Legionella pneumophila, the agent of Legionnaires' disease pneumonia, is transmitted to humans following the inhalation of contaminated water droplets. In aquatic systems, L. pneumophila survives much of time within multi-organismal biofilms. Therefore, we examined the ability of L. pneumophila (clinical isolate 130b) to persist within biofilms formed by various types of aquatic bacteria, using a bioreactor with flow, steel surfaces, and low-nutrient conditions. L. pneumophila was able to intercalate into and persist within a biofilm formed by Klebsiella pneumoniae, Flavobacterium sp. or Pseudomonas fluorescens. The levels of L. pneumophila within these biofilms were as much as 4×104 CFU per cm2 of steel coupon and lasted for at least 12 days. These data document that K. pneumoniae, Flavobacterium sp., and P. fluorescens can promote the presence of L. pneumophila in dynamic biofilms. In contrast to these results, L. pneumophila 130b did not persist within a biofilm formed by Pseudomonas aeruginosa, confirming that some bacteria are permissive for Legionella colonization whereas others are antagonistic. In addition to colonizing certain mono-species biofilms, L. pneumophila 130b persisted within a two-species biofilm formed by K. pneumoniae and Flavobacterium sp. Interestingly, the legionellae were also able to colonize a two-species biofilm formed by K. pneumoniae and P. aeruginosa, demonstrating that a species that is permissive for L. pneumophila can override the inhibitory effect(s) of a non-permissive species.

All studied octocoral mitochondrial genomes contain a gene from the MutS family, whose members code for proteins involved in DNA mismatch repair, other types of DNA repair, meiotic recombination, and other functions. Although mutS homologues are found in all domains of life as well as viruses, octocoral mt-mutS is the only such gene encoded in an organellar genome. While the function of mtMutS is not known, its domain architecture, conserved sequence, and presence of some characteristic residues suggest its involvement in mitochondrial DNA repair. This inference is supported by exceptionally low rates of mt-sequence evolution observed in octocorals. Previous studies of mt-mutS have been limited by the small number of octocoral mt-genomes available. We utilized sequence-capture data from the recent Quattrini et al. study to assemble complete mitochondrial genomes for 97 species of octocorals. Combined with sequences publicly available in GenBank, this resulted in a dataset of 184 complete mitochondrial genomes, which we used to re-analyze the conservation and evolution of mt-mutS. We discovered the first case of mt-mutS loss among octocorals in one of the two Pseudoanthomastus sp. assembled from Quattrini et al. data. This species displayed accelerated rate and and changed patterns of nucleotide substitutions in mt-genome, which we argue provide additional evidence for the role of mtMutS in DNA repair. In addition, we found accelerated mt-sequence evolution in the presence of mt-mutS in several octocoral lineages. This accelerated evolution did not appear to be the result of relaxed selection pressure and did not entail changes in patterns of nucleotide substitutions. Overall, our results support previously reported patterns of conservation in mt-mutS and suggest that mtMutS is involved in DNA repair in octocoral mitochondria. They also indicate that the presence of mt-mutS contributes to, but does not fully explain, the low rates of sequence evolution in octocorals

MutS is a key component of the Mismatch Repair (MMR) pathway. Members of the MutS protein family are present in prokaryotes, eukaryotes, and viruses. Six MutS homologues (MSH1-6) have been identified in yeast, of which three function in nuclear MMR, while MSH1 functions in mitochondrial DNA repair. MSH proteins are believed to be well conserved in animals, except for MSH1—which is thought to be lost. Two intriguing exceptions to this general picture have been found, both in the class Anthozoa within phylum Cnidaria. First, an orthologue of the yeast-MSH1 was reported in one hexacoral species. Second, a MutS homologue (mtMutS) has been found in the mitochondrial genome of all octocorals. To understand the origin and potential functional implications of these exceptions, we investigated the evolution of the MutS family both in Cnidaria and in animals in general. Our study confirmed the acquisition of octocoral mtMutS by a horizontal gene transfer from a giant virus. Surprisingly, we identified MSH1 in all hexacorals and several sponges and placozoans. By contrast, MSH1 orthologues were lacking in other cnidarians, ctenophores, and bilaterian animals. Furthermore, while we identified MSH2 and MSH6 in nearly all animals, MSH4, MSH5, and, especially, MSH3 were missing in multiple species. Overall, our analysis revealed a dynamic evolution of the MutS family in animals, with multiple losses of MSH1, MSH3, some losses of MSH4 and MSH5, and a gain of the octocoral mtMutS. We propose that octocoral mtMutS functionally replaced MSH1 that was present in the common ancestor of Anthozoa.

