Presence and activity of nitrogen-fixing bacteria in Scots pine needles in a boreal forest: a nitrogen-addition experiment

Endophytic nitrogen-fixing bacteria have been detected and isolated from the needles of conifer trees growing in North American boreal forests. Because boreal forests are nutrient-limited, these bacteria could provide an important source of nitrogen for tree species. This study aimed to determine their presence and activity in a Scandinavian boreal forest, using immunodetection of nitrogenase enzyme subunits and acetylene-reduction assays of native Scots pine (Pinus sylvestris L.) needles. The presence and rate of nitrogen fixation by endophytic bacteria were compared between control plots and fertilized plots in a nitrogen-addition experiment. In contrast to the expectation that nitrogen-fixation rates would decline in fertilized plots, as seen, for instance, with nitrogen-fixing bacteria associated with bryophytes, there was no difference in the presence or activity of nitrogen-fixing bacteria between the two treatments. The extrapolated calculated rate of nitrogen fixation relevant for the forest stand was 20 g N ha(-1) year(-1), which is rather low compared with Scots pine annual nitrogen use but could be important for the nitrogen-poor forest in the long term. In addition, of 13 colonies of potential nitrogen-fixing bacteria isolated from the needles on nitrogen-free media, 10 showed in vitro nitrogen fixation. In summary, 16S rRNA sequencing identified the species as belonging to the genera Bacillus, Variovorax, Novosphingobium, Sphingomonas, Microbacterium and Priestia, which was confirmed by Illumina whole-genome sequencing. Our results confirm the presence of endophytic nitrogen-fixing bacteria in Scots pine needles and suggest that they could be important for the long-term nitrogen budget of the Scandinavian boreal forest

Metabolomic Study of Heterotrophically Grown Chlorella sp. Isolated from Wastewater in Northern Sweden

There are numerous strains of Chlorella with a corresponding variety of metabolic pathways. A strain we previously isolated from wastewater in northern Sweden can grow heterotrophically as well as autotrophically in light and has higher lipid contents under heterotrophic growth conditions. The aims of the present study were to characterize metabolic changes associated with the higher lipid contents in order to enhance our understanding of lipid production in microalgae and potentially identify new compounds with utility in sustainable development. Inter alia, the amino acids glutamine and lysine were 7-fold more abundant under heterotrophic conditions, the key metabolic intermediate alpha-ketoglutarate was more abundant under heterotrophic conditions with glucose, and maltose was more abundant under heterotrophic conditions with glycerol than under autotrophic conditions. The metabolite 3-hydroxy-butyric acid, the direct precursor of the biodegradable plastic PHB (poly-3-hydroxy-butyric acid), was also more abundant under heterotrophic conditions. Our metabolomic analysis has provided new insights into the alga's lipid production pathways and identified metabolites with potential use in sustainable development, such as the production of renewable, biodegradable plastics, cosmetics, and nutraceuticals, with reduced pollution and improvements in both ecological and human health

The organization, regulation and phylogeny of uptake hydrogenase genes in Frankia

Frankia alni ACN14a, Frankia sp. CcI3 and Frankia sp. EAN1pec, which have different host specificity and geographical distribution, have two uptake hydrogenase syntons in their genome: hydrogenase synton#1 and hydrogenase synton#2. The organization of hydrogenase genes on these syntons also varies. Phylogenetic analysis of the structural genes of these syntons showed that they were significantly divergent and that hydrogenase synton#1 subunits of these Frankia strains were probably ancestral among the actinobacteria. Hydrogenase gene duplication might have occurred long before emergence of the three Frankia lineages. The structural subunits of hydrogenase HupS2 and HupL2 (synton#2) of F. alni ACN14a and Frankia sp. CcI3, which belong to phylogenetic Frankia cluster 1, were grouped closely together but away from Frankia sp. EAN1pec, which belongs to Frankia cluster 3. Phylogenetic analysis showed the occurrence of lateral transfer of hupL2 in Frankia sp. EAN1pec to or from Geobacter sulfurreducens. The transcript levels of hupS1 and hupL1 relative to hupS2 and hupL2 were higher in F. alni ACN14a grown under free-living conditions. Under symbiotic conditions, transcript levels of hupS2 and hupL2 were higher than those of hupS1 and hupL1. Hydrogenase subunits of synton#1 are more expressed under free-living conditions, whereas those of synton#2 are mainly involved in symbiotic interactions

The phylogeny of uptake hydrogenases in Frankia

Uptake hydrogenase is an enzyme that is believed to be beneficial for nitrogen fixation in bacteria. Recent studies have shown that there are two sets of uptake hydrogenase genes, named synton #1 and #2, in Frankia sp. Here, we have made a phylogenetic analysis of the structural subunits of hydrogenase synton #1 and #2, which showed a distinct clustering pattern between Frankia strains isolated from different host plants and non-Frankia organisms. The structural subunits of hydrogenase synton #1 of Frankia sp. CpI1, Frankia alni ACN14a and AvCI1 were grouped together while those of Frankia sp. CcI3, KB5, UGL140104 and UGL011102 formed another group. The structural subunits of hydrogenase synton #2 of F. alni ACN14a, Frankia sp. CcI3 and BCU110501 grouped together, but Frankia sp. KB5, CpI1, F. alni ArI3 and AvCI1 formed another group. The structural subunits of hydrogenase synton #1 and #2 of Frankia sp. EAN1pec were more closely related with those of non-Frankia bacteria Streptomyces avermitilis and Anaeromyxobacter sp., respectively, than they were to other Frankia strains, which suggested the occurrence of lateral gene transfer (LGT) involving these organisms. In addition, the accessory Hyp proteins of hydrogenase synton #1 and hydrogenase synton #2 of F. alni ACN14a and Frankia sp. CcI3 were shown to be phylogenetically more related to each other than they were to Frankia EAN1pec

Identification of Casuarina-Frankia strains by use of polymerase chain reaction (PCR) with arbitrary primers

Free-living N2-fixing Frankia strains isolated from Casuarina sp. were investigated for genomic polymorphism. We used six 10-mer oligonucleotides as single arbitrary primers (AP) for the polymerase chain reaction (PCR) in order to amplify random DNA fragments in the genome of free-living Frankia strains. Agarose-gels of the amplified genomic DNA revealed that two of the six arbitrary primers showed polymorphism in the eight different Frankia genomes. Analysis of the AP-PCR products showed 9 polymorphic bands ranging from 4.1-0.60 kb. We conclude that single arbitrary primers can be used to amplify genomic DNA, and that polymorphism can be detected between the amplification products of the different Frankia genomes