Jeffrey G. Gardner

N-Lysine Propionylation Controls the Activity of Propionyl-CoA Synthetase

Reversible protein acetylation is a ubiquitous means for the rapid control of diverse cellular processes. Acetyltransferase enzymes transfer the acetyl group from acetyl-CoA to lysine residues, while deacetylase enzymes catalyze removal of the acetyl group by hydrolysis or by an NAD+-dependent reaction. Propionyl-coenzyme A (CoA), like acetyl-CoA, is a high energy product of fatty acid metabolism and is produced through a similar chemical reaction. Because acetyl-CoA is the donor molecule for protein acetylation, we investigated whether proteins can be propionylated in vivo, using propionyl-CoA as the donor molecule. We report that the Salmonella enterica propionyl-CoA synthetase enzyme PrpE is propionylated in vivo at lysine 592; propionylation inactivates PrpE. The propionyl-lysine modification is introduced by bacterial Gcn-5-related N-acetyltransferase enzymes and can be removed by bacterial and human Sir2 enzymes (sirtuins). Like the sirtuin deacetylation reaction, sirtuin-catalyzed depropionylation is NAD+-dependent and produces a byproduct, O-propionyl ADP-ribose, analogous to the O-acetyl ADP-ribose sirtuin product of deacetylation. Only a subset of the human sirtuins with deacetylase activity could also depropionylate substrate. The regulation of cellular propionyl-CoA by propionylation of PrpE parallels regulation of acetyl-CoA by acetylation of acetyl-CoA synthetase and raises the possibility that propionylation may serve as a regulatory modification in higher organisms.

Control of Acetyl-Coenzyme A Synthetase (AcsA) Activity by Acetylation/Deacetylation without NAD+ Involvement in Bacillus subtilis

Posttranslational modification is an efficient mechanism for controlling the activity of structural proteins, gene expression regulators, and enzymes in response to rapidly changing physiological conditions. Here we report in vitro and in vivo evidence that the acuABC operon of the gram-positive soil bacterium Bacillus subtilis encodes a protein acetyltransferase (AcuA) and a protein deacetylase (AcuC), which may control the activity of acetyl-coenzyme A (CoA) synthetase (AMP-forming, AcsA) in this bacterium. Results from in vitro experiments using purified proteins show that AcsA is a substrate for the acetyl-CoA-dependent AcuA acetyltransferase. Mass spectrometry analysis of a tryptic digest of acetylated AcsA (AcsA(Ac)) identified residue Lys549 as the sole modification site in the protein. Unlike sirtuins, the AcuC protein did not require NAD(+) as cosubstrate to deacetylate AcsA(Ac). The function of the putative AcuB protein remains unknown.

In Bacillus subtilis, the Sirtuin Protein Deacetylase, Encoded by the srtN Gene (Formerly yhdZ), and Functions Encoded by the acuABC Genes Control the Activity of Acetyl Coenzyme A Synthetase

This report provides in vivo evidence for the posttranslational control of the acetyl coenzyme A (Ac-CoA) synthetase (AcsA) enzyme of Bacillus subtilis by the acuA and acuC gene products. In addition, both in vivo and in vitro data presented support the conclusion that the yhdZ gene of B. subtilis encodes a NAD(+)-dependent protein deacetylase homologous to the yeast Sir2 protein (also known as sirtuin). On the basis of this new information, a change in gene nomenclature, from yhdZ to srtN (for sirtuin), is proposed to reflect the activity associated with the YdhZ protein. In vivo control of B. subtilis AcsA function required the combined activities of AcuC and SrtN. Inactivation of acuC or srtN resulted in slower growth and cell yield under low-acetate conditions than those of the wild-type strain, and the acuC srtN strain grew under low-acetate conditions as poorly as the acsA strain. Our interpretation of the latter result was that both deacetylases (AcuC and SrtN) are needed to maintain AcsA as active (i.e., deacetylated) so the cell can grow with low concentrations of acetate. Growth of an acuA acuC srtN strain on acetate was improved over that of the acuA(+) acuC srtN strain, indicating that the AcuA acetyltransferase enzyme modifies (i.e., inactivates) AcsA in vivo, a result consistent with previously reported in vitro evidence that AcsA is a substrate of AcuA.

