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.     

Polysaccharide degradation systems of the saprophytic bacterium <Emphasis Type="Italic">Cellvibrio japonicus</Emphasis>

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.     

Characterization of Xylan Utilization and Discovery of a New Endoxylanase in Thermoanaerobacterium saccharolyticum through Targeted Gene Deletions

The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C₅ and C₆ sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of β-xylosidase xylD slowed the growth of T. saccharolyticum on birchwood xylan and led to an accumulation of short-chain xylo-oligomers, no other single deletion, including the deletion of the previously characterized endoxylanase XynA, had a phenotype distinct from that of the wild type. This result indicates a multiplicity of xylanase enzymes which facilitate xylan degradation in T. saccharolyticum. Growth on xylan was prevented only when a previously uncharacterized endoxylanase encoded by xynC was also deleted in conjunction with xynA. Sequence analysis of xynC indicates that this enzyme, a low-molecular-weight endoxylanase with homology to glycoside hydrolase family 11 enzymes, is secreted yet untethered to the cell wall. Together, these observations expand our understanding of the enzymatic basis of xylan hydrolysis by T. saccharolyticum.     

FLP-FRT-Based Method To Obtain Unmarked Deletions of CHU_3237 (porU) and Large Genomic Fragments of Cytophaga hutchinsonii

Cytophaga hutchinsonii is a widely distributed cellulolytic bacterium in the phylum Bacteroidetes. It can digest crystalline cellulose rapidly without free cellulases or cellulosomes. The mechanism of its cellulose utilization remains a mystery. We developed an efficient method based on a linear DNA double-crossover and FLP-FRT recombination system to obtain unmarked deletions of both single genes and large genomic fragments in C. hutchinsonii. Unmarked deletion of CHU_3237 (porU), an ortholog of the C-terminal signal peptidase of a type IX secretion system (T9SS), resulted in defects in colony spreading, cellulose degradation, and protein secretion, indicating that it is a component of the T9SS and that T9SS plays an important role in cellulose degradation by C. hutchinsonii. Furthermore, deletions of four large genomic fragments were obtained using our method, and the sizes of the excised fragments varied from 9 to 19 kb, spanning from 6 to 22 genes. The customized FLP-FRT method provides an efficient tool for more rapid progress in the cellulose degradation mechanism and other physiological aspects of C. hutchinsonii.     

A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality

A novel method to efficiently generate unmarked in-frame gene deletions in Rhodococcus equi was developed, exploiting the cytotoxic effect of 5-fluorocytosine (5-FC) by the action of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) enzymes. The opportunistic, intracellular pathogen R. equi is resistant to high concentrations of 5-FC. Introduction of Escherichia coli genes encoding CD and UPRT conferred conditional lethality to R. equi cells incubated with 5-FC. To exemplify the use of the codA::upp cassette as counter-selectable marker, an unmarked in-frame gene deletion mutant of R. equi was constructed. The supA and supB genes, part of a putative cholesterol catabolic gene cluster, were efficiently deleted from the R. equi wild-type genome. Phenotypic analysis of the generated ΔsupAB mutant confirmed that supAB are essential for growth of R. equi on cholesterol. Macrophage survival assays revealed that the ΔsupAB mutant is able to survive and proliferate in macrophages comparable to wild type. Thus, cholesterol metabolism does not appear to be essential for macrophage survival of R. equi. The CD-UPRT based 5-FC counter-selection may become a useful asset in the generation of unmarked in-frame gene deletions in other actinobacteria as well, as actinobacteria generally appear to be 5-FC resistant and 5-FU sensitive.     

N-Terminal Deletion of Peptide:N-Glycanase Results in Enhanced Deglycosylation Activity

Peptide:N-glycanase catalyzes the detachment of N-linked glycan chains from glycopeptides or glycoproteins by hydrolyzing the β-aspartylglucosaminyl bond. Peptide:N-glycanase in yeast binds to Rad23p through its N-terminus. In this study, the complex formed between Peptide:N-glycanase and Rad23p was found to exhibit enhanced deglycosylation activity, which suggests an important role for this enzyme in the misfolded glycoprotein degradation pathway in vivo. To investigate the role of this enzyme in this pathway, we made stepwise deletions of the N-terminal helices of peptide:N-glycanase. Enzymatic analysis of the deletion mutants showed that deletion of the N-terminal H1 helix (Png1p-ΔH1) enhanced the deglycosylation activity of N-glycanase towards denatured glycoproteins. In addition, this mutant exhibited high deglycosylation activity towards native glycoproteins. Dynamic simulations of the wild type and N-terminal H1 deletion mutant implied that Png1p-ΔH1 is more flexible than wild type Png1p. The efficient deglycosylation of Png1p-ΔH1 towards native and non-native glycoproteins offers a potential biotechnological application.     

