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Plant mannans are a component of lignocellulose that can have diverse compositions in terms of its backbone and side‐chain substitutions. Consequently, the degradation of mannan substrates requires a cadre of enzymes for complete reduction to substituent monosaccharides that can include mannose, galactose, and/or glucose. One bacterium that possesses this suite of enzymes is the Gram‐negative saprophyte Cellvibrio japonicus, which has 10 predicted mannanases from the Glycoside Hydrolase (GH) families 5, 26, and 27. Here we describe a systems biology approach to identify and characterize the essential mannan‐degrading components in this bacterium. The transcriptomic analysis uncovered significant changes in gene expression for most mannanases, as well as many genes that encode carbohydrate active enzymes (CAZymes) when mannan was actively being degraded. A comprehensive mutational analysis characterized 54 CAZyme‐encoding genes in the context of mannan utilization. Growth analysis of the mutant strains found that the man26C, aga27A, and man5D genes, which encode a mannobiohydrolase, α‐galactosidase, and mannosidase, respectively, were important for the deconstruction of galactomannan, with Aga27A being essential. Our updated model of mannan degradation in C. japonicus proposes that the removal of galactose sidechains from substituted mannans constitutes a crucial step for the complete degradation of this hemicellulose. [ABSTRACT FROM AUTHOR]

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.sup.+]-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.sup.+] 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.

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 (Acs[A.sup.Ac]) identified residue Lys549 as the sole modification site in the protein. Unlike sirtuins, the AcuC protein did not require NA[D.sup.+] as cosubstrate to deacetylate Acs[A.sup.Ac]. The function of the putative AcuB protein remains unknown.

Bacteria are major drivers of organic matter decomposition and play crucial roles in global nutrient cycling. Although the degradation of dead fungal biomass (necromass) is increasingly recognized as an important contributor to soil carbon (C) and nitrogen (N) cycling, the genes and metabolic pathways involved in necromass degradation are less characterized. In particular, how bacteria degrade necromass containing different quantities of melanin, which largely control rates of necromass decomposition in situ, is largely unknown. To address this gap, we conducted a multitimepoint transcriptomic analysis using three Gram-negative, bacterial species grown on low or high melanin necromass of Hyaloscypha bicolor. The bacterial species, Cellvibrio japonicus, Chitinophaga pinensis, and Serratia marcescens, belong to genera known to degrade necromass in situ. We found that while bacterial growth was consistently higher on low than high melanin necromass, the CAZyme-encoding gene expression response of the three species was similar between the two necromass types. Interestingly, this trend was not shared for genes encoding nitrogen utilization, which varied in C. pinensis and S. marcescens during growth on high vs low melanin necromass. Additionally, this study tested the metabolic capabilities of these bacterial species to grow on a diversity of C and N sources and found that the three bacteria have substantially different utilization patterns. Collectively, our data suggest that as necromass changes chemically over the course of degradation, certain bacterial species are favored based on their different ial metabolic capacities. IMPORTANCE Fungal necromass is a major component of the carbon (C) in soils as well as an important source of nitrogen (N) for plant and microbial growth. Bacteria associated with necromass represent a distinct subset of the soil microbiome and characterizing their functional capacities is the critical next step toward understanding how they influence necromass turnover. This is particularly important for necromass varying in melanin content, which has been observed to control the rate of necromass decomposition across a variety of ecosystems. Here we assessed the gene expression of three necromass-degrading bacteria grown on low or high melanin necromass and characterized their metabolic capacities to grow on different C and N substrates. These transcriptomic and metabolic studies provide the first steps toward assessing the physiological relevance of up-regulated CAZyme-encoding genes in necromass decomposition and provide foundational data for generating a predictive model of the molecular mechanisms underpinning necromass decomposition by soil bacteria. [ABSTRACT FROM AUTHOR]

Chitin utilization by microbes plays a significant role in biosphere carbon and nitrogen cycling, and studying the microbial approaches used to degrade chitin will facilitate our understanding of bacterial strategies to degrade a broad range of recalcitrant polysaccharides. The early stages of chitin depolymerization by the bacterium Cellvibrio japonicus have been characterized and are dependent on one chitin‐specific lytic polysaccharide monooxygenase and nonredundant glycoside hydrolases from the family GH18 to generate chito‐oligosaccharides for entry into metabolism. Here, we describe the mechanisms for the latter stages of chitin utilization by C. japonicus with an emphasis on the fate of chito‐oligosaccharides. Our systems biology approach combined transcriptomics and bacterial genetics using ecologically relevant substrates to determine the essential mechanisms for chito‐oligosaccharide transport and catabolism in C. japonicus. Using RNAseq analysis we found a coordinated expression of genes that encode polysaccharide‐degrading enzymes. Mutational analysis determined that the hex20B gene product, predicted to encode a hexosaminidase, was required for efficient utilization of chito‐oligosaccharides. Furthermore, two gene loci (CJA_0353 and CJA_1157), which encode putative TonB‐dependent transporters, were also essential for chito‐oligosaccharides utilization. This study further develops our model of C. japonicus chitin metabolism and may be predictive for other environmentally or industrially important bacteria. [ABSTRACT FROM AUTHOR]

