Jui-Hsing Chou

Proof that Burkholderia Strains Form Effective Symbioses with Legumes: a Study of Novel Mimosa-Nodulating Strains from South America

ABSTRACT Twenty Mimosa-nodulating bacterial strains from Brazil and Venezuela, together with eight reference Mimosa-nodulating rhizobial strains and two other β-rhizobial strains, were examined by amplified rRNA gene restriction analysis. They fell into 16 patterns and formed a single cluster together with the known β-rhizobia, Burkholderia caribensis, Burkholderia phymatum, and Burkholderia tuberum. The 16S rRNA gene sequences of 15 of the 20 strains were determined, and all were shown to belong to the genus Burkholderia; four distinct clusters could be discerned, with strains isolated from the same host species usually clustering very closely. Five of the strains (MAP3-5, Br3407, Br3454, Br3461, and Br3469) were selected for further studies of the symbiosis-related genes nodA, the NodD-dependent regulatory consensus sequences (nod box), and nifH. The nodA and nifH sequences were very close to each other and to those of B. phymatum STM815, B. caribensis TJ182, and Cupriavidus taiwanensis LMG19424 but were relatively distant from those of B. tuberum STM678. In addition to nodulating their original hosts, all five strains could also nodulate other Mimosa spp., and all produced nodules on Mimosa pudica that had nitrogenase (acetylene reduction) activities and structures typical of effective N2-fixing symbioses. Finally, both wild-type and green fluorescent protein-expressing transconjugant strains of Br3461 and MAP3-5 produced N2-fixing nodules on their original hosts, Mimosa bimucronata (Br3461) and Mimosa pigra (MAP3-5), and hence this confirms strongly that Burkholderia strains can form effective symbioses with legumes.

Burkholderia spp. are the most competitive symbionts of Mimosa, particularly under N‐limited conditions

Bacteria isolated from Mimosa nodules in Taiwan, Papua New Guinea, Mexico and Puerto Rico were identified as belonging to either the alpha- or beta-proteobacteria. The beta-proteobacterial Burkholderia and Cupriavidus strains formed effective symbioses with the common invasive species Mimosa diplotricha, M. pigra and M. pudica, but the alpha-proteobacterial Rhizobium etli and R. tropici strains produced a range of symbiotic phenotypes from no nodulation through ineffective to effective nodulation, depending on Mimosa species. Competition studies were performed between three of the alpha-proteobacteria (R. etli TJ167, R. tropici NGR181 and UPRM8021) and two of the beta-rhizobial symbionts (Burkholderia mimosarum PAS44 and Cupriavidus taiwanensis LMG19424) for nodulation of these invasive Mimosa species. Under flooded conditions, B. mimosarum PAS44 out-competed LMG19424 and all three alpha-proteobacteria to the point of exclusion. This advantage was not explained by initial inoculum levels, rates of bacterial growth, rhizobia-rhizobia growth inhibition or individual nodulation rate. However, the competitive domination of PAS44 over LMG19424 was reduced in the presence of nitrate for all three plant hosts. The largest significant effect was for M. pudica, in which LMG19424 formed 57% of the nodules in the presence of 0.5 mM potassium nitrate. In this host, ammonium also had a similar, but lesser, effect. Comparable results were also found using an N-containing soil mixture, and environmental N levels are therefore suggested as a factor in the competitive success of the bacterial symbiont in vivo.

Burkholderia sabiae sp. nov., isolated from root nodules of Mimosa caesalpiniifolia

Two rhizobial strains, Br3407(T) and Br3405, were isolated from nitrogen-fixing nodules on the roots of Mimosa caesalpiniifolia, a legume tree native to Brazil. On the basis of 16S rRNA gene sequence similarities, both strains were shown previously to belong to the genus Burkholderia. A polyphasic approach, including DNA-DNA hybridizations, pulsed-field gel electrophoresis of whole-genome DNA profiles, whole-cell protein analyses, fatty acid methyl ester analysis and extensive biochemical characterization, was used to clarify the taxonomic position of these strains further; the strains are here classified within a novel species, for which the name Burkholderia sabiae sp. nov. is proposed. The type strain is strain Br3407(T) (=LMG 24235(T) =BCRC 17587(T)).

