An extracellular polysaccharide is involved in the aluminum tolerance of Pullulanibacillus sp. CA42, a newly isolated strain from the Chinese water chestnut growing in an actual acid sulfate soil area in Vietnam.

A novel aluminum-tolerant bacterial strain CA42 was isolated from the aquatic plant Eleocharis dulcis, which grows in a highly acidic swamp in Vietnam. Inoculation with CA42 allowed Oryza sativa to grow in the presence of 300 µM AlCl3 at pH 3.5, and biofilms were observed around the roots. Using 16S rRNA gene sequencing analysis, the strain was identified as Pullulanibacillus sp. CA42. This strain secreted large amounts of an extracellular polysaccharide (CA42 EPS). Results from structural analyses on CA42 EPS, namely methylation analysis and nuclear magnetic resonance (NMR), indicated that the chemical structure of CA42 EPS was a glycogen-like α-glucan. Purified CA42 EPS and the commercially available oyster glycogen adsorbed aluminum ions up to 15-30 µmol/g dry weight. Digestion treatments with α-amylase and pullulanase completely attenuated the aluminum ion-adsorbing activity of purified CA42 EPS and oyster glycogen, suggesting that the glycogen-like structure adsorbed aluminum ions and that its branching structure played an important role in its aluminum adsorbing activity. Furthermore, the aluminum tolerance of CA42 cells was attenuated by pullulanase treatment directly on the live CA42 cells. These results suggest that CA42 EPS adsorbs aluminum ions and is involved in the aluminum tolerance mechanism of Pullulanibacillus sp. CA42. Thus, this strain may be a potential plant growth-promoting bacterium in acidic soils. In addition, this study is the first to report a glycogen-like polysaccharide that adsorbs aluminum ions.

Structural analysis of an aluminum-binding capsular polysaccharide produced by Acidocella aluminiidurans strain AL46, an aluminum-tolerant bacterium isolated from plant roots in a highly acidic swamp in actual acid sulfate soil.

Acid sulfate soil is found throughout Southeast Asia, and its strong acidity (pH 2-4) is accompanied by various plant growth-inhibiting factors that can reduce crop production. Among these factors, aluminum elution from the soil due to soil acidity strongly inhibits crop growth and is particularly problematic for agricultural production. We previously isolated Acidocella aluminiidurans strain AL46, a highly aluminum-tolerant bacterium, from the rhizospheres of the grass Panicum repens, inhabiting the acid sulfate soil in Vietnam. To elucidate the mechanism underlying the high aluminum tolerance of strain AL46, in the present study, we investigated the aluminum-adsorption ability of strain AL46 surface polysaccharides and confirmed the strong adsorption ability of the capsular polysaccharide (AL46CPS). Based on this finding, we further determined the chemical structure of AL46CPS using 1H and 13C NMR spectroscopy by conducting 2D DQF-COSY, TOCSY, HSQC, HMBC, and NOESY experiments. AL46CPS comprises a trisaccharide repeating unit with the following structure: [→2)-ß-d-Rhap-(1 â†’ 3)-α-d-Rhap-(1 â†’ 2)-α-d-Rhap-(1→]n. These findings highlight the potential application of AL46CPS as a new aluminum-adsorbing substance in acidic environments to prevent crop loss.

Characterization of the chemical structure and innate immune-stimulating activity of an extracellular polysaccharide from Rhizobium sp. strain M2 screened using a silkworm muscle contraction assay.

We screened innate immunostimulant-producing bacteria using a silkworm muscle contraction assay, and isolated Rhizobium sp. strain M2 from soil. We purified the innate immunostimulant from strain M2, and characterized the chemical structure by nuclear magnetic resonance spectroscopy and chemical analyses. The innate immunostimulant (M2 EPS) comprised glucose, galactose, pyruvic acid, and succinic acid with a molar ratio of 6.8:1.0:0.9:0.4, and had a succinoglycan-like high molecular-weight heteropolysaccharide structure. To determine the structural motif involved in the innate immunostimulating activity, we modified the M2 EPS structure chemically, and found that the activity was increased by removal of the succinic and pyruvic acid substitutions. Strong acid hydrolysis completely inactivated the M2 EPS. Unmasking of the ß-1,3/6-glucan structure of the side-chain by deacylation and depyruvylation may enhance the innate immune-stimulating activity of M2 EPS. These findings suggest that the succinoglycan-like polysaccharide purified from strain M2 has innate immune-stimulating activity, and its glycan structure is necessary for the activity.

