Production of a bacterial secretome highly efficient for the deconstruction of xylans.

Bacteria within the Paenibacillus genus are known to secrete a diverse array of enzymes capable of breaking down plant cell wall polysaccharides. We studied the extracellular xylanolytic activity of Paenibacillus xylanivorans and examined the complete range of secreted proteins when grown on carbohydrate-based carbon sources of increasing complexity, including wheat bran, sugar cane straw, beechwood xylan and sucrose, as control. Our data showed that the relative abundances of secreted proteins varied depending on the carbon source used. Extracellular enzymatic extracts from wheat bran (WB) or sugar cane straw (SCR) cultures had the highest xylanolytic activity, coincidently with the largest representation of carbohydrate active enzymes (CAZymes). Scaling-up to a benchtop bioreactor using WB resulted in a significant enhancement in productivity and in the overall volumetric extracellular xylanase activity, that was further concentrated by freeze-drying. The enzymatic extract was efficient in the deconstruction of xylans from different sources as well as sugar cane straw pretreated by alkali extrusion (SCRe), resulting in xylobiose and xylose, as primary products. The overall yield of xylose released from SCRe was improved by supplementing the enzymatic extract with a recombinant GH43 ß-xylosidase (EcXyl43) and a GH62 α-L-arabinofuranosidase (CsAbf62A), two activities that were under-represented. Overall, we showed that the extracellular enzymatic extract from P. xylanivorans, supplemented with specific enzymatic activities, is an effective approach for targeting xylan within lignocellulosic biomass.

In vitro effects of alginate lyase SG4 + produced by Paenibacillus lautus alone and combined with antibiotics on biofilm formation by mucoid Pseudomonas aeruginosa.

Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The Km and Vmax of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t 1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms.

A single mutation in cyclodextrin glycosyltransferase from Paenibacillus barengoltzii changes cyclodextrin and maltooligosaccharides production.

Cyclodextrin glycosyltransferases (CGTases) are bacterial enzymes that catalyze starch conversion into cyclodextrins, which have several biotechnological applications including solubilization of hydrophobic compounds, masking of unpleasant odors and flavors in pharmaceutical preparations, and removal of cholesterol from food. Additionally, CGTases produce maltooligosaccharides, which are linear molecules with potential benefits for human health. Current research efforts are concentrated in the development of engineered enzymes with improved yield and/or particular product specificity. In this work, we analyzed the role of four residues of the CGTase from Paenibacillus barengoltzii as determinants of product specificity. Single mutations were introduced in the CGTase-encoding gene to obtain mutants A137V, A144V, L280A and M329I and the activity of recombinant proteins was evaluated. The residue at position 137 proved to be relevant for CGTase activity. Molecular dynamics studies demonstrated additionally that mutation A137V produces a perturbation in the catalytic site of the CGTase, which correlates with a 10-fold reduction in its catalytic efficiency. Moreover, this mutant showed increased production of maltooligosaccharides with a high degree of polymerization, mostly maltopentaose to maltoheptaose. Our results highlight the role of residue 137 as a determinant of product specificity in this CGTase and may be applied to the rational design of saccharide-producing enzymes.

GH10 XynA is the main xylanase identified in the crude enzymatic extract of Paenibacillus sp. A59 when grown on xylan or lignocellulosic biomass.

