Computationally designed pyocyanin demethylase acts synergistically with tobramycin to kill recalcitrant Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa is an opportunistic human pathogen that develops difficult-to-treat biofilms in immunocompromised individuals, cystic fibrosis patients, and in chronic wounds. P. aeruginosa has an arsenal of physiological attributes that enable it to evade standard antibiotic treatments, particularly in the context of biofilms where it grows slowly and becomes tolerant to many drugs. One of its survival strategies involves the production of the redox-active phenazine, pyocyanin, which promotes biofilm development. We previously identified an enzyme, PodA, that demethylated pyocyanin and disrupted P. aeruginosa biofilm development in vitro. Here, we asked if this protein could be used as a potential therapeutic for P. aeruginosa infections together with tobramycin, an antibiotic typically used in the clinic. A major roadblock to answering this question was the poor yield and stability of wild-type PodA purified from standard Escherichia coli overexpression systems. We hypothesized that the insufficient yields were due to poor packing within PodA's obligatory homotrimeric interfaces. We therefore applied the protein design algorithm, AffiLib, to optimize the symmetric core of this interface, resulting in a design that incorporated five mutations leading to a 20-fold increase in protein yield from heterologous expression and purification and a substantial increase in stability to environmental conditions. The addition of the designed PodA with tobramycin led to increased killing of P. aeruginosa cultures under oxic and hypoxic conditions in both the planktonic and biofilm states. This study highlights the potential for targeting extracellular metabolites to assist the control of P. aeruginosa biofilms that tolerate conventional antibiotic treatment.

New AMP-forming acid:CoA ligases from Streptomyces lividans, some of which are posttranslationally regulated by reversible lysine acetylation.

In nature, organic acids are a commonly used source of carbon and energy. Many bacteria use AMP-forming acid:CoA ligases to convert organic acids into their corresponding acyl-CoA derivatives, which can then enter metabolism. The soil environment contains a broad diversity of organic acids, so it is not surprising that bacteria such as Streptomyces lividans can activate many of the available organic acids. Our group has shown that the activity of many acid:CoA ligases is posttranslationally controlled by acylation of an active-site lysine. In some cases, the modification is reversed by deacylases of different types. We identified eight new acid:CoA ligases in S. lividans TK24. Here, we report the range of organic acids that each of these enzymes can activate, and determined that two of the newly identified CoA ligases were under NAD+ -dependent sirtuin deacylase reversible lysine (de)acetylation control, four were not acetylated by two acetyltransferases used in this work, and two were acetylated but not deacetylated by sirtuin. This work provides insights into the broad organic-acid metabolic capabilities of S. lividans, and sheds light into the control of the activities of CoA ligases involved in the activation of organic acids in this bacterium.

Protein Acetylation in Bacteria.

Acetylation is a posttranslational modification conserved in all domains of life that is carried out by N-acetyltransferases. While acetylation can occur on Nα-amino groups, this review will focus on Nε-acetylation of lysyl residues and how the posttranslational modification changes the cellular physiology of bacteria. Up until the late 1990s, acetylation was studied in eukaryotes in the context of chromatin maintenance and gene expression. At present, bacterial protein acetylation plays a prominent role in central and secondary metabolism, virulence, transcription, and translation. Given the diversity of niches in the microbial world, it is not surprising that the targets of bacterial protein acetyltransferases are very diverse, making their biochemical characterization challenging. The paradigm for acetylation in bacteria involves the acetylation of acetyl-CoA synthetase, whose activity must be tightly regulated to maintain energy charge homeostasis. While this paradigm has provided much mechanistic detail for acetylation and deacetylation, in this review we discuss advances in the field that are changing our understanding of the physiological role of protein acetylation in bacteria.

In Salmonella enterica, OatA (Formerly YjgM) Uses O-Acetyl-Serine and Acetyl-CoA to Synthesize N,O-Diacetylserine, Which Upregulates Cysteine Biosynthesis.

