Rich came to Harvard from MIT in 1969 as a Junior Fellow, setting up shop on the third floor of the Biological Laboratories in space kindly provided by Jack Strominger.The third floor was an intense and exciting environment as it was the home to three Nobel Laureates and a training environment for five young investigators who would later win Nobel Prizes. Rich’s plan was to study the cell envelope of E. coli. But things quickly changed. Dick Burgess and Andrew Travers had just made the electrifying discovery down the hall in the Watson lab (in parallel with John Dunn and Ekke Bautz at Rutgers) that promoter recognition in E. coli is mediated by a subunit of RNA polymerase that they called sigma factor. The finding that RNA polymerase could be separated into a core catalytic component and a subunit needed for initiation at promoters ignited a search for alternative sigma factors that could recognize promoters for different sets of genes. Such alternative sigma factors would represent a major new mechanism for controlling gene expression in bacteria, acting at the level of the transcription machinery itself. But how to find such alternative sigma factors, if they existed?
Endonucleolytic cleavage within polycistronic mRNAs can lead to differential stability, and thus discordant abundance, among cotranscribed genes. RNase Y, the major endonuclease for mRNA decay in Bacillus subtilis, was originally identified for its cleavage activity toward the cggR-gapA operon, an event that differentiates the synthesis of a glycolytic enzyme from its transcriptional regulator. A three-protein Y-complex (YlbF, YmcA, and YaaT) was recently identified as also being required for this cleavage in vivo, raising the possibility that it is an accessory factor acting to regulate RNase Y. However, whether the Y-complex is broadly required for RNase Y activity is unknown. Here, we used end-enrichment RNA sequencing (Rend-seq) to globally identify operon mRNAs that undergo maturation posttranscriptionally by RNase Y and the Y-complex. We found that the Y-complex is required for the majority of RNase Y-mediated mRNA maturation events and also affects riboswitch abundance in B. subtilis. In contrast, noncoding RNA maturation by RNase Y often does not require the Y-complex. Furthermore, deletion of RNase Y has more pleiotropic effects on the transcriptome and cell growth than deletions of the Y-complex. We propose that the Y-complex is a specificity factor for RNase Y, with evidence that its role is conserved in Staphylococcus aureus.
Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a "mother machine"-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.
Lipoteichoic acid (LTA) is an anionic surface polymer that is essential for normal growth of Staphylococcus aureus, making the LTA polymerase, LTA synthase (LtaS), a proposed drug target for combating Staphylococcal infections. LtaS is a polytopic membrane protein with five membrane-spanning helices and an extracellular domain, and it uses phosphatidylglycerol to assemble a glycerol phosphate chain on a glycosylated diacylglycerol membrane anchor. We report here the first reconstitution of LtaS polymerization activity and show that the azo dye Congo red inhibits this enzyme both in vitro and in cells. Related azo dyes and the previously reported LtaS inhibitor 1771 have weak or no in vitro inhibitory activity. Synthetic lethality with mutant strains known to be nonviable in the absence of LTA confirms selective inhibition by Congo red. As the only validated LtaS inhibitor, Congo red can serve as a probe to understand how inhibiting lipoteichoic acid biosynthesis affects cell physiology and may also guide the discovery of more potent inhibitors for use in treating S. aureus infections.
Entry into sporulation in Bacillus subtilis is governed by a phosphorelay in which phosphoryl groups from a histidine kinase are successively transferred via relay proteins to the response regulator Spo0A. Spo0A~P, in turn, sets in motion events that lead to asymmetric division and activation of the cell-specific transcription factor σ(F), a hallmark for entry into sporulation. Here, we have used a microfluidics-based platform to investigate the activation of Spo0A and σ(F) in individual cells held under constant, sporulation-inducing conditions. The principal conclusions were that: (i) activation of σ(F) occurs with an approximately constant probability after adaptation to conditions of nutrient limitation; (ii) activation of σ(F) is tightly correlated with, and preceded by, Spo0A~P reaching a high threshold level; (iii) activation of Spo0A takes place abruptly just prior to asymmetric division; and (iv) the primary source of noise in the activation of Spo0A is the phosphorelay. We propose that cells exhibit a constant probability of attaining a high threshold level of Spo0A~P due to fluctuations in the flux of phosphoryl groups through the phosphorelay.
