Paenibacillus granivorans sp. nov., a new Paenibacillus species which degrades native potato starch granules

From a native potato starch-degrading enrichment culture, strain A30 had been isolated and had tentatively been identified as a member of the Bacillus firmus/lentus group (Wijbenga et al. Appl. Microbiol. Biotechnol. 35, 180-184, 1991). In this paper the isolate A30 is further characterized using phylogentic analysis of the 16S rDNA and determination of a number of additional phenotypic characteristics. These data are compared to those of Paenibacillus amylolyticus, P. chibensis, and P. thiaminolyticus. It is concluded that strain A30 is a new Paenibacillus species, for which the name Paenibacillus granivorans is suggested.     

Paenibacillus swuensis sp. nov., a bacterium isolated from soil

Strain DY6^T, a Gram-positive endospore-forming motile rod-shaped bacterium, was isolated from soil in South Korea and characterized to determine its taxonomic position. Phylogenetic analyses based on the 16S rRNA gene sequence of strain DY6^T revealed that strain DY6^T belongs to the genus Paenibacillus in the family Paenibacillaceae in the class Bacilli. The highest degree of sequence similarities of strain DY6^T were found with Paenibacillus gansuensis B518^T (97.9%), P. chitinolyticus IFO 15660^T (95.3%), P. chinjuensis WN9T (94.7%), and P. rigui WPCB173^T (94.7%). Chemotaxonomic data revealed that the predominant fatty acids were anteiso-C_15:0 (38.7%) and C_16:0 (18.0%). A complex polar lipid profile consisted of major amounts of diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. The predominant respiratory quinone was MK-7. Based on these phylogenetic, chemotaxonomic, and phenotypic data, strain DY6^T (=KCTC 33026^T =JCM 18491^T) should be classified as a type strain of a novel species, for which the name Paenibacillus swuensis sp. nov. is proposed.     

Paenibacillus nicotianae sp. nov., isolated from a tobacco sample

A Gram-stain positive, facultative anaerobic endospore-forming bacterium, designated strain YIM h-19^T, was isolated from a tobacco sample. Cells were observed to be motile rods by means of peritrichous flagella and colonies were observed to be convex, yellow, circular and showed catalase-positive and oxidase-negative reactions. Strain YIM h-19^T was able to grow at 4–45 °C, pH 6.0–8.0 and 0–3 % NaCl (w/v). The predominant respiratory quinone was identified as MK-7. Major fatty acids were identified as anteiso-C_15:0, anteiso-C_17:0 and C_16:0. The polar lipids were found to be phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and two unidentified polar lipids. The genomic DNA G+C content was determined to be 54 mol%. 16S rRNA gene sequence analysis showed the strain YIM h-19^T was most closely related to Paenibacillus hordei RH-N24^T and Paenibacillus hunanensis FeL05^T with similarities of 98.30 and 94.64 % respectively. However, DNA–DNA hybridization data indicated that the isolate represented a novel genomic species with the genus Paenibacillus. All data from genotypic and phenotypic analyses support the conclusion that strain YIM h-19^T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus nicotianae sp. nov. is proposed. The type strain is YIM h-19^T (=CGMCC1.12819^T = NRRL B-59112^T).     

Chitinase production by a newly isolated thermotolerant Paenibacillus sp. BISR-047

Chitinase is one of the important mycolytic enzymes with industrial significance, and is produced by a number of organisms, including bacteria. In this study, we describe isolation, characterization and media optimization for chitinase production from a newly isolated thermotolerant bacterial strain, BISR-047, isolated from desert soil and later identified as Paenibacillus sp. The production of extracellularly secreted chitinase by this strain was optimized by varying pH, temperature, incubation period, substrate concentrations, carbon and nitrogen source,etc. The maximum chitinase production was achieved at 45 °C with media containing (in g/l) chitin 2.0, yeast extract 1.5, glycerol 1.0, and ammonium sulphate 0.2 % (media pH 7.0). A three-fold increase in the chitinase production (712 IU/ml) was found at the optimized media conditions at 6 days of incubation. The enzyme showed activity at broad pH (3–10) and temperature (35–100 °C) ranges, with optimal activity displayed at pH 5.0 and 55 °C, respectively. The produced enzyme was found to be highly thermostable at higher temperatures, with a half-life of 4 h at 100 °C.     