L-Cysteine biosynthesis has been extensively analyzed in Salmonella enterica. The cysteine regulon contains the genes whose protein products are necessary to convert sulfate to sulfide, which is eventually reacted with O-acetyl-serine (OAS) to generate cysteine. The LysR type regulator, CysB, is required for activation of the cysteine regulon, and its interaction with various cys genes has been thoroughly characterized. Results from previous studies by others, suggested that OAS undergoes a spontaneous O- to N- migration to produce N-acetyl-serine (NAS), and that NAS is the true signal sensed by CysB. It was unclear, however, whether such migration occurred spontaneously in vivo or if NAS was generated enzymatically. Work reported herein characterizes a S. enterica N-acetyltransferase, OatA (formerly YjgM), which acetylates the N α-amino group of OAS, producing N,O-diacetyl-serine (DAS) at the expense of acetyl-CoA. We isolated OatA to homogeneity and performed its initial biochemical characterization. The product of the OatA reaction was isolated by HPLC and confirmed by mass spectrometry to be DAS; OatA did not acetylate NAS, consistent with the conclusion that OatA is an N-acetyltransferase, not an O-acetyltransferase. Binding of OAS to OatA appears to be positively cooperative with the apparent K0.5 for OAS determined to be 0.74 mM, the kcat was 1.05 s-1, and the catalytic efficiency of the enzyme (k cat/K0.5 ) was 1.4 × 103 M-1 s-1. Size exclusion chromatography indicated that OatA was a monomer in solution. In S. enterica, overexpression of oatA led to shorter lag times on sulfate-limiting medium and that these delayed lag times were due to increased expression of the cysteine regulon, as indicated by RT-qPCR results. OatA is the first Gcn5-related N-acetyltransferase (aka GNAT) involved in the regulation of amino acid biosynthetic genes in Salmonella. On the basis of results of transcriptomics studies performed by other investigators, we hypothesize that DAS may play a role in biofilm formation in S. enterica and other bacteria.

Small-Molecule Acetylation Controls the Degradation of Benzoate and Photosynthesis in Rhodopseudomonas palustris.

The degradation of lignin-derived aromatic compounds such as benzoate has been extensively studied in Rhodopseudomonas palustris, and the chemistry underpinning the conversion of benzoate to acetyl coenzyme A (acetyl-CoA) is well understood. Here we characterize the last unknown gene, badL, of the bad (benzoic acid degradation) cluster. BadL function is required for growth under photoheterotrophic conditions with benzoate as the organic carbon source (i.e., light plus anoxia). On the basis of bioinformatics and in vivo and in vitro data, we show that BadL, a Gcn5-related N-acetyltransferase (GNAT) (PF00583), acetylates aminobenzoates to yield acetamidobenzoates. The latter relieved repression of the badDEFGAB operon by binding to BadM, triggering the synthesis of enzymes that activate and dearomatize the benzene ring. We also show that acetamidobenzoates are required for the expression of genes encoding the photosynthetic reaction center light-harvesting complexes through a BadM-independent mechanism. The effect of acetamidobenzoates on pigment synthesis is new and different than their effect on the catabolism of benzoate.IMPORTANCE This work shows that the BadL protein of Rhodopseudomonas palustris has N-acetyltransferase activity and that this activity is required for the catabolism of benzoate under photosynthetic conditions in this bacterium. R. palustris occupies lignin-rich habitats, making its benzoate-degrading capability critical for the recycling of this important, energy-rich biopolymer. This work identifies the product of the BadL enzyme as acetamidobenzoates, which were needed to derepress genes encoding benzoate-degrading enzymes and proteins of the photosynthetic apparatus responsible for the generation of the proton motive force under anoxia in the presence of light. In short, acetamidobenzoates potentially coordinate the use of benzoate as a source of reducing power and carbon with the generation of a light-driven proton motive force that fuels ATP synthesis, motility, transport, and many other processes in the metabolically versatile bacterium R. palustris.

Rhodobacterales use a unique L-threonine kinase for the assembly of the nucleotide loop of coenzyme B12.