Bacteria naturally form communities of cells known as biofilms. However the physiological roles of biofilms produced by non-pathogenic microbiota remain largely unknown. To assess the impact of a biofilm on host physiology we explored the effect of several non-pathogenic biofilm-forming bacteria on Caenorhabditis elegans. We show that biofilm formation by Bacillus subtilis, Lactobacillus rhamnosus and Pseudomonas fluorescens induces C. elegans stress resistance. Biofilm also protects against pathogenic infection and prolongs lifespan. Total mRNA analysis identified a set of host genes that are upregulated in response to biofilm formation by B. subtilis. We further demonstrate that mtl-1 is responsible for the biofilm-mediated increase in oxidative stress resistance and lifespan extension. Induction of mtl-1 and hsp-70 promotes biofilm-mediated thermotolerance. ilys-2 activity accounts for biofilm-mediated resistance to Pseudomonas aeruginosa killing. These results reveal the importance of non-pathogenic biofilms for host physiology and provide a framework to study commensal biofilms in higher organisms.
Sigma (σ) factors direct gene transcription by binding to and determining the promoter recognition specificity of RNA polymerase (RNAP) in bacteria. Genes transcribed under the control of alternative sigma factors allow cells to respond to stress and undergo developmental processes, such as sporulation in Bacillus subtilis, in which gene expression is controlled by a cascade of alternative sigma factors. Binding of sigma factors to RNA polymerase depends on the coiled-coil (or clamp helices) motif of the β' subunit. We have identified an amino acid substitution (L257P) in the coiled coil that markedly inhibits the function of σ(H), the earliest-acting alternative sigma factor in the sporulation cascade. Cells with this mutant RNAP exhibited an early and severe block in sporulation but not in growth. The mutant was strongly impaired in σ(H)-directed gene expression but not in the activity of the stress-response sigma factor σ(B) Pulldown experiments showed that the mutant RNAP was defective in associating with σ(H) but could still associate with σ(A) and σ(B) The differential effects of the L257P substitution on sigma factor binding to RNAP are likely due to a conformational change in the β' coiled coil that is specifically detrimental for interaction with σ(H) This is the first example, to our knowledge, of an amino acid substitution in RNAP that exhibits a strong differential effect on a particular alternative sigma factor.IMPORTANCE In bacteria, all transcription is mediated by a single multisubunit RNA polymerase (RNAP) enzyme. However, promoter-specific transcription initiation necessitates that RNAP associates with a σ factor. Bacteria contain a primary σ factor that directs transcription of housekeeping genes and alternative σ factors that direct transcription in response to environmental or developmental cues. We identified an amino acid substitution (L257P) in the B. subtilis β' subunit whereby RNAP(L257P) associates with some σ factors (σ(A) and σ(B)) and enables vegetative cell growth but is defective in utilization of σ(H) and is consequently blocked for sporulation. To our knowledge, this is the first identification of an amino acid substitution within the core enzyme that affects utilization of a specific sigma factor.
Staphylococcus aureus is a leading cause of both nosocomial and community-acquired infection. Biofilm formation at the site of infection reduces antimicrobial susceptibility and can lead to chronic infection. During biofilm formation, a subset of cells liberate cytoplasmic proteins and DNA, which are repurposed to form the extracellular matrix that binds the remaining cells together in large clusters. Using a strain that forms robust biofilms in vitro during growth under glucose supplementation, we carried out a genome-wide screen for genes involved in the release of extracellular DNA (eDNA). A high-density transposon insertion library was grown under biofilm-inducing conditions, and the relative frequency of insertions was compared between genomic DNA (gDNA) collected from cells in the biofilm and eDNA from the matrix. Transposon insertions into genes encoding functions necessary for eDNA release were identified by reduced representation in the eDNA. On direct testing, mutants of some of these genes exhibited markedly reduced levels of eDNA and a concomitant reduction in cell clustering. Among the genes with robust mutant phenotypes were gdpP, which encodes a phosphodiesterase that degrades the second messenger cyclic-di-AMP, and xdrA, the gene for a transcription factor that, as revealed by RNA-sequencing analysis, influences the expression of multiple genes, including many involved in cell wall homeostasis. Finally, we report that growth in biofilm-inducing medium lowers cyclic-di-AMP levels and does so in a manner that depends on the gdpP phosphodiesterase gene.