Paenibacillus marinisediminis sp. nov., a bacterium isolated from marine sediment

A Gram-negative, nonmotile, endospore-forming, rod-shaped bacterial strain LHW35^T, which belonged to the genus Paenibacillus, was isolated from marine sediment collected from the south coast of the Republic of Korea. A phylogenetic analysis of 16S rRNA gene sequences indicated that strain LHW35^T was most closely related to Paenibacillus taiwanensis G-soil-2-3^T (97.2% similarity). The optimal growth conditions for strain LHW35^T were 37°C, pH 6.0, and 0% (w/v) NaCl. The main isoprenoid quinone was menaquinone-7 (MK-7) and the major polyamine was spermidine. The diamino acid present in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major fatty acids were anteiso-C_15:0 and C_16:0. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, unidentified amino-hospholipids, unidentified phospholipids, and unidentified polar lipids. A DNA-DNA hybridization experiment using the type strain of P. taiwanensis indicated <40% relatedness. The DNA G+C content was 45.0 mol%. Based on these phylogenetic, genomic, and phenotypic analyses, strain LHW35^T should be classified as a novel species within the genus Paenibacillus, for which the name Paenibacillus marinisediminis sp. nov. is proposed. The type strain is LHW35^T (=KACC 16317^T =JCM 17886^T).     

Paenibacillus cucumis sp. nov. isolated from greenhouse soil

Strain CO 4–7^T was isolated from greenhouse soil used for cultivation of cucumbers in Korea. The 16S rRNA gene sequence of strain CO 4–7^T showed the highest sequence similarity with Paenibacillus contaminans CKOBP-6^T (94.2%) among the type strains. Strain CO 4–7^T was a strictly aerobic, Gram-staining-positive, endospore-forming, and motile rodshaped bacterium. Strain CO 4–7^T grew at 10–45°C (optimum, 30°C), at pH 6.0–7.5 (optimum, pH 6.5) and in the presence of 0–5% NaCl (optimum, 0.5%). The DNA G+C content of strain CO 4–7^T was 48.5 mol%. It contained MK-7 as the major isoprenoid quinone and anteiso-C15:0 (51.8%), C16:0 (12.7%), and iso-C16:0 (8.6%) as the major fatty acids. The cell wall contained meso-diaminopimelic acid. Based on evidence from our polyphasic taxonomic study, it was concluded that strain CO 4-7T should be classified as a novel species of the genus Paenibacillus, for which, the name Paenibacillus cucumis sp. nov. is proposed. The type strain is CO 4–7^T (=KACC 17444^T=JCM 19515^T).     

Paenibacillus sp. Strain E18 Bifunctional Xylanase-Glucanase with a Single Catalytic Domain

Xylanases are utilized in a variety of industries for the breakdown of plant materials. Most native and engineered bifunctional/multifunctional xylanases have separate catalytic domains within the same polypeptide chain. Here we report a new bifunctional xylanase (XynBE18) produced by Paenibacillus sp. E18 with xylanase and β-1,3-1,4-glucanase activities derived from the same active center by substrate competition assays and site-directed mutagenesis of xylanase catalytic Glu residues (E129A and E236A). The gene consists of 981 bp, encodes 327 amino acids, and comprises only one catalytic domain that is highly homologous to the glycoside hydrolase family 10 xylanase catalytic domain. Recombinant XynBE18 purified from Escherichia coli BL21(DE3) showed specificity toward oat spelt xylan and birchwood xylan and β-1,3-1,4-glucan (barley β-glucan and lichenin). Homology modeling and molecular dynamic simulation were used to explore structure differences between XynBE18 and the monofunctional xylanase XynE2, which has enzymatic properties similar to those of XynBE18 but does not hydrolyze β-1,3-1,4-glucan. The cleft containing the active site of XynBE18 is larger than that of XynE2, suggesting that XynBE18 is able to bind larger substrates such as barley β-glucan and lichenin. Further molecular docking studies revealed that XynBE18 can accommodate xylan and β-1,3-1,4-glucan, but XynE2 is only accessible to xylan. These results indicate a previously unidentified structure-function relationship for substrate specificities among family 10 xylanases.Cellulose, hemicellulose, and lignin are the major components of plant cell walls. Hemicellulose and lignin provide a protective barrier against enzymatic attack of cellulose (15). Xylan is the major component of hemicellulose, the complete degradation of which requires a multistep process involving xylanases and various xylan debranching enzymes, such as β-xylosidase, acetylxylan-esterase, α-glucuronidase, and α-arabinofuranosidase (5, 28).Xylanases, in conjunction with other enzymes (14), such as cellulases, glucanases, and proteases, are widely used in animal feed, brewing, food processing, and waste treatment, as well as in the pulp and paper industries (25, 32). For example, combined application of xylanase and β-1,3-1,4-glucanase can reduce the intestinal viscosity of feed for higher nutrition availability and improve the filtration rate and extraction yield in the brewing industry (16, 21). The natural diversity of enzymes provides these industries with candidates having bifunctional activity, such as the xylanases from Aspergillus niger (12) and Marasmius sp. (26) that have xylanase and cellulase activities and the bifunctional xylanase-lichenase from Ruminococcus flavefaciens 17 (7). On the other hand, many artificial bifunctional xylanases have been synthesized for more efficient biodegradation of plant fiber (19). Except for the bifunctional xylanase-glucanase from A. niger A-25, which has not been subjected to sequence and structural analysis but is conjectured to have one catalytic domain only based on molecular weight and kinetic analysis (4), all other bi- or multifunctional enzymes have separate catalytic domains with distinct substrate specificities.Corn straw consists mainly of cellulose, hemicellulose, and lignin, and thus, the microorganisms present in corn straw often produce various enzymes such as endoglucanase, cellobiohydrolase, β-glucosidase, xylanase, and so on (33). In the present study, we selected corn ensilage as the microbial source for the isolation of multifunctional xylanases. A Paenibacillus sp. strain with high xylanolytic activity was isolated, and the xylanase gene was cloned. Analysis of the sequence and enzyme properties and kinetics revealed that the xylanase gene encodes a bifunctional xylanase-glucanase with a single catalytic domain. Further homology modeling and molecular dynamic (MD) studies confirmed that the bifunctional protein has a substrate binding cleft large enough to accommodate xylan or β-1,3-1,4-glucan. These results suggest that this enzyme is a new glycoside hydrolase (GH) family 10 (http://www.cazy.org/) bifunctional xylanase-glucanase with a single catalytic domain (3).     

