High Levels of Cyclic Diguanylate Interfere with Beneficial Bacterial Colonization.

During colonization of the Hawaiian bobtail squid (Euprymna scolopes), Vibrio fischeri bacteria undergo a lifestyle transition from a planktonic motile state in the environment to a biofilm state in host mucus. Cyclic diguanylate (c-di-GMP) is a cytoplasmic signaling molecule that is important for regulating motility-biofilm transitions in many bacterial species. V. fischeri encodes 50 proteins predicted to synthesize and/or degrade c-di-GMP, but a role for c-di-GMP regulation during host colonization has not been investigated. We examined strains exhibiting either low or high levels of c-di-GMP during squid colonization and found that while a low-c-di-GMP strain had no colonization defect, a high c-di-GMP strain was severely impaired. Expression of a heterologous c-di-GMP phosphodiesterase restored colonization, demonstrating that the effect is due to high c-di-GMP levels. In the constitutive high-c-di-GMP state, colonizing V. fischeri exhibited reduced motility, altered biofilm aggregate morphology, and a regulatory interaction where transcription of one polysaccharide locus is inhibited by the presence of the other polysaccharide. Our results highlight the importance of proper c-di-GMP regulation during beneficial animal colonization, illustrate multiple pathways regulated by c-di-GMP in the host, and uncover an interplay of multiple exopolysaccharide systems in host-associated aggregates. IMPORTANCE There is substantial interest in studying cyclic diguanylate (c-di-GMP) in pathogenic and environmental bacteria, which has led to an accepted paradigm in which high c-di-GMP levels promote biofilm formation and reduce motility. However, considerably less focus has been placed on understanding how this compound contributes to beneficial colonization. Using the Vibrio fischeri-Hawaiian bobtail squid study system, we took advantage of recent genetic advances in the bacterium to modulate c-di-GMP levels and measure colonization and track c-di-GMP phenotypes in a symbiotic interaction. Studies in the animal host revealed a c-di-GMP-dependent genetic interaction between two distinct biofilm polysaccharides, Syp and cellulose, that was not evident in culture-based studies: elevated c-di-GMP altered the composition and abundance of the in vivo biofilm by decreasing syp transcription due to increased cellulose synthesis. This study reveals important parallels between pathogenic and beneficial colonization and additionally identifies c-di-GMP-dependent regulation that occurs specifically in the squid host.

Genetic Manipulation of Vibrio fischeri.

Vibrio fischeri is a nonpathogenic organism related to pathogenic Vibrio species. The bacterium has been used as a model organism to study symbiosis in the context of its association with its host, the Hawaiian bobtail squid Euprymna scolopes. The genetic tractability of this bacterium has facilitated the mapping of pathways that mediate interactions between these organisms. The protocols included here describe methods for genetic manipulation of V. fischeri. Following these protocols, the researcher will be able to introduce linear DNA via transformation to make chromosomal mutations, to introduce plasmid DNA via conjugation and subsequently eliminate unstable plasmids, to eliminate antibiotic resistance cassettes from the chromosome, and to randomly or specifically mutagenize V. fischeri with transposons. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Transformation of V. fischeri with linear DNA Basic Protocol 2: Plasmid transfer into V. fischeri via conjugation Support Protocol 1: Removing FRT-flanked antibiotic resistance cassettes from the V. fischeri genome Support Protocol 2: Eliminating unstable plasmids from V. fischeri Alternate Protocol 1: Introduction of exogenous DNA using a suicide plasmid Alternate Protocol 2: Site-specific transposon insertion using a suicide plasmid Alternate Protocol 3: Random transposon mutagenesis using a suicide plasmid.

LapG mediates biofilm dispersal in Vibrio fischeri by controlling maintenance of the VCBS-containing adhesin LapV.