Sporulation in Bacillus subtilis is governed by a cascade of alternative RNA polymerase sigma factors. We previously identified a small protein Fin that is produced under the control of the sporulation sigma factor σ(F) to create a negative feedback loop that inhibits σ(F) -directed gene transcription. Cells deleted for fin are defective for spore formation and exhibit increased levels of σ(F) -directed gene transcription. Based on pull-down experiments, chemical crosslinking, bacterial two-hybrid experiments and nuclear magnetic resonance chemical shift analysis, we now report that Fin binds to RNA polymerase and specifically to the coiled-coil region of the β' subunit. The coiled-coil is a docking site for sigma factors on RNA polymerase, and evidence is presented that the binding of Fin and σ(F) to RNA polymerase is mutually exclusive. We propose that Fin functions by a mechanism distinct from that of classic sigma factor antagonists (anti-σ factors), which bind directly to a target sigma factor to prevent its association with RNA polymerase, and instead functions to inhibit σ(F) by competing for binding to the β' coiled-coil.
Bacteria use a variety of stress-sensing systems to sense and respond to diverse stressors and to ensure their survival under adverse conditions. The gram-positive bacterium Bacillus subtilis responds to energy stress (ATP depletion) and to environmental stressors using two distinct stress-sensing pathways that converge on the alternative sigma factor σB to provoke a general stress response. Past efforts to study the σB stress response in bulk culture and on agarose pads were unable to visualize the responses of individual cells under tightly controlled conditions for extended periods of time. Here we use a microfluidics-based strategy to discern the basic features of σB activation in single cells in response to energy and environmental stress, both immediately upon stressor exposure and for tens of generations thereafter. Upon energy stress at various levels of stressor, cells exhibited fast, transient, and amplitude-modulated responses but not frequency modulation as previously reported. Upon environmental stress, which is mediated by the stressosome complex, wild-type cells primarily exhibited a transient and amplitude-modulated response. However, mutant cells producing only one of the four paralogous RsbR stressosome proteins showed striking and previously unseen differences. Whereas RsbRA-only cells mimicked the wild type, RsbRC-only cells displayed a slower but sustained overall response composed of repeated activation events in single cells.
PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from Bacillus subtilis. Based on structural analyses and genetic and biochemical experiments, we identify an α-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor. Our analysis indicates that this switch is widely conserved among PP2C family members, serving as a platform to control phosphatase activity in response to diverse inputs. Remarkably, the switch is shared with proteasomal proteases, which we identify as evolutionary and structural relatives of PP2C phosphatases. Although these proteases use an unrelated catalytic mechanism, rotation of equivalent helices controls protease activity by movement of the equivalent carbonyl oxygen into the active site.
Bacterial type VI secretion is an offensive and defensive weapon that utilizes a molecular warhead to inject toxins into neighboring cells. In this issue of Cell, Whitney et al. report a new class of toxin that disrupts the core metabolism of recipient cells and uncover a surprising requirement for EF-Tu.
