Enhanced formation of shiga toxin-producing Escherichia coli persister variants in environments relevant to leafy greens production.

Bacterial persistence is a form of phenotypic heterogeneity in which a subpopulation, persisters, has high tolerance to antibiotics and other stresses. Persisters of enteric pathogens may represent the subpopulations capable of surviving harsh environments and causing human infections. Here we examined the persister populations of several shiga toxin-producing Escherichia coli (STEC) outbreak strains under conditions relevant to leafy greens production. The persister fraction of STEC in exponential-phase of culture varied greatly among the strains examined, ranging from 0.00003% to 0.0002% for O157:H7 strains to 0.06% and 0.08% for STEC O104:H4 strains. A much larger persister fraction (0.1-11.2%) was observed in STEC stationary cells grown in rich medium, which was comparable to the persister fractions in stationary cells grown in spinach lysates (0.6-3.6%). The highest persister fraction was measured in populations of cells incubated in field water (9.9-23.2%), in which no growth was detected for any of the STEC strains examined. Considering the high tolerance of persister cells to antimicrobial treatments and their ability to revert to normal cells, the presence of STEC persister cells in leafy greens production environments may pose a significant challenge in the development of effective control strategies to ensure the microbial safety of fresh vegetables.

Deciphering the Regulatory Circuitry That Controls Reversible Lysine Acetylation in Salmonella enterica.

UNLABELLED: In Salmonella enterica, the reversible lysine acetylation (RLA) system is comprised of the protein acetyltransferase (Pat) and sirtuin deacetylase (CobB). RLA controls the activities of many proteins, including the acetyl coenzyme A (acetyl-CoA) synthetase (Acs), by modulating the degree of Acs acetylation. We report that IolR, a myo-inositol catabolism repressor, activates the expression of genes encoding components of the RLA system. In vitro evidence shows that the IolR protein directly regulates pat expression. An iolR mutant strain displayed a growth defect in minimal medium containing 10 mM acetate, a condition under which RLA function is critical to control Acs activity. Increased levels of Pat, CobB, or Acs activity reversed the growth defect, suggesting the Pat/CobB ratio in an iolR strain is altered and that such a change affects the level of acetylated, inactive Acs. Results of quantitative reverse transcription-PCR (qRT-PCR) analyses of pat, cobB, and acs expression indicated that expression of the genes alluded to in the IolR-deficient strain was reduced 5-, 3-, and 2.6-fold, respectively, relative to the levels present in the strain carrying the iolR(+) allele. Acs activity in cell-free extracts from an iolR mutant strain was reduced ~25% relative to that of the iolR(+) strain. Glucose differentially regulated expression of pat, cobB, and acs. The catabolite repressor protein (Crp) positively regulated expression of pat while having no effect on cobB. IMPORTANCE: Reversible lysine acylation is used by cells of all domains of life to modulate the function of proteins involved in diverse cellular processes. Work reported herein begins to outline the regulatory circuitry that integrates the expression of genes encoding enzymes that control the activity of a central metabolic enzyme in C2 metabolism. Genetic analyses revealed effects on reversible lysine acylation that greatly impacted the growth behavior of the cell. This work provides the first insights into the complexities of the system responsible for controlling reversible lysine acylation at the transcriptional level in the enteropathogenic bacterium Salmonella enterica.

Candida albicans ethanol stimulates Pseudomonas aeruginosa WspR-controlled biofilm formation as part of a cyclic relationship involving phenazines.