Harvesting of Chlorella vulgaris using a bioflocculant from Paenibacillus sp. AM49

Microbial flocculants for harvesting mass cultured Chlorella vulgaris were screened and that from Paenibacillussp. AM49 was identified as the best. The flocculation efficiency of this bioflocculant increased with the pH within a range of pH 5–11 and was 83%, which was higher than the 72% and 78% produced by aluminum sulfate and polyacrylamide, respectively. The highest flocculation efficiency was with 6.8 mm CaCl₂ as co-flocculant. The bioflocculant from Paenibacillussp. AM49 can be used effectively to harvest C. vulgaris from large-scale cultures.     

Paenibacillus sinensis sp. nov., a nitrogen-fixing species isolated from plant rhizospheres

Two strains HN-1^T and 39 were isolated from rhizospheres of different plants grown in different regions of PR China. The two strains exhibited high nitrogenase activities and possessed almost identical 16S rRNA gene sequences. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between the two strains were 99.9 and 99.8%, respectively, suggesting that they belong to one species. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strains HN-1^T and 39 are the members of the genus Paenibacillus and both strains exhibited 99.5% similarity to Paenibacillus stellifer DSM 14472^T and the both strains represented a separate lineage from all other Paenibacillus species. However, the ANI of type strain HN-1^T with P. stellifer DSM 14472^T was 90.69, which was below the recommended threshold value (<?95–96% ANI). The dDDH showed 42.1% relatedness between strain HN-1^T and P. stellifer DSM 14472^T, which was lower than the recommended threshold value (dDDH?<?70%). The strain HN-1^T contain anteiso-C_15:0 as major fatty acids and MK-7 as predominant isoprenoid quinone. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four aminophospholipids and an unidentified glycolipid. Unlike the most closely related P. stellifer DSM 14472^T, strain HN-1^T or 39 was positive for catalase reaction. Distinct phenotypic and genomic characterisations from previously described taxa support the classification of strains HN-1^T or 39 as representatives of a novel species of the genus Paenibacillus, for which the name Paenibacillus sinensis is proposed, with type strains HN-1^T (=CGMCC 1.18902, JCM 34,620), and reference strain 39 (=CGMCC 1.18879, JCM 34,616), respectively.

     

Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤50°C) was 10°C higher than that for CITase-T3040 (≤40°C); the k(cat)/K(M) value of CITase-598K was approximately two times higher (32.2s(-1)mM(-1)) than that of CITase-T3040 (17.8s(-1)mM(-1)). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.     