A novel bacterial isolate with polysaccharides degrading activity was identified as Paenibacillus sp., and named Paenibacillus sp. A59. Even though it is a strict mesophile, optimal xylanase activity of the crude enzymatic extract was achieved between 50°C and 70°C and more than 60% of the activity was retained after incubation for 48h at 50°C, indicating thermotolerance of the enzymes involved. The extract was also active on pre-treated sugarcane residue (SCR) and wheat straw, releasing xylobiose and xylose as the main products, therefore confirming its predominantly xylanolytic activity. By zymograms and mass spectrometry of crude enzymatic extracts of xylan or SCR cultures, a 32kDa GH10 beta- 1,4- endoxylanase with xylanase and no CMCase activity was identified. We named this enzyme XynA and it was the only xylanase identified under both conditions assayed, suggesting that it is a good candidate for recombinant expression and evaluation in hemicelluloses deconstruction applications. Also, a protein with two S-layer homology domains (SLH) and a large uncharacterized C-terminal domain as well as an ABC substrate binding protein were identified in crude extracts of SCR cultures. We propose that Paenibacillus sp. A59 uses a system similar to anaerobic and other Gram positive bacteria, with SLH-domain proteins anchoring polysaccharide-degrading enzymes close to the membrane and the substrate binding protein assisting translocation of simple sugars to the cell interior.

Screening of endoglucanase-producing bacteria in the saline rhizosphere of Rhizophora mangle.

In screening the culturable endoglucanase-producing bacteria in the rhizosphere of Rhizophora mangle, we found a prevalence of genera Bacillus and Paenibacillus. These bacteria revealed different activities in endoglucolysis and biofilm formation when exposed to specific NaCl concentrations, indicating modulated growth under natural variations in mangrove salinity.

A glucuronoxylan-specific xylanase from a new Paenibacillus favisporus strain isolated from tropical soil of Brazil.

A new xylanolytic strain, Paenibacillus favisporus CC02-N2, was isolated from sugarcane plantation fields in Brazil. The strain had a xylan-degrading system with multiple enzymes, one of which, xylanase Xyn30A, was identified and characterized. The enzyme is a single-domain xylanase belonging to family 30 of the glycosyl hydrolases (GH30). Xyn30A shows high activity on glucuronoxylans, with a Vmax of 267.2 U mg⁻¹, a Km of 4.0 mg/ml, and a kcat of 13,333 min⁻¹ on beechwood xylan, but it does not hydrolyze arabinoxylans. The three-dimensional structure of Xyn30A consists of a common (ß/α)8 barrel linked to a side-chain-associated ß-structure, similar to previously characterized GH30 xylanases. The hydrolysis products from glucuronoxylan were methylglucuronic-acid-substituted xylooligomers (acidic xylooligosaccharides). The enzyme bound to insoluble xylan but not to crystalline cellulose. Our results suggest a specific role for Xyn30A in xylan biodegradation in natural habitats. The enzyme is a good candidate for the production of tailored xylooligosaccharides for use in the food industry and in the biotechnological transformation of biomass.

Production of enzymes by Paenibacillus chitinolyticus and Paenibacillus ehimensis to obtain chitooligosaccharides.

Obtaining oligosaccharides from chitosan has been the focus of several studies in the pharmaceutical, chemical, food, and medical areas, due to their functional properties. Here, we evaluated the production potential of biologically functional chitooligosaccharides using enzymes extracts produced by Paenibacillus chitinolyticus and Paenibacillus ehimensis. After 48 h of fermentation, these microorganisms were able to produce chitosanases, which generated oligomers with a degree of polymerization between dimers and hexamers. The maximum conversion of chitosan to oligomers was 99.2 %, achieved after 12 h incubation of chitosan with enzymes produced by P. ehimensis. The chitooligosaccharides generated were capable of scavenging the 2,2-diphenyl-1-picrylhydrazyl radical, reaching a maximum scavenging rate of 61 and 39 % when produced with P. ehimensis and P. chitinolyticus enzymes, respectively. The use of these enzymes in the crude form could facilitate their use in industrial applications.

Additive effect of single amino acid replacements on the kinetic stability of ß-glucosidase B.

