Paenibacillus sp. Strain JDR-2 and XynA1: a Novel System for Methylglucuronoxylan Utilization

Environmental and economic factors predicate the need for efficient processing of renewable sources of fuels and chemicals. To fulfill this need, microbial biocatalysts must be developed to efficiently process the hemicellulose fraction of lignocellulosic biomass for fermentation of pentoses. The predominance of methylglucuronoxylan (MeGAX_n), a β-1,4 xylan in which 10% to 20% of the xylose residues are substituted with α-1,2-4-O-methylglucuronate residues, in hemicellulose fractions of hardwood and crop residues has made this a target for processing and fermentation. A Paenibacillus sp. (strain JDR-2) has been isolated and characterized for its ability to efficiently utilize MeGAX_n. A modular xylanase (XynA₁) of glycosyl hydrolase family 10 (GH 10) was identified through DNA sequence analysis that consists of a triplicate family 22 carbohydrate binding module followed by a GH 10 catalytic domain followed by a single family 9 carbohydrate binding module and concluding with C-terminal triplicate surface layer homology (SLH) domains. Immunodetection of the catalytic domain of XynA₁ (XynA₁ CD) indicates that the enzyme is associated with the cell wall fraction, supporting an anchoring role for the SLH modules. With MeGAX_n as substrate, XynA₁ CD generated xylobiose and aldotetrauronate (MeGAX₃) as predominant products. The inability to detect depolymerization products in medium during exponential growth of Paenibacillus sp. strain JDR-2 on MeGAX_n, as well as decreased growth rate and yield with XynA₁ CD-generated xylooligosaccharides and aldouronates as substrates, indicates that XynA₁ catalyzes a depolymerization process coupled to product assimilation. This depolymerization/assimilation system may be utilized for development of biocatalysts to efficiently convert MeGAX_n to alternative fuels and biobased products.     

Complete genome sequence of Paenibacillus sp. strain JDR-2

Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from sweetgum (Liquidambar styraciflua) wood, is able to efficiently depolymerize, assimilate and metabolize 4-O-methylglucuronoxylan, the predominant structural component of hardwood hemicelluloses. A basis for this capability was first supported by the identification of genes and characterization of encoded enzymes and has been further defined by the sequencing and annotation of the complete genome, which we describe. In addition to genes implicated in the utilization of β-1,4-xylan, genes have also been identified for the utilization of other hemicellulosic polysaccharides. The genome of Paenibacillus sp. JDR-2 contains 7,184,930 bp in a single replicon with 6,288 protein-coding and 122 RNA genes. Uniquely prominent are 874 genes encoding proteins involved in carbohydrate transport and metabolism. The prevalence and organization of these genes support a metabolic potential for bioprocessing of hemicellulose fractions derived from lignocellulosic resources.     

Paenibacillus sp. strain JDR-2 and XynA1: a novel system for methylglucuronoxylan utilization

Environmental and economic factors predicate the need for efficient processing of renewable sources of fuels and chemicals. To fulfill this need, microbial biocatalysts must be developed to efficiently process the hemicellulose fraction of lignocellulosic biomass for fermentation of pentoses. The predominance of methylglucuronoxylan (MeGAXn), a beta-1,4 xylan in which 10% to 20% of the xylose residues are substituted with alpha-1,2-4-O-methylglucuronate residues, in hemicellulose fractions of hardwood and crop residues has made this a target for processing and fermentation. A Paenibacillus sp. (strain JDR-2) has been isolated and characterized for its ability to efficiently utilize MeGAXn. A modular xylanase (XynA1) of glycosyl hydrolase family 10 (GH 10) was identified through DNA sequence analysis that consists of a triplicate family 22 carbohydrate binding module followed by a GH 10 catalytic domain followed by a single family 9 carbohydrate binding module and concluding with C-terminal triplicate surface layer homology (SLH) domains. Immunodetection of the catalytic domain of XynA1 (XynA1 CD) indicates that the enzyme is associated with the cell wall fraction, supporting an anchoring role for the SLH modules. With MeGAXn as substrate, XynA1 CD generated xylobiose and aldotetrauronate (MeGAX3) as predominant products. The inability to detect depolymerization products in medium during exponential growth of Paenibacillus sp. strain JDR-2 on MeGAXn, as well as decreased growth rate and yield with XynA1 CD-generated xylooligosaccharides and aldouronates as substrates, indicates that XynA1 catalyzes a depolymerization process coupled to product assimilation. This depolymerization/assimilation system may be utilized for development of biocatalysts to efficiently convert MeGAXn to alternative fuels and biobased products.     