Efficient symbiotic colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri depends on bacterial biofilm formation on the surface of the squid's light organ. Subsequently, the bacteria disperse from the biofilm via an unknown mechanism and enter through pores to reach the interior colonization sites. Here, we identify a homolog of Pseudomonas fluorescens LapG as a dispersal factor that promotes cleavage of a biofilm-promoting adhesin, LapV. Overproduction of LapG inhibited biofilm formation and, unlike the wild-type parent, a ΔlapG mutant formed biofilms in vitro. Although V. fischeri encodes two putative large adhesins, LapI (near lapG on chromosome II) and LapV (on chromosome I), only the latter contributed to biofilm formation. Consistent with the Pseudomonas Lap system model, our data support a role for the predicted c-di-GMP-binding protein LapD in inhibiting LapG-dependent dispersal. Furthermore, we identified a phosphodiesterase, PdeV, whose loss promotes biofilm formation similar to that of the ΔlapG mutant and dependent on both LapD and LapV. Finally, we found a minor defect for a ΔlapD mutant in initiating squid colonization, indicating a role for the Lap system in a relevant environmental niche. Together, these data reveal new factors and provide important insights into biofilm dispersal by V. fischeri.

Vibrio fischeri: Laboratory Cultivation, Storage, and Common Phenotypic Assays.

Vibrio fischeri is a nonpathogenic organism related to pathogenic Vibrio species that can be readily grown and stored with common laboratory equipment. In this article, protocols for routine growth, storage, and phenotypic assessment of V. fischeri, as well as recipes for useful media, are included. Specifically, this article describes procedures and considerations for growth of this microbe in complex and minimal media. It also describes assays for biofilm formation, motility, and bioluminescence, three commonly assessed phenotypes of V. fischeri. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Growth of V. fischeri from frozen stocks Basic Protocol 2: Growth of V. fischeri in rich, undefined liquid medium Alternate Protocol 1: Growth of V. fischeri in minimal medium Basic Protocol 3: Storage of V. fischeri in frozen stocks Basic Protocol 4: Biofilm assay on solid agar Alternate Protocol 2: Biofilm assay in shaking liquid culture Alternate Protocol 3: Biofilm assay in static liquid culture Basic Protocol 5: Motility assay Basic Protocol 6: Luminescence assay.

Post-translational Protein Acetylation: An Elegant Mechanism for Bacteria to Dynamically Regulate Metabolic Functions.

Post-translational modifications (PTM) decorate proteins to provide functional heterogeneity to an existing proteome. The large number of known PTMs highlights the many ways that cells can modify their proteins to respond to diverse stimuli. Recently, PTMs have begun to receive increased interest because new sensitive proteomics workflows and structural methodologies now allow researchers to obtain large-scale, in-depth and unbiased information concerning PTM type and site localization. However, few PTMs have been extensively assessed for functional consequences, leaving a large knowledge gap concerning the inner workings of the cell. Here, we review understanding of N-𝜀-lysine acetylation in bacteria, a PTM that was largely ignored in bacteria until a decade ago. Acetylation is a modification that can dramatically change the function of a protein through alteration of its properties, including hydrophobicity, solubility, and surface properties, all of which may influence protein conformation and interactions with substrates, cofactors and other macromolecules. Most bacteria carry genes predicted to encode the lysine acetyltransferases and lysine deacetylases that add and remove acetylations, respectively. Many bacteria also exhibit acetylation activities that do not depend on an enzyme, but instead on direct transfer of acetyl groups from the central metabolites acetyl coenzyme A or acetyl phosphate. Regardless of mechanism, most central metabolic enzymes possess lysines that are acetylated in a regulated fashion and many of these regulated sites are conserved across the spectrum of bacterial phylogeny. The interconnectedness of acetylation and central metabolism suggests that acetylation may be a response to nutrient availability or the energy status of the cell. However, this and other hypotheses related to acetylation remain untested.

Global Lysine Acetylation in Escherichia coli Results from Growth Conditions That Favor Acetate Fermentation.

Lysine acetylation is thought to provide a mechanism for regulating metabolism in diverse bacteria. Indeed, many studies have shown that the majority of enzymes involved in central metabolism are acetylated and that acetylation can alter enzyme activity. However, the details regarding this regulatory mechanism are still unclear, specifically with regard to the signals that induce lysine acetylation. To better understand this global regulatory mechanism, we profiled changes in lysine acetylation during growth of Escherichia coli on the hexose glucose or the pentose xylose at both high and low sugar concentrations using label-free mass spectrometry. The goal was to see whether lysine acetylation differed during growth on these two different sugars. No significant differences, however, were observed. Rather, the initial sugar concentration was the principal factor governing changes in lysine acetylation, with higher sugar concentrations causing more acetylation. These results suggest that acetylation does not target specific metabolic pathways but rather simply targets accessible lysines, which may or may not alter enzyme activity. They further suggest that lysine acetylation principally results from conditions that favor accumulation of acetyl phosphate, the principal acetate donor in E. coliIMPORTANCE Bacteria alter their metabolism in response to nutrient availability, growth conditions, and environmental stresses using a number of different mechanisms. One is lysine acetylation, a posttranslational modification known to target many metabolic enzymes. However, little is known about this regulatory mode. We investigated the factors inducing changes in lysine acetylation by comparing growth on glucose and xylose. We found that the specific sugar used for growth did not alter the pattern of acetylation; rather, the amount of sugar did, with more sugar causing more acetylation. These results imply that lysine acetylation is a global regulatory mechanism that is responsive not to the specific carbon source per se but rather to the accumulation of downstream metabolites.

