Crucial Role for Lipoteichoic Acid Assembly in the Metabolic Versatility and Antibiotic Resistance of Staphylococcus aureus.

Staphylococcus aureus is a public health threat due to the prevalence of antibiotic resistance and the capacity of this organism to infect numerous organs in vertebrates. To generate energy needed to proliferate within tissues, S. aureus transitions between aerobic respiration and fermentation. Fermentation results in a distinct colony morphology called the small-colony variant (SCV) due to decreased membrane potential and ATP production. These traits promote increased resistance to aminoglycoside antibiotics. Consequently, SCVs are associated with persistent infections. We hypothesize that dedicated physiological pathways support fermentative growth of S. aureus that represent potential targets for treatment of resistant infections. Lipoteichoic acid (LTA) is an essential component of the Gram-positive cell envelope that functions to maintain ion homeostasis, resist osmotic stress, and regulate autolytic activity. Previous studies revealed that perturbation of LTA reduces viability of metabolically restricted S. aureus, but the mechanism by which LTA supports S. aureus metabolic versatility is unknown. Though LTA is essential, the enzyme that synthesizes the modified lipid anchor, YpfP, is dispensable. However, ypfP mutants produce altered LTA, leading to elongation of the polymer and decreased cell association. We demonstrate that viability of ypfP mutants is significantly reduced upon environmental and genetic induction of fermentation. This anaerobic viability defect correlates with decreased membrane potential and is restored upon cation supplementation. Additionally, ypfP suppressor mutants exhibiting restored anaerobic viability harbor compensatory mutations in the LTA biosynthetic pathway that restore membrane potential. Overall, these results demonstrate that LTA maintains membrane potential during fermentative proliferation and promotes S. aureus metabolic versatility.

Polyamines and linear DNA mediate bacterial threat assessment of bacteriophage infection.

Monitoring the extracellular environment for danger signals is a critical aspect of cellular survival. However, the danger signals released by dying bacteria and the mechanisms bacteria use for threat assessment remain largely unexplored. Here, we show that lysis of Pseudomonas aeruginosa cells releases polyamines that are subsequently taken up by surviving cells via a mechanism that relies on Gac/Rsm signaling. While intracellular polyamines spike in surviving cells, the duration of this spike varies according to the infection status of the cell. In bacteriophage-infected cells, intracellular polyamines are maintained at high levels, which inhibits replication of the bacteriophage genome. Many bacteriophages package linear DNA genomes and linear DNA is sufficient to trigger intracellular polyamine accumulation, suggesting that linear DNA is sensed as a second danger signal. Collectively, these results demonstrate how polyamines released by dying cells together with linear DNA allow P. aeruginosa to make threat assessments of cellular injury.

Assessing the Role of Cold-Shock Protein C: a Novel Regulator of Acinetobacter baumannii Biofilm Formation and Virulence.

Acinetobacter baumannii is a formidable opportunistic pathogen that is notoriously difficult to eradicate from hospital settings. This resilience is often attributed to a proclivity for biofilm formation, which facilitates a higher tolerance toward external stress, desiccation, and antimicrobials. Despite this, little is known regarding the mechanisms orchestrating A. baumannii biofilm formation. Here, we performed RNA sequencing (RNA-seq) on biofilm and planktonic populations for the multidrug-resistant isolate AB5075 and identified 438 genes with altered expression. To assess the potential role of genes upregulated within biofilms, we tested the biofilm-forming capacity of their respective mutants from an A. baumannii transposon library. In so doing, we uncovered 24 genes whose disruption led to reduced biofilm formation. One such element, cold shock protein C (cspC), had a highly mucoid colony phenotype, enhanced tolerance to polysaccharide degradation, altered antibiotic tolerance, and diminished adherence to abiotic surfaces. RNA-seq of the cspC mutant revealed 201 genes with altered expression, including the downregulation of pili and fimbria genes and the upregulation of multidrug efflux pumps. Using transcriptional arrest assays, it appears that CspC mediates its effects, at least in part, through RNA chaperone activity, influencing the half-life of several important transcripts. Finally, we show that CspC is required for survival during challenge by the human immune system and is key for A. baumannii dissemination and/or colonization during systemic infection. Collectively, our work identifies a cadre of new biofilm-associated genes within A. baumannii and provides unique insight into the global regulatory network of this emerging human pathogen.

Bacteriophage-Bacteria Interactions in the Gut: From Invertebrates to Mammals.

Bacteria and their viruses (bacteriophages or phages) interact antagonistically and beneficially in polymicrobial communities such as the guts of animals. These interactions are multifaceted and are influenced by environmental conditions. In this review, we discuss phage-bacteria interactions as they relate to the complex environment of the gut. Within the mammalian and invertebrate guts, phages and bacteria engage in diverse interactions including genetic coexistence through lysogeny, and phages directly modulate microbiota composition and the immune system with consequences that are becoming recognized as potential drivers of health and disease. With greater depth of understanding of phage-bacteria interactions in the gut and the outcomes, future phage therapies become possible.

Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes in Bacillus subtilis.

Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet-to-be-discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, and G132E) that rendered the mutated YpsA nontoxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS.IMPORTANCEBacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria.

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

Deciphering the Role of a SLOG Superfamily Protein YpsA in Gram-Positive Bacteria.

Bacteria adapt to different environments by regulating cell division and several conditions that modulate cell division have been documented. Understanding how bacteria transduce environmental signals to control cell division is critical in understanding the global network of cell division regulation. In this article we describe a role for Bacillus subtilis YpsA, an uncharacterized protein of the SLOG superfamily of nucleotide and ligand-binding proteins, in cell division. We observed that YpsA provides protection against oxidative stress as cells lacking ypsA show increased susceptibility to hydrogen peroxide treatment. We found that the increased expression of ypsA leads to filamentation and disruption of the assembly of FtsZ, the tubulin-like essential protein that marks the sites of cell division in B. subtilis. We also showed that YpsA-mediated filamentation is linked to the growth rate. Using site-directed mutagenesis, we targeted several conserved residues and generated YpsA variants that are no longer able to inhibit cell division. Finally, we show that the role of YpsA is possibly conserved in Firmicutes, as overproduction of YpsA in Staphylococcus aureus also impairs cell division.

An essential Staphylococcus aureus cell division protein directly regulates FtsZ dynamics.

Binary fission has been well studied in rod-shaped bacteria, but the mechanisms underlying cell division in spherical bacteria are poorly understood. Rod-shaped bacteria harbor regulatory proteins that place and remodel the division machinery during cytokinesis. In the spherical human pathogen Staphylococcus aureus, we found that the essential protein GpsB localizes to mid-cell during cell division and co-constricts with the division machinery. Depletion of GpsB arrested cell division and led to cell lysis, whereas overproduction of GpsB inhibited cell division and led to the formation of enlarged cells. We report that S. aureus GpsB, unlike other Firmicutes GpsB orthologs, directly interacts with the core divisome component FtsZ. GpsB bundles and organizes FtsZ filaments and also stimulates the GTPase activity of FtsZ. We propose that GpsB orchestrates the initial stabilization of the Z-ring at the onset of cell division and participates in the subsequent remodeling of the divisome during cytokinesis.