Vibrio cholerae Response Regulator VxrB Controls Colonization and Regulates the Type VI Secretion System

Two-component signal transduction systems (TCS) are used by bacteria to sense and respond to their environment. TCS are typically composed of a sensor histidine kinase (HK) and a response regulator (RR). The Vibrio cholerae genome encodes 52 RR, but the role of these RRs in V. cholerae pathogenesis is largely unknown. To identify RRs that control V. cholerae colonization, in-frame deletions of each RR were generated and the resulting mutants analyzed using an infant mouse intestine colonization assay. We found that 12 of the 52 RR were involved in intestinal colonization. Mutants lacking one previously uncharacterized RR, VCA0566 (renamed VxrB), displayed a significant colonization defect. Further experiments showed that VxrB phosphorylation state on the predicted conserved aspartate contributes to intestine colonization. The VxrB regulon was determined using whole genome expression analysis. It consists of several genes, including those genes that create the type VI secretion system (T6SS). We determined that VxrB is required for T6SS expression using several in vitro assays and bacterial killing assays, and furthermore that the T6SS is required for intestinal colonization. vxrB is encoded in a four gene operon and the other vxr operon members also modulate intestinal colonization. Lastly, though ΔvxrB exhibited a defect in single-strain intestinal colonization, the ΔvxrB strain did not show any in vitro growth defect. Overall, our work revealed that a small set of RRs is required for intestinal colonization and one of these regulators, VxrB affects colonization at least in part through its regulation of T6SS genes.     

Characterisation of microbial floras and functional gene levels in an anaerobic/aerobic bio-reactor for the degradation of carboxymethyl cellulose

The current study determined the carboxymethyl cellulose (CMC) degradation efficiency, dominant microbial flora, eubacteria and archaebacteria characteristics, and expression levels of genes cel5A, cel6B, and bglC in an anaerobic/aerobic bio-reactor consisting of two-stage UASB (U1 and U2) and two-stage BAF (B1 and B2). The results showed that under three CMC loads, the CMC degradation efficiency of the UASB-BAF system was 91.25 %, 80.44 %, and 78.73 %, respectively. At higher CMC loads, the degradation of cellulose and transformation to cellobiose in U1 was higher, while the transformation to glucose was lower. The results of DGGE and real-time PCR indicated that cellulose degradation bacteria are dominant in U1, cellulose degradation bacteria and cellulose degradation symbiosis bacteria are dominant in B1, and non-cellulose degradation symbiosis bacteria are dominant in both U2 and B2. The rate-limiting enzyme gene of cellulose degradation in U1, B1, and B2 is cel6B, but it is cel5A in U2.     

Role of the gerP Operon in Germination and Outgrowth of Bacillus anthracis Spores

Germination of Bacillus anthracis spores occurs when nutrients such as amino acids or purine nucleosides stimulate specific germinant receptors located in the spore inner membrane. The gerP_ABCDEF operon has been suggested to play a role in facilitating the interaction between germinants and their receptors in spores of Bacillus subtilis and Bacillus cereus. B. anthracis mutants containing deletions in each of the six genes belonging to the orthologue of the gerP_ABCDEF operon, or deletion of the entire operon, were tested for their ability to germinate. Deletion of the entire gerP operon resulted in a significant delay in germination in response to nutrient germinants. These spores eventually germinated to levels equivalent to wild-type, suggesting that an additional entry point for nutrient germinants may exist. Deletions of each individual gene resulted in a similar phenotype, with the exception of ΔgerPF, which showed no obvious defect. The removal of two additional gerPF-like orthologues was necessary to achieve the germination defect observed for the other mutants. Upon physical removal of the spore coat, the mutant lacking the full gerP operon no longer exhibited a germination defect, suggesting that the GerP proteins play a role in spore coat permeability. Additionally, each of the gerP mutants exhibited a severe defect in calcium-dipicolinic acid (Ca-DPA)–dependent germination, suggesting a role for the GerP proteins in this process. Collectively, these data implicate all GerP proteins in the early stages of spore germination.     

Dockerin‐containing protease inhibitor protects key cellulosomal cellulases from proteolysis in Clostridium cellulolyticum

Cellulosomes are key for lignocellulosic biomass degradation in cellulolytic Clostridia. Better understanding of the mechanism of cellulosome regulation would allow us to improve lignocellulose hydrolysis. It is hypothesized that cellulosomal protease inhibitors would regulate cellulosome architecture and then lignocellulose hydrolysis. Here, a dockerin‐containing protease inhibitor gene (dpi) in Clostridium cellulolyticum H10 was characterized by mutagenesis and physiological analyses. The dpi mutant had a decreased cell yield on glucose, cellulose and xylan, lower cellulose utilization efficiency, and a 70% and 52% decrease of the key cellulosomal components, Cel48F and Cel9E respectively. The decreased cellulolysis is caused by the proteolysis of major cellulosomal components, such as Cel48F and Cel9E. Disruption of cel9E severely impaired cell growth on cellulose while loss of cel48F completely abolished cellulolytic activity. These observations are due to the combinational results of gene inactivation and polar effects caused by intron insertion. Purified recombinant Dpi showed inhibitory activity against cysteine protease. Taken together, Dpi protects key cellulosomal cellulases from proteolysis in H10. This study identified the physiological importance of cellulosome‐localized protease inhibitors in Clostridia.     

