Pulsed Electromagnetic Fields Disrupt Staphylococcus epidermidis Biofilms and Enhance the Antibiofilm Efficacy of Antibiotics.

Staphylococcus epidermidis is implicated in a multitude of human infections and is one of the major causes of clinical infections in hospitals, especially at surgical sites and on indwelling medical devices, such as orthopedic implants. These infections are especially dangerous because of the S. epidermidis propensity to form biofilms, which increases resistance to antibiotics and the natural immune response. This study investigated pulsed electromagnetic fields (PEMF) as a potential treatment to combat such infections, as PEMF exposure was expected to disrupt the electrostatic forces that adhere staphylococcal cells to surfaces and to one another. To test the effect of PEMF on biofilms, S. epidermidis cultures were exposed to PEMF at various durations either during the growth phase or after a full biofilm had formed. In addition, cells were exposed to PEMF and concomitant antibiotic treatment. Biofilm viability was quantified by both crystal violet and alamarBlue assays and scanning electron microscopy. The results demonstrated that PEMF significantly inhibited biofilm formation and disrupted preformed biofilms in vitro while also showing synergistic biofilm inhibition when combined with antibiotics. These combined results indicate that PEMF should be considered a promising novel technique for treating S. epidermidis biofilm infections and undergo further testing in vivo. IMPORTANCE Antibiotic resistance and biofilm infections are major issues in health care because of the lack of a successful treatment modality and poor patient outcomes. These infections are a particular issue following orthopedic surgery or trauma wherein an infection may form on an orthopedic implant or patient's bone. The presented study demonstrates that pulsed electromagnetic fields may be a promising novel treatment for such infections and can overcome the medical challenges presented by biofilm formation. Furthermore, the effects demonstrated are even greater when combining pulsed electromagnetic field therapy with traditional antibiotics.

Meta-Tyrosine Induces Cytotoxic Misregulation of Metabolism in Escherichia coli.

The non-protein amino acid meta-Tyrosine (m-Tyr) is produced in cells under conditions of oxidative stress, and m-Tyr has been shown to be toxic to a broad range of biological systems. However, the mechanism by which m-Tyr damages cells is unclear. In E. coli, the quality control (QC) function of phenyalanyl-tRNA synthetase (PheRS) is required for resistantce to m-Tyr. To determine the mechanism of m-Tyr toxicity, we utilitized a strain of E. coli that expresses a QC-defective PheRS. The global responses of E. coli cells to m-Tyr were assessed by RNA-seq, and >500 genes were differentially expressed after the addition of m-Tyr. The most strongly up-regulated genes are involved in unfolded-protein stress response, and cells exposed to m-Tyr contained large, electron-dense protein aggregates, indicating that m-Tyr destabilized a large fraction of the proteome. Additionally, we observed that amino acid biosynthesis and transport regulons, controlled by ArgR, TrpR, and TyrR, and the stringent-response regulon, controlled by DksA/ppGpp, were differentially expressed. m-Tyr resistant mutants were isolated and found to have altered a promoter to increase expression of the enzymes for Phe production or to have altered transporters, which likely result in less uptake or increased efflux of m-Tyr. These findings indicate that when m-Tyr has passed the QC checkpoint by the PheRS, this toxicity of m-Tyr may result from interfering with amino acid metabolism, destabalizing a large number of proteins, and the formation of protein aggregates.

Quality Control by Isoleucyl-tRNA Synthetase of Bacillus subtilis Is Required for Efficient Sporulation.

Isoleucyl-tRNA synthetase (IleRS) is an aminoacyl-tRNA synthetase whose essential function is to aminoacylate tRNAIle with isoleucine. Like some other aminoacyl-tRNA synthetases, IleRS can mischarge tRNAIle and correct this misacylation through a separate post-transfer editing function. To explore the biological significance of this editing function, we created a ileS(T233P) mutant of Bacillus subtilis that allows tRNAIle mischarging while retaining wild-type Ile-tRNAIle synthesis activity. As seen in other species defective for aminoacylation quality control, the growth rate of the ileS(T233P) strain was not significantly different from wild-type. When the ileS(T233P) strain was assessed for its ability to promote distinct phenotypes in response to starvation, the ileS(T233P) strain was observed to exhibit a significant defect in formation of environmentally resistant spores. The sporulation defect ranged from 3-fold to 30-fold and was due to a delay in activation of early sporulation genes. The loss of aminoacylation quality control in the ileS(T233P) strain resulted in the inability to compete with a wild-type strain under selective conditions that required sporulation. These data show that the quality control function of IleRS is required in B. subtilis for efficient sporulation and suggests that editing by aminoacyl-tRNA synthetases may be important for survival under starvation/nutrient limitation conditions.