Understanding the complex growth and metabolic dynamics in microorganisms requires advanced kinetic models containing both metabolic reactions and enzymatic regulation to predict phenotypic behaviors under different conditions and perturbations. Most current kinetic models lack gene expression dynamics and are separately calibrated to distinct media, which consequently makes them unable to account for genetic perturbations or multiple substrates. This challenge limits our ability to gain a comprehensive understanding of microbial processes towards advanced metabolic optimizations that are desired for many biotechnology applications. Here, we present an integrated computational and experimental approach for the development and optimization of mechanistic kinetic models for microbial growth and metabolic and enzymatic dynamics. Our approach integrates growth dynamics, gene expression, protein secretion, and gene‐deletion phenotypes. We applied this methodology to build a dynamic model of the growth kinetics in batch culture of the bacterium Cellvibrio japonicus grown using either cellobiose or glucose media. The model parameters were inferred from an experimental data set using an evolutionary computation method. The resulting model was able to explain the growth dynamics of C. japonicus using either cellobiose or glucose media and was also able to accurately predict the metabolite concentrations in the wild‐type strain as well as in β‐glucosidase gene deletion mutant strains. We validated the model by correctly predicting the non‐diauxic growth and metabolite consumptions of the wild‐type strain in a mixed medium containing both cellobiose and glucose, made further predictions of mutant strains growth phenotypes when using cellobiose and glucose media, and demonstrated the utility of the model for designing industrially‐useful strains. Importantly, the model is able to explain the role of the different β‐glucosidases and their behavior under genetic perturbations. This integrated approach can be extended to other metabolic pathways to produce mechanistic models for the comprehensive understanding of enzymatic functions in multiple substrates. [ABSTRACT FROM AUTHOR]

The ability of Cellvibrio japonicus, a sequenced Gram-negative cellulolytic bacterium, to degrade bioenergy-related feedstocks is examined. A method is developed for constructing directed gene disruptions in Cellvibrio japonicus and has shown that efficient cellular secretion and growth on biomass are prevented by disruption of the type II secretion system.

Summary: Lignocellulose degradation by microbes plays a central role in global carbon cycling, human gut metabolism and renewable energy technologies. While considerable effort has been put into understanding the biochemical aspects of lignocellulose degradation, much less work has been done to understand how these enzymes work in an in vivo context. Here, we report a systems level study of xylan degradation in the saprophytic bacterium Cellvibrio japonicus. Transcriptome analysis indicated seven genes that encode carbohydrate active enzymes were up‐regulated during growth with xylan containing media. In‐frame deletion analysis of these genes found that only gly43F is critical for utilization of xylo‐oligosaccharides, xylan, and arabinoxylan. Heterologous expression of gly43F was sufficient for the utilization of xylo‐oligosaccharides in Escherichia coli. Additional analysis found that the xyn11A, xyn11B, abf43L, abf43K, and abf51A gene products were critical for utilization of arabinoxylan. Furthermore, a predicted transporter (CJA_1315) was required for effective utilization of xylan substrates, and we propose this unannotated gene be called xntA (xylan transporter A). Our major findings are (i) C. japonicus employs both secreted and surface associated enzymes for xylan degradation, which differs from the strategy used for cellulose degradation, and (ii) a single cytoplasmic β‐xylosidase is essential for the utilization of xylo‐oligosaccharides. [ABSTRACT FROM AUTHOR]