Effects of pH on Lignin and Cellulose Degradation by Streptomyces viridosporus

Lignocellulose degradation by Streptomyces viridosporus results in the oxidative depolymerization of lignin and the production of a water-soluble lignin polymer, acid-precipitable polymeric lignin (APPL). The effects of the culture pH on lignin and cellulose metabolism and APPL production by S. viridosporus are reported. Dry, ground, hot-water-extracted corn (Zea mays) lignocellulose was autoclaved in 1-liter reagent bottles (5 g per bottle) and inoculated with 50-ml volumes of S. viridosporus cells suspended in buffers of specific pH (pH 6.0 to 9.2 at 0.4 pH unit intervals). Four replicates of inoculated cultures and of uninoculated controls at each pH were incubated as solid-state fermentations at 37°C. After 6 weeks of incubation the percent loss of lignocellulose, lignin, and carbohydrate and the amount of APPL produced were determined for each replicate. Optimal lignocellulose degradation, as shown by substrate weight loss, was observed in the pH range of 8.4 to 8.8. Only minor differences were seen in the Klason lignin, carbohydrate, protein, and ash contents of the APPLS produced by cultures at each pH. The effects of pH on the degradation of a spruce (Picea pungens) [¹⁴C-lignin]lignocellulose and a Douglas fir (Pseudotsuga menziesii) [¹⁴C-glucan]-lignocellulose were also determined at pH values between 6.5 and 9.5 (0.5 pH unit intervals). The incubations were carried out for 3 weeks at 37°C with bubbler-tube cultures. The percentage of initial ¹⁴C recovered as ¹⁴CO₂, ¹⁴C-labeled water-soluble products, and [¹⁴C]APPL was then determined. The mineralization of lignin and cellulose to CO₂ was optimal at pHs 6.5 and 7.0, respectively. However, the optimum for lignin and cellulose solubilization was pH 8.5, which correlated with the pH 8.5 optimum for APPL production. Overall, the data show that, whereas lignin mineralization is optimal at neutral to slightly acidic pHs, lignocellulose degradation with lignin solubilization and APPL production is promoted by alkaline pHs. These findings indicate that lignin-solubilizing actinomycetes may play an important role in the metabolism of lignin in neutral to alkaline soils in which ligninolytic fungi are not highly competitive.     

Comparative genomic hybridization detects secondary chromosomal deletions in Escherichia coli K-12 MG1655 mutants and highlights instability in the flhDC region

The use of whole-genome microarrays for monitoring mutagenized or otherwise engineered genetic derivatives is a potentially powerful tool for checking genomic integrity. Using comparative genomic hybridization of a number of unrelated, directed deletion mutants in Escherichia coli K-12 MG1655, we identified unintended secondary genomic deletions in the flhDC region in Δfnr, Δcrp, and ΔcreB mutants. These deletions were confirmed by PCR and phenotypic tests. Our findings show that nonmotile progeny are found in some MG1655 directed deletion mutants, and studies on the effects of gene knockouts should be viewed with caution when the mutants have not been screened for the presence of secondary deletions or confirmed by other methods.     

Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as “transceptors” with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.     

Deletion of Dystrophin In-Frame Exon 5 Leads to a Severe Phenotype: Guidance for Exon Skipping Strategies

Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes.     

DdrA,DdrD, and PprA: Components of UV and Mitomycin C Resistance in Deinococcus radiodurans R1

Mutants created by deleting the ddrA, ddrB, ddrC, ddrD, and pprA loci of Deinococcus radiodurans R1alone and in all possible combinations of pairs revealed that the encoded gene products contribute to this species’ resistance to UV light and/or mitomycin C. Deleting pprA from an otherwise wild type cell sensitizes the resulting strain to UV irradiation, reducing viability by as much as eight fold relative to R1. If this deletion is introduced into a ΔddrA or ΔddrD background, the resulting strains become profoundly sensitive to the lethal effects of UV light. At a fluence of 1000 Jm^-2, the ΔddrA ΔpprA and ΔddrD ΔpprA strains are 100- and 1000-fold more sensitive to UV relative to the strain that has only lost pprA. Deletion of ddrA results in a 100 fold increase in strain sensitivity to mitomycin C, but in backgrounds that combine a deletion of ddrA with deletions of either ddrC or ddrD, mitomycin resistance is restored to wild type levels. Inactivation of ddrB also increases D. radiodurans sensitivity to mitomycin, but unlike the ddrA mutant deleting ddrC or ddrD from a ΔddrB background further increases that sensitivity. Despite the effect that loss of these gene products has on DNA damage resistance, none appear to directly affect either excision repair or homologous recombination suggesting that they participate in novel processes that facilitate tolerance to UV light and interstrand crosslinks in this species.     

clpC operon regulates cell architecture and sporulation in Bacillus anthracis

The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knockout strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B. anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and open up further interest on this operon, which might be of importance to success of B. anthracis as pathogen.