A new quorum-sensing system (TprA/PhrA) for Streptococcus pneumoniae†D39 that regulates a lantibiotic biosynthesis gene cluster.

The Phr peptides of the Bacillus species mediate quorum sensing, but their identification and function in other species of bacteria have not been determined. We have identified a Phr peptide quorum-sensing system (TprA/PhrA) that controls the expression of a lantibiotic gene cluster in the Gram-positive human pathogen, Streptococcus pneumoniae. Lantibiotics are highly modified peptides that are part of the bacteriocin family of antimicrobial peptides. We have characterized the basic mechanism for a Phr-peptide signaling system in S. pneumoniae and found that it induces the expression of the lantibiotic genes when pneumococcal cells are at high density in the presence of galactose, a main sugar of the human nasopharynx, a highly competitive microbial environment. Activity of the Phr peptide system is not seen when pneumococcal cells are grown with glucose, the preferred carbon source and the most prevalent sugar encountered by S. pneumoniae during invasive disease. Thus, the lantibiotic genes are expressed under the control of both cell density signals via the Phr peptide system and nutritional signals from the carbon source present, suggesting that quorum sensing and the lantibiotic machinery may help pneumococcal cells compete for space and resources during colonization of the nasopharynx.

Oxidation of cellular amino acid pools leads to cytotoxic mistranslation of the genetic code.

Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress can lead to the accumulation of non-canonical amino acids that must be excluded from the proteome in order to maintain cellular viability.

Identification of Bacillus subtilis SipW as a bifunctional signal peptidase that controls surface-adhered biofilm formation.

Biofilms of microbial cells encased in an exopolymeric matrix can form on solid surfaces, but how bacteria sense a solid surface and upregulate biofilm genes is largely unknown. We investigated the role of the Bacillus subtilis signal peptidase, SipW, which has a unique role in forming biofilms on a solid surface and is not required at an air-liquid interface. Surprisingly, we found that the signal peptidase activity of SipW was not required for solid-surface biofilms. Furthermore, a SipW mutant protein was constructed that lacks the ability to form a solid-surface biofilm but still retains signal peptidase activity. Through genetic and gene expression tests, the non-signal peptidase role of SipW was found to activate biofilm matrix genes specifically when cells were on a solid surface. These data provide the first evidence that a signal peptidase is bifunctional and that SipW has a regulatory role in addition to its role as a signal peptidase.

Assembly of minicellulosomes on the surface of Bacillus subtilis.

To cost-efficiently produce biofuels, new methods are needed to convert lignocellulosic biomass into fermentable sugars. One promising approach is to degrade biomass using cellulosomes, which are surface-displayed multicellulase-containing complexes present in cellulolytic Clostridium and Ruminococcus species. In this study we created cellulolytic strains of Bacillus subtilis that display one or more cellulase enzymes. Proteins containing the appropriate cell wall sorting signal are covalently anchored to the peptidoglycan by coexpressing them with the Bacillus anthracis sortase A (SrtA) transpeptidase. This approach was used to covalently attach the Cel8A endoglucanase from Clostridium thermocellum to the cell wall. In addition, a Cel8A-dockerin fusion protein was anchored on the surface of B. subtilis via noncovalent interactions with a cell wall-attached cohesin module. We also demonstrate that it is possible to assemble multienzyme complexes on the cell surface. A three-enzyme-containing minicellulosome was displayed on the cell surface; it consisted of a cell wall-attached scaffoldin protein noncovalently bound to three cellulase-dockerin fusion proteins that were produced in Escherichia coli. B. subtilis has a robust genetic system and is currently used in a wide range of industrial processes. Thus, grafting larger, more elaborate minicellulosomes onto the surface of B. subtilis may yield cellulolytic bacteria with increased potency that can be used to degrade biomass.

Cellular mechanisms that control mistranslation.

Mistranslation broadly encompasses the introduction of errors during any step of protein synthesis, leading to the incorporation of an amino acid that is different from the one encoded by the gene. Recent research has vastly enhanced our understanding of the mechanisms that control mistranslation at the molecular level and has led to the discovery that the rates of mistranslation in vivo are not fixed but instead are variable. In this Review we describe the different steps in translation quality control and their variations under different growth conditions and between species though a comparison of in vitro and in vivo findings. This provides new insights as to why mistranslation can have both positive and negative effects on growth and viability.

Identification of residues important for cleavage of the extracellular signaling peptide CSF of Bacillus subtilis from its precursor protein.