Formation of a division septum near a randomly chosen pole during sporulation in B. subtilis creates unequal sized daughter cells with dissimilar programs of gene expression. An unanswered question is how polar septation activates a transcription factor (σ(F)) selectively in the small cell. We present evidence that the upstream regulator of σ(F), the phosphatase SpoIIE, is compartmentalized in the small cell by transfer from the polar septum to the adjacent cell pole where SpoIIE is protected from proteolysis and activated. Polar recognition, protection from proteolysis, and stimulation of phosphatase activity are linked to oligomerization of SpoIIE. This mechanism for initiating cell-specific gene expression is independent of additional sporulation proteins; vegetative cells engineered to divide near a pole sequester SpoIIE and activate σ(F) in small cells. Thus, a simple model explains how SpoIIE responds to a stochastically-generated cue to activate σ(F) at the right time and in the right place.
Biofilm formation by Bacillus subtilis is largely governed by a circuit in which the response regulator Spo0A turns on the gene for the anti-repressor SinI. SinI, in turn, binds to and inactivates SinR, a dedicated repressor of genes for matrix production. Mutants of the genes ylbF, ymcA, and yaaT are blocked in biofilm formation, but the mechanism by which they act has been mysterious. A recent report attributed their role in biofilm formation to stimulating Spo0A activity. However, we detect no measurable effect on the transcription of sinI. Instead, we find that the block in biofilm formation is caused by an increase in the levels of SinR and of its mRNA. Evidence is presented that YlbF, YmcA and YaaT interact with, and control the activity of, RNase Y, which is known to destabilize sinR mRNA. We show that the processing of another target of RNase Y, cggR-gapA mRNA, similarly depends on YlbF and YmcA. Our work suggests that sinR mRNA stability is an additional posttranscriptional control mechanism governing the switch to multicellularity and raises the possibility that YlbF, YmcA, and YaaT broadly regulate mRNA stability as part of an RNase Y-containing, multi-subunit complex.
Pseudomonas aeruginosa is an opportunistic human pathogen whose survival is aided by forming communities known as biofilms, in which cells are encased in a self-produced matrix. . We devised a mutant screen based on colony morphology to identify additional genes with previously unappreciated roles in biofilm formation. Our screen, which identified most known biofilm-related genes, also uncovered PA14_16550 and PA14_69700, deletions of which abrogated and augmented biofilm formation, respectively. We also identified ptsP, which encodes enzyme I of the nitrogen-regulated phosphotransferase (PTS(Ntr) ) system, as being important for cyclic-di-GMP production and for biofilm formation. Further experiments showed that biofilm formation is hindered in the absence of phosphotransfer through the PTS(Ntr) , but only in the presence of enzyme II (PtsN), the putative regulatory module of the PTS(Ntr) . These results implicate unphosphorylated PtsN as a negative regulator of biofilm formation and establish one of the first known roles of the PTS(Ntr) in P. aeruginosa.
Staphylococcus aureus is an important human pathogen that can form biofilms on various surfaces. These cell communities are protected from the environment by a self-produced extracellular matrix composed of proteins, DNA and polysaccharide. The exact composition and the role of the different components are not fully understood. In this study we investigate the role of extracellular DNA (eDNA) and its interaction with the recently identified cytoplasmic proteins that have a moonlighting role in the biofilm matrix. These matrix proteins associate with the cell surface upon the drop in pH that naturally occurs during biofilm formation, and we found here that this association is independent of eDNA. Conversely, the association of eDNA with the matrix was dependent on matrix proteins. Both proteinase treatment and DNase treatment severely reduced clumping of resuspended biofilms; highlighting the importance of both proteins and eDNA in connecting cells together. By adding an excess of exogenous DNA to DNase treated biofilm, clumping was partially restored, confirming the crucial role of eDNA for the interconnection of cells. Based on our results we propose that eDNA acts as an electrostatic net, interconnecting cells surrounded by positively charged matrix proteins at low pH.
IMPORTANCE: Extracellular DNA (eDNA) is an important component of the biofilm matrix in diverse bacteria but its role in biofilm formation is not well understood. Here we report that in Staphylococcus aureus eDNA associates with cells in a manner that depends on matrix proteins and that eDNA is required for linking cells together in the biofilm. These results confirm previous studies that eDNA is an important component of the S. aureus biofilm matrix and also suggest that eDNA acts as an electrostatic net that tethers cells together via the proteinaceous layer of the biofilm matrix.
