Gil Segal, Howard Shuman and colleagues sequence the genomes of 38 Legionella species and analyze 5,885 predicted effector proteins. Their analysis identifies a core set of seven effectors shared by all 38 species and numerous previously unidentified conserved effector domains. Infection by the human pathogen Legionella pneumophila relies on the translocation of ∼300 virulence proteins, termed effectors, which manipulate host cell processes. However, almost no information exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species and predicted their effector repertoires using a previously validated machine learning approach. This analysis identified 5,885 predicted effectors. The effector repertoires of different Legionella species were found to be largely non-overlapping, and only seven core effectors were shared by all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from the natural protozoan hosts of these species. Furthermore, we detected numerous new conserved effector domains and discovered new domain combinations, which allowed the inference of as yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution.

Metallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals. We report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5). Sequence alignments of all known Tetrahymena metallothioneins have allowed us to rationalize the structure of these proteins. We now formally subdivide the known metallothioneins from the ciliate genus Tetrahymena into two well defined subfamilies, 7a and 7b, based on phylogenetic analysis, on the pattern of clustering of Cys residues, and on the pattern of inducibility by the heavy metals Cd and Cu. Sequence alignment also reveals a remarkably regular, conserved and hierarchical modular structure of all five subfamily 7a MTs, which include MTT3 and MTT5. The former has three modules, while the latter has only two. Induction levels of the three T. thermophila genes were determined using quantitative real time RT-PCR. Various stressors (including heavy metals) brought about dramatically different fold-inductions for each gene; MTT5 showed the highest fold-induction. Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs. EST evidence for alternative splicing in the 3′ UTR of the MTT5 mRNA with potential regulatory activity is reported. The small number and remarkably regular structure of Tetrahymena MTs, coupled with the experimental tractability of this model organism for studies of in vivo function, make it an attractive system for the experimental dissection of the roles, structure/function relationships, regulation of gene expression, and adaptive evolution of these proteins, as well as for the development of biotechnological applications for the environmental monitoring of toxic substances.

Summary: Heavy metals are among the most serious pollutants, and thus there is a need to develop sensitive and rapid biomonitoring methods for heavy metals in the environment. Critical parameters such as bioavailability, toxicity and genotoxicity cannot be tested using chemical analysis, but only can be assayed using living cells. A whole‐cell biosensor uses the whole cell as a single reporter incorporating both bioreceptor and transducer elements. In the present paper, we report results with two gene constructs using the Tetrahymena thermophila MTT1 and MTT5 metallothionein promoters linked with the eukaryotic luciferase gene as a reporter. This is the first report of a ciliated protozoan used as a heavy metal whole‐cell biosensor. T. thermophila transformed strains were created as heavy metal whole‐cell biosensors, and turn on bioassays were designed to detect, in about 2 h, the bioavailable heavy metals in polluted soil or aquatic samples. Validation of these whole‐cell biosensors was carried out using both artificial and natural samples, including methods for detecting false positives and negatives. Comparison with other published cell biosensors indicates that the Tetrahymena metallothionein promoter‐based biosensors appear to be the most sensitive eukaryotic metal biosensors and compare favourably with some prokaryotic biosensors as well.

It is generally accepted that selection for resistance to grazing by protists has contributed to the evolution of Legionella pneumophila as a pathogen. Grazing resistance is becoming more generally recognized as having an important role in the ecology and evolution of bacterial pathogenesis. However, selection for grazing resistance presupposes the existence of protist grazers that provide the selective pressure. To determine whether there are protists that graze on pathogenic Legionella species, we investigated the existence of such organisms in a variety of environmental samples. We isolated and characterized diverse protists that graze on L. pneumophila and determined the effects of adding L. pneumophila on the protist community structures in microcosms made from these environmental samples. Several unrelated organisms were able to graze efficiently on L. pneumophila. The community structures of all samples were markedly altered by the addition of L. pneumophila. Surprisingly, some of the Legionella grazers were closely related to species that are known hosts for L. pneumophila, indicating the presence of unknown specificity determinants for this interaction. These results provide the first direct support for the hypothesis that protist grazers exert selective pressure on Legionella to acquire and retain adaptations that contribute to survival, and that these properties are relevant to the ability of the bacteria to cause disease in people. We also report a novel mechanism of killing of amoebae by one Legionella species that requires an intact Type IV secretion system but does not involve intracellular replication. We refer to this phenomenon as ‘food poisoning’.