Several of the enzymes involved in the conversion of adenosylcobyric acid (AdoCby) to adenosylcobamide (AdoCba) are yet to be identified and characterized in some cobamide (Cba)-producing prokaryotes. Using a bioinformatics approach, we identified the bluE gene (locus tag RSP_0788) of Rhodobacter sphaeroides 2.4.1 as a putative functional homolog of the L-threonine kinase enzyme (PduX, EC 2.7.1.177) of S. enterica. In AdoCba, (R)-1-aminopropan-2-ol O-phosphate (AP-P) links the nucleotide loop to the corrin ring; most known AdoCba producers derive AP-P from L-Thr-O-3-phosphate (L-Thr-P). Here, we show that RsBluE has L-Thr-independent ATPase activity in vivo and in vitro. We used 31 P-NMR spectroscopy to show that RsBluE generates L-Thr-P at the expense of ATP and is unable to use L-Ser as a substrate. BluE from R. sphaeroides or Rhodobacter capsulatus restored AdoCba biosynthesis in S. enterica ΕpduX and R. sphaeroides ΕbluE mutant strains. R. sphaeroides ΕbluE strains exhibited a decreased pigment phenotype that was restored by complementation with BluE. Finally, phylogenetic analyses revealed that bluE was restricted to the genomes of a few Rhodobacterales that appear to have a preference for a specific form of Cba, namely Coá´½-(á´½-5,6-dimethylbenzimidazolyl-Coᵦ-adenosylcobamide (a.k.a. adenosylcobalamin, AdoCbl; coenzyme B12 , CoB12 ).

In Streptomyces lividans, acetyl-CoA synthetase activity is controlled by O-serine and Nɛ -lysine acetylation.

Protein acetylation is a rapid mechanism for control of protein function. Acetyl-CoA synthetase (AMP-forming, Acs) is the paradigm for the control of metabolic enzymes by lysine acetylation. In many bacteria, type I or II protein acetyltransferases acetylate Acs, however, in actinomycetes type III protein acetyltransferases control the activity of Acs. We measured changes in the activity of the Streptomyces lividans Acs (SlAcs) enzyme upon acetylation by PatB using in vitro and in vivo analyses. In addition to the acetylation of residue K610, residue S608 within the acetylation motif of SlAcs was also acetylated (PKTRSGK610 ). S608 acetylation rendered SlAcs inactive and non-acetylatable by PatB. It is unclear whether acetylation of S608 is enzymatic, but it was clear that this modification occurred in vivo in Streptomyces. In S. lividans, an NAD+ -dependent sirtuin deacetylase from Streptomyces, SrtA (a homologue of the human SIRT4 protein) was needed to maintain SlAcs function in vivo. We have characterized a sirtuin-dependent reversible lysine acetylation system in Streptomyces lividans that targets and controls the Acs enzyme of this bacterium. These studies raise questions about acetyltransferase specificity, and describe the first Acs enzyme in any organism whose activity is modulated by O-Ser and Nɛ -Lys acetylation.

A Toxin Involved in Salmonella Persistence Regulates Its Activity by Acetylating Its Cognate Antitoxin, a Modification Reversed by CobB Sirtuin Deacetylase.

Bacterial toxin-antitoxin systems trigger the onset of a persister state by inhibiting essential cellular processes. The TacT toxin of Salmonella enterica is known to induce a persister state in macrophages through the acetylation of aminoacyl-tRNAs. Here, we show that the TacT toxin and the TacA antitoxin work as a complex that modulates TacT activity via the acetylation state of TacA. TacT acetylates TacA at residue K44, a modification that is removed by the NAD+-dependent CobB sirtuin deacetylase. TacA acetylation increases the activity of TacT, downregulating protein synthesis. TacA acetylation altered binding to its own promoter, although this did not change tacAT expression levels. These claims are supported by results from in vitro protein synthesis experiments used to monitor TacT activity, in vivo growth analyses, electrophoretic mobility shift assays, and quantitative reverse transcription-PCR (RT-qPCR) analysis. TacT is the first example of a Gcn5-related N-acetyltransferase that modifies nonprotein and protein substrates.IMPORTANCE During host infection, pathogenic bacteria can modulate their physiology to evade host defenses. Some pathogens use toxin-antitoxin systems to modulate a state of self-toxicity that can decrease their cellular activity, triggering the onset of a persister state. The lower metabolic activity of persister cells allows them to escape host defenses and antibiotic treatments. Hence a better understanding of the mechanisms used by pathogens to ingress and egress the persister state is of relevance to human health.