Microbes transiently differentiate into distinct, specialized cell types to generate functional diversity and cope with changing environmental conditions. Though alternate programs often entail radically different physiological and morphological states, recent single-cell studies have revealed that these crucial decisions are often left to chance. In these cases, the underlying genetic circuits leverage the intrinsic stochasticity of intracellular chemistry to drive transition between states. Understanding how these circuits transform transient gene expression fluctuations into lasting phenotypic programs will require a combination of quantitative modeling and extensive, time-resolved observation of switching events in single cells. In this article, we survey microbial cell fate decisions demonstrated to involve a random element, describe theoretical frameworks for understanding stochastic switching between states, and highlight recent advances in microfluidics that will enable characterization of key dynamic features of these circuits. Expected final online publication date for the Annual Review of Microbiology Volume 69 is October 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
The ability to form multicellular communities known as biofilms is a widespread adaptive behavior of bacteria. Members of the Bacillus group of bacteria have been found to form biofilms on plant roots, where they protect against pathogens and promote growth. In the case of the model bacterium Bacillus subtilis the genetic pathway controlling biofilm formation and the production of an extracellular matrix is relatively well understood. However, it is unclear whether other members of this genus utilize similar mechanisms. We determined that a plant-associated strain of Bacillus cereus (905) can form biofilms by two seemingly independent pathways. In one mode involving the formation of floating biofilms (pellicles) B. cereus 905 appears to rely on orthologs of many of the genes known to be important for B. subtilis biofilm formation. We report that B. cereus 905 also forms submerged, surface-associated biofilms and in a manner that resembles biofilm formation by the pathogen Staphylococcus aureus. This alternative mode, which does not rely on B. subtilis-like genes for pellicle formation, takes place under conditions of glucose fermentation and depends on a drop in the pH of the medium.
Bacteria produce d-amino acids for incorporation into the peptidoglycan and certain nonribosomally produced peptides. However, d-amino acids are toxic if mischarged on tRNAs or misincorporated into protein. Common strains of the Gram-positive bacterium Bacillus subtilis are particularly sensitive to the growth-inhibitory effects of d-tyrosine due to the absence of d-aminoacyl tRNA deacylase, an enzyme that prevents misincorporation of d-tyrosine and other d-amino acids into nascent proteins. We isolated spontaneous mutants of B. subtilis that survive in the presence of a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine. Whole genome sequencing revealed that these strains harbored mutations affecting tRNA(Tyr) charging. Three of the most potent mutations enhanced the expression of the gene (tyrS) for tyrosyl-tRNA synthetase. In particular, resistance was conferred by mutations that destabilized the terminator hairpin of the tyrS riboswitch, as well as by a mutation that transformed a tRNA(Phe) into a tyrS riboswitch ligand. The most potent mutation, a substitution near the tyrosine-recognition site of tyrosyl-tRNA synthetase, improved enzyme stereoselectivity. We conclude that these mutations promote the proper charging of tRNA(Tyr), thus facilitating the exclusion of d-tyrosine from protein biosynthesis in cells that lack d-aminoacyl tRNA deacylase.
IMPORTANCE: Proteins are composed of l-amino acids. Mischarging of tRNAs with d-amino acids or the misincorporation of d-amino acids into proteins causes toxicity. This work reports on mutations that confer resistance to d-amino acids and their mechanisms of action.
My career in science was launched when I was an undergraduate at Princeton University and reinforced by graduate training at the Massachusetts Institute of Technology. However, it was only after I moved to Harvard University as a junior fellow that my affections were captured by a seemingly mundane soil bacterium. What Bacillus subtilis offered was endless fascinating biological problems (alternative sigma factors, sporulation, swarming, biofilm formation, stochastic cell fate switching) embedded in a uniquely powerful genetic system. Along the way, my career in science became inseparably interwoven with teaching and mentoring, which proved to be as rewarding as the thrill of discovery.