In chronic infections, pathogens are often in the presence of other microbial species. For example, Pseudomonas aeruginosa is a common and detrimental lung pathogen in individuals with cystic fibrosis (CF) and co-infections with Candida albicans are common. Here, we show that P. aeruginosa biofilm formation and phenazine production were strongly influenced by ethanol produced by the fungus C. albicans. Ethanol stimulated phenotypes that are indicative of increased levels of cyclic-di-GMP (c-di-GMP), and levels of c-di-GMP were 2-fold higher in the presence of ethanol. Through a genetic screen, we found that the diguanylate cyclase WspR was required for ethanol stimulation of c-di-GMP. Multiple lines of evidence indicate that ethanol stimulates WspR signaling through its cognate sensor WspA, and promotes WspR-dependent activation of Pel exopolysaccharide production, which contributes to biofilm maturation. We also found that ethanol stimulation of WspR promoted P. aeruginosa colonization of CF airway epithelial cells. P. aeruginosa production of phenazines occurs both in the CF lung and in culture, and phenazines enhance ethanol production by C. albicans. Using a C. albicans adh1/adh1 mutant with decreased ethanol production, we found that fungal ethanol strongly altered the spectrum of P. aeruginosa phenazines in favor of those that are most effective against fungi. Thus, a feedback cycle comprised of ethanol and phenazines drives this polymicrobial interaction, and these relationships may provide insight into why co-infection with both P. aeruginosa and C. albicans has been associated with worse outcomes in cystic fibrosis.

A positive selection approach identifies residues important for folding of Salmonella enterica Pat, an N(ε)-lysine acetyltransferase that regulates central metabolism enzymes.

In Salmonella enterica, the protein acetyltransferase (Pat) enzyme is part of the sirtuin-dependent acylation/deacylation system (SDPADS) that modulates the activity of several proteins via the acylation of lysine residues critical to their activities. Pat is a ~98 kDa protein with two distinct domains, an N-terminal acyl-CoA synthetase (NDP-forming) domain (~700 aa) and a C-terminal acetyltransferase domain (~160 aa), with homology to proteins of the Gcn5-related N-acetyltransferase (GNAT) superfamily. Although the role of the GNAT-like domain is likely responsible for the catalytic activity of Pat, the role of the N-terminal domain remains unclear. Here we report the use of positive selection for identification of residues critical for Pat enzyme activity. This approach revealed seven residues that, when changed, resulted in drastic loss of Pat activity in vitro which caused a discernable loss-of-function phenotype. Five of the seven residues were located in the N-terminal region of Pat and two were located in the GNAT-like domain. Each single-amino-acid variant had a circular dichroism spectrum that differed from that of the wild-type Pat protein, suggesting that loss of enzymatic activity in the mutant proteins was likely due to an inability to acquire its biologically active fold.

Biochemical and thermodynamic analyses of Salmonella enterica Pat, a multidomain, multimeric N(ε)-lysine acetyltransferase involved in carbon and energy metabolism.

In the bacterium Salmonella enterica, the CobB sirtuin protein deacetylase and the Gcn5-related N(ε)-acetyltransferase (GNAT) Pat control carbon utilization and metabolic flux via N(ε)-lysine acetylation/deacetylation of metabolic enzymes. To date, the S. enterica Pat (SePat) acetyltransferase has not been biochemically characterized. Here we report the kinetic and thermodynamic characterization of the SePat enzyme using two of its substrates, acetyl coenzyme A (Ac-CoA) synthetase (Acs; AMP forming, EC 6.2.1.1) and Ac-CoA. The data showed typical Michaelis-Menten kinetic behavior when Ac-CoA was held at a saturating concentration while Acs was varied, and a sigmoidal kinetic behavior was observed when Acs was saturating and the Ac-CoA concentration was varied. The observation of sigmoidal kinetics and positive cooperativity for Ac-CoA is an unusual feature of GNATs. Results of isothermal titration calorimetry (ITC) experiments showed that binding of Ac-CoA to wild-type SePat produced a biphasic curve having thermodynamic properties consistent with two distinct sites. Biphasicity was not observed in ITC experiments that analyzed the binding of Ac-CoA to a C-terminal construct of SePat encompassing the predicted core acetyltransferase domain. Subsequent analytical gel filtration chromatography studies showed that in the presence of Ac-CoA, SePat oligomerized to a tetrameric form, whereas in the absence of Ac-CoA, SePat behaved as a monomer. The positive modulation of SePat activity by Ac-CoA, a product of the Acs enzyme that also serves as a substrate for SePat-dependent acetylation, is likely a layer of metabolic control. IMPORTANCE For decades, N(ε)-lysine acetylation has been a well-studied mode of regulation of diverse proteins involved in almost all aspects of eukaryotic physiology. Until recently, N(ε)-lysine acetylation was not considered a widespread phenomenon in bacteria. Recent studies have indicated that N(ε)-lysine acetylation and its impact on cellular metabolism may be just as diverse in bacteria as they are in eukaryotes. The S. enterica Pat enzyme, specifically, has recently been implicated in the modulation of many metabolic enzymes. Understanding the molecular mechanisms of how this enzyme controls the activity of diverse enzymes by N(ε)-lysine acetylation will advance our understanding of how the prokaryotic cell responds to its changing environment in order to meet its metabolic needs.

