Genetic and Biochemical Analysis of CodY-Mediated Cell Aggregation in Staphylococcus aureus Reveals an Interaction between Extracellular DNA and Polysaccharide in the Extracellular Matrix.

The global regulator CodY links nutrient availability to the regulation of virulence factor gene expression in Staphylococcus aureus, including many genes whose products affect biofilm formation. Antithetical phenotypes of both biofilm deficiency and accumulation have been reported for codY-null mutants; thus, the role of CodY in biofilm development remains unclear. codY mutant cells of a strain producing a robust biofilm elaborate proaggregation surface-associated features not present on codY mutant cells that do not produce a robust biofilm. Biochemical analysis of the clinical isolate SA564, which aggregates when deficient for CodY, revealed that these features are sensitive to nuclease treatment and are resistant to protease exposure. Genetic analyses revealed that disrupting lgt (the diacylglycerol transferase gene) in codY mutant cells severely weakened aggregation, indicating a role for lipoproteins in the attachment of the biofilm matrix to the cell surface. An additional and critical role of IcaB in producing functional poly-N-acetylglucosamine (PIA) polysaccharide in extracellular DNA (eDNA)-dependent biofilm formation was shown. Moreover, overproducing PIA is sufficient to promote aggregation in a DNA-dependent manner regardless of source of nucleic acids. Taken together, our results point to PIA synthesis as the primary determinant of biofilm formation when CodY activity is reduced and suggest a modified electrostatic net model for matrix attachment whereby PIA associates with eDNA, which interacts with the cell surface via covalently attached membrane lipoproteins. This work counters the prevailing view that polysaccharide- and eDNA/protein-based biofilms are mutually exclusive. Rather, we demonstrate that eDNA and PIA can work synergistically to form a biofilm.IMPORTANCEStaphylococcus aureus remains a global health concern and exemplifies the ability of an opportunistic pathogen to adapt and persist within multiple environments, including host tissue. Not only does biofilm contribute to persistence and immune evasion in the host environment, it also may aid in the transition to invasive disease. Thus, understanding how biofilms form is critical for developing strategies for dispersing biofilms and improving biofilm disease-related outcomes. Using biochemical, genetic, and cell biology approaches, we reveal a synergistic interaction between PIA and eDNA that promotes cell aggregation and biofilm formation in a CodY-dependent manner in S. aureus We also reveal that envelope-associated lipoproteins mediate attachment of the biofilm matrix to the cell surface.

Differential adenine methylation analysis reveals increased variability in 6mA in the absence of methyl-directed mismatch repair.

IMPORTANCE: Methylation greatly influences the bacterial genome by guiding DNA repair and regulating pathogenic and stress-response phenotypes. But, the rate of epigenetic changes and their consequences on molecular phenotypes are underexplored. Through a detailed characterization of genome-wide adenine methylation in a commonly used laboratory strain of Escherichia coli, we reveal that mismatch repair deficient populations experience an increase in epimutations resulting in a genome-wide reduction of 6mA methylation in a manner consistent with genetic drift. Our findings highlight how methylation patterns evolve and the constraints on epigenetic evolution due to post-replicative DNA repair, contributing to a deeper understanding of bacterial genome evolution and how epimutations may introduce semi-permanent variation that can influence adaptation.

Complex Ecotype Dynamics Evolve in Response to Fluctuating Resources.