Previously, we applied in vitro evolution to generate the thermoresistant triple mutant H62R/N223Y/M319I of ß-glucosidase B (BglB) from Paenibacillus polymyxa. In order to dissect the energetic contributions to protein stabilization achieved by these mutations, we measured the kinetic constants of the heat denaturation of wild type BglB, the triple mutant and the three single mutants (H62R, N223Y, M319I) by circular dichroism at various temperatures. Our results show that all four mutants delayed the denaturation process. Based on the Transition State theory, the increase of the activation barrier for the thermal denaturation of the triple mutant (ΔΔG ( N→TS )) is equivalent to that produced by the sum of the contributions from the three single mutants, whose C ( ß ) s are located at least 18 Å apart. This analysis provides a formal demonstration of the generally accepted idea that protein thermal stability can be increased through sequential addition of individual mutations. Each of the mutations described here contribute in part to the overall effect, which in this case affects the unfolding barrier.

Thermal denaturation of ß-glucosidase B from Paenibacillus polymyxa proceeds through a Lumry-Eyring mechanism.

ß-glucosidase B (BglB), 1,4-ß-D: -glucanohydrolase, is an enzyme with various technological applications for which some thermostable mutants have been obtained. Because BglB denatures irreversibly with heating, the stabilities of these mutants are assessed kinetically. It, therefore, becomes relevant to determine whether the measured rate constants reflect one or several elementary kinetic steps. We have analyzed the kinetics of heat denaturation of BglB from Paenibacillus polymyxa under various conditions by following the loss of secondary structure and enzymatic activity. The denaturation is accompanied by aggregation and an initial reversible step at low temperatures. At T ≥ T ( m ), the process follows a two-state irreversible mechanism for which the kinetics does not depend on the enzyme concentration. This behavior can be explained by a Lumry-Eyring model in which the difference between the rates of the irreversible and the renaturation steps increases with temperature. Accordingly, at high scan rates (≥1 °C min(-1)) or temperatures (T ≥ T ( m )), the measurable activation energy involves only the elementary step of denaturation.

Metalloprotease production by Paenibacillus larvae during the infection of honeybee larvae.

American foulbrood is a bacterial disease of worldwide distribution that affects larvae of the honeybee Apis mellifera. The causative agent is the Gram-positive, spore-forming bacterium Paenibacillus larvae. Several authors have proposed that P. larvae secretes metalloproteases that are involved in the larval degradation that occurs after infection. The aim of the present work was to evaluate the production of a metalloprotease by P. larvae during larval infection. First, the complete gene encoding a metalloprotease was identified in the P. larvae genome and its distribution was evaluated by PCR in a collection of P. larvae isolates from different geographical regions. Then, the complete gene was amplified, cloned and overexpressed, and the recombinant metalloprotease was purified and used to generate anti-metalloprotease antibodies. Metalloprotease production was evaluated by immunofluorescence and fluorescence in situ hybridization. The gene encoding a P. larvae metalloprotease was widely distributed in isolates from different geographical origins in Uruguay and Argentina. Metalloprotease was detected inside P. larvae vegetative cells, on the surface of P. larvae spores and secreted to the external growth medium. Its production was also confirmed in vivo, during the infection of honeybee larvae. This protein was able to hydrolyse milk proteins as described for P. larvae, suggesting that could be involved in larval degradation. This work contributes to the knowledge of the pathogenicity mechanisms of a bacterium of great economic significance and is one step in the characterization of potential P. larvae virulence factors.

Paenibacillus larvae enolase as a virulence factor in honeybee larvae infection.

Paenibacillus larvae is a gram-positive spore-forming bacteria, causative agent of American Foulbrood (AFB), a severe disease affecting larvae of the honeybee Apis mellifera. In an attempt to detect potential virulence factors secreted by P. larvae, we identified an enolase among different secreted proteins. Although this protein is a cytosolic enzyme involved in glycolytic pathways, it has been related to virulence. The aim of the present work was to evaluate its role during the infection of honeybee larvae. Toxicity assays showed that enolase was highly toxic and immunogenic to honeybee larvae. Its production was detected inside P. larvae vegetative cells, on the surface of P. larvae spores and secreted to the external growth medium. P. larvae enolase production was also confirmed in vivo, during the infection of honeybee larvae. This protein was able to hydrolyze milk proteins as described for P. larvae, suggesting that could be involved in larval degradation, maybe through the plasmin(ogen) system. These results suggest that P. larvae enolase may have a role in virulence and could contribute to a general insight about insect-pathogen interaction mechanisms.

