Crystal structures of a putative periplasmic cystine-binding protein from Candidatus Liberibacter asiaticus: insights into an adapted mechanism of ligand binding.

The amino acid-binding receptors, a component of ABC transporters, have evolved to cater to different specificities and functions. Of particular interest are cystine-binding receptors, which have shown broad specificity. In the present study, a putative periplasmic cystine-binding protein from Candidatus Liberibacter asiaticus (CLasTcyA) was characterized. Analysis of the CLasTcyA sequence and crystal structures in the ligand-bound state revealed novel features of CLasTcyA in comparison to related proteins. One of the unique features found in CLasTcyA structure was the positioning of the C-terminal extended loop of one chain very close to the substrate-binding site of the adjacent monomer in the asymmetric unit. The presence of a disulphide bond, unique to Candidatus Liberibacter family, holds the C-terminal extended loop in position. Analysis of the substrate-binding pocket of CLasTcyA suggested a broad specificity and a completely different orientation of the bound substrates in comparison to related protein structures. The open conformation for one of the two chains of the asymmetric unit in the Arg-bound structure revealed a limited open state (18.4°) for CLasTcyA as compared to open state of other related proteins (~ 60°). The strong interaction between Asp126 on helix-α5 of small domain and Arg82 (bigger domain) restricts the degree of opening in ligand-free open state. The dissociation constant of 1.26 µm by SPR and 3.7 µm by MST exhibited low affinity for the cystine. This is the first structural characterization of an l-cystine ABC transporter from plant pathogen and our results suggest that CLasTcyA may have evolved to cater to its specific needs for its survival in the host.

Polyphasic taxonomic characterisation of a novel strain as Pararhizobium haloflavum sp. nov., isolated from soil samples near a sewage treatment tank.

A Gram-stain negative, aerobic, motile and ovoid- to rod-shaped bacteria strain, designated XC0140T, was isolated from soil samples near the sewage treatment tank of a chemical factory in Zhejiang Province, China, and subjected to polyphasic taxonomic investigation. Strain XC0140T grew at 10-37 °C and pH 6.0-9.0 (optimum, 35 °C and pH 7.5) and with 0-17% (w/v) NaCl (optimum, 1%). According to phylogenetic analysis based on 16S rRNA gene sequences, strain XC0140T was assigned to the genus Pararhizobium with high 16S rRNA gene sequence similarity of 95.97% to "Pararhizobium helanshanense CCNWQTX14T", followed by Pararhizobium sphaerophysae CCNWGS0238T (95.95%). Chemotaxonomic analysis showed that strain XC0140T contains ubiquinone-10 as the predominant respiratory quinone and possessed summed feature 8 (comprising C18: 1 ω7c and/or ω6c), 11-methyl C18:1 ω7c, C18: 0 and C16: 0 as predominant forms of fatty acids. The polar lipids of strain XC0140T consisted of seven phospholipids (PL), two aminolipids (AL), one glycolipid (GL) and three unidentified lipids (L1, L2 and L3). The DNA G+C content was 62.7 mol%. Based on the polyphasic taxonomic characterization, strain XC0140T is considered to represent a novel species of the genus Pararhizobium, for which the name Pararhizobium haloflavum sp. nov. is proposed. (type strain XC0140T = MCCC 1K03228T = KCTC 52582T).

Kaistia granuli sp. nov., isolated from anaerobic granules in an upflow anaerobic sludge blanket reactor.

A Gram-negative, chemo-organotrophic, non-motile, non-spore-forming, rod-shaped bacterium (designated strain Ko04(T)) was isolated from anaerobic granules in an upflow anaerobic sludge blanket reactor, and was investigated using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Ko04(T) belongs to the order Rhizobiales in the Alphaproteobacteria. Comparative 16S rRNA gene sequence analysis showed that strain Ko04(T) was most closely related to Kaistia adipata (97.5 %) and that sequence similarities with other species of Rhizobiales with validly published names were less than 92.5 %. The predominant ubiquinone was Q-10 and the major fatty acids were C(18 : 1)omega7c/omega9t/omega12t, C(19 : 0 )cyclo omega8c and C(18 : 0). The G+C content of the genomic DNA of strain Ko04(T) was 67.8 mol%. The level of DNA-DNA relatedness with K. adipata Chj404(T) was 15 %. The results of the genotypic analyses in combination with chemotaxonomic and physiological data demonstrated that strain Ko04(T) represents a novel species within the genus Kaistia, for which the name Kaistia granuli sp. nov. is proposed. The type strain is Ko04(T) (=KCTC 12575(T)=LMG 23410(T)).