Requirement of the Type II Secretion System for Utilization of Cellulosic Substrates by Cellvibrio japonicus

ABSTRACT Cellulosic biofuels represent a powerful alternative to petroleum but are currently limited by the inefficiencies of the conversion process. While Gram-positive and fungal organisms have been widely explored as sources of cellulases and hemicellulases for biomass degradation, Gram-negative organisms have received less experimental attention. We investigated the ability of Cellvibrio japonicus, a recently sequenced Gram-negative cellulolytic bacterium, to degrade bioenergy-related feedstocks. Using a newly developed biomass medium, we showed that C. japonicus is able to utilize corn stover and switchgrass as sole sources of carbon and energy for growth. We also developed tools for directed gene disruptions in C. japonicus and used this system to construct a mutant in the gspD gene, which is predicted to encode a component of the type II secretion system. The gspD::pJGG1 mutant displayed a greater-than-2-fold decrease in endoglucanase secretion compared to wild- type C. japonicus. In addition, the mutant strain showed a pronounced growth defect in medium with biomass as a carbon source, yielding 100-fold fewer viable cells than the wild type. To test the potential of C. japonicus to undergo metabolic engineering, we constructed a strain able to produce small amounts of ethanol from biomass. Collectively, these data suggest that C. japonicus is a useful platform for biomass conversion and biofuel production.

Biochemical and Mutational Analyses of AcuA, the Acetyltransferase Enzyme That Controls the Activity of the Acetyl Coenzyme A Synthetase (AcsA) in Bacillus subtilis

The acuABC genes of Bacillus subtilis comprise a putative posttranslational modification system. The AcuA protein is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, the AcuC protein is a class I histone deacetylase, and the role of the AcuB protein is not known. AcuA controls the activity of acetyl coenzyme A synthetase (AcsA; EC 6.2.1.1) in this bacterium by acetylating residue Lys549. Here we report the kinetic analysis of wild-type and variant AcuA proteins. We contrived a genetic scheme for the identification of AcuA residues critical for activity. Changes at residues H177 and G187 completely inactivated AcuA and led to its rapid turnover. Changes at residues R42 and T169 were less severe. In vitro assay conditions were optimized, and an effective means of inactivating the enzyme was found. The basic kinetic parameters of wild-type and variant AcuA proteins were obtained and compared to those of eukaryotic GNATs. Insights into how the isolated mutations may exert their deleterious effect were investigated by using the crystal structure of an AcuA homolog.

Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio Japonicus

Cellvibrio japonicus is a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO, CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function. Characterization of the latter module revealed tight and specific binding to chitin, thereby unraveling a new family of chitin-binding modules (classified as CBM73). X-ray crystallographic elucidation of the CjLPMO10A catalytic module revealed that the active site of the enzyme combines structural features previously only observed in either cellulose or chitin-active LPMO10s. Analysis of the copper-binding site by EPR showed a signal signature more similar to those observed for cellulose-cleaving LPMOs. The full-length LPMO shows no activity toward cellulose but is able to bind and cleave both α- and β-chitin. Removal of the chitin-binding modules reduced LPMO activity toward α-chitin compared with the full-length enzyme. Interestingly, the full-length enzyme and the individual catalytic LPMO module boosted the activity of an endochitinase equally well, also yielding similar amounts of oxidized products. Finally, gene deletion studies show that CjLPMO10A is needed by C. japonicus to obtain efficient growth on both purified chitin and crab shell particles.

Systems biology defines the biological significance of redox-active proteins during cellulose degradation in an aerobic bacterium

Microbial depolymerization of plant cell walls contributes to global carbon balance and is a critical component of renewable energy. The genomes of lignocellulose degrading microorganisms encode diverse classes of carbohydrate modifying enzymes, although currently there is a paucity of knowledge on the role of these proteins in vivo. We report the comprehensive analysis of the cellulose degradation system in the saprophytic bacterium Cellvibrio japonicus. Gene expression profiling of C. japonicus demonstrated that three of the 12 predicted β‐1,4 endoglucanases (cel5A, cel5B, and cel45A) and the sole predicted cellobiohydrolase (cel6A) showed elevated expression during growth on cellulose. Targeted gene disruptions of all 13 predicted cellulase genes showed that only cel5B and cel6A were required for optimal growth on cellulose. Our analysis also identified three additional genes required for cellulose degradation: lpmo10B encodes a lytic polysaccharide monooxygenase (LPMO), while cbp2D and cbp2E encode proteins containing carbohydrate binding modules and predicted cytochrome domains for electron transfer. CjLPMO10B oxidized cellulose and Cbp2D demonstrated spectral properties consistent with redox function. Collectively, this report provides insight into the biological role of LPMOs and redox proteins in cellulose utilization and suggests that C. japonicus utilizes a combination of hydrolytic and oxidative cleavage mechanisms to degrade cellulose.

