b-Chitin has important ecological and physiological roles and potential for widespread applications, but the characterization of chitin-related enzymes from b-chitin producers was rarely reported. Querying against the Tara Oceans Gene Atlas, 4,939 chitin-related unique sequences from 12 Pfam accessions were found in Bacillariophyta metatranscriptomes. Putative chitin synthase (CHS) sequences are decreasingly present in Crustacea (39%), Stramenopiles (16%) and Insecta (14%) from the Marine Atlas of Tara Oceans Unigenes version 1 Metatranscriptomes (MATOUv11T) database. A CHS gene from the model diatom Thalassiosira pseudonana (Thaps3_J4413, designated TpCHS1) was identified. Homology analysis of TpCHS1 in Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP), PhycoCosm, and the PLAZA diatom omics data set showed that Mediophyceae and Thalassionemales species were potential new b-chitin producers besides Thalassiosirales. TpCHS1 was overexpressed in Saccharomyces cerevisiae and Phaeodactylum tricornutum. In transgenic P. tricornutum lines, TpCHS1-eGFP localizes to the Golgi apparatus and plasma membrane and predominantly accumulates in the cleavage furrow during cell division. Enhanced TpCHS1 expression could induce abnormal cell morphology and reduce growth rates in P. tricornutum, which might be ascribed to the inhibition of the G2/M phase. S. cerevisiae was proved to be a better system for expressing large amounts of active TpCHS1, which effectively incorporates UDP-N-acetylglucosamine in radiometric in vitro assays. Our study expands the knowledge on chitin synthase taxonomic distribution in marine eukaryotic microbes, and is the first to collectively characterize an active marine diatom CHS which may play an important role during cell division.

Rhizobia nodulating native Astragalus and Oxytropis spp. in Northern Europe are not well-studied. In this study, we isolated bacteria from nodules of four Astragalus spp. and two Oxytropis spp. from the arctic and subarctic regions of Sweden and Russia. The phylogenetic analyses were performed by using sequences of three housekeeping genes (16S rRNA, rpoB and recA) and two accessory genes (nodC and nifH). The results of our multilocus sequence analysis (MLSA) of the three housekeeping genes tree showed that all the 13 isolates belonged to the genus Mesorhizobium and were positioned in six clades. Our concatenated housekeeping gene tree also suggested that the isolates nodulating Astragalus inopinatus, Astragalus frigidus, Astragalus alpinus ssp. alpinus and Oxytropis revoluta might be designated as four new Mesorhizobium species. The 13 isolates were grouped in three clades in the nodC and nifH trees. N-15 analysis suggested that the legumes in association with these isolates were actively fixing nitrogen. (C) 2016 Elsevier GmbH. All rights reserved.

Despite the recognition that Rhizobium leguminosarum sv. viciae is the most common symbiont of Vicia species worldwide, there is no available information on rhizobia nodulating native Vicia species in Sweden. We have therefore studied the genetic diversity and phylogeny of root nodule bacteria isolated from V. cracca, V. hirsuta, V. sepium, V. tetrasperma and V. sylvatica growing in different locations in Sweden as well as an isolate each from V. cracca in Tromso, Norway, and V. multicaulis in Siberia, Russia. Out of 25 isolates sampled from the six Vicia species in 12 different locations, there were 14 different genotypes based on the atpD, recA and nodA gene phylogenies. All isolates were classified into Rhizobium leguminosarum sv. viciae group based on the concatenated atpD and recA phylogeny and the nodA phylogeny. (C) 2016 Elsevier GmbH. All rights reserved.

Competitiveness for nodulation is a desirable trait in rhizobia strains used as inoculant. In Sinorhizobium meliloti 1021 mutation in either of the trehalose utilization genes thuA or thuB influences its competitiveness for root colonization and nodule occupancy depending on the interacting host. We have therefore investigated whether mutation in the thuA ortholog in Mesorhizobium loti MAFF303099 also leads to a similar competitive phenotype on its hosts. The results show that M. loti thuA mutant Ml7023 was symbiotically effective and was as competitive as the wild type in colonization and nodule occupancy on Lotus corniculatus and Lotus japonicus. The thuA gene in M. loti was not induced during root colonization or in the infection threads unlike in S. meliloti, despite its induction by trehalose and high osmolarity in in vitro assays.

