Azoarcus olearius BH72 is an endophyte capable of biological nitrogen fixation (BNF) and of supplying nitrogen to its host plant. Our previous microarray approach provided insights into the transcriptome of strain BH72 under N2-fixation in comparison to ammonium-grown conditions, which already indicated the induction of genes not related to the BNF process. Due to the known limitations of the technique, we might have missed additional differentially expressed genes (DEGs). Thus, we used directional RNA-Seq to better comprehend the transcriptional landscape under these growth conditions. RNA-Seq detected almost 24% of the annotated genes to be regulated, twice the amount identified by microarray. In addition to confirming entire regulated operons containing known DEGs, the new approach detected the induction of genes involved in carbon metabolism and flagellar and twitching motility. This may support N2-fixation by increasing energy production and by finding suitable microaerobic niches. On the other hand, energy expenditures were reduced by suppressing translation and vitamin biosynthesis. Nonetheless, strain BH72 does not appear to be content with N2-fixation but is primed for alternative economic N-sources, such as nitrate, urea or amino acids; a strong gene induction of machineries for their uptake and assimilation was detected. RNA-Seq has thus provided a better understanding of a lifestyle under limiting nitrogen sources by elucidating hitherto unknown regulated processes.

Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the transfer of terminal sialic acids (Sia) from sialoglycoconjugates to terminal galactose or N-acetylgalactosamine residues of oligosaccharides without the requirement of CMP-Neu5Ac, the activated Sia used by typical sialyltransferases. Enzymes comprise a N-terminal catalytic domain (CD) followed by a lectin-like domain (LD). Most work on trypanosomal TS has been done on enzymatic activities focusing on the CD of TS from Trypanosoma cruzi (causing Chagas disease in Latin America), subspecies of Trypanosoma brucei, (causing human sleeping sickness in Africa) and Trypanosoma congolense (causing African Animal Trypanosomosis in livestock). Previously, we demonstrated that T. congolense TS (TconTS)-LD binds to several carbohydrates, such as 1,4-β-mannotriose. In this study we investigated the influence of TconTS3-LD on Sia transfer efficiency of TconTS1a-CD by swapping domains. in silico analysis on structure models of TconTS enzymes revealed the potential of domain swaps between TconTS1a and TconTS3 without structural disruptions of the enzymes overall topologies. Recombinant domain swapped TconTS1a/TS3 showed clear Sia transfer activity, when using fetuin and lactose as Sia donor and acceptor substrates, respectively. While Sia transfer activity remained unchanged from the level of TconTS1a, hydrolytic release of free Neu5Ac as a side product was suppressed resulting in increased transfer efficiency. Presence of 1,4-β-mannotriose during TS reactions modulates enzyme activities enhancing transfer efficiency possibly due to occupation of the binding site in TconTS1a-LD. Interestingly this effect was in the same range as that observed when swapping TconTS1a-CD and TconTS3-LD. In summary, this study demonstrate the proof-of-principle for swapping CDs and LDs of TconTS and that TconTS3-LD influences enzymatic activity of TconTS1a-CD providing evidence that LDs play pivotal roles in modulating activities and biological functions of TconTS and possibly other TS.