Utilization of Ganglioside-Degrading Paenibacillus sp. Strain TS12 for Production of Glucosylceramide

Gangliosides, sialic acid-containing glycosphingolipids, are membrane constituents of vertebrates and are known to have important roles in cellular differentiation, adhesion, and recognition. We report here the isolation of a bacterium capable of degrading gangliotetraose-series gangliosides and a new method for the production of glucosylceramide with this bacterium. GM1a ganglioside was found to be sequentially degraded by Paenibacillus sp. strain TS12, which was isolated from soil, as follows: GM1a → asialo GM1 → asialo GM2 → lactosylceramide → glucosylceramide. TS12 was found to produce a series of ganglioside-degrading enzymes, such as sialidases, β-galactosidases, and β-hexosaminidases. TS12 also produced β-glucosidases, but glucosylceramide was somewhat resistant to the bacterial enzyme under the conditions used. Taking advantage of the specificity, we developed a new method for the production of glucosylceramide using TS12 as a biocatalyst. The method involves the conversion of crude bovine brain gangliosides to glucosylceramide by coculture with TS12 and purification of the product by chromatography with Wakogel C-300 HG.     

Structure, function, and regulation of the aldouronate utilization gene cluster from Paenibacillus sp. strain JDR-2

Direct bacterial conversion of the hemicellulose fraction of hardwoods and crop residues to biobased products depends upon extracellular depolymerization of methylglucuronoxylan (MeGAX_n), followed by assimilation and intracellular conversion of aldouronates and xylooligosaccharides to fermentable xylose. Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium, secretes a multimodular cell-associated GH10 endoxylanase (XynA1) that catalyzes depolymerization of MeGAX_n and rapidly assimilates the principal products, β-1,4-xylobiose, β-1,4-xylotriose, and MeGAX₃, the aldotetrauronate 4-O-methylglucuronosyl-α-1,2-xylotriose. Genomic libraries derived from this bacterium have now allowed cloning and sequencing of a unique aldouronate utilization gene cluster comprised of genes encoding signal transduction regulatory proteins, ABC transporter proteins, and the enzymes AguA (GH67 α-glucuronidase), XynA2 (GH10 endoxylanase), and XynB (GH43 β-xylosidase/α-arabinofuranosidase). Expression of these genes, as well as xynA1 encoding the secreted GH10 endoxylanase, is induced by growth on MeGAX_n and repressed by glucose. Sequences in the yesN, lplA, and xynA2 genes within the cluster and in the distal xynA1 gene show significant similarity to catabolite responsive element (cre) defined in Bacillus subtilis for recognition of the catabolite control protein (CcpA) and consequential repression of catabolic regulons. The aldouronate utilization gene cluster in Paenibacillus sp. strain JDR-2 operates as a regulon, coregulated with the expression of xynA1, conferring the ability for efficient assimilation and catabolism of the aldouronate product generated by a multimodular cell surface-anchored GH10 endoxylanase. This cluster offers a desirable metabolic potential for bacterial conversion of hemicellulose fractions of hardwood and crop residues to biobased products.     

Cooperative Degradation of Chitin by Extracellular and Cell Surface-Expressed Chitinases from Paenibacillus sp. Strain FPU-7

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.     

Genome Analysis Coupled with Physiological Studies Reveals a Diverse Nitrogen Metabolism in Methylocystis sp. Strain SC2

Background

Methylocystis sp. strain SC2 can adapt to a wide range of methane concentrations. This is due to the presence of two isozymes of particulate methane monooxygenase exhibiting different methane oxidation kinetics. To gain insight into the underlying genetic information, its genome was sequenced and found to comprise a 3.77 Mb chromosome and two large plasmids.

Principal Findings

We report important features of the strain SC2 genome. Its sequence is compared with those of seven other methanotroph genomes, comprising members of the Alphaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. While the pan-genome of all eight methanotroph genomes totals 19,358 CDS, only 154 CDS are shared. The number of core genes increased with phylogenetic relatedness: 328 CDS for proteobacterial methanotrophs and 1,853 CDS for the three alphaproteobacterial Methylocystaceae members, Methylocystis sp. strain SC2 and strain Rockwell, and Methylosinus trichosporium OB3b. The comparative study was coupled with physiological experiments to verify that strain SC2 has diverse nitrogen metabolism capabilities. In correspondence to a full complement of 34 genes involved in N₂ fixation, strain SC2 was found to grow with atmospheric N₂ as the sole nitrogen source, preferably at low oxygen concentrations. Denitrification-mediated accumulation of 0.7 nmol ³⁰N₂/hr/mg dry weight of cells under anoxic conditions was detected by tracer analysis. N₂ production is related to the activities of plasmid-borne nitric oxide and nitrous oxide reductases.