A Complex Gene Locus Enables Xyloglucan Utilization in the Model Saprophyte Cellvibrio Japonicus.

The degradation of plant biomass by saprophytes is an ecologically important part of the global carbon cycle, which has also inspired a vast diversity of industrial enzyme applications. The xyloglucans (XyGs) constitute a family of ubiquitous and abundant plant cell wall polysaccharides, yet the enzymology of XyG saccharification is poorly studied. Here, we present the identification and molecular characterization of a complex genetic locus that is required for xyloglucan utilization by the model saprophyte Cellvibrio japonicus. In harness, transcriptomics, reverse genetics, enzyme kinetics, and structural biology indicate that the encoded cohort of an α‐xylosidase, a β‐galactosidase, and an α‐l‐fucosidase is specifically adapted for efficient, concerted saccharification of dicot (fucogalacto)xyloglucan oligosaccharides following import into the periplasm via an associated TonB‐dependent receptor. The data support a biological model of xyloglucan degradation by C. japonicus with striking similarities – and notable differences – to the complex polysaccharide utilization loci of the Bacteroidetes.

Polysaccharide degradation systems of the saprophytic bacterium Cellvibrio japonicus

Study of recalcitrant polysaccharide degradation by bacterial systems is critical for understanding biological processes such as global carbon cycling, nutritional contributions of the human gut microbiome, and the production of renewable fuels and chemicals. One bacterium that has a robust ability to degrade polysaccharides is the Gram-negative saprophyte Cellvibrio japonicus. A bacterium with a circuitous history, C. japonicus underwent several taxonomy changes from an initially described Pseudomonas sp. Most of the enzymes described in the pre-genomics era have also been renamed. This review aims to consolidate the biochemical, structural, and genetic data published on C. japonicus and its remarkable ability to degrade cellulose, xylan, and pectin substrates. Initially, C. japonicus carbohydrate-active enzymes were studied biochemically and structurally for their novel polysaccharide binding and degradation characteristics, while more recent systems biology approaches have begun to unravel the complex regulation required for lignocellulose degradation in an environmental context. Also included is a discussion for the future of C. japonicus as a model system, with emphasis on current areas unexplored in terms of polysaccharide degradation and emerging directions for C. japonicus in both environmental and biotechnological applications.

In-frame deletions allow functional characterization of complex cellulose degradation phenotypes in Cellvibrio japonicus

ABSTRACT The depolymerization of the recalcitrant polysaccharides found in lignocellulose has become an area of intense interest due to the role of this process in global carbon cycling, human gut microbiome nutritional contributions, and bioenergy production. However, underdeveloped genetic tools have hampered study of bacterial lignocellulose degradation, especially outside model organisms. In this report, we describe an in-frame deletion strategy for the Gram-negative lignocellulose-degrading bacterium Cellvibrio japonicus. This method leverages optimized growth conditions for conjugation and sacB counterselection for the generation of markerless in-frame deletions. This method produces mutants in as few as 8 days and allows for the ability to make multiple gene deletions per strain. It is also possible to remove large sections of the genome, as shown in this report with the deletion of the nine-gene (9.4-kb) gsp operon in C. japonicus. We applied this system to study the complex phenotypes of cellulose degradation in C. japonicus. Our data indicated that a Δcel5B Δcel6A double mutant is crippled for cellulose utilization, more so than by either single mutation alone. Additionally, we deleted individual genes in the two-gene cbp2ED operon and showed that both genes contribute to cellulose degradation in C. japonicus. Overall, these described techniques substantially enhance the utility of C. japonicus as a model system to study lignocellulose degradation.