Burkholderia diazotrophica sp. nov., isolated from root nodules of Mimosa spp.

Five strains, JPY461(T), JPY359, JPY389, DPU-3 and STM4206 were isolated from nitrogen-fixing nodules on the roots of Mimosa spp. and their taxonomic positions were investigated using a polyphasic approach. All five strains grew at 15-40 °C (optimum, 30-37 °C), at pH 4.0-8.0 (optimum, pH 6.0-7.0) and with 0-1 % (w/v) NaCl [optimum, 0 % (w/v)]. On the basis of 16S rRNA gene sequence analysis, a representative strain (JPY461(T)) showed 97.2 % sequence similarity to the closest related species Burkholderia acidipaludis SA33(T), a similarity of 97.2 % to Burkholderia terrae KMY02(T), 97.1 % to Burkholderia phymatum STM815(T) and 97.1 % to Burkholderia hospita LMG 20598(T). The predominant fatty acids of the five novel strains were summed feature 2 (comprising C(16 : 1) iso I and/or C(14 : 0) 3-OH), summed feature 3 (comprising C(16 : 1)ω7c and/or C(16 : 1)ω6c), C(16 : 0) , C(16 : 0) 3-OH, C(17 : 0) cyclo, C(18 : 1)ω7c and C(19 : 0) cyclo ω8c. The major isoprenoid quinone was Q-8 and the DNA G+C content of the strains was 63.0-65.0 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid, an unidentified aminolipid and several unidentified phospholipids. The DNA-DNA relatedness of the novel strain with respect to recognized species of the genus Burkholderia was less than 54 %. On the basis of 16S rRNA and recA gene sequence similarities, chemotaxonomic and phenotypic data, the five strains represent a novel species in the genus Burkholderia, for which the name Burkholderia diazotrophica sp. nov. is proposed with the type strain, JPY461(T) ( = LMG 26031(T) = BCRC 80259(T) = KCTC 23308(T)).

Burkholderia symbiotica sp. nov., isolated from root nodules of Mimosa spp. native to north-east Brazil

Four strains, designated JPY-345(T), JPY-347, JPY-366 and JPY-581, were isolated from nitrogen-fixing nodules on the roots of two species of Mimosa, Mimosa cordistipula and Mimosa misera, that are native to North East Brazil, and their taxonomic positions were investigated by using a polyphasic approach. All four strains grew at 15-43 °C (optimum 35 °C), at pH 4-7 (optimum pH 5) and with 0-2 % (w/v) NaCl (optimum 0 % NaCl). On the basis of 16S rRNA gene sequence analysis, strain JPY-345(T) showed 97.3 % sequence similarity to the closest related species Burkholderia soli GP25-8(T), 97.3 % sequence similarity to Burkholderia caryophylli ATCC25418(T) and 97.1 % sequence similarity to Burkholderia kururiensis KP23(T). The predominant fatty acids of the strains were C(18 : 1)ω7c (36.1 %), C(16 : 0) (19.8 %) and summed feature 3, comprising C(16 : 1)ω7c and/or C(16 : 1)ω6c (11.5 %). The major isoprenoid quinone was Q-8 and the DNA G+C content of the strains was 64.2-65.7 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several uncharacterized aminophospholipids and phospholipids. DNA-DNA hybridizations between the novel strain and recognized species of the genus Burkholderia yielded relatedness values of <51.8 %. On the basis of 16S rRNA and recA gene sequence similarities and chemotaxonomic and phenotypic data, the four strains represent a novel species in the genus Burkholderia, for which the name Burkholderia symbiotica sp. nov. is proposed. The type strain is JPY-345(T) (= LMG 26032(T) = BCRC 80258(T) = KCTC 23309(T)).