Conclusions/Perspectives

Presence of a complete denitrification pathway in strain SC2, including the plasmid-encoded nosRZDFYX operon, is unique among known methanotrophs. However, the exact ecophysiological role of this pathway still needs to be elucidated. Detoxification of toxic nitrogen compounds and energy conservation under oxygen-limiting conditions are among the possible roles. Relevant features that may stimulate further research are, for example, absence of CRISPR/Cas systems in strain SC2, high number of iron acquisition systems in strain OB3b, and large number of transposases in strain Rockwell.     

Utilization of ganglioside-degrading Paenibacillus sp. strain TS12 for production of glucosylceramide

Gangliosides, sialic acid-containing glycosphingolipids, are membrane constituents of vertebrates and are known to have important roles in cellular differentiation, adhesion, and recognition. We report here the isolation of a bacterium capable of degrading gangliotetraose-series gangliosides and a new method for the production of glucosylceramide with this bacterium. GM1a ganglioside was found to be sequentially degraded by Paenibacillus sp. strain TS12, which was isolated from soil, as follows: GM1a --> asialo GM1 --> asialo GM2 --> lactosylceramide --> glucosylceramide. TS12 was found to produce a series of ganglioside-degrading enzymes, such as sialidases, beta-galactosidases, and beta-hexosaminidases. TS12 also produced beta-glucosidases, but glucosylceramide was somewhat resistant to the bacterial enzyme under the conditions used. Taking advantage of the specificity, we developed a new method for the production of glucosylceramide using TS12 as a biocatalyst. The method involves the conversion of crude bovine brain gangliosides to glucosylceramide by coculture with TS12 and purification of the product by chromatography with Wakogel C-300 HG.     

Paenibacillus sp. Strain SB-6 Induces Weathering of Ca-montmorillonite: Illitization and Formation of Calcite

Investigation of the weathering of silicate minerals is helpful to understand the process of soil development, cycling of nutrient elements, and potential applications in fixation of carbon dioxide from the atmosphere through carbonate precipitation. In this study, weathering experiments of calcium-montmorillonite were conducted using Paenibacillus sp. strain SB-6 for 70 days. The results indicated that the Si⁴⁺, Al³⁺, Ca²⁺ and Na⁺ concentrations in the medium of the biotic experiments were evidently higher than those of the abiotic experiments, and that Paenibacillus sp. could help the transformation of partial montmorillonite into an illite–montmorillonite mixed-layer. In the process of illitization, K⁺ went into the interlayer of montmorillonite and hydrated Ca²⁺ and Na⁺ released from it. In the late stage of the experiments, the Ca²⁺ released from montmorillonite combined with carbonate ions generated by the bacterial metabolism, forming calcite.     

Novel Intermediates of Acenaphthylene Degradation by Rhizobium sp. Strain CU-A1: Evidence for Naphthalene-1,8-Dicarboxylic Acid Metabolism

The acenaphthylene-degrading bacterium Rhizobium sp. strain CU-A1 was isolated from petroleum-contaminated soil in Thailand. This strain was able to degrade 600 mg/liter acenaphthylene completely within three days. To elucidate the pathway for degradation of acenaphthylene, strain CU-A1 was mutagenized by transposon Tn5 in order to obtain mutant strains deficient in acenaphthylene degradation. Metabolites produced from Tn5-induced mutant strains B1, B5, and A53 were purified by thin-layer chromatography and silica gel column chromatography and characterized by mass spectrometry. The results suggested that this strain cleaved the fused five-membered ring of acenaphthylene to form naphthalene-1,8-dicarboxylic acid via acenaphthenequinone. One carboxyl group of naphthalene-1,8-dicarboxylic acid was removed to form 1-naphthoic acid which was transformed into salicylic acid before metabolization to gentisic acid. This work is the first report of complete acenaphthylene degradation by a bacterial strain.     

Atropine Metabolism by Pseudomonas sp. Strain AT3: Evidence for Nortropine as an Intermediate in Tropine Breakdown and Reactions Leading to Succinate

Pseudomonas strain AT3, isolated by elective culture with atropine, hydrolyzed atropine and grew diauxically, first on the tropic acid and then on the tropine. Tropine was also used as a sole carbon and energy source. The methyl group of tropine was eliminated as formaldehyde, and the nortropine thus formed was a precursor of 6-hydroxycyclohepta-1,4-dione. Ammonia was detected as a product of nitrogen elimination. 6-Hydroxycyclohepta-1,4-dione was oxidized to cyclohepta-1,3,5-trione by an induced NAD(sup+)-specific dehydrogenase. Although cyclohepta-1,3,5-trione is a (beta)-diketone with two potential hydrolytic cleavage sites, an induced hydrolase was specific for one of these sites, with 4,6-dioxoheptanoate as the only hydrolysis product. Unlike the alternative cleavage product (3,6-dioxoheptanoate), this compound is also a (beta)-diketone, and a second hydrolytic cleavage formed succinate and acetone. Although Pseudomonas strain AT3 was not capable of growth with acetone, the compound was not detected in the culture medium and may have been lost to the atmosphere. Exhaustive experimentation with a wide range of conditions did not result in detection of the enzymes required for cleavage of the carbon-nitrogen bonds leading to the formation of nortropine and 6-hydroxycyclohepta-1,4-dione.     