Extracellular Phr pentapeptides produced by gram-positive, spore-forming bacteria regulate processes during the transition from exponential- to stationary-phase growth. Phr pentapeptides are produced by cleavage of their precursor proteins. We determined the residues that direct this cleavage for the Bacillus subtilis Phr peptide, CSF, which is derived from the C terminus of PhrC. Strains expressing PhrC with substitutions in residues -1 to -5 relative to the cleavage site had a defect in CSF production. The mutant PhrC proteins retained a functional signal sequence for secretion, as assessed by secretion of PhrC-PhoA fusions. To determine whether the substitutions directly affected cleavage of PhrC to CSF, we tested cleavage of synthetic pro-CSF peptides that corresponded to the C terminus of PhrC and had an amino acid substitution at the -2, -3, or -4 position. The mutant pro-CSF peptides were cleaved less efficiently to CSF than the wild-type pro-CSF peptide whether they were incubated with whole cells, cell wall material, or the processing protease subtilisin or Vpr. To further define the range of amino acids that support CSF production, the amino acid at the -4 position of PhrC was replaced by the 19 canonical amino acids. Only four substitutions resulted in a >2-fold defect in CSF production, indicating that this position is relatively immune to mutational perturbations. These data revealed residues that direct cleavage of CSF and laid the groundwork for testing whether other Phr peptides are processed in a similar manner.

CSF, a species-specific extracellular signaling peptide for communication among strains of Bacillus subtilis and Bacillus mojavensis.

ComX and CSF are Bacillus subtilis extracellular signaling peptides. Many different strains of B. subtilis do not communicate due to strain-specific variation of ComX. We demonstrate that CSF is a species-specific signaling molecule that partially compensates for the lack of ComX-mediated communication between different strains of B. subtilis.

Identification of subtilisin, Epr and Vpr as enzymes that produce CSF, an extracellular signalling peptide of Bacillus subtilis.

Cell-cell communication regulates many important processes in bacteria. Gram-positive bacteria use peptide signals for communication, such as the Phr pentapeptides of Bacillus subtilis. The Phr pentapeptides are secreted with a pro domain that is cleaved to produce an active signalling peptide. To identify the protease(s) involved in production of the mature Phr signalling peptides, we developed assays for detecting cleavage of one of the B. subtilis Phr pentapeptides, CSF, from the proCSF precursor. Using both a cellular and a mass spectrometric approach, we determined that a sigma-H-regulated, secreted, serine protease(s) cleaved proCSF to CSF. Mutants lacking the three proteases that fit these criteria, subtilisin, Epr and Vpr, had a defect in CSF production. Purified subtilisin and Vpr were shown to be capable of processing proCSF as well as at least one other Phr peptide produced by B. subtilis, PhrA, but they were not able to process the PhrE signalling peptide of B. subtilis, indicating that there are probably other unidentified proteases involved in Phr peptide production. Subtilisin, Epr and Vpr are members of the subtilisin family of proteases that are widespread in bacteria, suggesting that many bacterial species may be capable of producing Phr signalling peptides.

Defining the genetic differences between wild and domestic strains of Bacillus subtilis that affect poly-gamma-dl-glutamic acid production and biofilm formation.

Biofilms are communities of microbial cells that are encased in a self-produced, polymeric matrix and are adherent to a surface. For several species of bacteria, an enhanced ability to form biofilms has been linked with an increased capability to produce exopolymers. To identify exopolymers of Bacillus subtilis that can contribute to biofilm formation, we transferred the genetic determinants that control exopolymer production from a wild, exopolymer-positive strain to a domesticated, exopolymer-negative strain. Mapping these genetic determinants led to the identification of gamma-poly-dl-glutamic acid (gamma-PGA) as an exopolymer that increases biofilm formation, possibly through enhancing cell-surface interactions. Production of gamma-PGA by Bacillus subtilis was known to be dependent on the two-component regulator ComPA; this study highlighted the additional dependence on the DegS-DegU, DegQ and SwrA regulator proteins. The inability of the domestic strain of B. subtilis to produce gamma-PGA was mapped to two base pairs; a single base pair change in the promoter region of degQ and a single base pair insertion in the coding region of swrA. Introduction of alleles of degQ and swrA from the wild strain into the domestic strain was sufficient to allow gamma-PGA production. In addition to controlling gamma-PGA production, ComPA and DegSU were also shown to activate biofilm formation through an as yet undefined pathway. The identification of these regulators as affecting gamma-PGA production and biofilm formation suggests that these processes are regulated by osmolarity, high cell density and phase variation.