Genome sequence determination and metagenomic characterization of a Dehalococcoides mixed culture grown on cis-1,2-dichloroethene.

A Dehalococcoides-containing bacterial consortium that performed dechlorination of 0.20 mM cis-1,2-dichloroethene to ethene in 14 days was obtained from the sediment mud of the lotus field. To obtain detailed information of the consortium, the metagenome was analyzed using the short-read next-generation sequencer SOLiD 3. Matching the obtained sequence tags with the reference genome sequences indicated that the Dehalococcoides sp. in the consortium was highly homologous to Dehalococcoides mccartyi CBDB1 and BAV1. Sequence comparison with the reference sequence constructed from 16S rRNA gene sequences in a public database showed the presence of Sedimentibacter, Sulfurospirillum, Clostridium, Desulfovibrio, Parabacteroides, Alistipes, Eubacterium, Peptostreptococcus and Proteocatella in addition to Dehalococcoides sp. After further enrichment, the members of the consortium were narrowed down to almost three species. Finally, the full-length circular genome sequence of the Dehalococcoides sp. in the consortium, D. mccartyi IBARAKI, was determined by analyzing the metagenome with the single-molecule DNA sequencer PacBio RS. The accuracy of the sequence was confirmed by matching it to the tag sequences obtained by SOLiD 3. The genome is 1,451,062 nt and the number of CDS is 1566, which includes 3 rRNA genes and 47 tRNA genes. There exist twenty-eight RDase genes that are accompanied by the genes for anchor proteins. The genome exhibits significant sequence identity with other Dehalococcoides spp. throughout the genome, but there exists significant difference in the distribution RDase genes. The combination of a short-read next-generation DNA sequencer and a long-read single-molecule DNA sequencer gives detailed information of a bacterial consortium.

Evasion of Innate Immune Responses by the Highly Virulent Cryptococcus gattii by Altering Capsule Glucuronoxylomannan Structure.

Cryptococcus neoformans causes life-threatening diseases mainly in immunosuppressed hosts such as AIDS patients; C. gattii causes disseminated infections even in healthy hosts. To identify the possible molecular mechanisms underlying this difference in virulence, we investigated the survival and histopathology of lung tissue in wild-type and CD4-depleted mice infected with C. neoformans H99 and C. gattii JP02 (the highly virulent strain isolated in Japan); we then compared dendritic cell (DC) cytokine release responses to different cell fractions from these two strains. JP02-infected mice exhibited shorter survival and fewer inflammatory cells in the lung than H99-infected control mice. Depletion of CD4-related cellular immunity reduced survival of H99-infected mice but had no effect on the survival or inflammatory cell infiltration in JP02-infected mice, suggesting that JP02 evades immune detection. To identify the molecule(s) conferring this difference, we measured cytokine production from murine DCs co-cultured with H99 and JP02 in vitro. The levels of inflammatory cytokines from DCs treated with intact JP02 cells, the extracted capsule, secreted extracellular polysaccharides, and purified glucuronoxylomannan (GXM) were markedly lower than those induced by intact H99 cells and corresponding H99 fractions. Structural analysis of GXM indicated that JP02 altered one of two O-acetyl groups detected in the H99 GXM. Deacetylated GXM lost the ability to induce inflammatory cytokine release from DCs, implicating these O-acetyl groups in immune recognition. We conclude that the highly virulent C. gattii processes a structural alteration in GXM that allows this pathogen to evade the immune response and therefore elimination.

Proteomic characterization of seeds from yellow lupin (Lupinus luteus L.).