Paenibacillus polymyxa,a Jack of all trades

The bacterium Paenibacillus polymyxa is found naturally in diverse niches. Microbiome analyses have revealed enrichment in the genus Paenibacillus in soils under different adverse conditions, which is often accompanied by improved growth conditions for residing plants. Furthermore, Paenibacillus is a member of the core microbiome of several agriculturally important crops, making its close association with plants an interesting research topic. This review covers the versatile interaction possibilities of P. polymyxa with plants and its applicability in industry and agriculture. Thanks to its array of produced compounds and traits, P. polymyxa is likely an efficient plant growth-promoting bacterium, with the potential of biofertilization, biocontrol and protection against abiotic stresses. By contrast, cases of phytotoxicity of P. polymyxa have been described as well, in which growth conditions seem to play a key role. Because of its adjustable character, we propose this bacterial species as an outstanding model for future studies on host–microbe communications and on the manner how the environment can influence these interactions.     

Paenibacillus hemolyticus, the first hemolytic Paenibacillus with growth-promoting activities discovered

Paenibacillus spp. are Gram-positive, facultatively aerobic, bacilli-shaped endospore-forming bacteria. They have been detected in a variety of environments, such as soil, water, forage, insect larvae, and even clinical samples. The strain 139SI (GenBank accession No.: JF825470.1) from three strains of Paenibacillus isolates investigated here was chosen as the type strain of the proposed novel species. The other two similar strain isolates investigated were 140SI (JF825471.1) and 141SI (JQ734548.1). These strains were identified as members of the genus Paenibacillus on the basis of phenotypic characteristics, phylogenetic analysis and 16S rRNA G+C content. Surprisingly, these strains exhibited a strong hemolytic activity on 5% sheep blood agar. Their crude extracts also showed positive growth-promoting activities in colon cancer and Vero cell lines. To our knowledge, this is the first Paenibacillus with hemolytic and growth-promoting activities reported, and the name Paenibacillus hemolyticus for this novel species is proposed. The capability of this novel species in hemolytic and cell growth activities suggests its potential in both clinical and pharmacological implications.     

Molecular cloning and characterization of a novel glucocerebrosidase of Paenibacillus sp. TS12

We report here the molecular cloning and characterization of a glucocerebrosidase [EC 3.2.1.45] from Paenibacillus sp. TS12. The open reading frame of the glucocerebrosidase gene consisted of 2,493 bp nucleotides and encoded 831 amino acid residues. The enzyme exhibited no sequence similarity with a classical glucocerebrosidase belonging to glycoside hydrolase (GH) family 30, but rather showed significant similarity with GH family 3 beta-glucosidases from Clostridium thermocellum, Ruminococcus albus, and Aspergillus aculeateus. The recombinant enzyme, expressed in Escherichia coli BL21(DE3)pLysS, had a molecular weight of 90.7 kDa and hydrolyzed NBD-labeled glucosylceramide, but not galactosylceramide, GM1a or sphingomyelin. The enzyme was most active at pH 6.5, and its apparent Km and Vmax values for NBD-labeled glucosylceramide and p-nitrophenyl-beta-glucopyranoside were 223 microM and 1.60 micromol/min/mg of protein, and 593 microM and 112 micromol/min/mg of protein, respectively. Site-directed mutagenesis indicated that Asp-223 is an essential amino acid for the catalytic reaction and possibly functions a catalytic nucleophile, as in GH family 3 beta-glucosidases. This is the first report of the molecular cloning and characterization of a glucocerebrosidase from a procaryote.     

Paenibacillus beijingensis sp. nov., a nitrogen-fixing species isolated from wheat rhizosphere soil

A novel nitrogen-fixing bacterium, BJ-18^T, was isolated from wheat rhizosphere soil. Strain BJ-18^T was observed to be Gram-positive, facultatively anaerobic, motile and rod-shaped (0.4–0.9 μm × 2.0–2.9 μm). Phylogenetic analysis based on a partial nifH gene sequence and an assay for nitrogenase activity showed its nitrogen-fixing capacity. Phylogenetic analysis based on full 16S rRNA gene sequences suggested that strain BJ-18^T is a member of the genus Paenibacillus. High similarity of 16S rRNA gene sequence was found between BJ-18^T and Paenibacillus peoriae DSM 8320^T (99.05 %), Paenibacillus jamilae DSM 13815^T (98.86 %), Paenibacillus brasiliensis DSM 13188^T (98.55 %), Paenibacillus polymyxa DSM 36^T (98.74 %), Paenibacillus terrae DSM 15891^T (97.99 %) and Paenibacillus kribbensis JCM 11465^T (97.92 %), whereas the similarity was below 96.0 % between BJ-18^T and the other Paenibacillus species. DNA–DNA relatedness between strain BJ-18^T and P. peoriae DSM 8320^T, P. jamilae DSM 13815^T, P. brasiliensis DSM 13188^T, P. polymyxa DSM 36^T, P. kribbensis JCM 11465^T and P. terrae DSM 15891^T was determined to be 43.6 ± 2.7, 34.2 ± 5.3, 47.9 ± 6.6, 36.8 ± 3.5, 27.4 ± 4.3 and 23.6 ± 4.1 % respectively. The DNA G+C content of BJ-18^T was determined to be 45.8 mol %. The major fatty acid was identified as anteiso-C_15:0 (67.1 %). The polar lipids present in strain BJ-18^T were identified as diphosphatidylglycerol, phosphatidyl methylethanolamine, phosphatidylethanolamine and phosphatidylglycerol. The phenotypic and genotypic characteristics, and DNA–DNA relatedness data, suggest that BJ-18^T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus beijingensis sp. nov. (Type strain BJ-18^T=DSM25425^T=CGMCC 1.12045^T) is proposed.     

