G C A T T A C G G C A T genes Article The Peptidoglycan Biosynthesis Gene murC in Frankia: Actinorhizal vs. Plant Type Fede Berckx 1, Daniel Wibberg 2, Jörn Kalinowski 2 and Katharina Pawlowski 2,* 1 Department of Ecology, Environment, and Plant Sciences, Stockholm University, 10691 Stockholm, Sweden; [email protected] 2 Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany; [email protected] (D.W.); [email protected] (J.K.) * Correspondence: [email protected]; Tel.: +46-8-16-3772; Fax: +46-8-16-5525 Received: 2 March 2020; Accepted: 8 April 2020; Published: 16 April 2020 Abstract: Nitrogen-fixing Actinobacteria of the genus Frankia can be subdivided into four phylogenetically distinct clades; members of clusters one to three engage in nitrogen-fixing root nodule symbioses with actinorhizal plants. Mur enzymes are responsible for the biosynthesis of the peptidoglycan layer of bacteria. The four Mur ligases, MurC, MurD, MurE, and MurF, catalyse the addition of a short polypeptide to UDP-N-acetylmuramic acid. Frankia strains of cluster-2 and cluster-3 contain two copies of murC, while the strains of cluster-1 and cluster-4 contain only one. Phylogenetically, the protein encoded by the murC gene shared only by cluster-2 and cluster-3, termed MurC1, groups with MurC proteins of other Actinobacteria. The protein encoded by the murC gene found in all Frankia strains, MurC2, shows a higher similarity to the MurC proteins of plants than of Actinobacteria. MurC2 could have been either acquired via horizontal gene transfer or via gene duplication and convergent evolution, while murC1 was subsequently lost in the cluster-1 and cluster-4 strains. In the nodules induced by the cluster-2 strains, the expression levels of murC2 were significantly higher than those of murC1. Thus, there is clear sequence divergence between both types of Frankia MurC, and Frankia murC1 is in the process of being replaced by murC2, indicating selection in favour of murC2. Nevertheless, protein modelling showed no major structural differences between the MurCs from any phylogenetic group examined. Keywords: Peptidoglycan; MurC; Frankia; nitrogen fixation; actinorhizal symbiosis; root nodules 1. Introduction Bacteria are exposed to various environmental stresses against which the cell wall has to provide protection, while also maintaining the cell shape. Intracellular symbiotic bacteria need to survive both inside and outside the host, which can create the need for cell wall modifications. The cell walls of Gram-positive bacteria are characterized by a ca. 30 nm thick peptidoglycan (PG) layer. As a much thinner layer in the cell wall, PG can also be found in Gram-negative bacteria (3–6 nm), cyanobacteria (10 nm), and moss chloroplasts (5 nm) [1–4]. PG is synthesized by the activity of Mur ligases, which add a number of amino acids to UDP-N-acetylmuramic acid to connect different strands. In a series of ATP-dependent reactions, L-alanine (MurC), D-glutamate (MurD), L-lysine (MurE), and D-alanyl-D-alanine (MurF) are added sequentially. The precise sequence of the reactions catalysed by these enzymes was reviewed by Barreteau et al. [5] and tested in Mycobacterium tuberculosis [6], as they represent an interesting target for antibiotic drug discovery. Several reports on the activity of Mur ligases have been published for non-pathogenic bacteria. In the cyanobacterium Anabena sp., murC and murB play an important role in heterocyst differentiation [7]. Although, so far, PG layers have not been identified for the chloroplasts of Spermatophyta, it is known that Arabidopsis thaliana Genes 2020, 11, 432; doi:10.3390/genes11040432 www.mdpi.com/journal/genes Genes 2020, 11, 432 2 of 12 contains a copy of murE, and the mutation of this gene leads to the reduced chlorophyll content of leaves, indicating an essential role of murE for chloroplast development [8]. Yet, a murE mutant of the moss Physcomitrella patens could be complemented with a cyanobacterial murE gene, but not with Arabidopsis murE, indicating functional divergence. At any rate, the occurrence of Mur ligase genes in diverse eukaryote genomes from green algae to angiosperms have made them the subject of study into plastid development, endosymbiosis, and the multiple events of horizontal gene transfer (HGT) that led to the evolution of land plants. Frankia is a genus of nitrogen-fixing soil actinobacteria. They engage in root nodule endosymbiosis with a diverse group of host plants within the orders of the Fagales, Rosales, and Cucurbitales, collectively referred to as actinorhizal plants (reviewed by Pawlowski and Demchenko [9]). The genus can be split into four phylogenetically distinct clades, known as cluster-1 to cluster-4; the first three clusters contain the symbiotic strains [10]. The phylogenetic relationships between the clusters has been a point of discussion based on use of single marker genes [11,12], but recent studies using multiple phylogenetic markers or the core genome have shown that cluster-2 is the earliest divergent symbiotic clade [13–16]. Strains belonging to cluster-2 have a particular low saprotrophic potential. Within cluster two, so far, only two strains could be grown in culture, namely: Frankia coriariae BMG5.1 [17] and the closely related strain BMG5.30 [18]. Recent studies on the Frankia genome sequences isolated from whole nodules, or from Frankia vesicles isolated from nodules, have led to the elucidation of the phylogeny of cluster-2 and the identification of several lineages within this cluster [13–15]. Analysis of these genomes revealed that murC, encoding the enzyme that adds L-alanine as the first amino acid to UDP-N-actetylmuramic acid in the course of cell wall PG biosynthesis, was present in two divergent copies in all of the genomes of the Frankia cluster-2 strains available. No gene duplication has been reported for murC thus far. Therefore, we were interested in the function and the origin of both murC copies. The presence of two copies of a given gene in a genome can be explained via two different scenarios. One scenario is gene duplication (reviewed by Hurles [19]), which can occur through non-homologous recombination or through retrotransposition. If the newly acquired copy of the gene becomes fixed, three options are possible. First, either one of the copies can lose its function and become a pseudogene. Second, one of the copies can gain a new function, a process referred to as neofunctionalization. Third, the gene function can be split over the two copies, referred to as subfunctionalization. In this case, normally, the two genes are expressed under different conditions. The other scenario to explain two copies of a given gene in the genome would be horizontal gene transfer (HGT). For example, the intracellular accommodation of cyanobacteria led to HGT events between the cyanobacteria and host, ultimately leading to the evolution of the plastid, although the origin of the plastid envelope is not yet fully understood [20]. In other words, the second copy of murC in Frankia could have been acquired through HGT, either from another soil bacterium or from the host plastid via the host nucleus. This study aimed to answer the questions if one of the two Frankia murC copies originated from gene duplication or from HGT, and whether there is evidence for subfunctionalization. A thorough a phylogenetic analysis of Frankia MurC proteins revealed that the strains from cluster-2 and cluster-3 contained two types of MurC, MurC1 and MurC2, while the strains from cluster-1 and cluster-4 contained only MurC2. Protein structural modelling was performed for different types of MurC in order to analyse the structural conservation between the proteins, and the expression of the two versions of murC was studied in representatives of cluster-2 and cluster-3 using RT-qPCR. 2. Materials and Methods 2.1. Biological Material The expression of murC1 and murC2 was analysed in the nodules of Datisca glomerata (C. Presl) Paill. induced by Candidatus Frankia californiensis Dg2 [21] and by Candidatus Frankia datiscae Cm1 [14], respectively. It was analysed in the nodules of Coriaria nepalensis Wall. induced by Candiatus Genes 2020, 11, 432 3 of 12 Frankia datiscae Dg1 [15], in the nodules of Coriaria arborea Linds. induced by Candidatus Frankia meriodionalis Cppng1 [14], and in the nodules of Coriaria myrtifolia induced by Frankia coriariae BMG5.1 [17]. The expression was also analysed in the nodules of Discaria trinervis (Gillies ex hook.) Reiche induced by Frankia discariae BCU110501 [22]. Plants were germinated from seeds and grown in a greenhouse under a 16/8 h light/dark regime at 23/18 ◦C. Plants were supplied with a quarter strength Hoagland’s solution with 10 mM KNO3 [23] once a week, and with deionized water twice a week. Eight weeks after germination, the plants were inoculated with crushed nodules of D. glomerate, which were then used for the propagation of Dg1, Dg2, and Cm1, separately. Crushed nodules of C. arborea induced by Cppng1 [14] were used for inoculation with Cppng1. For inoculation with F. coriariae BMG5.1 [17] and F. discariae BCU110501 [22], in vitro cultures of Frankia were used, which had been grown in a BAP medium without nitrogen [24], adjusted to pH 9 for BMG5.1, at 28 ◦C for 35 and 28 d, respectively. For inoculation, the cells were pelleted and washed twice with sterile double-distilled H2O. At the time of inoculation, the washed root systems of the plants were evaluated to confirm that no plant was already nodulated. After inoculation, plants were supplied once per week with a quarter Hoagland’s medium without nitrogen, and twice a week with deionized water. Nodules of D. glomerata were harvested twelve weeks after inoculation, while nodules of C. arborea, C. myrtifolia, and of D.