Yellow lupin (Lupinus luteus L.) is a legume crop containing a large amount of protein in its seeds. In this study, we constructed a seed-protein catalog to provide a foundation for further study of the seeds. A total of 736 proteins were identified in 341 2DE spots by nano-LC-MS/MS. Eight storage proteins were found as multiple spots in the 2DE gels. The 736 proteins correspond to 152 unique proteins as shown by UniRef50 clustering. Sixty-seven of the 152 proteins were associated with KEGG-defined pathways. Of the remaining proteins, 57 were classified according to a GO term. The functions of the remaining 28 proteins have yet to be determined. This is the first yellow lupin seed-protein catalog, and it contains considerably more data than previously reported for white lupin (L. albus L.).

Identification of a low digestibility δ-Conglutin in yellow lupin (Lupinus luteus L.) seed meal for atlantic salmon (Salmo salar L.) by coupling 2D-PAGE and mass spectrometry.

The need of quality protein in the aquaculture sector has forced the incorporation of alternative plant proteins into feeding diets. However, most plant proteins show lower digestibility levels than fish meal proteins, especially in carnivorous fishes. Manipulation of protein content by plant breeding can improve the digestibility rate of plant proteins in fish, but the identification of low digestibility proteins is essential. A reduction of low digestibility proteins will not only increase feed efficiency, but also reduce water pollution. Little is known about specific digestible protein profiles and/or molecular identification of more bioavailable plant proteins in fish diets. In this study, we identified low digestibility L. luteus seed proteins using Atlantic salmon (Salmo salar) crude digestive enzymes in an in vitro assay. Low digestibility proteins were identified by comparing SDS-PAGE banding profiles of digested and non-digested lupin seed proteins. Gel image analysis detected a major 12 kDa protein band in both lupin meal and protein isolate digested products. The 12 kDa was confirmed by 2D-PAGE gels and the extracted protein was analyzed with an ion trap mass spectrometer in tandem mass mode. The MS/MS data showed that the 12 kDa low digestibility protein was a large chain δconglutin, a common seed storage protein of yellow lupin. Comparison of the protein band profiles between lupin meal and protein isolates showed that the isolatation process did not affect the low digestibility of the 12 kDa protein.

Complete mitochondrial genome of the aluminum-tolerant fungus Rhodotorula taiwanensis RS1 and comparative analysis of Basidiomycota mitochondrial genomes.

The complete mitochondrial genome of Rhodotorula taiwanensis RS1, an aluminum-tolerant Basidiomycota fungus, was determined and compared with the known mitochondrial genomes of 12 Basidiomycota species. The mitochondrial genome of R. taiwanensis RS1 is a circular DNA molecule of 40,392 bp and encodes the typical 15 mitochondrial proteins, 23 tRNAs, and small and large rRNAs as well as 10 intronic open reading frames. These genes are apparently transcribed in two directions and do not show syntenies in gene order with other investigated Basidiomycota species. The average G+C content (41%) of the mitochondrial genome of R. taiwanensis RS1 is the highest among the Basidiomycota species. Two introns were detected in the sequence of the atp9 gene of R. taiwanensis RS1, but not in that of other Basidiomycota species. Rhodotorula taiwanensis is the first species of the genus Rhodotorula whose full mitochondrial genome has been sequenced; and the data presented here supply valuable information for understanding the evolution of fungal mitochondrial genomes and researching the mechanism of aluminum tolerance in microorganisms.

Burkholderia bannensis sp. nov., an acid-neutralizing bacterium isolated from torpedo grass (Panicum repens) growing in highly acidic swamps.

