Person:

Losick, Richard

Biofilms are often associated with human bacterial infections, and the natural tolerance of biofilms to antibiotics challenges treatment. Compounds with antibiofilm activity could become useful adjuncts to antibiotic therapy. We used norspermidine, a natural trigger for biofilm disassembly in the developmental cycle of Bacillus subtilis, to develop guanidine and biguanide compounds with up to 20-fold increased potency in preventing biofilm formation and breaking down existing biofilms. These compounds also were active against pathogenic Staphylococcus aureus. An integrated approach involving structure–activity relationships, protonation constants, and crystal structure data on a focused synthetic library revealed that precise spacing of positively charged groups and the total charge at physiological pH distinguish potent biofilm inhibitors.

The peptidoglycan precursor, Lipid II, produced in the model Gram-positive bacterium Bacillus subtilis differs from Lipid II found in Gram-negative bacteria such as Escherichia coli by a single amidation on the peptide side chain. How this difference affects the cross-linking activity of penicillin-binding proteins (PBPs) that assemble peptidoglycan in cells has not been investigated because B. subtilis Lipid II was not previously available. Here we report the synthesis of B. subtilis Lipid II and its use by purified B. subtilis PBP1 and E. coli PBP1A. While enzymes from both organisms assembled B. subtilis Lipid II into glycan strands, only the B. subtilis enzyme cross-linked the strands. Furthermore, B. subtilis PBP1 catalyzed the exchange of both d-amino acids and d-amino carboxamides into nascent peptidoglycan, but the E. coli enzyme only exchanged d-amino acids. We exploited these observations to design a fluorescent d-amino carboxamide probe to label B. subtilis PG in vivo and found that this probe labels the cell wall dramatically better than existing reagents.

A model system for investigating how developmental regulatory networks determine cell fate is spore formation in Bacillus subtilis. The master regulator for sporulation is Spo0A, which is activated by phosphorylation via a phosphorelay that is subject to three positive feedback loops. The ultimate decision to sporulate is, however, stochastic in that only a portion of the population sporulates even under optimal conditions. It was previously assumed that activation of Spo0A and hence entry into sporulation is subject to a bistable switch mediated by one or more feedback loops. Here we reinvestigate the basis for bimodality in sporulation. We show that none of the feedback loops is rate limiting for the synthesis and phosphorylation of Spo0A. Instead, the loops ensure a just-in-time supply of relay components for rising levels of phosphorylated Spo0A, with phosphate flux through the relay being limiting for Spo0A activation and sporulation. In addition, genes under Spo0A control did not exhibit a bimodal pattern of expression as expected for a bistable switch. In contrast, we observed a highly heterogeneous pattern of Spo0A activation that increased in a nonlinear manner with time. We present a computational model for the nonlinear increase and propose that the phosphorelay is a noise generator and that only cells that attain a threshold level of phosphorylated Spo0A sporulate.

ABSTRACT Biofilm formation by Staphylococcus aureus involves the formation of an extracellular matrix, but the composition of this matrix has been uncertain. Here we report that the matrix is largely composed of cytoplasmic proteins that reversibly associate with the cell surface in a manner that depends on pH. We propose a model for biofilm formation in which cytoplasmic proteins are released from cells in stationary phase. These proteins associate with the cell surface in response to decreasing pH during biofilm formation. Rather than utilizing a dedicated matrix protein, S. aureus appears to recycle cytoplasmic proteins that moonlight as components of the extracellular matrix.

We report the discovery of a simple environmental sensing mechanism for biofilm formation in the bacterium Bacillus subtilis that operates without the involvement of a dedicated RNA or protein. Certain serine codons, the four TCN codons, in the gene for the biofilm repressor SinR caused a lowering of SinR levels under biofilm-inducing conditions. Synonymous substitutions of these TCN codons with AGC or AGT impaired biofilm formation and gene expression. Conversely, switching AGC or AGT to TCN codons upregulated biofilm formation. Genome-wide ribosome profiling showed that ribosome density was higher at UCN codons than at AGC or AGU during biofilm formation. Serine starvation recapitulated the effect of biofilm-inducing conditions on ribosome occupancy and SinR production. As serine is one of the first amino acids to be exhausted at the end of exponential phase growth, reduced translation speed at serine codons may be exploited by other microbes in adapting to stationary phase. DOI: http://dx.doi.org/10.7554/eLife.01501.001

Genetically identical cells sharing an environment can display markedly different phenotypes. It is often unclear how much of this variation derives from chance, external signals, or attempts by individual cells to exert autonomous phenotypic programs. By observing thousands of cells for hundreds of consecutive generations under constant conditions, we dissect the stochastic decision between a solitary, motile state and a chained, sessile state in Bacillus subtilis. The motile state is memoryless, exhibiting no autonomous control over the time spent in the state, whereas chaining is tightly timed. Timing enforces coordination among related cells in the multicellular state. Further, we show that the three-protein regulatory circuit governing the decision is modular, as initiation and maintenance of chaining are genetically separable functions. As stimulation of the same initiating pathway triggers biofilm formation, we argue that autonomous timing allows a trial commitment to multicellularity that external signals could extend.

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.

The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine and was reported to inhibit biofilm formation via the incorporation of these d-amino acids into the cell wall. Here, we show that l-amino acids were able to specifically reverse the inhibitory effects of their cognate d-amino acids. We also show that d-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding d-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of d-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of d-amino acids without losing the ability to incorporate at least one noncanonical d-amino acid, d-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of d-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis.

Asymmetric division during sporulation by Bacillus subtilis generates a mother cell that undergoes a 5-h program of differentiation. The program is governed by a hierarchical cascade consisting of the transcription factors: σ^E, σ^K, GerE, GerR, and SpoIIID. The program consists of the activation and repression of 383 genes. The σ^E factor turns on 262 genes, including those for GerR and SpoIIID. These DNA-binding proteins downregulate almost half of the genes in the σ^E regulon. In addition, SpoIIID turns on ten genes, including genes involved in the appearance of σK. Next, σ^K activates 75 additional genes, including that for GerE. This DNA-binding protein, in turn, represses half of the genes that had been activated by σ^K while switching on a final set of 36 genes. Evidence is presented that repression and activation contribute to proper morphogenesis. The program of gene expression is driven forward by its hierarchical organization and by the repressive effects of the DNA-binding proteins. The logic of the program is that of a linked series of feed-forward loops, which generate successive pulses of gene transcription. Similar regulatory circuits could be a common feature of other systems of cellular differentiation.

Sporulene, a C35-terpenoid hydrocarbon with an unusual pentacyclic structure, is produced by Bacillus subtilis during sporulation.