The thu operon (thuEFGKAB) in Sinorhizobium meliloti codes for transport and utilization functions of the disaccharide trehalose. Sequenced genomes of members of the Rhizobiaceae reveal that some rhizobia and Agrobacterium possess the entire thu operon in similar organizations and that Mesorhizobium loti MAFF303099 lacks the transport (thuEFGK) genes. In this study, we show that this operon is dedicated to the transport and assimilation of maltitol and isomers of sucrose (leucrose, palatinose, and trehalulose) in addition to trehalulose, not only in S. meliloti but also in Agrobacterium tumefaciens. By using genetic complementation, we show that the thuAB genes of S. meliloti, M. loti, and A. tumefaciens are functionally equivalent. Further, we provide both genetic and biochemical evidence to show that these bacteria assimilate these disaccharides by converting them to their respective 3-keto derivatives and that the thuAB genes code for this ketodisaccharide-forming enzyme(s). Formation of 3-ketotrehalose in real time in live S. meliloti is shown through Raman spectroscopy. The presence of an additional ketodisaccharide- forming pathway(s) in A. tumefaciens is also indicated. To our knowledge, this is the first report to identify the genes that code for the conversion of disaccharides to their 3-ketodisaccharide derivatives in any organism.

The ability of wild indigenous legumes to form root nodules capable of biological nitrogen (N 2) fixation has rarely been demonstrated for species in natural ecosystems in large parts of Europe. In order to understand and manage these ecosystems, it is important to demonstrate nodulation across a diverse range of environments, sites and climates. This study surveyed nodulation at a number of sites in Scotland and Sweden. Presence of nodules was noted and nodule structure and indicators of nitrogen fixation capacity were assessed using light and transmission electron microscopy. Soils from several sites were also sampled for carbon and nitrogen analysis. The collections comprised 24 species in Scotland, and 30 taxa in Sweden; 17 of these in common for both countries. Highest species numbers occurred in meadows, farmland margins, hedgerows, roadsides and wasteland. Coastal sites and sites in the mountainous region above the Arctic Circle hosted several rare species. All sampled species had features of N 2-fixing nodules such as pink colour (leghaemoglobin) when dissected and bacteroids. Nodule structure for a number of species is here reported for the first time and presence of the N 2-fixing enzyme nitrogenase is demonstrated in three previously not studied Swedish legume species. North European legumes may make significant contributions to the N-budgets of their ecosystems. Such species (and their symbionts) represent unique germplasm that may be adopted to empower advances in agriculture and conservation aimed at mitigation and adaptation to the effects of climate change.

Very little is known about the genetic diversity and phylogeny of rhizobia nodulating Lotus species in northern temperate regions. We have therefore studied the genetic diversity among a total of 61 root nodule bacteria isolated from Lotus corniculatus and Anthyllis vulneraria from different geographic sites and habitats in Sweden by restriction fragment length polymorphism (RFLP) of the internal transcribed spacer between their 16S rRNA and 23S rRNA (IGS) region. A high diversity consisting of 26 IGS types from 54 L. corniculatus isolates and five IGS types from seven A. vulneraria isolates was found. The 16S rRNA sequences and phylogeny of representatives of the different IGS types showed four interesting exceptions from the majority of the isolates belonging to the genus Mesorhizobium: Two isolates were both found to be closely related to Rhodococcus spp., and two other isolates showed close relationship with Geobacillus spp. and Paenibacillus spp., respectively. The nodA sequences and phylogeny showed that all the isolates, including those not belonging to the traditional rhizobia genera, harbored nodA sequences which were typical of Mesorhizobium loti. Generally, the 16S rRNA and nodA phylogenetic trees were not congruent in that isolates with similar 16S rRNA sequences were associated with isolates harboring different nodA sequences. All the isolates were confirmed to nodulate L. corniculatus in an inoculation test. This is the first report of members of these non-rhizobia genera being able to nodulate legumes, and we suggest that they may have acquired their nodulating properties through lateral gene transfer.

Very little is known about rhizobia that form nodules on Thermopsis spp. We report the isolation of a Mesorhizobium huakuii strain with a unique nodA gene that form nodules on Thermopsis lupinoides in Kamtchatka, Russia. The isolate did not form nodules on Thermopsis chinensis or Thermopsis caroliniana, which suggests it may be host specific.