Two strains of acid-neutralizing bacteria, E25(T) and E21, were isolated from torpedo grass (Panicum repens) growing in highly acidic swamps (pH 2-4) in actual acid sulfate soil areas of Thailand. Cells of the strains were gram-negative, aerobic, non-spore-forming rods, 0.6-0.8 µm wide and 1.6-2.1 µm long. The strains showed good growth at pH 4.0-8.0 and 17-37 °C. The organisms contained ubiquinone Q-8 as the predominant isoprenoid quinone and C(16 : 0), C(17 : 0) cyclo and C(18 : 1)ω7c as the major fatty acids. Their fatty acid profiles were similar to those reported for other Burkholderia species. The DNA G+C content of the strains was 65 mol%. On the basis of 16S rRNA gene sequence similarity, the strains were shown to belong to the genus Burkholderia. Although the calculated 16S rRNA gene sequence similarity of E25(T) to strain E21 and the type strains of Burkholderia unamae, B. tropica, B. sacchari, B. nodosa and B. mimosarum was 100, 98.7, 98.6, 97.6, 97.4 and 97.3 %, respectively, strains E25(T) and E21 formed a group that was distinct in the phylogenetic tree; the DNA-DNA relatedness of E25(T) to E21 and B. unamae CIP 107921(T), B. tropica LMG 22274(T), B. sacchari LMG 19450(T), B. nodosa LMG 23741(T) and B. mimosarum LMG 23256(T) was 90, 42, 42, 42, 45 and 35 %, respectively. The results of physiological and biochemical tests including whole-cell protein pattern analysis allowed phenotypic differentiation of these strains from previously described Burkholderia species. Therefore, strains E25(T) and E21 represent a novel species, for which the name Burkholderia bannensis sp. nov. is proposed. The type strain is E25(T) ( = NBRC 103871(T)  = BCC 36998(T)).

Burkholderia heleia sp. nov., a nitrogen-fixing bacterium isolated from an aquatic plant, Eleocharis dulcis, that grows in highly acidic swamps in actual acid sulfate soil areas of Vietnam.

Nitrogen-fixing bacteria, strains SA41(T), SA42 and SA53, were isolated from an aquatic plant, Eleocharis dulcis, that grows in highly acidic swamps (pH 2-4) in actual acid sulfate soil areas of Vietnam. The isolates were Gram-negative, aerobic, non-spore-forming, rod-shaped bacteria, having a cell width of 0.6-0.7 microm and a length of 1.5-1.7 microm. They showed good growth between pH 3.0 and 7.0, and between 17 and 37 degrees C. The organisms contained ubiquinone Q-8 as the predominant isoprenoid quinone, and C(16 : 0), C(17 : 0) cyclo, C(18 : 1) omega7c and summed feature 3 (C(16 : 1) omega7c and/or iso-C(15 : 0) 2-OH) as major fatty acids. Their fatty acid profiles are similar to those reported for other Burkholderia species. The DNA G+C content of these strains was 64 mol%. On the basis of 16S rRNA gene sequence similarity, these strains were shown to belong to the genus Burkholderia. Although their calculated 16S rRNA gene sequence similarity values to Burkholderia silvatlantica, Burkholderia mimosarum, Burkholderia ferrariae and Burkholderia tropica were 98.5, 98.2, 98.0 and 97.0 %, respectively, the isolates formed a distinct group in phylogenetic trees, and the DNA-DNA relatedness values of strain SA41(T) to these species were 39, 41, 39 and 33 %, respectively. The results of physiological and biochemical tests, including whole-cell protein pattern analysis, allowed phenotypic differentiation of these strains from the published Burkholderia species. Therefore, strains SA41(T), SA42 and SA53 represent a novel species for which the name Burkholderia heleia sp. nov. is proposed. The type strain is SA41(T) (=NBRC 101817(T)=VTCC-D6-7(T)).

Acidocella aluminiidurans sp. nov., an aluminium-tolerant bacterium isolated from Panicum repens grown in a highly acidic swamp in actual acid sulfate soil area of Vietnam.