Control of protein function by reversible Nɛ-lysine acetylation in bacteria.

Recently published work indicates that reversible N(ɛ)-lysine (N(ɛ)-Lys) acetylation of proteins in bacteria may be as diverse, and as important for cellular function, as it has been reported in eukaryotes for the last five decades. In addition to biochemical and genetic approaches, proteomic studies have identified N(ɛ)-Lys acetylation of proteins and enzymes involved in diverse cellular activities such as transcription, translation, stress response, detoxification, and especially carbohydrate and energy metabolism. These findings provide a platform for elucidating the molecular mechanisms behind modulation of enzyme activity by N(ɛ)-Lys acetylation, as well as for understanding how the prokaryotic cell maintains homeostasis in a changing environment.

Nε-lysine acetylation of a bacterial transcription factor inhibits Its DNA-binding activity.

Evidence suggesting that eukaryotes and archaea use reversible N(ε)-lysine (N(ε)-Lys) acetylation to modulate gene expression has been reported, but evidence for bacterial use of N(ε)-Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs). We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat). Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD(+)-dependent Sir2 (sirtuin)-like protein deacetylase (CobB) deacetylated acetylated RcsB (RcsB(Ac)), demonstrating that N(ε)-Lys acetylation of RcsB is reversible. Analysis of RcsB(Ac) and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible N(ε)-Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells.

Production in two-liter beverage bottles of proteins for NMR structure determination labeled with either 15N- or 13C-15N.

The use of 2-L polyethylene terephthalate beverage bottles as a bacterial culture vessel has been recently introduced as an enabling technology for high-throughput structural biology [Sanville Millard, C. et al., 2003. Protein Express. Purif. 29, 311-320]. In the article following this one [Stols et al., this issue, pp. 95-102], this approach was elaborated for selenomethionine labeling used for multiwavelength anomalous dispersion phasing in the X-ray crystallographic determinations of protein structure. Herein, we report an effective and reproducible schedule for uniform 15N- and 13C-labeling of recombinant proteins in 2-L beverage bottles for structural determination by NMR spectroscopy. As an example, three target proteins selected from Arabidopsis thaliana were expressed in Escherichia coli Rosetta (DE3)/pLysS from a T7-based expression vector, purified, and characterized by electrospray ionization mass spectrometry and NMR analysis by 1H-15N heteronuclear single quantum correlation spectroscopy. The results show that expressions in the unlabeled medium provide a suitable control for estimation of the level of production of the labeled protein. Mass spectral characterizations show that the purified proteins contained a level of isotopic incorporation equivalent to the isotopically labeled materials initially present in the growth medium, while NMR analysis of the [U-15N]-labeled proteins provided a convenient method to assess the solution state properties of the target protein prior to production of a more costly double-labeled sample.

Results from high-throughput DNA cloning of Arabidopsis thaliana target genes using site-specific recombination.

The Center for Eukaryotic Structural Genomics (CESG) was founded as a collaborative effort to develop technologies for the rapid and economic determination of protein three-dimensional structures. The initial focus was on the genome of the model plant Arabidopsis thaliana. Protocols for high-throughput cloning of Arabidopsis open reading frames into Escherichia coli expression vectors are presented along with an analysis of results from approximately 2000 cloning experiments. Open reading frames were chosen on the likelihood that they would represent important unknown regions of protein conformation and fold space or that they would elucidate novel fold-function relationships. The chosen open reading frames were amplified from a cDNA pool created by reverse transcription of RNA isolated from an Arabidopsis callus culture. A novel Gateway protocol was developed to insert the amplified open reading frames into an entry vector for storage and sequence determination. Sequence verified entry clones were then used to create expression vectors again via the Gateway system.