Search

The par locus of the Enterococcus faecalis plasmid pAD1 is an RNA-regulated addiction module encoding the peptide toxin Fst. Homology searches revealed that Fst belongs to a family of at least nine related peptides encoded on the chromosomes and plasmids of six different Gram-positive bacterial species. Comparison of an alignment of these peptides with the results of a saturation mutagenesis analysis indicated regions of the peptides important for biological function. Examination of the genetic context of the fst genes revealed that all of these peptides are encoded within par-like loci with conserved features similar to pAD1 par. All four Ent. faecalis family members were demonstrated to produce the expected toxin-encoding and regulatory RNA products. The locus from the Ent. faecalis plasmid pAMSl was demonstrated to function as an addiction module and Fst was shown to be toxic to Staphylococcus aureus, suggesting that a plasmid-encoded module in that species is performing the same function. Thus, the pAD1-encoded par locus appears to be the prototype of a family of related loci found in several Gram-positive species.

Numerous bacteria accumulate poly(3-hydroxybutyrate) (PHB) as an intracellular reservoir of carbon and energy in response to imbalanced nutritional conditions. In Bacillus spp., where PHB biosynthesis precedes the formation of the dormant cell type called the spore (sporulation), the direct link between PHB accumulation and efficiency of sporulation was observed in multiple studies. Although the idea of PHB as an intracellular carbon and energy source fueling sporulation was proposed several decades ago, the mechanisms underlying PHB contribution to sporulation have not been defined. Here, we demonstrate that PHB deficiency impairs Bacillus anthracis sporulation through diminishing the energy status of the cells and by reducing carbon flux into the tricarboxylic acid (TCA) cycle and de novo lipid biosynthesis. Consequently, this metabolic imbalance decreased biosynthesis of the critical components required for spore integrity and resistance, such as dipicolinic acid (DPA) and the spore's inner membrane. Supplementation of the PHB deficient mutant with exogenous fatty acids overcame these sporulation defects, highlighting the importance of the TCA cycle and lipid biosynthesis during sporulation. Combined, the results of this work reveal the molecular mechanisms of PHB contribution to B. anthracis sporulation and provide valuable insight into the metabolic requirements for this developmental process in Bacillus species. [ABSTRACT FROM AUTHOR]

Recent studies have demonstrated that expression of the S taphylococcus aureus lrgAB operon is specifically localized within tower structures during biofilm development. To gain a better understanding of the mechanisms underlying this spatial control of lrgAB expression, we carried out a detailed analysis of the LytSR two-component system. Specifically, a conserved aspartic acid ( Asp53) of the LytR response regulator was shown to be the target of phosphorylation, which resulted in enhanced binding to the lrgAB promoter and activation of transcription. In addition, we identified His390 of the LytS histidine kinase as the site of autophosphorylation and Asn394 as a critical amino acid involved in phosphatase activity. Interestingly, LytS-independent activation of LytR was observed during planktonic growth, with acetyl phosphate acting as a phosphodonor to LytR. In contrast, mutations disrupting the function of LytS prevented tower-specific lrgAB expression, providing insight into the physiologic environment within these structures. In addition, overactivation of LytR led to increased lrgAB promoter activity during planktonic and biofilm growth and a change in biofilm morphology. Overall, the results of this study are the first to define the LytSR signal transduction pathway, as well as determine the metabolic context within biofilm tower structures that triggers these signaling events. [ABSTRACT FROM AUTHOR]

Genome sequencing has demonstrated that Staphylococcus aureus encodes arginine biosynthetic genes argDCJBFGH synthesizing proteins that mediate arginine biosynthesis using glutamate as a substrate. Paradoxically, however, S. aureus does not grow in a defined, glutamate-replete medium lacking arginine and glucose (CDM-R). Studies from our laboratory have found that specific mutations are selected by S. aureus that facilitate growth in CDM-R. However, these selected mutants synthesize arginine utilizing proline as a substrate rather than glutamate. In this study, we demonstrate that the ectopic expression of the argDCJB operon supports the growth of S. aureus in CDM-R, thus documenting the functionality of this pathway. Furthermore, suppressor mutants of S. aureus JE2 putA::Tn, which is defective in synthesizing arginine from proline, were selected on CDM-R agar. Genome sequencing revealed that these mutants had compensatory mutations within both spoVG, encoding an ortholog of the Bacillus subtilis stage V sporulation protein, and sarA, encoding the staphylococcal accessory regulator. Transcriptional studies document that argD expression is significantly increased when JE2 spoVG sarA was grown in CDM-R. Lastly, we found that a mutation in ahrC was required to induce argD expression in JE2 spoVG sarA when grown in an arginine-replete medium (CDM), suggesting that AhrC also functions to repress argDCJB in an arginine-dependent manner. In conclusion, these data indicate that the argDCJB operon is functional when transcribed in vitro and that SNPs within potential putative regulatory proteins are required to alleviate the repression. [ABSTRACT FROM AUTHOR]

