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Interactions between eukaryotic hosts and their bacterial symbionts drive key ecological and evolutionary processes, from regulating ecosystems to the evolution of complex molecular machines and processes. Over time, endosymbionts generally evolve reduced genomes, and their relationship with their host tends to stabilize. However, host–bacteria relationships may be heavily influenced by environmental changes. Here, we review these effects on one of the most ancient and diverse endosymbiotic groups, formed by—among others—Legionellales, Francisellaceae, and Piscirickettsiaceae. This group is referred to as Deep-branching Intracellular Gammaproteobacteria (DIG), whose last common ancestor presumably emerged about 2 Ga ago. We show that DIGs are globally distributed, but generally at very low abundance, and are mainly identified in aquatic biomes. Most DIGs harbour a type IVB secretion system, critical for host-adaptation, but its structure and composition vary. Finally, we review the different types of microbial interactions that can occur in diverse environments, with direct or indirect effects on DIG populations. The increased use of omics technologies on environmental samples will allow a better understanding of host–bacterial interactions and help unravel the definition of DIGs as a group from an ecological, molecular, and evolutionary perspective.

Legionella pneumophila are host-adapted bacteria that infect and reproduce primarily in amoeboid protists. Using similar infection mechanisms, they infect human macrophages, and cause Legionnaires’ disease, an atypical pneumonia, and the milder Pontiac fever. We hypothesized that, despite the similarities in infection mechanisms, the hosts are different enough that there exist high-selective value mutations that would dramatically increase the fitness of Legionella inside the human host. By comparing a large number of isolates from independent infections, we identified two genes, mutated in three unrelated patients, despite the short duration of the incubation period (2–14 days). One is a gene coding for an outer membrane protein (OMP) belonging to the OmpP1/FadL family. The other is a gene coding for an EAL-domain-containing protein involved in cyclic-di-GMP regulation, which in turn modulates flagellar activity. The clinical strain, carrying the mutated EAL-domain-containing homologue, grows faster in macrophages than the wild-type strain, and thus appears to be better adapted to the human host. As human-to-human transmission is very rare, fixation of these mutations into the population and spread into the environment is unlikely. Therefore, parallel evolution – here mutations in the same genes observed in independent human infections – could point to adaptations to the accidental human host. These results suggest that despite the ability of L. pneumophila to infect, replicate in and exit from macrophages, its human-specific adaptations are unlikely to be fixed in the population.

Legionella pneumophila is the causative agent of Legionnaires' disease, a severe pneumonia. Cooling towers are a major source of large outbreaks of the disease. The growth of L. pneumophila in these habitats is influenced by the resident microbiota. Consequently, the aim of this study was to isolate and characterize bacterial species from cooling towers capable of inhibiting several strains of L. pneumophila and one strain of L. quinlivanii. Two cooling towers were sampled to isolate inhibiting bacterial species. Seven inhibitory isolates were isolated, through serial dilution plating and streaking on agar plates, belonging to seven distinct species. The genomes of these isolates were sequenced to identify potential genetic elements that could explain the inhibitory effect. The results showed that the bacterial isolates were taxonomically diverse and that one of the isolates may be a novel species. Genome analysis showed a high diversity of antimicrobial gene products identified in the genomes of the bacterial isolates. Finally, testing different strains of Legionella demonstrated varying degrees of susceptibility to the antimicrobial activity of the antagonistic species. This may be due to genetic variability between the Legionella strains. The results demonstrate that though cooling towers are breeding grounds for L. pneumophila, the bacteria must contend with various antagonistic species. Potentially, these species could be used to create an inhospitable environment for L. pneumophila, and thus decrease the probability of outbreaks occurring.

Legionella pneumophila is a natural inhabitant of water systems. From there, it can be transmitted to humans by aerosolization resulting in severe pneumonia. Most large outbreaks are caused by cooling towers colonized with L. pneumophila. The resident microbiota of the cooling tower is a key determinant for the colonization and growth of L. pneumophila. In our preceding study, the genus Pseudomonas correlated negatively with the presence of L. pneumophila in cooling towers, but it was not clear which species was responsible. Therefore, we identified the Pseudomonas species inhabiting 14 cooling towers using a Pseudomonas-specific 16S rRNA amplicon sequencing strategy. We found that cooling towers that are free of L. pneumophila contained a high relative abundance of members from the Pseudomonas alcaliphila/oleovorans phylogenetic cluster. P. alcaliphila JCM 10630 inhibited the growth of L. pneumophila on agar plates. Analysis of the P. alcaliphila genome revealed the presence of a gene cluster predicted to produce toxoflavin. L. pneumophila growth was inhibited by pure toxoflavin and by extracts from P. alcaliphila culture found to contain toxoflavin by liquid chromatography coupled with mass spectrometry. In addition, toxoflavin inhibits the growth of Vermameoba vermiformis, a host cell of L. pneumophila. Our study indicates that P. alcaliphila may be important to restrict growth of L. pneumophila in water systems through the production of toxoflavin. A sufficiently high concentration of toxoflavin is likely not achieved in the bulk water but might have a local inhibitory effect such as near or in biofilms.