Lessons from DNA microarray analysis: the gene expression profile of biofilms.

DNA microarray technology has been used to identify the global gene expression profile of biofilm cells. This is an interesting case study in how DNA microarray technology has advanced the molecular understanding of an understudied research area. DNA microarray analyses have suggested that there may be common responses upon biofilm formation, such as the repression of flagella genes and hyper-expression of genes for adhesion and ribosomal protein. They have also assisted in the identification of transcription factors that affect the formation of biofilms and indicated that there may not be biofilm-specific genes, arguing against biofilm formation being a developmental process. Instead, the DNA microarray data suggest that biofilms may have a unique pattern of gene expression, in which sub-sets of genes expressed in biofilms are also expressed under different planktonic conditions, but only in the biofilm are they all expressed simultaneously.

Environmental signals and regulatory pathways that influence biofilm formation.

In nature, bacteria often exist as biofilms. Here, we discuss the environmental signals and regulatory proteins that affect both the initiation of bacterial biofilm formation and the nature of the mature biofilm structure. Current research suggests that the environmental signals regulating whether bacterial cells will initiate a biofilm differ from one bacterial species to another. This may allow each bacterial species to colonize its preferred environment efficiently. In contrast, some of the environmental signals that have currently been identified to regulate the structure of a mature biofilm are nutrient availability and quorum sensing, and are not species specific. These environmental signals evoke changes in the nature of the mature biofilm that may ensure optimal nutrient acquisition. Nutrient availability regulates the depth of the biofilm in such a way that the maximal number of cells in a biofilm appears to occur at suboptimal nutrient concentrations. At either extreme, nutrient-rich or very nutrient-poor conditions, greater numbers of cells are in the planktonic phase where they have greater access to the local nutrients or can be distributed to a new environment. Similarly, quorum-sensing control of the formation of channels and pillar-like structures may ensure efficient nutrient delivery to cells in a biofilm.

Identification of AbrB-regulated genes involved in biofilm formation by Bacillus subtilis.

Bacillus subtilis is a ubiquitous soil bacterium that forms biofilms in a process that is negatively controlled by the transcription factor AbrB. To identify the AbrB-regulated genes required for biofilm formation by B. subtilis, genome-wide expression profiling studies of biofilms formed by spo0A abrB and sigH abrB mutant strains were performed. These data, in concert with previously published DNA microarray analysis of spo0A and sigH mutant strains, led to the identification of 39 operons that appear to be repressed by AbrB. Eight of these operons had previously been shown to be repressed by AbrB, and we confirmed AbrB repression for a further six operons by reverse transcription-PCR. The AbrB-repressed genes identified in this study are involved in processes known to be regulated by AbrB, such as extracellular degradative enzyme production and amino acid metabolism, and processes not previously known to be regulated by AbrB, such as membrane bioenergetics and cell wall functions. To determine whether any of these AbrB-regulated genes had a role in biofilm formation, we tested 23 mutants, each with a disruption in a different AbrB-regulated operon, for the ability to form biofilms. Two mutants had a greater than twofold defect in biofilm formation. A yoaW mutant exhibited a biofilm structure with reduced depth, and a sipW mutant exhibited only surface-attached cells and did not form a mature biofilm. YoaW is a putative secreted protein, and SipW is a signal peptidase. This is the first evidence that secreted proteins have a role in biofilm formation by Bacillus subtilis.

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.

The extracellular Phr peptide-Rap phosphatase signaling circuit of Bacillus subtilis.

In the field of cell-cell communication, an emerging class of extracellular signaling peptides that function intracellularly has been identified in Gram-positive bacteria. One illustrative member of this group is the Phr family of extracellular signaling peptides of Bacillus subtilis. The Phr signaling peptides are secreted by the bacterium, and then, despite the presence of intracellular peptidases, they are actively transported into the cell where they interact with intracellular receptors to regulate gene expression. The intracellular receptors are members of a family of aspartyl-phosphate phosphatases, the Rap phosphatases. These phosphatases cause the dephosphorylation of response regulator proteins, ubiquitous regulatory proteins in bacteria. Immediately downstream of the genes for the Rap phosphatases are the genes for the Phr peptides, forming rap phr signaling cassettes. There are at least seven rap phr signaling cassettes in B. subtilis, and the genome sequence of other Gram-positive, endospore-forming bacteria suggests that similar cassettes may also function in these bacteria. In B. subtilis, the rap phr cassettes regulate sporulation, genetic competence, and genes comprising the quorum response (i.e. the response to high cell density). This review will address the mechanism of extracellular Phr signaling peptide production, transport, response, and their role in quorum sensing.