Fast and reliable identification of pathogenic bacteria is of upmost importance to human health and safety. Methods that are currently used in clinical practice are often time consuming, require expensive equipment, trained personnel, and therefore have limited applications in low resource environments. Molecular identification methods address some of these shortcomings. At the same time, they often use antibodies, their fragments, or other biomolecules as recognition units, which makes such tests specific to a particular target. In contrast, array-based methods use a combination of reporters that are not specific to a single pathogen. These methods provide a more data-rich and universal response that can be used for identification of a variety of bacteria of interest. In this report, we demonstrate the application of the excitation-emission spectroscopy of an environmentally sensitive fluorescent dye for identification of pathogenic bacterial species. 2-(4'-Dimethylamino)-3-hydroxyflavone (DMAF) interacts with the bacterial cell envelope resulting in a distinct spectral response that is unique to each bacterial species. The dynamics of dye-bacteria interaction were thoroughly investigated, and the limits of detection and identification were determined. Neural network classification algorithm was used for pattern recognition analysis and classification of spectral data. The sensor successfully discriminated between eight representative pathogenic bacteria, achieving a classification accuracy of 85.8% at the species level and 98.3% at the Gram status level. The proposed method based on excitation-emission spectroscopy of an environmentally sensitive fluorescent dye is a powerful and versatile diagnostic tool with high accuracy in identification of bacterial pathogens., Competing Interests: The authors declare no conflicts., (This journal is © The Royal Society of Chemistry.)

The Staphylococcus aureus cidABC and lrgAB operons encode members of a well-conserved family of proteins thought to be involved in programmed cell death (PCD). Based on the structural similarities that CidA and LrgA share with bacteriophage holins, we have hypothesized that these proteins function by forming pores within the cytoplasmic membrane. To test this, we utilized a "lysis cassette" system that demonstrated the abilities of the cidA and lrgA genes to support bacteriophage endolysin-induced cell lysis. Typical of holins, CidA- and LrgA-induced lysis was dependent on the coexpression of endolysin, consistent with the proposed holin-like functions of these proteins. In addition, the CidA and LrgA proteins were shown to localize to the surface of membrane vesicles and cause leakage of small molecules, providing direct evidence of their hole-forming potential. Consistent with recent reports demonstrating a role for the lrgAB homologues in other bacterial and plant species in the transport of by-products of carbohydrate metabolism, we also show that lrgAB is important for S. aureus to utilize pyruvate during microaerobic and anaerobic growth, by promoting the uptake of pyruvate under these conditions. Combined, these data reveal that the CidA and LrgA membrane proteins possess holin-like properties that play an important role in the transport of small by-products of carbohydrate metabolism. IMPORTANCE The Staphylococcus aureus cidABC and lrgAB operons represent the founding members of a large, highly conserved family of genes that span multiple kingdoms of life. Despite the fact that they have been shown to be involved in bacterial PCD, very little is known about the molecular/biochemical functions of the proteins they encode. The results presented in this study reveal that the cidA and lrgA genes encode proteins with bacteriophage holin-like functions, consistent with their roles in cell death. However, these studies also demonstrate that these operons are involved in the transport of small metabolic by-products of carbohydrate metabolism, suggesting an intriguing link between these two seemingly disparate processes.

Oxygen consumption is a fundamental characteristic of staphylococcal physiology reflecting the energy and metabolic state of the bacterial cell. During aerobic growth, oxygen consumption rates (OCR) depend on nutrient availability and vary at different growth stages. The measurement of oxygen consumption rates provides a versatile tool to characterize the impact of various mutations, environmental cues, and antibiotics on bacterial growth and fitness. In this chapter, we describe a MitoXpress ® Xtra-based oxygen consumption assay for fast and reliable determination of respiration rates in Staphylococcus aureus. This highly reproducible and simple method requires a minimal set of reagents and allows rapid screening of multiple samples through real-time determination of the OCR with an oxygen-sensing probe and fluorescence plate reader., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Transposon mutagenesis is a genetic process that involves the random insertion of transposons into a genome resulting in the disruption of function of the genes in which they insert. Identification of the insertion sites through DNA sequencing allows for the identification of the genes disrupted and the creation of "libraries" containing a collection of mutants in which a large number of the nonessential genes have been disrupted. These mutant libraries have been a great resource for investigators to understand the various biological functions of individual genes, including those involved in metabolism, antibiotic susceptibility, and pathogenesis. Here, we describe the detailed methodologies for constructing a sequence defined transposon mutant library in both Staphylococcus aureus and S. epidermidis using the mariner-based transposon, bursa aurealis.

Transposon mutant libraries are valuable resources to investigators studying bacterial species, including Staphylococcus epidermidis, which are difficult to genetically manipulate. Although sequence-defined transposon mutant libraries have been constructed in Staphylococcus aureus, no such library exists for S. epidermidis. Nevertheless, the study of Tn917-mediated mutations has been paramount in discovering unique aspects of S. epidermidis biology including initial adherence and accumulation during biofilm formation. Herein, we describe modifications to the methodology first described by Bae et al. to utilize the mariner-based transposon bursa aurealis to generate mutants in S. epidermidis strain 1457.