Extremely thermoacidophilic Crenarchaeota belonging to the order Sulfolobales flourish in hot acidic habitats that are strongly oxidizing. The pH extremes of these habitats, however, often exceed the acid tolerance of type species and strains. Here, adaptive laboratory evolution was used over a 3-year period to test whether such organisms harbor additional thermoacidophilic capacity. Three distinct cell lines derived from a single type species were subjected to high-temperature serial passage while culture acidity was gradually increased. A 178-fold increase in thermoacidophily was achieved after 29 increments of shifted culture pH resulting in growth at pH 0.8 and 80°C. These strains were named super-acid-resistant Crenarchaeota (SARC). Mathematical modeling using growth parameters predicted the limits of acid resistance, while genome resequencing and transcriptome resequencing were conducted for insight into mechanisms responsible for the evolved trait. Among the mutations that were detected, a set of eight nonsynonymous changes may explain the heritability of increased acid resistance despite an unexpected lack of transposition. Four multigene components of the SARC transcriptome implicated oxidative stress as a primary challenge accompanying growth at acid extremes. These components included accelerated membrane biogenesis, induction of the mer operon, and an increased capacity for the generation of energy and reductant.

Current use of microbes for metabolic engineering suffers from loss of metabolic output due to natural selection. Rather than combat the evolution of bacterial populations, we chose to embrace what makes biological engineering unique among engineering fields – evolving materials. We harnessed bacteria to compute solutions to the biological problem of metabolic pathway optimization. Our approach is called Programmed Evolution to capture two concepts. First, a population of cells is programmed with DNA code to enable it to compute solutions to a chosen optimization problem. As analog computers, bacteria process known and unknown inputs and direct the output of their biochemical hardware. Second, the system employs the evolution of bacteria toward an optimal metabolic solution by imposing fitness defined by metabolic output. The current study is a proof-of-concept for Programmed Evolution applied to the optimization of a metabolic pathway for the conversion of caffeine to theophylline in E. coli. Introduced genotype variations included strength of the promoter and ribosome binding site, plasmid copy number, and chaperone proteins. We constructed 24 strains using all combinations of the genetic variables. We used a theophylline riboswitch and a tetracycline resistance gene to link theophylline production to fitness. After subjecting the mixed population to selection, we measured a change in the distribution of genotypes in the population and an increased conversion of caffeine to theophylline among the most fit strains, demonstrating Programmed Evolution. Programmed Evolution inverts the standard paradigm in metabolic engineering by harnessing evolution instead of fighting it. Our modular system enables researchers to program bacteria and use evolution to determine the combination of genetic control elements that optimizes catabolic or anabolic output and to maintain it in a population of cells. Programmed Evolution could be used for applications in energy, pharmaceuticals, chemical commodities, biomining, and bioremediation.

The alternative sigma factor σ54 has been shown to regulate the expression of a wide array of virulence-associated genes, as well as central metabolism, in bacterial pathogens. In Gram-positive organisms, the σ54 is commonly associated with carbon metabolism. In this study, we show that the Enterococcus faecalis alternative sigma factor σ54 (RpoN) and its cognate enhancer binding protein MptR are essential for mannose utilization and are primary contributors to glucose uptake through the Mpt phosphotransferase system. To gain further insight into how RpoN contributes to global transcriptional changes, we performed microarray transcriptional analysis of strain V583 and an isogenic rpoN mutant grown in a chemically defined medium with glucose as the sole carbon source. Transcripts of 340 genes were differentially affected in the rpoN mutant; the predicted functions of these genes mainly related to nutrient acquisition. These differentially expressed genes included those with predicted catabolite-responsive element (cre) sites, consistent with loss of repression by the major carbon catabolite repressor CcpA. To determine if the inability to efficiently metabolize glucose/mannose affected infection outcome, we utilized two distinct infection models. We found that the rpoN mutant is significantly attenuated in both rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI). Here, we examined a ccpA mutant in the CAUTI model and showed that the absence of carbon catabolite control also significantly attenuates bacterial tissue burden in this model. Our data highlight the contribution of central carbon metabolism to growth of E. faecalis at various sites of infection. IMPORTANCE Hospital-acquired infections account for 2 billion dollars annually in increased health care expenses and cause more than 100,000 deaths in the United States alone. Enterococci are the second leading cause of hospital-acquired infections. They form biofilms at surgical sites and are often associated with infections of the urinary tract following catheterization. Nutrient uptake and growth are key factors that influence their ability to cause disease. Our research identified a large set of genes that illuminate nutrient uptake pathways in enterococci. Perturbation of the metabolic circuit reduces virulence in a rabbit endocarditis model, as well as in catheter-associated urinary tract infection in mice. Targeting metabolic pathways that are important in infection may lead to new treatments against multidrug-resistant enterococcal infections.