Cyclodextrinases are carbohydrate-active enzymes involved in the linearization of circular amylose oligosaccharides. Primarily thought to function as part of starch metabolism, there have been previous reports of bacterial cyclodextrinases also having additional enzymatic activities on linear malto-oligosaccharides. This substrate class also includes environmentally rare α-diglucosides such as kojibiose (α-1,2), nigerose (α-1,3), and isomaltose (α-1,6), all of which have valuable properties as prebiotics or low-glycemic index sweeteners. Previous genome sequencing of three Cellvibrio japonicus strains adapted to utilize these α-diglucosides identified multiple, but uncharacterized, mutations in each strain. One of the mutations identified was in the amy13E gene, which was annotated to encode a neopullulanase. In this report, we functionally characterized this gene and determined that it in fact encodes a cyclodextrinase with additional activities on α-diglucosides. Deletion analysis of amy13E found that this gene was essential for kojibiose and isomaltose metabolism in C. japonicus. Interestingly, a Δamy13E mutant was not deficient for cyclodextrin or pullulan utilization in C. japonicus; however, heterologous expression of the gene in E. coli was sufficient for cyclodextrin-dependent growth. Biochemical analyses found that CjAmy13E cleaved multiple substrates but preferred cyclodextrins and maltose, but had no activity on pullulan. Our characterization of the CjAmy13E cyclodextrinase is useful for re fining functional enzyme predictions in related bacteria and for engineering enzymes for biotechnology or biomedical applications. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 C ellvibrio 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Posttranslational regulation of protein function by acetylation is present throughout nature. Regulation of protein function by Sir2 protein (sirtuin) deacetylases is conserved in all domains of life. In the prokaryote Salmonella enterica, the metabolic enzyme acetyl-coenzyme A synthetese (Acs) is regulated by a Sir2-dependent protein acetylation/deacetylation system (SDPADS). The recent identification of the acetyltransferase enzyme responsible for the acetylation of Acs defined the SDPADS in prokaryotes. This report identifies one residue in Acs, Leu-641, which is critical for the acetylation of Acs by the protein acetyltransferase enzyme. In vivo and in vitro evidence shows that mutations at Leu-641 prevent the acetylation of Acs by protein acetyltransferase, maintain the Acs enzyme in its active state, and bypass the need for sirtuin deacetylase activity during growth on acetate. [ABSTRACT FROM AUTHOR]

We describe a method for containing insoluble particulates for use as substrates in either bacterial growth or enzyme assays. This method was designed for high-throughput screening of environmental or engineered bacteria. Benchmarking this method with several model bacteria uncovered phenotypes not observable with the particulate substrates alone. [Display omitted] • Measuring the degradation of insoluble polysaccharides is challenging due to high costs and low-resolution assays. • The agar-based capture system for containing insoluble particulate matter described allows for high-throughput screening. • Agar-captured substrates remain accessible for bacterial growth and enzymatic assays and simplify experimental protocols. [ABSTRACT FROM AUTHOR]

Background: Xyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls. Due to its structural complexity, XyG requires a combination of backbone-cleaving and sidechain-debranching enzymes for complete deconstruction into its component monosaccharides. The soil saprophyte Cellvibrio japonicus has emerged as a genetically tractable model system to study biomass saccharification, in part due to its innate capacity to utilize a wide range of plant polysaccharides for growth. Whereas the downstream debranching enzymes of the xyloglucan utilization system of C. japonicus have been functionally characterized, the requisite backbone-cleaving endo-xyloglucanases were unresolved. Results: Combined bioinformatic and transcriptomic analyses implicated three glycoside hydrolase family 5 subfamily 4 (GH5_4) members, with distinct modular organization, as potential keystone endo-xyloglucanases in C. japonicus. Detailed biochemical and enzymatic characterization of the GH5_4 modules of all three recombinant proteins confirmed particularly high specificities for the XyG polysaccharide versus a panel of other cell wall glycans, including mixed-linkage beta-glucan and cellulose. Moreover, product analysis demonstrated that all three enzymes generated XyG oligosaccharides required for subsequent saccharification by known exo-glycosidases. Crystallographic analysis of GH5D, which was the only GH5_4 member specifically and highly upregulated during growth on XyG, in free, product-complex, and active-site affinity-labelled forms revealed the molecular basis for the exquisite XyG specificity among these GH5_4 enzymes. Strikingly, exhaustive reverse-genetic analysis of all three GH5_4 members and a previously biochemically characterized GH74 member failed to reveal a growth defect, thereby indicating functional compensation in vivo, both among members of this cohort and by other, yet unidentified, xyloglucanases in C. japonicus. Our systems-based analysis indicates distinct substrate-sensing (GH74, GH5E, GH5F) and attack-mounting (GH5D) functions for the endo-xyloglucanases characterized here. Conclusions: Through a multi-faceted, molecular systems-based approach, this study provides a new insight into the saccharification pathway of xyloglucan utilization system of C. japonicus. The detailed structural–functional characterization of three distinct GH5_4 endo-xyloglucanases will inform future bioinformatic predictions across species, and provides new CAZymes with defined specificity that may be harnessed in industrial and other biotechnological applications. [ABSTRACT FROM AUTHOR]