Phosphinothricin Acetyltransferases Identified Using In Vivo, In Vitro, and Bioinformatic Analyses.

Acetylation of small molecules is widespread in nature, and in some cases, cells use this process to detoxify harmful chemicals. Streptomyces species utilize a Gcn5 N-acetyltransferase (GNAT), known as Bar, to acetylate and detoxify a self-produced toxin, phosphinothricin (PPT), a glutamate analogue. Bar homologues, such as MddA from Salmonella enterica, acetylate methionine analogues such as methionine sulfoximine (MSX) and methionine sulfone (MSO), but not PPT, even though Bar homologues are annotated as PPT acetyltransferases. S. enterica was used as a heterologous host to determine whether or not putative PPT acetyltransferases from various sources could acetylate PPT, MSX, and MSO. In vitro and in vivo analyses identified substrates acetylated by putative PPT acetyltransferases from Deinococcus radiodurans (DR_1057 and DR_1182) and Geobacillus kaustophilus (GK0593 and GK2920). In vivo, synthesis of DR_1182, GK0593, and GK2920 blocked the inhibitory effects of PPT, MSX, and MSO. In contrast, DR_1057 did not detoxify any of the above substrates. Results of in vitro studies were consistent with the in vivo results. In addition, phylogenetic analyses were used to predict the functionality of annotated PPT acetyltransferases in Burkholderia xenovorans, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baylyi, and Escherichia coli IMPORTANCE: The work reported here provides an example of the use of a heterologous system for the identification of enzyme function. Many members of this superfamily of proteins do not have a known function, or it has been annotated solely on the basis of sequence homology to previously characterized enzymes. The critical role of Gcn5 N-acetyltransferases (GNATs) in the modulation of central metabolic processes, and in controlling metabolic stress, necessitates approaches that can reveal their physiological role. The combination of in vivo, in vitro, and bioinformatics approaches reported here identified GNATs that can acetylate and detoxify phosphinothricin.

Formate Metabolism in Shewanella oneidensis Generates Proton Motive Force and Prevents Growth without an Electron Acceptor.

UNLABELLED: Shewanella oneidensis strain MR-1 is a facultative anaerobe that thrives in redox-stratified environments due to its ability to utilize a wide array of terminal electron acceptors. Conversely, the electron donors utilized by S. oneidensis are more limited and include products of primary fermentation such as lactate, pyruvate, formate, and hydrogen. Lactate, pyruvate, and hydrogen metabolisms inS. oneidensis have been described previously, but little is known about the role of formate oxidation in the ecophysiology of these bacteria. Formate is produced by S. oneidensis through pyruvate formate lyase during anaerobic growth on carbon sources that enter metabolism at or above the level of pyruvate, and the genome contains three gene clusters predicted to encode three complete formate dehydrogenase complexes. To determine the contribution of each complex to formate metabolism, strains lacking one, two, or all three annotated formate dehydrogenase gene clusters were generated and examined for growth rates and yields on a variety of carbon sources. Here, we report that formate oxidation contributes to both the growth rate and yield of S. oneidensis through the generation of proton motive force. Exogenous formate also greatly accelerated growth on N-acetylglucosamine, a carbon source normally utilized very slowly by S. oneidensis under anaerobic conditions. Surprisingly, deletion of all three formate dehydrogenase gene clusters enabled growth of S. oneidensis using pyruvate in the absence of a terminal electron acceptor, a mode of growth never before observed in these bacteria. Our results demonstrate that formate oxidation is a fundamental strategy under anaerobic conditions for energy conservation inS. oneidensis. IMPORTANCE: Shewanella species have garnered interest in biotechnology applications for their ability to respire extracellular terminal electron acceptors, such as insoluble iron oxides and electrodes. While much effort has gone into studying the proteins for extracellular electron transport, how electrons generated through the oxidation of organic carbon sources enter this pathway remains understudied. Here, we quantify the role of formate oxidation in the anaerobic physiology of Shewanella oneidensis Formate oxidation contributes to both the growth rate and yield on a variety of carbon sources through the generation of proton motive force. Advances in our understanding of the anaerobic metabolism of S. oneidensis are important for our ability to utilize and engineer this organism for applications in bioenergy, biocatalysis, and bioremediation.