Alan D. Grossman

Regulation of noise in the expression of a single gene

Stochastic mechanisms are ubiquitous in biological systems. Biochemical reactions that involve small numbers of molecules are intrinsically noisy, being dominated by large concentration fluctuations. This intrinsic noise has been implicated in the random lysis/lysogeny decision of bacteriophage-λ, in the loss of synchrony of circadian clocks and in the decrease of precision of cell signals. We sought to quantitatively investigate the extent to which the occurrence of molecular fluctuations within single cells (biochemical noise) could explain the variation of gene expression levels between cells in a genetically identical population (phenotypic noise). We have isolated the biochemical contribution to phenotypic noise from that of other noise sources by carrying out a series of differential measurements. We varied independently the rates of transcription and translation of a single fluorescent reporter gene in the chromosome of Bacillus subtilis, and we quantitatively measured the resulting changes in the phenotypic noise characteristics. We report that of these two parameters, increased translational efficiency is the predominant source of increased phenotypic noise. This effect is consistent with a stochastic model of gene expression in which proteins are produced in random and sharp bursts. Our results thus provide the first direct experimental evidence of the biochemical origin of phenotypic noise, demonstrating that the level of phenotypic variation in an isogenic population can be regulated by genetic parameters.

Identification and Characterization of a Bacterial Chromosome Partitioning Site

We have identified a DNA site involved in chromosome partitioning in B. subtilis. This site was identified in vivo as the binding site for the chromosome partitioning protein Spo0J, a member of the ParB family of partitioning proteins. Spo0J is a site-specific DNA-binding protein that recognizes a 16 bp sequence found in spo0J. Allowing two mismatches, this sequence occurs ten times in the entire B. subtilis chromosome, all in the origin-proximal approximately 20%. Eight of the ten sequences are bound to Spo0J in vivo. The presence of a site on an otherwise unstable plasmid stabilized the plasmid in a Spo0J-dependent manner, demonstrating that this site, called parS, can function as a partitioning site. This site and Spo0J are conserved in a wide range of bacterial species.

Biochemical and genetic characterization of a competence pheromone from B. subtilis.

We have purified and characterized a modified peptide pheromone that accumulates in culture medium as B. subtilis grows to high density. This pheromone is required for the development of genetic competence. When added to cells at low density, the pheromone induces the premature development of competence. The peptide moiety of the pheromone matches nine of the last ten amino acids predicted from a 55 codon open reading frame, comX. comX and comQ, the gene immediately upstream of comX, are required for production of the pheromone. Response to the pheromone requires the comP-comA two-component regulatory system and the oligopeptide permease encoded by spo0K. Spo0K could transport the pheromone into the cell, or function as a receptor, binding the pheromone and sending a transmembrane signal, leading to activation of the ComA transcription factor and induction of competence development.

Bipolar Localization of the Replication Origin Regions of Chromosomes in Vegetative and Sporulating Cells of B. subtilis

To investigate chromosome segregation in B. subtilis, we introduced tandem copies of the lactose operon operator into the chromosome near the replication origin or terminus. We then visualized the position of the operator cassettes with green fluorescent protein fused to the Lac1 repressor. In sporulating bacteria, which undergo asymmetric cell division, origins localized near each pole of the cell whereas termini were restricted to the middle. In growing cells, which undergo binary fission, origins were observed at various positions but preferentially toward the poles early in the cell cycle. In contrast, termini showed little preference for the poles. These results indicate the existence of a mitotic-like apparatus that is responsible for moving the origin regions of newly formed chromosomes toward opposite ends of the cell.

Genome-Wide Analysis of the Stationary-Phase Sigma Factor (Sigma-H) Regulon of Bacillus subtilis

Sigma-H is an alternative RNA polymerase sigma factor that directs the transcription of many genes that function at the transition from exponential growth to stationary phase in Bacillus subtilis. Twenty-three promoters, which drive transcription of 33 genes, are known to be recognized by sigma-H-containing RNA polymerase. To identify additional genes under the control of sigma-H on a genome-wide basis, we carried out transcriptional profiling experiments using a DNA microarray containing >99% of the annotated B. subtilis open reading frames. In addition, we used a bioinformatics-based approach aimed at the identification of promoters recognized by RNA polymerase containing sigma-H. This combination of approaches was successful in confirming most of the previously described sigma-H-controlled genes. In addition, we identified 26 putative promoters that drive expression of 54 genes not previously known to be under the direct control of sigma-H. Based on the known or inferred function of most of these genes, we conclude that, in addition to its previously known roles in sporulation and competence, sigma-H controls genes involved in many physiological processes associated with the transition to stationary phase, including cytochrome biogenesis, generation of potential nutrient sources, transport, and cell wall metabolism.