Extracellular Acidic pH Inhibits Acetate Consumption by Decreasing Gene Transcription of the Tricarboxylic Acid Cycle and the Glyoxylate Shunt.

Escherichia coli produces acetate during aerobic growth on various carbon sources. After consuming the carbon substrate, E. coli can further grow on the acetate. This phenomenon is known as the acetate switch, where cells transition from producing acetate to consuming it. In this study, we investigated how pH governs the acetate switch. When E. coli was grown on a glucose-supplemented medium initially buffered to pH 7, the cells produced and then consumed the acetate. However, when the initial pH was dropped to 6, the cells still produced acetate but were only able to consume it when little (<10 mM) acetate was produced. When significant acetate was produced in acidic medium, which occurs when the growth medium contains magnesium, amino acids, and sugar, the cells were unable to consume the acetate. To determine the mechanism, we characterized a set of metabolic mutants and found that those defective in the tricarboxylic acid (TCA) cycle or glyoxylate shunt exhibited reduced rates of acetate consumption. We further found that the expression of the genes in these pathways was reduced during growth in acidic medium. The expression of the genes involved in the AckA-Pta pathway, which provides the principal route for both acetate production and consumption, was also inhibited in acidic medium but only after glucose was depleted, which correlates with the acetate consumption phase. On the basis of these results, we conclude that growth in acidic environments inhibits the expression of the acetate catabolism genes, which in turn prevents acetate consumption.IMPORTANCE Many microorganisms produce fermentation products during aerobic growth on sugars. One of the best-known examples is the production of acetate by Escherichia coli during aerobic growth on sugars. In E. coli, acetate production is reversible: once the cells consume the available sugar, they can consume the acetate previously produced during aerobic fermentation. We found that pH affects the reversibility of acetate production. When the cells produce significant acetate during growth in acidic environments, they are unable to consume it. Unconsumed acetate may accumulate in the cell and inhibit the expression of pathways required for acetate catabolism. These findings demonstrate how acetate alters cell metabolism; they also may be useful for the design of aerobic fermentation processes.

Identification of Novel Protein Lysine Acetyltransferases in Escherichia coli.

Posttranslational modifications, such as Nε-lysine acetylation, regulate protein function. Nε-lysine acetylation can occur either nonenzymatically or enzymatically. The nonenzymatic mechanism uses acetyl phosphate (AcP) or acetyl coenzyme A (AcCoA) as acetyl donor to modify an Nε-lysine residue of a protein. The enzymatic mechanism uses Nε-lysine acetyltransferases (KATs) to specifically transfer an acetyl group from AcCoA to Nε-lysine residues on proteins. To date, only one KAT (YfiQ, also known as Pka and PatZ) has been identified in Escherichia coli Here, we demonstrate the existence of 4 additional E. coli KATs: RimI, YiaC, YjaB, and PhnO. In a genetic background devoid of all known acetylation mechanisms (most notably AcP and YfiQ) and one deacetylase (CobB), overexpression of these putative KATs elicited unique patterns of protein acetylation. We mutated key active site residues and found that most of them eliminated enzymatic acetylation activity. We used mass spectrometry to identify and quantify the specificity of YfiQ and the four novel KATs. Surprisingly, our analysis revealed a high degree of substrate specificity. The overlap between KAT-dependent and AcP-dependent acetylation was extremely limited, supporting the hypothesis that these two acetylation mechanisms play distinct roles in the posttranslational modification of bacterial proteins. We further showed that these novel KATs are conserved across broad swaths of bacterial phylogeny. Finally, we determined that one of the novel KATs (YiaC) and the known KAT (YfiQ) can negatively regulate bacterial migration. Together, these results emphasize distinct and specific nonenzymatic and enzymatic protein acetylation mechanisms present in bacteria.IMPORTANCENε-Lysine acetylation is one of the most abundant and important posttranslational modifications across all domains of life. One of the best-studied effects of acetylation occurs in eukaryotes, where acetylation of histone tails activates gene transcription. Although bacteria do not have true histones, Nε-lysine acetylation is prevalent; however, the role of these modifications is mostly unknown. We constructed an E. coli strain that lacked both known acetylation mechanisms to identify four new Nε-lysine acetyltransferases (RimI, YiaC, YjaB, and PhnO). We used mass spectrometry to determine the substrate specificity of these acetyltransferases. Structural analysis of selected substrate proteins revealed site-specific preferences for enzymatic acetylation that had little overlap with the preferences of the previously reported acetyl-phosphate nonenzymatic acetylation mechanism. Finally, YiaC and YfiQ appear to regulate flagellum-based motility, a phenotype critical for pathogenesis of many organisms. These acetyltransferases are highly conserved and reveal deeper and more complex roles for bacterial posttranslational modification.