Tenacibaculum aiptasiae sp. nov., isolated from a sea anemone Aiptasia pulchella.

A novel bacterial strain, designated a4T, isolated from a sea anemone (Aiptasia pulchella) in Taiwan, was characterized using a polyphasic taxonomic approach. Strain a4T was aerobic, Gram-negative, pale-yellow-pigmented and rod-shaped. It grew optimally at 30-35 degrees C, in the presence of 3-4 % (w/v) NaCl and at pH 8.0. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain belonged to the genus Tenacibaculum (family Flavobacteriaceae, phylum Bacteroidetes). The closest neighbours were Tenacibaculum lutimaris TF-26T (97.6 % similarity) and Tenacibaculum aestuarii SMK-4T (97.7 % similarity). The novel isolate could be distinguished from all Tenacibaculum species by several phenotypic characteristics. The major fatty acids were summed feature 3 (comprising C16 : 1 omega 7c and/or iso-C15 : 0 2-OH, 19.6 %), iso-C15 : 0 (12.9 %), iso-C16 : 0 3-OH (10.2 %), iso-C17 : 0 3-OH (9.9 %) and iso-C15 : 1 (9.5 %). The DNA G+C content was 35.0 mol%. Hence, genotypic and phenotypic data demonstrate that strain a4(T) should be classified as a representative of a novel species in the genus Tenacibaculum, for which the name Tenacibaculum aiptasiae sp. nov. is proposed. The type strain is a4T (=BCRC 17655T =LMG 24004T).

Sphingomonas formosensis sp. nov., a polycyclic aromatic hydrocarbon-degrading bacterium isolated from agricultural soil

In the present study, a yellow-pigmented, Gram-negative, short rod-shaped novel bacterium that was capable of degrading a wide range of polycyclic aromatic hydrocarbons (naphthalene, phenanthrene and pyrene) was isolated from agricultural soil located in Yunlin County, Taiwan. Comparative 16S rRNA gene sequence analysis positioned the novel strain in the genus Sphingomonas as an independent lineage adjacent to a subclade containing Sphingomonas fennica K101(T), Sphingomonas histidinilytica UM2(T), Sphingomonas wittichii RW1(T) and Sphingomonas haloaromaticamans A175(T). 16S rRNA gene sequence analysis of strain CC-Nfb-2(T) showed highest sequence similarity to S. fennica K101(T) (96.2%), S. histidinilytica UM2(T) (96.1%), S. wittichii RW1(T) (95.9%), S. haloaromaticamans A175(T) (95.7%), and Sphingobium ummariense RL-3(T) (94.7%); lower sequence similarities were observed with strains of all other Sphingomonas species. The strain contained phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid and diphosphatidylglycerol. The predominant fatty acids were summed feature 8 (C(18:1)ω7c and/or C(18:1)ω6c) C(16:0) and 11-methyl C(18:1)ω7c; C(14:0) 2-OH was the major 2-hydroxy fatty acid. Previously, these lipids have been found to be characteristic of members of the genus Sphingomonas. The serine palmitoyl transferase gene (spt) was also detected and sphingolipid synthesis was confirmed. The predominant isoprenoid quinone system was ubiquinone (Q-10) and the isolate contained sym-homospermidine as the major polyamine. The DNA G+C content of the isolate was 62.8±0.8 mol%. On the basis of chemotaxonomic, phenotypic and phylogenetic data, strain CC-Nfb-2(T) represents a novel species within the genus Sphingomonas, for which the name Sphingomonas formosensis sp. nov. is proposed; the type strain is CC-Nfb-2(T) (=BCRC 80272(T)=DSM 24164(T)).