Ecotypic diversification and its associated cooperative behaviors are frequently observed in natural microbial populations whose access to resources is often sporadic. However, the extent to which fluctuations in resource availability influence the emergence of cooperative ecotypes is not fully understood. To determine how exposure to repeated resource limitation affects the establishment and long-term maintenance of ecotypes in a structured environment, we followed 32 populations of Escherichia coli evolving to either 1-day or 10-day feast/famine cycles for 900 days. Population-level analysis revealed that compared to populations evolving to 1-day cycles, 10-day populations evolved increased biofilm density, higher parallelism in mutational targets, and increased mutation rates. As previous investigations of evolution in structured environments have identified biofilm formation as the earliest observable phenotype associated with diversification of ecotypes, we revived cultures midway through the evolutionary process and conducted additional genomic, transcriptional, and phenotypic analyses of clones isolated from these evolving populations. We found not only that 10-day feast/famine cycles support multiple ecotypes but also that these ecotypes exhibit cooperative behavior. Consistent with the black queen hypothesis, or evolution of cooperation by gene loss, transcriptomic evidence suggests the evolution of bidirectional cross-feeding behaviors based on essential resources. These results provide insight into how analogous cooperative relationships may emerge in natural microbial communities. IMPORTANCE Despite regular feast and famine conditions representing an environmental pressure that is commonly encountered by microbial communities, the evolutionary outcomes of repeated cycles of feast and famine have been less studied. By experimentally evolving initially isogenic Escherichia coli populations to 10-day feast/famine cycles, we observed rapid diversification into ecotypes with evidence of bidirectional cross-feeding on costly resources and frequency-dependent fitness. Although unidirectional cross-feeding has been repeatedly observed to evolve in laboratory culture, most investigations of bidirectional cooperative behaviors in microbial populations have been conducted in engineered communities. This work demonstrates the de novo evolution of black queen relationships in a microbial population originating from a single ancestor, providing a model for investigation of the eco-evolutionary processes leading to mutualistic cooperation.

Regulation of the Sae Two-Component System by Branched-Chain Fatty Acids in Staphylococcus aureus.

Staphylococcus aureus is a ubiquitous Gram-positive bacterium and an opportunistic human pathogen. S. aureus pathogenesis relies on a complex network of regulatory factors that adjust gene expression. Two important factors in this network are CodY, a repressor protein responsive to nutrient availability, and the SaeRS two-component system (TCS), which responds to neutrophil-produced factors. Our previous work revealed that CodY regulates the secretion of many toxins indirectly via Sae through an unknown mechanism. We report that disruption of codY results in increased levels of phosphorylated SaeR (SaeR~P) and that codY mutant cell membranes contain a higher percentage of branched-chain fatty acids (BCFAs) than do wild-type membranes, prompting us to hypothesize that changes to membrane composition modulate the activity of the SaeS sensor kinase. Disrupting the lpdA gene encoding dihydrolipoyl dehydrogenase, which is critical for BCFA synthesis, significantly reduced the abundance of SaeR, phosphorylated SaeR, and BCFAs in the membrane, resulting in reduced toxin production and attenuated virulence. Lower SaeR levels could be explained in part by reduced stability. Sae activity in the lpdA mutant could be complemented genetically and chemically with exogenous short- or full-length BCFAs. Intriguingly, lack of lpdA also alters the activity of other TCSs, suggesting a specific BCFA requirement managing the basal activity of multiple TCSs. These results reveal a novel method of posttranscriptional virulence regulation via BCFA synthesis, potentially linking CodY activity to multiple virulence regulators in S. aureus. IMPORTANCE Two-component systems (TCSs) are an essential way that bacteria sense and respond to their environment. These systems are usually composed of a membrane-bound histidine kinase that phosphorylates a cytoplasmic response regulator. Because most of the histidine kinases are embedded in the membrane, lipids can allosterically regulate the activity of these sensors. In this study, we reveal that branched-chain fatty acids (BCFAs) are required for the activation of multiple TCSs in Staphylococcus aureus. Using both genetic and biochemical data, we show that the activity of the virulence activator SaeS and the phosphorylation of its response regulator SaeR are reduced in a branched-chain keto-acid dehydrogenase complex mutant and that defects in BCFA synthesis have far-reaching consequences for exotoxin secretion and virulence. Finally, we show that mutation of the global nutritional regulator CodY alters BCFA content in the membrane, revealing a potential mechanism of posttranscriptional regulation of the Sae system by CodY.