Quantitative evaluation of acidity tolerance of root nodule bacteria

Quantification of acidity tolerance in the laboratory may be the first step in rhizobial strain selection for the Amazon region. The present method evaluated rhizobia in Petri dishes with YMA medium at pH 6.5 (control) and 4.5, using scores of 1.0 (sensitive, "no visible" growth) to 4.0 (tolerant, maximum growth). Growth evaluations were done at 6, 9, 12, 15 and 18 day periods. This methods permits preliminary selection of root nodule bacteria from Amazonian soils with statistical precision. Among the 31 rhizobia strains initially tested, the INPA strains 048, 078, and 671 presented scores of 4.0 at both pHs after 9 days of growth. Strain analyses using a less rigorous criterion (growth scores higher than 3.0) include in this highly tolerant group the INPA strains 511, 565, 576, 632, 649, and 658, which grew on the most diluted zone (zone 4) after 9 days. Tolerant strains still must be tested for nitrogen fixation effectiveness, competitiveness or nodules sites, and soil persistence before their recommendations as inoculants

Characterization of tropical tree rhizobia and description of Mesorhizobium plurifarium sp. nov.

A collection of strains isolated from root nodules of Acacia species in Senegal was analysed previously by electrophoresis of total cell protein, auxanographic tests, rRNA-DNA hydridization, 16S rRNA gene sequencing, DNA base composition and DNA-DNA hybridization [de Lajudie, P., Willems, A., Pot, B. & 7 other authors (1994). Int J Syst Bacteriol 44, 715-733]. Strains from Acacia were shown to belong to two groups, Sinorhizobium terangae, and a so-called gel electrophoretic cluster U, which also included some reference strains from Brazil. Further taxonomic characterization of this group using the same techniques plus repetitive extragenic palindromic-PCR and nodulation tests is presented in this paper. Reference strains from Sudan and a number of new rhizobia isolated from nodules of Acacia senegal, Acacia tortilis subsp. raddiana and Prosopis juliflora in Senegal were included. As a result of this polyphasic approach, the creation of a new species, Mesorhizobium plurifarium, is proposed for a genotypically and phenotypically distinct group corresponding to the former cluster U and containing strains isolated from Acacia, Leucaena, Prosopis and Chamaecrista in West Africa (Senegal), East Africa (Sudan) and South America (Brazil). The type strain of Mesorhizobium plurifarium ORS 1032 has been deposited in the LMG collection as LMG 11892.

Replacement of Pro109 by His in TlpA, a thioredoxin-like protein from Bradyrhizobium japonicum, alters its redox properties but not its in vivo functions.

TlpA, the membrane-anchored, thioredoxin-like protein from Bradyrhizobium japonicum, is essential for cytochrome aa3 biogenesis. The periplasmic domain of TlpA was previously shown to have protein thiol:disulfide oxidoreductase activity and reducing properties similar to those of cytoplasmic thioredoxins. Here, we replaced the proline-109 in its active-site sequence C107 V108 P109 C110 by a histidine residue. The resulting active-site motif (CVHC) resembles that of oxidizing thiol:disulfide oxidoreductases such as protein disulfide isomerase (PDI) and DsbA. Indeed, the TlpA variant P109H was by 66 mV more oxidizing than the wild-type protein. Nevertheless, the altered protein was even more efficient in catalyzing the reduction of insulin disulfides by dithiothreitol than the wild-type due to a faster recycling of its catalytically active, reduced form. Cells of B. japonicum expressing only the mutated tlpA gene had the same phenotypes as wild-type cells, suggesting that the change in the redox potential of TlpA was not critical for its in vivo function.