The ability of Enterococcus faecalis to use a variety of carbon sources enables colonization at various anatomic sites within a mammalian host. N-acetylglucosamine (GlcNAc) is one of the most abundant natural sugars and provides bacteria with a source of carbon and nitrogen when metabolized. N-acetylglucosamine is also a component of bacterial peptidoglycan, further highlighting the significance of N-acetylglucosamine utilization. In this study, we show that CcpA-regulated enzymes are required for growth on the poly-β1,4-linked GlcNAc substrate, chitopentaose (β1,4-linked GlcNAc 5 ). We also show that EF0114 (EndoE) is required for growth on chitobiose (β1,4-linked GlcNAc 2 ), and that the GH20 domain of EndoE is required for the conversion of GlcNAc 2 to N-acetylglucosamine. GlcNAc is transported into the cell via two separate PTS systems, either the PTS IICBA system encoded by ef1516 ( nagE ) or the Mpt glucose/mannose permease complex (MptBACD). The Mpt PTS system is also the primary glucosamine transporter. In order for N-acetylglucosamine to be utilized as a carbon source, phosphorylated N-acetylglucosamine (GlcNAc-6-P) must be deacetylated and here we show that this activity is mediated by EF1317 (an N-acetylglucosamine-6-phosphate deacetylase; NagA homolog), as a deletion of ef1317 is unable to grow on GlcNAc as the carbon source. Deamination of glucosamine to fructose-6-phosphate is required for entry into glycolysis and we show that growth on glucosamine is dependent on EF0466 (a glucosamine-6-phosphate deaminase; NagB homolog). Collectively, our data highlight the chitinolytic machinery required for breaking down exogenous chitinous substrates, as well as the uptake and cytosolic enzymes needed for metabolizing N-acetylglucosamine. Importance Enterococcus faecalis causes life-threatening healthcare-associated infections in part due to its intrinsic and acquired antibiotic resistance, its ability to form biofilms, as well as its nutrient versatility. Alternative nutrient acquisition systems are key factors that contribute to enterococcal colonization at biologically unique host anatomic sites. Although E. faecalis can metabolize an array of carbon sources, little is known of how this bacterium acquires these secondary nutrient sources in mammalian hosts. Our research identifies the glycosidase machinery required for degrading exogenous chitinous substrates into N-acetylglucosamine monomers for transport and metabolism of one of the most abundant naturally occurring sugars, N-acetylglucosamine. Disrupting the function of this N-acetylglucosamine acquisition pathway may lead to new treatments against multidrug resistant enterococcal infections.

The ability of Enterococcus faecalis to colonize host anatomic sites is dependent on its adaptive response to host conditions. Three glycosyl hydrolase gene clusters, each belonging to the GH18 family ( ef0114 , ef0361 , and ef2863 ) in E. faecalis were previously found to be upregulated under glucose-limiting conditions. The GH18 catalytic domain is present in proteins that are classified as either chitinases or ENGases based on their β-1,4 endo-N-acetyl-beta-D-glucosaminidase activity, and ENGase activity is commonly associated with cleaving N-linked glycoproteins, an abundant glycan structure on host epithelial surfaces. Here we show that all three hydrolases are negatively regulated by the transcriptional regulator Carbon Catabolite Protein A (CcpA). Additionally, we demonstrate that a constitutively active CcpA variant represses the expression of CcpA-regulated genes irrespective of glucose availability. Previous studies showed that the GH18 catalytic domain of EndoE (EF0114) and EfEndo18A (EF2863) were capable of deglycosylating RNase B, a model high-mannose type glycoprotein. However, it remained uncertain which glycosidase is primarily responsible for deglycosylation of high-mannose type glycoproteins. In this study, we show by mutation analysis as well as a dose-dependent analysis of recombinant protein expression that EfEndo18A is primarily responsible for deglycosylating high-mannose glycoproteins and the glycans removed by EfEndo18A support growth under nutrient-limiting conditions, in vitro . In contrast, IgG is representative of a complex type glycoprotein, and we demonstrate that the GH18 domain of EndoE is primarily responsible for removal of this glycan decoration. Lastly, our data highlight the combined contribution of glycosidases to virulence of E. faecalis , in vivo. Importance Enterococcus faecalis has emerged as a multidrug-resistant (MDR) nosocomial pathogen that causes life-threatening healthcare-associated infections. Nutrient acquisition systems and immune evasions strategies are key factors that contribute to enterococcal colonization and influence the overall pathogenic potential of this organism. The regulation of these factors is governed by metabolic cues, specifically the availability of glucose as a preferred carbon source. Our research identifies CcpA as a major regulator of secondary nutrient acquisition and expands on the importance of GH18 family glycosyl hydrolases in E. faecalis . These hydrolases contribute to the direct targeting of host glycoproteins both for nutrient acquisition, as well as potentially evading both the innate and adaptive immune response. Disrupting the function of these microbial enzymes may lead to new treatments against multidrug resistant enterococcal infections.