Characterization of a Marine Diatom Chitin Synthase Using a Combination of Meta-Omics, Genomics, and Heterologous Expression Approaches.

ß-Chitin has important ecological and physiological roles and potential for widespread applications, but the characterization of chitin-related enzymes from ß-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 (MATOUv1+T) 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 ß-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. IMPORTANCE As the most abundant biopolymer in the oceans, the significance of chitin and its biosynthesis is rarely demonstrated in diatoms, which are the main contributors to the primary productivity of the oceans, ascribed to their huge biomass and efficient photosynthesis. We retrieved genes involved in chitin-based metabolism against the Tara Oceans Gene Atlas to expand our knowledge about their diversity and distribution in the marine environment. Potential new producers of chitin were found from the analysis of various algal transcriptome and genome databases. Heterologous expression confirms that Thalassiosira pseudonana contains an active chitin synthase (CHS) which may play an important role in the cell division process of diatoms. This study provides new insight into CHS geographic and taxonomic distribution in marine eukaryotic microbes, as well as into a new CHS functioning in the biosynthesis of ß-chitin in diatoms.

Diverse Mesorhizobium bacteria nodulate native Astragalus and Oxytropis in arctic and subarctic areas in Eurasia.

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. 15N analysis suggested that the legumes in association with these isolates were actively fixing nitrogen.

Genetic diversity of rhizobia nodulating native Vicia spp. in Sweden.

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 Tromsø, 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.

The trehalose utilization gene thuA ortholog in Mesorhizobium loti does not influence competitiveness for nodulation on Lotus spp.

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 thuEFGKAB operon of rhizobia and agrobacterium tumefaciens codes for transport of trehalose, maltitol, and isomers of sucrose and their assimilation through the formation of their 3-keto derivatives.

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.

Nodulation of Thermopsis lupinoides by a Mesorhizobium huakuii strain with a unique nodA gene in Kamtchatka, Russia.

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.

Genetic diversity of root nodule bacteria nodulating Lotus corniculatus and Anthyllis vulneraria in Sweden.

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.

Lack of trehalose catabolism in Sinorhizobium species increases their nodulation competitiveness on certain host genotypes.

The role of host and bacterial genotypes in determining the competitiveness of trehalose utilization mutants of Sinorhizobium meliloti and Sinorhizobium medicae was investigated here. Trehalose utilization mutants of S. meliloti and S. medicae were obtained by mutagenesis of their trehalose utilization gene thuB. The mutant strains and the wild type were coinoculated on three cultivars of alfalfa (Medicago sativa) and two cultivars of Medicago truncatula and assessed for competitiveness in root colonization, and nodule occupancy. The thuB mutants formed more nodules than their parent strains on two of the three alfalfa lines tested and on one of the two M. truncatula lines tested. They were not more competitive on the other alfalfa and M. truncatula lines. Their competitiveness for nodule occupancy did not correlate positively with their ability to colonize these roots but correlated with the extent of thuB induction in the infection threads. Induction of thuB was shown to be dependent on the concentration of trehalose in the environment. These results suggest a direct role for host trehalose metabolism in early plant-symbiont interactions and show that the ability to manage host-induced stresses during infection, rather than the ability to colonize the root, is critical for competitive nodulation.

Role of trehalose transport and utilization in Sinorhizobium meliloti--alfalfa interactions.

Genes thuA and thuB in Sinorhizobium meliloti Rm1021 code for a major pathway for trehalose catabolism and are induced by trehalose but not by related structurally similar disaccharides like sucrose or maltose. S. meliloti strains mutated in either of these two genes were severely impaired in their ability to grow on trehalose as the sole source of carbon. ThuA and ThuB show no homology to any known enzymes in trehalose utilization. ThuA has similarity to proteins of unknown function in Mesorhizobium loti, Agrobacterium tumefaciens, and Brucella melitensis, and ThuB possesses homology to dehydrogenases containing the consensus motif AGKHVXCEKP. thuAB genes are expressed in bacteria growing on the root surface and in the infection threads but not in the symbiotic zone of the nodules. Even though thuA and thuB mutants were impaired in competitive colonization of Medicago sativa roots, these strains were more competitive than the wild-type Rml021 in infecting alfalfa roots and forming nitrogen-fixing nodules. Possible reasons for their increased competitiveness are discussed.