Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal.

Legionella and Coxiella are intracellular pathogens that use the virulence-related Icm/Dot type-IVB secretion system to translocate effector proteins into host cells during infection. These effectors were previously shown to contain a C-terminal secretion signal required for their translocation. In this research, we implemented a hidden semi-Markov model to characterize the amino acid composition of the signal, thus providing a comprehensive computational model for the secretion signal. This model accounts for dependencies among sites and captures spatial variation in amino acid composition along the secretion signal. To validate our model, we predicted and synthetically constructed an optimal secretion signal whose sequence is different from that of any known effector. We show that this signal efficiently translocates into host cells in an Icm/Dot-dependent manner. Additionally, we predicted in silico and experimentally examined the effects of mutations in the secretion signal, which provided innovative insights into its characteristics. Some effectors were found to lack a strong secretion signal according to our model. We demonstrated that these effectors were highly dependent on the IcmS-IcmW chaperons for their translocation, unlike effectors that harbor a strong secretion signal. Furthermore, our model is innovative because it enables searching ORFs for secretion signals on a genomic scale, which led to the identification and experimental validation of 20 effectors from Legionella pneumophila, Legionella longbeachae, and Coxiella burnetii. Our combined computational and experimental methodology is general and can be applied to the identification of a wide spectrum of protein features that lack sequence conservation but have similar amino acid characteristics.

Native homing endonucleases can target conserved genes in humans and in animal models.

In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assays.

Contribution of lateral gene transfer to the gene repertoire of a gut-adapted methanogen.

Methanobrevibacter smithii is the most abundant archaeon in the human colon. As most of its neighbors are bacterial species, it is expected that lateral gene acquisition from bacteria might have contributed to the evolution and adaptation of this archaeon. We performed a tree-based genome-wide survey of putative lateral gene transfer products in M. smithii, using a phylogenetic pipeline. Over 15% of the coding genes of M. smithii are inferred to be bacterial in origin, based on this analysis. Laterally acquired genes have had the largest contribution to surface functions, and encode glycosyl-transferases and adhesin-like proteins. In addition, several important ABC transporters, especially metal transporters are of bacterial origin. Thus, bacterial genes contributed to the host-adaptation by allowing a larger variety of surface structures and increasing the efficiency of metal ion uptake in the competitive gut niche.

High-resolution modeling of the selection on local mRNA folding strength in coding sequences across the tree of life.

BACKGROUND: mRNA can form local secondary structure within the protein-coding sequence, and the strength of this structure is thought to influence gene expression regulation. Previous studies suggest that secondary structure strength may be maintained under selection, but the details of this phenomenon are not well understood. RESULTS: We perform a comprehensive study of the selection on local mRNA folding strengths considering variation between species across the tree of life. We show for the first time that local folding strength selection tends to follow a conserved characteristic profile in most phyla, with selection for weak folding at the two ends of the coding region and for strong folding elsewhere in the coding sequence, with an additional peak of selection for strong folding located downstream of the start codon. The strength of this pattern varies between species and organism groups, and we highlight contradicting cases. To better understand the underlying evolutionary process, we show that selection strengths in the different regions are strongly correlated, and report four factors which have a clear predictive effect on local mRNA folding selection within the coding sequence in different species. CONCLUSIONS: The correlations observed between selection for local secondary structure strength in the different regions and with the four genomic and environmental factors suggest that they are shaped by the same evolutionary process throughout the coding sequence, and might be maintained under direct selection related to optimization of gene expression and specifically translation regulation.

A possible universal role for mRNA secondary structure in bacterial translation revealed using a synthetic operon.

In bacteria, translation re-initiation is crucial for synthesizing proteins encoded by genes that are organized into operons. The mechanisms regulating translation re-initiation remain, however, poorly understood. We now describe the ribosome termination structure (RTS), a conserved and stable mRNA secondary structure localized immediately downstream of stop codons, and provide experimental evidence for its role in governing re-initiation efficiency in a synthetic Escherichia coli operon. We further report that RTSs are abundant, being associated with 18%-65% of genes in 128 analyzed bacterial genomes representing all phyla, and are selectively depleted when translation re-initiation is advantageous yet selectively enriched so as to insulate translation when re-initiation is deleterious. Our results support a potentially universal role for the RTS in controlling translation termination-insulation and re-initiation across bacteria.

Extensive Inter-Domain Lateral Gene Transfer in the Evolution of the Human Commensal Methanosphaera stadtmanae.

Methanosphaera stadtmanae is a commensal methanogenic archaeon found in the human gut. As most of its niche-neighbors are bacteria, it is expected that lateral gene transfer (LGT) from bacteria might have contributed to the evolutionary history of this organism. We performed a phylogenomic survey of putative LGT events in M. stadtmanae, using a phylogenetic pipeline. Our analysis indicates that a substantial fraction of the proteins of M. stadtmanae are inferred to have been involved in inter-domain LGT. Laterally acquired genes have had a large contribution to surface functions, by providing novel glycosyltransferase functions. In addition, several ABC transporters seem to be of bacterial origin, including the molybdate transporter. Thus, bacterial genes contributed to the adaptation of M. stadtmanae to a host-dependent lifestyle by allowing a larger variation in surface structures and increasing transport efficiency in the gut niche which is diverse and competitive.