The α-diglucoside trehalose has historically been known as a component of bacterial stress response, though more recently has been studied for its relevance in human gut health and biotechnology development. The utilization of trehalose as a nutrient source by bacteria relies on carbohydrate active enzymes, specifically those of the glycoside hydrolase family 37, to degrade the disaccharide into substituent glucose moieties for entry into metabolism. Environmental bacteria using oligosaccharides for nutrients often possess multiple carbohydrate active enzymes predicted to have the same biochemical activity, and therefore are thought to be functionally redundant. In this study, we characterized trehalose degradation by the biotechnologically important saprophytic bacterium Cellvibrio japonicus. This bacterium possesses two predicted α-α-trehalase genes, tre37A and tre37B, and our investigation using mutational analysis found that only the former is essential for trehalose utilization by C. japonicus. Heterologous expression experiments found that only the expression of the C. japonicus tre37A gene in an E. coli treA mutant strain allowed for full utilization of trehalose. Biochemical characterization of C. japonicus GH37 activity determined that the tre37A gene product is solely responsible for cleaving trehalose, and is an acidic α-α-trehalase. Bioinformatic and mutational analysis indicate that Tre37A directly cleaves trehalose to glucose in the periplasm, as C. japonicus does not possess a phosphotransferase system. This study facilitates the development of a comprehensive metabolic model for α-linked disaccharides in C. japonicus, and more broadly expands our understanding of the strategies that saprophytic bacteria employ to capture diverse carbohydrates from the environment. [ABSTRACT FROM AUTHOR]

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 (AcsAAc) identified residue Lys549 as the sole modification site in the protein. Unlike sirtuins, the AcuC protein did not require NAD+ as cosubstrate to deacetylate AcsAAc. The function of the putative AcuB protein remains unknown. [ABSTRACT FROM AUTHOR]

Physiological studies of recalcitrant polysaccharide degradation are challenging for several reasons, one of which is the difficulty in obtaining a reproducibly accurate real-time measurement of bacterial growth using insoluble substrates. Current methods suffer from several problems including (i) high background noise due to the insoluble material interspersed with cells, (ii) high consumable and reagent cost and (iii) significant time delay between sampling and data acquisition. A customizable substrate and cell separation device would provide an option to study bacterial growth using optical density measurements. To test this hypothesis we used 3-D printing to create biomass containment devices that allow interaction between insoluble substrates and microbial cells but do not interfere with spectrophotometer measurements. Evaluation of materials available for 3-D printing indicated that UV-cured acrylic plastic was the best material, being superior to nylon or stainless steel when examined for heat tolerance, reactivity, and ability to be sterilized. Cost analysis of the 3-D printed devices indicated they are a competitive way to quantitate bacterial growth compared to viable cell counting or protein measurements, and experimental conditions were scalable over a 100-fold range. The presence of the devices did not alter growth phenotypes when using either soluble substrates or insoluble substrates. We applied biomass containment to characterize growth of Cellvibrio japonicus on authentic lignocellulose (non-pretreated corn stover), and found physiological evidence that xylan is a significant nutritional source despite an abundance of cellulose present. [ABSTRACT FROM AUTHOR]

Understanding the strategies used by bacteria to degrade polysaccharides constitutes an invaluable tool for biotechnological applications. Bacteria are major mediators of polysaccharide degradation in nature; however, the complex mechanisms used to detect, degrade, and consume these substrates are not well-understood, especially for recalcitrant polysaccharides such as chitin. It has been previously shown that the model bacterial saprophyte Cellvibrio japonicus is able to catabolize chitin, but little is known about the enzymatic machinery underlying this capability. Previous analyses of the C. japonicus genome and proteome indicated the presence of four glycoside hydrolase family 18 (GH18) enzymes, and studies of the proteome indicated that all are involved in chitin utilization. Using a combination of in vitro and in vivo approaches, we have studied the roles of these four chitinases in chitin bioconversion. Genetic analyses showed that only the chi18D gene product is essential for the degradation of chitin substrates. Biochemical characterization of the four enzymes showed functional differences and synergistic effects during chitin degradation, indicating nonredundant roles in the cell. Transcriptomic studies revealed complex regulation of the chitin degradation machinery of C. japonicus and confirmed the importance of CjChi18D and CjLPMO10A, a previously characterized chitin-active enzyme. With this systems biology approach, we deciphered the physiological relevance of the glycoside hydrolase family 18 enzymes for chitin degradation in C. japonicus, and the combination of in vitro and in vivo approaches provided a comprehensive understanding of the initial stages of chitin degradation by this bacterium. [ABSTRACT FROM AUTHOR]