An aluminium-tolerant bacterium, strain AL46(T), was isolated from a waterweed, Panicum repens, grown in a highly acidic swamp (pH 3) at an actual acid sulfate soil area of Vietnam. Cells were Gram-negative, aerobic, non-spore-forming, non-motile rods (0.3 microm wide and 1.2-1.6 microm long). 16S rRNA gene sequence analysis indicated that strain AL46(T) belongs to the genus Acidocella, class Alphaproteobacteria. Strain AL46(T) was related most closely to the type strains of Acidocella facilis and Acidocella aminolytica (99.4 and 97.8 % 16S rRNA gene sequence similarity, respectively). Levels of DNA-DNA relatedness between strain AL46(T) and the above type strains were 40 %. The results of physiological and biochemical tests allowed the novel strain to be differentiated phenotypically from the two recognized Acidocella species. Data for predominant cellular fatty acids (cyclopropyl C(19 : 0) and C(18 : 1)), major isoprenoid quinone (Q-10) and DNA G+C content (65.6 mol%) were in accordance with those reported for the genus Acidocella. Therefore, strain AL46(T) is considered to represent a novel species of the genus Acidocella, for which the name Acidocella aluminiidurans sp. nov. is proposed. The type strain is AL46(T) (=NBRC 104303(T) =VTCC-D9-1(T)).

Burkholderia acidipaludis sp. nov., aluminum-tolerant bacteria isolated from Chinese water chestnut (Eleocharis dulcis) growing in highly acidic swamps in South-East Asia.

Two strains of aluminium-tolerant bacteria, SA33(T) and 7A078, were isolated from Chinese water chestnut (Eleocharis dulcis) growing in highly acidic swamps (pH 2-4) in actual acid sulfate soil areas of Vietnam (SA33(T)) and Thailand (7A078). The strains were Gram-negative, aerobic, non-spore-forming rods, 0.6-0.7 mum wide and 1.3-1.7 mum long. These strains showed good growth at pH 3.0-8.0 and 17-37 degrees C. The organisms contained ubiquinone Q-8 as the predominant isoprenoid quinone and C(16 : 0), C(18 : 1) ω 7c and C(17 : 0) cyclo as the major fatty acids. Their fatty acid profiles were similar to those reported for other Burkholderia species. The DNA G+C content of these strains was 64 mol%. On the basis of 16S rRNA gene sequence similarity, the strains were shown to belong to the genus Burkholderia. Although the 16S rRNA gene sequence similarity values calculated for strain SA33(T) to 7A078 and the type strains of Burkholderia kururiensis, B. sacchari and B. tuberum were 100, 97.3, 97.1 and 97.0 %, respectively, strains SA33(T) and 7A078 formed a group that was distinct in the phylogenetic trees; the DNA-DNA relatedness of strain SA33(T) to 7A078 and these three type strains were respectively 90, 47, 46 and 45 %. The results of physiological and biochemical tests, including whole-cell protein pattern analysis, allowed phenotypic differentiation of these strains from described Burkholderia species. Therefore, strains SA33(T) and 7A078 represent a novel species, for which the name Burkholderia acidipaludis sp. nov. is proposed. The type strain is SA33(T) (=NBRC 101816(T) =VTCC-D6-6(T)). Strain 7A078 (=NBRC 103872 =BCC 36999) is a reference strain.

Bacillus trypoxylicola sp. nov., xylanase-producing alkaliphilic bacteria isolated from the guts of Japanese horned beetle larvae (Trypoxylus dichotomus septentrionalis).

Three xylanase-producing alkaliphilic strains, SU1(T), 36AC4 and 36AC6, were isolated from the guts of larvae of the Japanese horned beetle (Trypoxylus dichotomus septentrionalis). The isolates stained Gram-positive and were aerobic, spore-forming, non-motile and rod-shaped and grew optimally at 30 degrees C and pH 9. They contained MK-7 as the major isoprenoid quinone and iso-C(15 : 0), anteiso-C(15 : 0), anteiso-C(17 : 0) and iso-C(17 : 0) as the major fatty acids. The DNA G+C contents of the strains were 37.4-37.7 mol%. On the basis of 16S rRNA gene sequence similarity, these strains were shown to belong to the genus Bacillus. Although their 16S rRNA gene sequence similarity to the type strains of the alkaliphilic species Bacillus pseudalcaliphilus and B. alcalophilus was 97 %, the novel isolates formed a distinct group in the phylogenetic trees and DNA-DNA relatedness values to the type strains of these species were less than 30 %. Results of physiological and biochemical tests, including salt preference, enabled these strains to be differentiated phenotypically from described Bacillus species. Therefore, strains SU1(T), 36AC4 and 36AC6 represent a novel species for which the name Bacillus trypoxylicola sp. nov. is proposed; the type strain is SU1(T) (=NBRC 102646(T) =KCTC 13244(T)).

