outer membrane only in the gram-negative organisms. The similarity of certain essential competence proteins to those required for the assembly of type-4 pili and for type-2 protein secretion is discussed. Finally several hypotheses for the biological role of transformation are presented and evaluated.

Transformation and conjugation permit the passage of DNA through the bacterial membranes and represent dominant modes for the transfer of genetic information between bacterial cells or between bacterial and eukaryotic cells. As such, they are responsible for the spread of fitnessenhancing traits, including antibiotic resistance. Both processes usually involve the recognition of double-stranded DNA, followed by the transfer of single strands. Elaborate molecular machines are responsible for negotiating the passage of macromolecular DNA through the layers of the cell surface. All or nearly all the machine components involved in transformation and conjugation have been identified, and here we present models for their roles in DNA transport.In bacteria, transformation and conjugation usually mediate the transport of single-stranded DNA (ssDNA) across one or more membranes. Transformation involves the uptake of environmental DNA, whereas conjugation permits the direct transfer of DNA between cells (Fig. 1). Other DNA-transport phenomena in bacteria, such as the passage of DNA through the bacterial division septa and those carried out by many bacteriophages (1), involve the movement of double-stranded DNA (dsDNA) and will not be discussed here. Transformation and conjugation probably evolved for the acquisition of fitness-enhancing genetic information, but other mutually nonexclusive theories posit that transformation might have evolved to provide templates for DNA repair or to supply nutrition for bacteria (2). Today, both processes are recognized as important mechanisms for horizontal gene transfer and genome plasticity over evolutionary history, and they are largely responsible for the rapid spread of antibiotic resistance among pathogenic bacteria (3, 4). Bacterial TransformationNaturally transformable bacteria acquire a physiological state known as "competence" through the regulated expression of genes for protein components of the uptake machinery. Natural transformation has been most studied in Bacillus subtilis, Streptococcus pneumoniae, Neisseria gonorrhoeae, and Haemophilus influenzae. These and other competent bacteria use similar proteins for DNA uptake, with few differences between species. An interesting exception is Helicobacter pylori, which uses a conjugation-like system for transformation (5). Here, we will discuss the DNA uptake systems of B. subtilis and N. gonorrhoeae as representative of those in Gram-positive and -negative bacteria, respectively (Fig. 1A). The main distinction between these cell types is that Gram-negative bacteria are enclosed by cytoplasmic and outer membranes, with an intervening periplasmic space and thin layer of peptidoglycan (~3 to 7 nm) (6). Gram-positive bacteria lack an outer membrane, and their cytoplasmic membrane is surrounded by a ~22-nm periplasmic space and a thick layer of peptidoglycan (~33 nm) (7).* To whom correspondence should be addressed. Peter.J.Christie@uth.tmc.edu (P.J.C.); dubnau@phri.org (D.D.). (8,9). In the absence of ComEA, 20% residual DN...

SummaryHigh expression of the transcriptional activator ComK occurs in 10-20% of the cells in stationary phase cultures of Bacillus subtilis strain 168. ComK drives the expression of more than 100 genes constituting the semidormant K-state, distinct from sporulation and vegetative growth. Among the genes so activated are those that permit competence for genetic transformation. We have addressed the origin of bistability in expression of ComK. We show that bistability requires positive autoregulation at the promoter of comK , but not a potential toggle switch, in which ComK represses the promoter of rok and Rok represses the promoter of comK . We further address the source of the noise that results in the stochastic selection of cells that will express comK . A revised model for the regulation of comK expression is proposed that partially explains bistability.

Summary In Bacillus subtilis, the competence transcription factor ComK activates its own transcription as well as the transcription of genes that encode DNA transport proteins. ComK is expressed in about 10% of the cells in a culture grown to competence. Using DNA microarrays representing ≈ 95% of the protein‐coding open reading frames in B. subtilis, we compared the expression profiles of wild‐type and comK strains, as well as of a mecA mutant (which produces active ComK in all the cells of the population) and a comK mecA double mutant. In these comparisons, we identified at least 165 genes that are upregulated by ComK and relatively few that are downregulated. The use of reporter fusions has confirmed these results for several genes. Many of the ComK‐regulated genes are organized in clusters or operons, and 23 of these clusters are preceded by apparent ComK‐box promoter motifs. In addition to those required for DNA uptake, other genes that are upregulated in the presence of ComK are probably involved in DNA repair and in the uptake and utilization of nutritional sources. From this and previous work, we conclude that the ComK regulon defines a growth‐arrested state, distinct from sporulation, of which competence for genetic transformation is but one notable feature. We suggest that this is a unique adaptation to stress and that it be termed the ‘K‐state’.

Development of genetic competence in