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media.

Complex media are routinely used to cultivate diverse bacteria. However, this complexity can obscure the factors that govern cell growth. While studying protein acetylation in buffered tryptone broth supplemented with glucose (TB7-glucose), we observed that Escherichia coli did not fully consume glucose prior to stationary phase. However, when we supplemented this medium with magnesium, the glucose was completely consumed during exponential growth, with concomitant increases in cell number and biomass but reduced cell size. Similar results were observed with other sugars and other peptide-based media, including lysogeny broth. Magnesium also limited cell growth for Vibrio fischeri and Bacillus subtilis in TB7-glucose. Finally, magnesium supplementation reduced protein acetylation. Based on these results, we conclude that growth in peptide-based media is magnesium limited. We further conclude that magnesium supplementation can be used to tune protein acetylation without genetic manipulation. These results have the potential to reduce potentially deleterious acetylated isoforms of recombinant proteins without negatively affecting cell growth.IMPORTANCE Bacteria are often grown in complex media. These media are thought to provide the nutrients necessary to grow bacteria to high cell densities. In this work, we found that peptide-based media containing a sugar are magnesium limited for bacterial growth. In particular, magnesium supplementation is necessary for the bacteria to use the sugar for cell growth. Interestingly, in the absence of magnesium supplementation, the bacteria still consume the sugar. However, rather than use it for cell growth, the bacteria instead use the sugar to acetylate lysines on proteins. As lysine acetylation may alter the activity of proteins, this work demonstrates how lysine acetylation can be tuned through magnesium supplementation. These findings may be useful for recombinant protein production, when acetylated isoforms are to be avoided. They also demonstrate how to increase bacterial growth in complex media.

Positioning ganglioside D3 as an immunotherapeutic target in lymphangioleiomyomatosis.

Tumors that develop in lymphangioleiomyomatosis (LAM) as a consequence of biallelic loss of TSC1 or TSC2 gene function express melanoma differentiation antigens. However, the percentage of LAM cells expressing these melanosomal antigens is limited. Here, we report the overexpression of ganglioside D3 (GD3) in LAM. GD3 is a tumor-associated antigen otherwise found in melanoma and neuroendocrine tumors; normal expression is largely restricted to neuronal cells in the brain. We also observed markedly reduced serum antibody titers to GD3, which may allow for a population of GD3-expressing LAM cells to expand within patients. This is supported by the demonstrated sensitivity of cultured LAM cells to complement mediated cytotoxicity via GD3 antibodies. GD3 can serve as a natural killer T (NKT) cell antigen when presented on CD1d molecules expressed on professional antigen-presenting cells. Although CD1d-expressing monocyte derivatives were present in situ, enhanced NKT-cell recruitment to LAM lung was not observed. Cultured LAM cells retained surface expression of GD3 over several passages and also expressed CD1d, implying that infiltrating NKT cells can be directly cytotoxic toward LAM lung lesions. Immunization with antibodies to GD3 may thus be therapeutic in LAM, and enhancement of existing NKT-cell infiltration may be effective to further improve antitumor responses. Overall, we hereby establish GD3 as a suitable target for immunotherapy of LAM.