Identification of ß-propeller phytase-encoding genes in culturable Paenibacillus and Bacillus spp. from the rhizosphere of pasture plants on volcanic soils.

Phytate is one of the most abundant sources of organic phosphorus (P) in soils, but must be mineralized by phytase-producing bacteria to release P for plant uptake. Microbial inoculants based on Bacillus spp. have been developed commercially, but few studies have evaluated the ecology of these bacteria in the rhizosphere or the types of enzymes that they produce. Here, we studied the diversity of aerobic endospore-forming bacteria (EFB) with the ability to mineralize phytate in the rhizosphere of pasture plants grown in volcanic soils of southern Chile. PCR methods were used to detect candidate phytase-encoding genes and to identify EFB bacteria that carry these genes. This study revealed that the phytate-degrading EFB populations of pasture plants included species of Paenibacillus and Bacillus, which carried genes encoding ß-propeller phytase (BPP). Assays of enzymatic activity confirmed the ability of these rhizosphere isolates to degrade phytate. The phytase-encoding genes described here may prove valuable as molecular markers to evaluate the role of EFB in organic P mobilization in the rhizosphere.

Secreted and immunogenic proteins produced by the honeybee bacterial pathogen, Paenibacillus larvae.

American Foulbrood is a severe disease affecting larvae of honeybee Apis mellifera, causing significant decrease in the honeybee population, beekeeping industries and agricultural production. In spite of its importance, little is known about the virulence factors secreted by Paenibacillus larvae during larval infection. The aim of the present work was to perform a first approach to the identification and characterization of P. larvae secretome. P. larvae secreted proteins were analyzed by SDS-PAGE and identified by MALDI-TOF. Protein toxicity was evaluated using an experimental model based on feeding of A. mellifera larvae and immunogenicity was evaluated by Western blot, using an antiserum raised against cells and spores of P. larvae. Ten different proteins were identified among P. larvae secreted proteins, including proteins involved in transcription, metabolism, translation, cell envelope, transport, protein folding, degradation of polysaccharides and motility. Although most of these proteins are cytosolic, many of them have been previously detected in the extracellular medium of different Bacillus spp. cultures and have been related to virulence. The secreted proteins resulted highly toxic and immunogenic when larvae were exposed using an experimental model. This is the first description of proteins secreted by the honeybee pathogen P. larvae. This information may be relevant for the elucidation of bacterial pathogenesis mechanisms.

Characterization of secreted proteases of Paenibacillus larvae, potential virulence factors involved in honeybee larval infection.

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), the most severe bacterial disease that affects honeybee larvae. AFB causes a significant decrease in the honeybee population affecting the beekeeping industry and agricultural production. After infection of larvae, P. larvae secretes proteases that could be involved in the pathogenicity. In the present article, we present the secretion of different proteases by P. larvae. Inhibition assays confirmed the presence of metalloproteases. Two different proteases patterns (PP1 and PP2) were identified in a collection of P. larvae isolates from different geographic origin. Forty nine percent of P. larvae isolates showed pattern PP1 while 51% exhibited pattern PP2. Most isolates belonging to genotype ERIC I - BOX A presented PP2, most isolates belonging to ERIC I - BOX C presented PP1 although relations were not significant. Isolates belonging to genotypes ERIC II and ERIC III presented PP2. No correlation was observed between the secreted proteases patterns and geographic distribution, since both patterns are widely distributed in Uruguay. According to exposure bioassays, isolates showing PP2 are more virulent than those showing PP1, suggesting that difference in pathogenicity could be related to the secretion of proteases.