Movement of Replicating DNA through a Stationary Replisome

We found that DNA is replicated at a central stationary polymerase, and each replicated region moves away from the replisome. In Bacillus subtilis, DNA polymerase is predominantly located at or near midcell. When replication was blocked in a specific chromosomal region, that region was centrally located with DNA polymerase. Upon release of the block, each copy of the duplicated region was located toward opposite cell poles, away from the central replisome. In a roughly synchronous population of cells, a region of chromosome between origin and terminus moved to the replisome prior to duplication. Thus, the polymerase at the replication forks is stationary, and the template is pulled in and released outward during duplication. We propose that B. subtilis, and probably many bacteria, harness energy released during nucleotide condensation by a stationary replisome to facilitate chromosome partitioning.

Identification of Catabolite Repression as a Physiological Regulator of Biofilm Formation by Bacillus subtilis by Use of DNA Microarrays

Biofilms are structured communities of cells that are encased in a self-produced polymeric matrix and are adherent to a surface. Many biofilms have a significant impact in medical and industrial settings. The model gram-positive bacterium Bacillus subtilis has recently been shown to form biofilms. To gain insight into the genes involved in biofilm formation by this bacterium, we used DNA microarrays representing >99% of the annotated B. subtilis open reading frames to follow the temporal changes in gene expression that occurred as cells transitioned from a planktonic to a biofilm state. We identified 519 genes that were differentially expressed at one or more time points as cells transitioned to a biofilm. Approximately 6% of the genes of B. subtilis were differentially expressed at a time when 98% of the cells in the population were in a biofilm. These genes were involved in motility, phage-related functions, and metabolism. By comparing the genes differentially expressed during biofilm formation with those identified in other genomewide transcriptional-profiling studies, we were able to identify several transcription factors whose activities appeared to be altered during the transition from a planktonic state to a biofilm. Two of these transcription factors were Spo0A and sigma-H, which had previously been shown to affect biofilm formation by B. subtilis. A third signal that appeared to be affecting gene expression during biofilm formation was glucose depletion. Through quantitative biofilm assays and confocal scanning laser microscopy, we observed that glucose inhibited biofilm formation through the catabolite control protein CcpA.

Spx-dependent global transcriptional control is induced by thiol-specific oxidative stress in Bacillus subtilis

The Spx protein of Bacillus subtilis represses activator-stimulated transcription by interacting with the C-terminal domain of RNA polymerase (RNAP) α subunit. Its concentration increases in cells lacking the ATP-dependent protease, ClpXP, resulting in severe effects on growth and developmental processes. Microarray analysis was undertaken to identify genes that are induced or repressed when Spx interacts with RNAP. The induced genes included those encoding products known to function in maintaining thiol homeostasis. Two genes, thioredoxin (trxA) and thioredoxin reductase (trxB), are transcriptionally induced under conditions of thiol-specific oxidative (disulfide) stress by a mechanism involving Spx-RNAP interaction. Disulfide stress also results in an increase in Spx-dependent transcriptional repression. The increase in Spx activity in cells encountering disulfide stress is due in part to a posttranscriptional mechanism of spx control resulting in an increase in Spx concentration. An spx null mutant and a strain bearing an allele of rpoA that prevents Spx-RNAP interaction show hypersensitivity to disulfide stress. From these results, it is proposed that Spx is an activator that mobilizes the operations necessary to reverse the effects of oxidative damage, but it also serves as a negative regulator that causes the postponement of developmental programs and energy-consuming growth-related functions while the cell copes with the period of stress.

An Exported Peptide Functions Intracellularly to Contribute to Cell Density Signaling in B. subtilis

Competence development and sporulation in B. subtilis are partly controlled by peptides that accumulate in culture medium as cells grow to high density. We constructed two genes that encode mature forms of two different signaling molecules, the PhrA peptide that stimulates sporulation, and CSF, the competence- and sporulation-stimulating factor. Both pentapeptides are normally produced by secretion and processing of precursor molecules. The mature pentapeptides were functional when expressed inside the cell, indicating that they normally need to be imported to function. Furthermore, at physiological concentrations (10 nM), CSF was transported into the cell by the oligopeptide permease encoded by spo0K (opp). CSF was shown to have at least three different targets corresponding to its three activities: stimulating competence gene expression at low concentrations, and inhibiting competence gene expression and stimulating sporulation at high concentrations.

Nutritional Control of Elongation of DNA Replication by (p)ppGpp

DNA replication is highly regulated in most organisms. Although much research has focused on mechanisms that regulate initiation of replication, mechanisms that regulate elongation of replication are less well understood. We characterized a mechanism that regulates replication elongation in the bacterium Bacillus subtilis. Replication elongation was inhibited within minutes after amino acid starvation, regardless of where the replication forks were located on the chromosome. We found that small nucleotides ppGpp and pppGpp, which are induced upon starvation, appeared to inhibit replication directly by inhibiting primase, an essential component of the replication machinery. The replication forks arrested with (p)ppGpp did not recruit the recombination protein RecA, indicating that the forks are not disrupted. (p)ppGpp appear to be part of a surveillance mechanism that links nutrient availability to replication by rapidly inhibiting replication in starved cells, thereby preventing replication-fork disruption. This control may be important for cells to maintain genomic integrity.