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.

Control of type III protein secretion using a minimal genetic system.

Gram-negative bacteria secrete proteins using a type III secretion system (T3SS), which functions as a needle-like molecular machine. The many proteins involved in T3SS construction are tightly regulated due to its role in pathogenesis and motility. Here, starting with the 35 kb Salmonella pathogenicity island 1 (SPI-1), we eliminated internal regulation and simplified the genetics by removing or recoding genes, scrambling gene order and replacing all non-coding DNA with synthetic genetic parts. This process results in a 16 kb cluster that shares no sequence identity, regulation or organizational principles with SPI-1. Building this simplified system led to the discovery of essential roles for an internal start site (SpaO) and small RNA (InvR). Further, it can be controlled using synthetic regulatory circuits, including under SPI-1 repressing conditions. This work reveals an incredible post-transcriptional robustness in T3SS assembly and aids its control as a tool in biotechnology.

Programming a Human Commensal Bacterium, Bacteroides thetaiotaomicron, to Sense and Respond to Stimuli in the Murine Gut Microbiota.

Engineering commensal organisms for challenging applications, such as modulating the gut ecosystem, is hampered by the lack of genetic parts. Here, we describe promoters, ribosome-binding sites, and inducible systems for use in the commensal bacterium Bacteroides thetaiotaomicron, a prevalent and stable resident of the human gut. We achieve up to 10,000-fold range in constitutive gene expression and 100-fold regulation of gene expression with inducible promoters and use these parts to record DNA-encoded memory in the genome. We use CRISPR interference (CRISPRi) for regulated knockdown of recombinant and endogenous gene expression to alter the metabolic capacity of B. thetaiotaomicron and its resistance to antimicrobial peptides. Finally, we show that inducible CRISPRi and recombinase systems can function in B. thetaiotaomicron colonizing the mouse gut. These results provide a blueprint for engineering new chassis and a resource to engineer Bacteroides for surveillance of or therapeutic delivery to the gut microbiome.

The structure of S. lividans acetoacetyl-CoA synthetase shows a novel interaction between the C-terminal extension and the N-terminal domain.

The adenosine monoposphate-forming acyl-CoA synthetase enzymes catalyze a two-step reaction that involves the initial formation of an acyl adenylate that reacts in a second partial reaction to form a thioester between the acyl substrate and CoA. These enzymes utilize a Domain Alternation catalytic mechanism, whereby a ∼ 110 residue C-terminal domain rotates by 140° to form distinct catalytic conformations for the two partial reactions. The structure of an acetoacetyl-CoA synthetase (AacS) is presented that illustrates a novel aspect of this C-terminal domain. Specifically, several acetyl- and acetoacetyl-CoA synthetases contain a 30-residue extension on the C-terminus compared to other members of this family. Whereas residues from this extension are disordered in prior structures, the AacS structure shows that residues from this extension may interact with key catalytic residues from the N-terminal domain.

Insights into the specificity of lysine acetyltransferases.

Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. Here we report the structure of a GNAT in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs.

Determinants within the C-terminal domain of Streptomyces lividans acetyl-CoA synthetase that block acetylation of its active site lysine in vitro by the protein acetyltransferase (Pat) enzyme.

Reversible lysine acetylation (RLA) is a widespread regulatory mechanism that modulates the function of proteins involved in diverse cellular processes. A strong case has been made for RLA control exerted by homologues of the Salmonella enterica protein acetyltransferase (SePat) enzyme on the broadly distributed AMP-forming CoA ligase (a.k.a. acyl-CoA synthetases) family of metabolic enzymes, with acetyl-CoA synthetase (Acs) being the paradigm in the field. Here we investigate why the Acs homologue in Streptomyces lividans (SlAcs) is poorly acetylated in vitro by the S. lividans protein acetyltransferase (SlPat) enzyme. Chimeras of S. enterica Acs (SeAcs) and S. lividans Acs (SlAcs) constructed during the course of this work were acetylated by SlPatA in vitro, retained most of their activity, and were under RLA control in a heterologous host. We identified SeAcs residues N- and C-terminal to the target lysine that when introduced into SlAcs, rendered the latter under RLA control. These results lend further support to the idea that Pat enzymes interact with extensive surfaces of their substrates. Finally, we suggest that acetylation of SlAcs depends on factors or conditions other than those present in our in vitro system. We also discuss possible explanations why SlAcs is not controlled by RLA as defined in other bacterial species.

Acetoacetyl-CoA synthetase activity is controlled by a protein acetyltransferase with unique domain organization in Streptomyces lividans.