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 (CAZymes). Several putative chitin degrading enzymes are also found amongst these CAZymes, such as chitinases, chitobiases and lytic polysaccharide monooxygenases (LPMOs). In the present 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 hitherto 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 towards cellulose, but is able to bind and cleave both α- and β-chitin. Removal of the chitin-binding modules reduced LPMO activity towards α-chitin compared to 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. [ABSTRACT FROM AUTHOR]

Cellvibrio japonicus is a saprophytic bacterium proficient at environmental polysaccharide degradation for carbon and energy acquisition. Genetic, enzymatic, and structural characterization of C. japonicus carbohydrate active enzymes, specifically those that degrade plant and animal-derived polysaccharides, demonstrated that this bacterium is a carbohydrate-bioconversion specialist. Structural analyses of these enzymes identified highly specialized carbohydrate binding modules that facilitate activity. Steady progress has been made in developing genetic tools for C. japonicus to better understand the function and regulation of the polysaccharide-degrading enzymes it possesses, as well as to develop it as a biotechnology platform to produce renewable fuels and chemicals.

Importance: Understanding the bacterial metabolism of starch is important as this polysaccharide is a ubiquitous ingredient in foods, supplements, and medicines, all of which influence gut microbiome composition and health. Our RNAseq and growth data set provides a valuable resource to those who want to better understand the regulation of starch utilization in Gram-negative bacteria. These data are also useful as they provide an example of how to approach studying a starch-utilizing bacterium that has many putative amylases by coupling transcriptomic data with growth assays to overcome the potential challenges of functional redundancy. The RNAseq data can also be used as a part of larger meta-analyses to compare how C. japonicus regulates carbohydrate active enzymes, or how this bacterium compares to gut microbiome constituents in terms of starch utilization potential., Competing Interests: The authors declare no conflict of interest.

Serratia marcescens is a Gram-negative bacterium found in terrestrial and aquatic environments and studied for its polysaccharide utilization capabilities as part of larger efforts to discover novel carbohydrate-active enzymes. Here, we announce the genome sequence of an S. marcescens strain (PIC3611) that is able to utilize complex polysaccharide substrates.

Cellvibrio japonicus is a saprophytic bacterium that has been studied for its substantial carbohydrate degradation capability. We announce the genome sequences of three strains with improved growth characteristics when utilizing α-diglucosides. These data provide additional insight into the metabolic flexibility of a biotechnologically relevant bacterium., (Copyright © 2019 Garcia et al.)

A variety of strategies now exist for the extracellular expression of recombinant proteins using laboratory strains of Escherichia coli . However, secreted proteins often accumulate in the culture medium at levels that are too low to be practically useful for most synthetic biology and metabolic engineering applications. The situation is compounded by the lack of generalized screening tools for optimizing the secretion process. To address this challenge, we developed a genetic approach for studying and engineering protein-secretion pathways in E. coli . Using the YebF pathway as a model, we demonstrate that direct fluorescent labeling of tetracysteine-motif-tagged secretory proteins with the biarsenical compound FlAsH is possible in situ without the need to recover the cell-free supernatant. High-throughput screening of a bacterial strain library yielded superior YebF expression hosts capable of secreting higher titers of YebF and YebF-fusion proteins into the culture medium. We also show that the method can be easily extended to other secretory pathways, including type II and type III secretion, directly in E. coli . Thus, our FlAsH-tetracysteine-based genetic assay provides a convenient, high-throughput tool that can be applied generally to diverse secretory pathways. This platform should help to shed light on poorly understood aspects of these processes as well as to further assist in the construction of engineered E. coli strains for efficient secretory-protein production.

Microbial degradation of plant cell walls is a critical contributor to the global carbon cycle, and enzymes derived from microbes play a key role in the sustainable biofuels industry. Despite its biological and biotechnological importance, relatively little is known about how microbes degrade plant cell walls. Much of this gap in knowledge has resulted from difficulties in extending modern molecular tools to the study of plant cell wall-degrading microbes. The bacterium Cellvibrio japonicus has recently emerged as a powerful model system for the study of microbial plant cell wall degradation. C. japonicus is unique among microbial model systems in that it possesses the ability to carry out the complete degradation of plant cell wall polysaccharides. Furthermore, an extensive array of genetic and molecular tools exists for functional genomic analysis. In this review, we describe progress in the development of methodology for the functional genomic study of plant cell wall degradation by this microbe, and discuss future directions for research., (Copyright © 2012 Elsevier Inc. All rights reserved.)