Mucilaginibacter kameinonensis sp., nov., isolated from garden soil.

An extracellular polysaccharide-producing bacterium, strain SCK(T), was isolated from a soil sample taken from Kameino, Fujisawa, Japan. The isolate was Gram-negative and cells were non-motile, irregular-shaped rods that grew optimally at 25 degrees C and grew between pH 5 and 8. Strain SCK(T) contained MK-7 as the major isoprenoid quinone, iso-C(15:0) and C(16:1)omega7c and/or iso-C(15:0) 2-OH as the major fatty acids and sphingolipids, with d-17:0 as the main dihydrosphingosine. Flexirubin-type pigments were also present. The DNA G+C content was 43.7 mol%. On the basis of 16S rRNA gene sequence analysis, strain SCK(T) was shown to belong to the genus Mucilaginibacter. The 16S rRNA gene sequence similarity between strain SCK(T) and the two type strains of Mucilaginibacter was 93%. The results of physiological and biochemical tests allowed phenotypic differentiation of the strain from published Mucilaginibacter species. Therefore, strain SCK(T) represents a novel species, for which the name Mucilaginibacter kameinonensis sp. nov. is proposed. The type strain is SCK(T) (=NBRC 102645(T) =KCTC 22227(T)).

Curtobacterium ammoniigenes sp. nov., an ammonia-producing bacterium isolated from plants inhabiting acidic swamps in actual acid sulfate soil areas of Vietnam.

The ammonia-producing bacteria B55(T), CA73, SA69 and SA72 were isolated from the waterweeds Ludwigia adscendens (B55(T)) and Eleocharis dulcis (CA73, SA69 and SA72) grown in highly acidic swamps (pH 2-4) in actual acid sulfate soil areas of Vietnam. The isolates were Gram-positive, irregular rod-shaped, non-spore-forming bacteria. On the basis of 16S rRNA gene sequence similarity, strain B55(T) was shown to belong to the genus Curtobacterium of the class Actinobacteria. Chemotaxonomic data (MK-9 as major isoprenoid quinone, d-ornithine as cell-wall diamino acid, acetyl as the acyl type of peptidoglycan) supported the affiliation of all four strains to this genus. Although their 16S rRNA gene sequence similarity was 99 % to species with validly published names within the genus, they formed a group that was distinct in the phylogenetic tree, and DNA-DNA relatedness values to these established species were less than 10 %. The results of physiological and biochemical tests and major fatty acids (cyclohexyl-C(17 : 0), anteiso-C(17 : 0) and cyclohexyl-C(19 : 0)) allowed phenotypic differentiation of these strains from the species of Curtobacterium with validly published names. Therefore, strains B55(T), CA73, SA69 and SA72 represent a novel species, for which the name Curtobacterium ammoniigenes sp. nov. is proposed. The type strain is B55(T) (=NBRC 101786(T)=VTCC D6-11(T)=JCM 14609(T)).

Structural analysis of an extracellular polysaccharide produced by a benzene tolerant bacterium, Rhodococcus sp. 33.

Rhodococcus sp. 33 can tolerate and efficiently degrade various concentrations of benzene, one of the most toxic and prevailing environmental pollutants. This strain produces a large quantity of extracellular polysaccharide (33 EPS), which plays an important role in the benzene tolerance in Rhodococcus sp. 33, especially by helping the cells to survive an initial challenge with benzene. This EPS has been reported to be composed of D-galactose, D-glucose, D-mannose, D-glucuronic acid, and pyruvic acid at a molar ratio of 1:1:1:1:1. To understand the protective effect of 33 EPS, we determined its chemical structure by using 1H and 13C NMR spectroscopy including 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The polysaccharide was shown to consist of tetrasaccharide repeating units with the following structure: [structure: see text].