Luteimonas aquatica sp. nov., isolated from fresh water from Southern Taiwan

A yellow-pigmented bacterial strain, designated RIB1-20(T), isolated from fresh water was investigated by means of a polyphasic taxonomic approach. The cells were Gram-negative, rod-shaped and non-spore-forming. Phylogenetic analyses with the 16S rRNA gene sequence showed that the strain formed a monophyletic branch towards the periphery of the evolutionary radiation occupied by the genus Luteimonas, its two closest neighbours being Luteimonas composti CC-YY255(T) (96.1% sequence similarity) and Luteimonas mephitis B1953/27.1(T) (95.8%). Strain RIB1-20(T) was clearly distinguished from both of those type strains using phylogenetic analysis, DNA-DNA hybridization, fatty acid composition data and a range of physiological and biochemical characteristics. It is evident from the genotypic and phenotypic data that strain RIB1-20(T) represents a novel species of the genus Luteimonas, for which the name Luteimonas aquatica sp. nov. is proposed. The type strain is RIB1-20(T) (=BCRC 17731(T) =LMG 24212(T)).

Comamonas composti sp nov., isolated from food waste compost

A bacterial strain designated YY287(T), isolated from food waste compost, was investigated by polyphasic taxonomic approach. The cells were rod-shaped, Gram-negative, non-pigmented, non-spore-forming and non-fermentative. Phylogenetic analyses using the 16S rRNA gene sequence showed that the strain formed a monophyletic branch towards the periphery of the evolutionary radiation occupied by the genus Comamonas; its closest neighbours were the type strains Comamonas testosteroni DSM 50244(T) (96.5%), Comamonas terrigena DSM 7099(T) (95.4%), Comamonas odontotermitis Dant 3-8(T) (95.2%) and Comamonas koreensis KCTC 12005(T) (94.6%). Strain YY287(T) was clearly distinguished from all of these strains using phylogenetic analysis, DNA-DNA hybridization, fatty acid composition data and a range of physiological and biochemical characteristics. The major fatty acids were 16:0 (33%), 18:1omega7c (13%) and summed feature 3 (16:1omega7c and/or 15:0 iso 2-OH; 41%). The DNA G+C content of the genomic DNA was 62.8 mol%. It is evident from the genotypic and phenotypic data that strain YY287(T) represents a novel species in the genus Comamonas, for which the name Comamonas composti sp. nov. is proposed. The type strain is YY287(T) (=BCRC 17659(T)=LMG 24008(T)).

Azonexus hydrophilus sp. nov., a nifH gene-harbouring bacterium isolated from freshwater

Three Gram-negative, non-pigmented, rod-shaped, facultatively aerobic bacterial strains, designated d8-1T, d8-2 and IMCC1716, were isolated from a freshwater spring sample and a eutrophic freshwater pond. Based on characterization using a polyphasic approach, the three strains showed highly similar phenotypic, physiological and genetic characteristics. All of the strains harboured the nitrogenase gene nifH, but nitrogen-fixing activities could not be detected in nitrogen-free culture media. The three strains shared 99.6-99.7 % 16S rRNA gene sequence similarity and showed 89-100 % DNA-DNA relatedness, suggesting that they represent a single genomic species. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains d8-1T, d8-2 and IMCC1716 formed a monophyletic branch in the periphery of the evolutionary radiation occupied by the genus Azonexus. Their closest neighbours were Azonexus caeni Slu-05T (96.7-96.8 % similarity) and Azonexus fungiphilus BS5-8T (96.3-96.6 %). The DNA-DNA relatedness of the novel strains to these two species of the genus Azonexus was less than 70 %. The isolates could also be differentiated from recognized members of the genus Azonexus on the basis of phenotypic and biochemical characteristics. It is evident, therefore, that the three strains represent a novel species of the genus Azonexus, for which the name Azonexus hydrophilus sp. nov. is proposed. The type strain is d8-1T (=LMG 24005T=BCRC 17657T).