Mitochondria require~1,500 proteins for their maintenance and proper functionality, which constitute the mitochondrial proteome (mt-proteome). Although a few of these proteins, mostly subunits of the electron transport chain complexes, are encoded in mitochondrial DNA (mtDNA), the vast majority are encoded in the nuclear genome and imported to the organelle. Previous studies have shown a continuous and complex evolution of mt-proteome among eukaryotes. However, there was less attention paid to mt-proteome evolution within Metazoa, presumably because animal mtDNA and, by extension, animal mitochondria are often considered to be uniform. In this analysis, two bioinformatic approaches (Orthologue-detection and Mitochondrial Targeting Sequence prediction) were used to identify mt-proteins in 23 species from four nonbilaterian phyla: Cnidaria, Ctenophora, Placozoa, and Porifera, as well as two choanoflagellates, the closest animal relatives. Our results revealed a large variation in mt-proteome in nonbilaterian animals in size and composition. Myxozoans, highly reduced cnidarian parasites, possessed the smallest inferred mitochondrial proteomes, while calcareous sponges possessed the largest. About 513 mitochondrial orthologous groups were present in all nonbilaterian phyla and human. Interestingly, 42 human mitochondrial proteins were not identified in any nonbilaterian species studied and represent putative innovations along the bilaterian branch. Several of these proteins were involved in apoptosis and innate immunity, two processes known to evolve within Metazoa. Conversely, several proteins identified as mitochondrial in nonbilaterian phyla and animal outgroups were absent in human, representing cases of possible loss. Finally, a few human cytosolic proteins, such as histones and cytosolic ribosomal proteins, were predicted to be targeted to mitochondria in nonbilaterian animals. Overall, our analysis provides the first step in characterization of mt-proteomes in nonbilaterian animals and understanding evolution of animal mt-proteome.

MutS is a key component of the Mismatch Repair (MMR) pathway. Members of the MutS family of proteins are present in bacteria, archaea, eukaryotes, and viruses. Six MutS homologues (MSH1-6), have been identified in yeast, three of which function in nuclear MMR, while MSH1 has been associated with mitochondrial DNA repair. MSH1 is believed to be lacking in animals, potentially reflecting the loss of MMR in animal mitochondria, and correlated with higher rates of mitochondrial sequence evolution. An intriguing exception has been found in octocorals, a group of marine animals from phylum Cnidaria, which encode a MutS-homologue (mtMutS) in their mitochondrial genome. It has been suggested that this protein functions in mitochondrial DNA repair, which would explain some of the lowest rates of mitochondrial sequence evolution observed in this group. To place the acquisition of mtMutS in a functional context, we investigated the evolution of the whole MutS family in animals. Our study confirmed the acquisition of octocoral mtMutS by horizontal gene transfer from a giant virus. Surprisingly, we found orthologues of yeast MSH1 in all hexacorals (the sister group of octocorals) and several sponges and placozoans. By contrast, MSH1 orthologues were lacking in octocorals, medusozoan cnidarians, ctenophores, and bilaterian animals. Furthermore, while we were able to identify MSH2 and MSH6 in all animals, MSH4, MSH5, and, especially, MSH3 were missing in multiple species. Overall, our analysis reveals a dynamic evolution of MSH family in animals, with multiple losses of MSH1, MSH3, some losses of MSH4 and MSH5, and a gain of octocoral mtMutS.