Kinetics of Acetate Utilization by Two Thermophilic Acetotrophic Methanogens: Methanosarcina sp. Strain CALS-1 and Methanothrix sp. Strain CALS-1

The kinetics of acetate utilization were examined for washed concentrated cell suspensions of two thermophilic acetotrophic methanogens isolated from a 58°C anaerobic digestor. Progress curves for acetate utilization by cells of Methanosarcina sp. strain CALS-1 showed that the utilization rate was concentration independent (zero order) above concentrations near 3 mM and that acetate utilization ceased when a threshold concentration near 1 mM was reached. Acetate utilization by cells of Methanothrix sp. strain CALS-1 was concentration independent down to 0.1 to 0.2 mM, and threshold values of 12 to 21 μM were observed. Typical utilization rates in the concentration-independent stage were 210 and 130 nmol min⁻¹ mg of protein⁻¹ for the methanosarcina and the methanothrix, respectively. These results are in agreement with a general model in which high acetate concentrations favor Methanosarcina spp., while low concentrations favor Methanothrix spp. However, acetate utilization by these two strains did not follow simple Michaelis-Menton kinetics.     

Nitrogen Control of Atrazine Utilization in Pseudomonas sp. Strain ADP

Pseudomonas sp. strain ADP uses the herbicide atrazine as the sole nitrogen source. We have devised a simple atrazine degradation assay to determine the effect of other nitrogen sources on the atrazine degradation pathway. The atrazine degradation rate was greatly decreased in cells grown on nitrogen sources that support rapid growth of Pseudomonas sp. strain ADP compared to cells cultivated on growth-limiting nitrogen sources. The presence of atrazine in addition to the nitrogen sources did not stimulate degradation. High degradation rates obtained in the presence of ammonium plus the glutamine synthetase inhibitor MSX and also with an Nas⁻ mutant derivative grown on nitrate suggest that nitrogen regulation operates by sensing intracellular levels of some key nitrogen-containing metabolite. Nitrate amendment in soil microcosms resulted in decreased atrazine mineralization by the wild-type strain but not by the Nas⁻ mutant. This suggests that, although nitrogen repression of the atrazine catabolic pathway may have a strong impact on atrazine biodegradation in nitrogen-fertilized soils, the use of selected mutant variants may contribute to overcoming this limitation.     

Isolation and Partial Characterization of Antagonistic Peptides Produced by Paenibacillus sp. Strain B2 Isolated from the Sorghum Mycorrhizosphere

Paenibacillus sp. strain B2, isolated from the mycorrhizosphere of sorghum colonized by Glomus mosseae, produces an antagonistic factor. This factor has a broad spectrum of activity against gram-positive and gram-negative bacteria and also against fungi. The antagonistic factor was isolated from the bacterial culture medium and purified by cation-exchange, reverse-phase, and size exclusion chromatography. The purified factor could be separated into three active compounds following characterization by amino acid analysis and by combined reverse-phase chromatography and mass spectrometry (liquid chromatography-mass spectrometry and mass spectrometry-mass spectrometry). The first compound had the same retention time as polymyxin B₁, whereas the two other compounds were more hydrophobic. The molecular masses of the latter compounds are 1,184.7 and 1,202.7 Da, respectively, and their structure is similar to that of polymyxin B₁, with a cyclic heptapeptide moiety attached to a tripeptide side chain and a fatty acyl residue. They both contain threonine, phenylalanine, leucine, and 2,4-diaminobutyric acid residues. The peptide with a molecular mass of 1,184.7 contains a 2,3-didehydrobutyrine residue with a molecular mass of 101 Da replacing a threonine at the A2 position of the polymyxin side chain. This modification could explain the broader range of antagonistic activity of this peptide compared to that of polymyxin B.     

Physiological Regulation of Isocitrate Dehydrogenase and the Role of 2-Oxoglutarate in Prochlorococcus sp. Strain PCC 9511

The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus.     

Purification and Properties of an Extracellular Agarase from Alteromonas sp. Strain C-1

A marine bacterial strain isolated from the Bay of San Vicente, Chile, was identified as Alteromonas sp. strain C-1. In the presence of agar, this strain produced high levels of an extracellular agarase. The production of agarase was repressed by glucose, with a parallel decrease in bacterial growth. The enzyme was purified to homogeneity by anion-exchange chromatography and gel filtration, with an overall yield of 45%. The enzyme has a molecular weight of 52,000, is salt sensitive, and hydrolyzes agar, yielding neoagarotetraose as the main product, with an optimum pH of about 6.5.