Photosynthetic symbionts of Aeschynomene spp. form a cluster with bradyrhizobia on the basis of fatty acid and rRNA analyses.

The relationship between photosynthetic rhizobia that nodulate 10 Aeschynomene species (Aeschynomene afraspera, Aeschynomene denticulata, Aeschynomene evenia, Aeschynomene indica, Aeschynomene nilotica, Aeschynomene pratensis, Aeschynomene rudis, Aeschynomene scabra, Aeschynomene schimperi, and Aeschynomene sensitiva) and reference strains of the genera Bradyrhizobium, Rhizobium, and Azorhizobium was investigated by analyzing cellular fatty acid methyl esters (FAME) and 16S rRNA sequences. The members of each genus produced very distinct FAME patterns, and the photosynthetic rhizobia formed a subcluster in the Bradyrhizobium cluster. The absence of the cyc C19:0 type of fatty acid in all of the photosynthetic rhizobium strains isolated from 10 Aeschynomene species distinguished these microorganisms from other known rhizobia, including strain BTAi 1, a photosynthetic symbiont of A. indica. We sequenced a 264-base segment of the 16S rRNA genes of selected strains after amplification by the PCR and compared the results with previously published sequences for species of rhizobia and related photosynthetic bacteria. Photosynthetic strains IRBG 2 (from A. afraspera), IRBG 230 (from A. nilotica), and ORS 322 (from A. afraspera) had identical sequences but were distinct from strain BTAi (from A. indica) and from strain IRBG 231 (from A. denticulata), which is similar to the type strain (DNA homology group Ia) of Bradyrhizobium japonicum. Nonphotosynthetic strain IRBG 274 (from A. afraspera) was closely related to Bradyrhizobium elkanii (DNA homology group II). All of the photosynthetic rhizobia clearly fell into the Bradyrhizobium cluster. Although the results of the FAME and 16S rRNA analyses were in excellent agreement, our placement of the photosynthetic rhizobia is in apparent conflict with phenotypic data, as determined by numerical taxonomy (Ladha and So, Int. J. Syst. Bacteriol., in press) which placed the photosynthetic rhizobia in a coherent cluster that is as far from the genus Bradyrhizobium as the genera Rhizobium and Azorhizobium are. While the FAME and 16S rRNA data probably provide a more reliable indication of phylogeny, the degree of phenotypic divergence observed raises questions concerning the polyphasic approach to bacterial systematics.

The NodL and NodJ proteins from Rhizobium and Bradyrhizobium strains are similar to capsular polysaccharide secretion proteins from gram-negative bacteria.

The NodL and NodJ nodulation proteins have been described in different Rhizobium and Bradyrhizobium species. The nodLJ genes belong to the nod regulon. Other genes from this regulon are involved in the biosynthesis and modification of lipo-oligosaccharide molecule(s) which are morphogenic signals when acting on legume roots. It has been proposed that the NodL and NodJ proteins belong to a bacterial inner membrane transport system of small molecules. Nucleotide sequencing of Mudll PR13 insertions in the nodulation region of the symbiotic plasmid from a Rhizobium leguminosarum bv. phaseoli strain CE3 has revealed the presence of nodL and nodJ-related sequences downstream of nodC. Computer nucleotide sequence analysis of the entire NodL and NodJ sequences from R. leguminosarum bv. viciae and Bradyrhizobium japonicum strains show that both proteins are similar to the KpsT and KpsM proteins from Escherichia coli K1 and K5 strains, to the BexB and BexA proteins from Haemophilis influenzae and to the CtrD and CtrC proteins from Neisseria meningitidis, respectively. Except for the NodL and NodJ proteins, all of them have been involved in the mechanism of secretion of polysaccharides in each of their harbouring species. On the basis of the similarity found, we propose that the NodL and the NodJ proteins could be involved in the export of a lipo-oligosaccharide.