GCN5-type N-acetyltransferases (GNATs) are enzymes that catalyse the transfer of the acetyl group from acetyl-CoA to a primary amine. GNATs are conserved in all domains of life. Some members of this family of enzymes acetylate the side-chain of specific lysine residues in proteins of diverse function. In bacteria, GNAT-catalysed protein acetylation regulates carbon metabolism, RNA metabolism and transcriptional regulation. Metabolic regulation in Streptomyces species is of interest due to the role of these organisms in natural product synthesis. Here we identify SlPatA, a GNAT in Streptomyces lividans with unique domain organization, and a new acetylation target, namely acetoacetyl-CoA synthetase (SlAacS). The latter has homologues in all domains of life. In vitro and in vivo evidence show that SlAacS is a bona fide acetoacetyl-CoA synthetase. SlPatA acetylates SlAacS more efficiently than it does acetyl-CoA synthetase, an enzyme known to be under acetylation control. SlPatA acetylates SlAacS at the active-site residue Lys617 and acetylation inactivates SlAacS. Acetylated SlAacS was deacetylated by a sirtuin-type protein deacetylase. SlAacS acetylation/deacetylation may represent a conserved mechanism for regulation of acetoacetyl-CoA synthetase activity in all domains of life.

PanM, an acetyl-coenzyme A sensor required for maturation of L-aspartate decarboxylase (PanD).

UNLABELLED: Coenzyme A (CoA) is essential for cellular chemistry in all forms of life. The pantothenate moiety of CoA is generated from the condensation of pantoate and ß-alanine. ß-Alanine is formed by decarboxylation of l-aspartate catalyzed by PanD, a pyruvoyl enzyme that is synthesized by the cell as an inactive precursor (pro-PanD). Maturation of pro-PanD into PanD occurs via a self-cleavage event at residue Ser25, which forms the catalytic pyruvoyl moiety. We recently reported that Salmonella enterica PanM was necessary for pro-PanD maturation, both in vitro and in vivo. Notably, PanM is annotated as a Gcn5-like N-acetyltransferase (GNAT), which suggested that lysine acetylation might be part of the mechanism of maturation. Here we show that PanM lacks acetyltransferase activity and that acetyl-CoA stimulates its activity. Results of experiments with nonhydrolyzable ethyl-CoA and genetically encoded acetyl-lysine-containing PanD support the conclusion that PanM-dependent pro-PanD maturation does not involve an acetyl transfer event. We also show that CoA binding to PanM is needed for in vivo activity and that disruption of CoA binding prevents PanM from interacting with PanD. We conclude that PanM is a GNAT homologue that lost its acetyltransferase activity and evolved a new function as an acetyl-CoA sensor that can trigger the maturation of pro-PanD. IMPORTANCE: Nε-lysine acetylation is increasingly being recognized as a widespread and important form of posttranslational regulation in bacteria. The acetyltransferases that catalyze these reactions are poorly characterized in bacteria. Based on annotation, most bacterial genomes contain several acetyltransferases, but the physiological roles of only a handful have been determined. Notably, a subset of putative acetyltransferases lack residues that are critical for activity in most biochemically characterized acetyltransferases. We show that one such putative acetyltransferase, PanM (formerly YhhK), lacks acetyltransferase activity but functions instead as an acetyl-coenzyme A (CoA) sensor. This work establishes the possibility that, like PanM, other putative acetyltransferases may have evolved new functions while retaining the ability to sense acetyl-CoA.

Biologically active isoforms of CobB sirtuin deacetylase in Salmonella enterica and Erwinia amylovora.

Sirtuins are NAD(+)-dependent protein deacylases that are conserved in all domains of life and are involved in diverse cellular processes, including control of gene expression and central metabolism. Eukaryotic sirtuins have N-terminal extensions that have been linked to protein multimerization and cellular localization. Here the first evidence of sirtuin isoforms in bacteria is reported. The enterobacterium Salmonella enterica synthesizes two isoforms of CobB sirtuin, a shorter 236-amino-acid isoform (here CobB(S)) and a longer 273-amino-acid isoform (here CobB(L)). The N-terminal 37-amino-acid extension of CobB(L) is amphipathic, containing 18 basic amino acids (12 of which are Arg) and 13 hydrophobic ones; both isoforms were active in vivo and in vitro. Northern blot and transcription start site analyses revealed that cobB is primarily expressed as two monocistronic cobB mRNAs from two transcription start sites, one of which was mapped within the neighboring ycfX gene and the other of which was located within cobB. Additionally, a low-abundance ycfX-cobB bicistronic mRNA was observed which could encode up to three proteins (YcfX, CobB(L), and CobB(S)). CobB(L) isoforms are common within the family Enterobacteriaceae, but species of the genus Erwinia (including the plant pathogen Erwinia amylovora) encode only the CobB(L) isoform. The CobB(L) isoform from E. amylovora restored growth of as S. enterica cobB mutant strain on low acetate.