Characterization of a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39

Aims:  The aims of this study were to identify and characterize the novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39.
Methods and Results:  Strain B39 was closely related to Paenibacillus cookii in 16S rRNA gene sequence. Nonetheless, this isolate can be identified as a novel Paenibacillus sp. with respect to its physiological characteristics, biochemical reactions, and profiles of fatty acid compositions. A cellulase with both CMCase and avicelase activities was secreted from strain B39 and purified by ion-exchange chromatography. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis, the molecular weight of B39 cellulase was determined as 148 kDa, which was much higher than other cellulases currently reported from Paenibacillus species. The enzyme showed a maximum CMCase activity at 60°C and pH 6·5. Addition of 1 mmol l⁻¹ of Ca²⁺ markedly enhanced both CMCase and avicelase activities of the enzyme.
Conclusions:  We have identified and characterized a novel thermophilic Paenibacillus sp. strain B39 which produced a high-molecular weight cellulase with both CMCase and avicelase activities.
Significance and Impact of the Study:  Based on the ability to hydrolyse CMC and avicel, the cellulase produced by Paenibacillus sp. strain B39 would have potential applications in cellulose biodegradation.     

Endoglucanases from Paenibacillus spp. form a new clan in glycoside hydrolase family 5

Endoglucanase (Egl)-producing bacteria from soil samples were screened using insoluble cellulosic substrates as sole carbon sources at alkaline pH (pH 9-10). Four Egls with Avicelase activity at alkaline pH were found in the culture broth of each isolate. The Egl genes of the isolates (all Paenibacillus spp.) were shotgun cloned and sequenced-all had a 1752bp open reading frame (584 amino acids) with a putative signal sequence (33 amino acids), and encoded mature enzymes of 551 amino acids (58,360-58,672Da). The mature enzymes showed a high degree of similarity to each other (>93% identity), with the next closest similarity to Egl3a of a patented strain of Paenibacillus lautus NCIMB 40250 (81.5-87.3% identity). These enzymes showed low similarity to other known Egls with less than 50% identity. A representative recombinant enzyme degraded lichenan, carboxymethylcellulose (CMC), glucomannan, acid or alkaline swollen celluloses, and microcrystalline cellulose (Avicel). The optimal pH and temperature of the recombinant enzyme for degrading CMC and Avicel were pH 6.0-8.5 and 45-55 degrees C, respectively. Egls belong to glycoside hydrolase family 5 and form a distinct clan based on the phylogenetic analysis of their amino acid sequences.     

解木聚糖类芽孢杆菌木葡聚糖酶的分离纯化及酶学性质研究

解木聚糖类芽孢杆菌(Paenibacillus xylanilyticus)发酵液经硫酸铵分级沉淀、HiPrep26/10 Desalting柱脱盐、HiPrepDEAE FF16/10阴离子交换柱、HiPrep 16/60 Sephacryl S-100凝胶柱、HiPrep 16/10 Source 30S阳离子交换柱等,最终纯化出单一组分的木葡聚糖酶,经过SDS-PAGE电泳分析,此木葡聚糖酶相对分子量约为39 kD。该菌所产木葡聚糖酶的最适反应温度是50℃,在60℃以下较稳定;最适反应pH是7.0,在pH5.0-10.0范围内酶活力较为稳定。酶的动力学研究显示Km为65 g/L,Vmax为6.49μmol/min,kcat=10.86 s-1。底物特异性研究表明对木葡聚糖具有较高比活力。酶蛋白经质谱分析,比对结果显示与来源于Paenibacillus pabuli的木葡聚糖酶有较高同源性。本研究为首次报道解木聚糖类芽孢杆菌(P.xylanilyticus)产木葡聚糖酶。