Translation initiation is a major rate-limiting step for protein synthesis. However, recent studies strongly suggest that the efficiency of protein synthesis is additionally regulated by multiple factors that impact the elongation phase. To assess the influence of early elongation on protein synthesis, we employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays. Here we report that the identity of the amino acids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure. Single-molecule measurements of translation kinetics revealed pausing of the ribosome and aborted protein synthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions. Finally, introduction of preferred sequence motifs only at specific codon positions improves protein synthesis efficiency for recombinant proteins. Collectively, our data underscore the critical role of early elongation events in translational control of gene expression.

In this work, we report likely recurrent horizontal (lateral) gene transfer events of genes encoding pore-forming toxins of the aerolysin family between species belonging to different kingdoms of life. Clustering based on pairwise similarity and phylogenetic analysis revealed several distinct aerolysin sequence groups, each containing proteins from multiple kingdoms of life. These results strongly support at least six independent transfer events between distantly related phyla in the evolutionary history of one protein family and discount selective retention of ancestral genes as a plausible explanation for this patchy phylogenetic distribution. We discuss the possible roles of these proteins and show evidence for a convergent new function in two extant species. We hypothesize that certain gene families are more likely to be maintained following horizontal gene transfer from commensal or pathogenic organism to its host if they 1) can function alone; and 2) are immediately beneficial for the ecology of the organism, as in the case of pore-forming toxins which can be utilized in multicellular organisms for defense and predation.

Conserved poly(A) tracks in coding regions are attenuators of translation.

Oscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g., Lactococcus , Lactobacillus , Leuconostoc , Streptococcus , and Enterococcus , identified by all three methods, other, subdominant bacteria belonging to the families Bifidobacteriaceae and Moraxellaceae (mostly Enhydrobacter ), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures.

A number of bacterial virulence factors have been observed to adopt structures similar to that of aerolysin, the principal toxin of Aeromonas species. However, a comprehensive description of architecture and structure of the aerolysin-like superfamily has not been determined. In this study, we define a more compact aerolysin-like domain – or aerolysin fold – and show that this domain is far more widely spread than anticipated since it can be found throughout kingdoms. The aerolysin-fold could be found in very diverse domain and functional contexts, although a toxic function could often be assigned. Due to this diversity, the borders of the superfamily could not be set on a sequence level. As a border-defining member, we therefore chose pXO2-60 – a protein from the pathogenic pXO2 plasmid of Bacillus anthracis. This fascinating protein, which harbors a unique ubiquitin-like fold domain at the C-terminus of the aerolysin-domain, nicely illustrates the diversity of the superfamily. Its putative role in the virulence of B. anthracis and its three dimensional model are discussed.

Motivation: Trimeric autotransporter adhesins (TAAs), such as Yersinia YadA, Neisseria NadA, Moraxella UspAs, Haemophilus Hia and Bartonella BadA, are important pathogenicity factors of proteobacteria. Their high sequence diversity and distinct mosaic-like structure lead to difficulties in the annotation of their sequences. These stem from the large number of short repeats, the presence of compositionally unusual coiled-coils, fuzzy domain boundaries and regions of seemingly low sequence complexity. Results: We have developed a workflow, named daTAA, for the accurate domain annotation of TAAs. Its core consists of manually curated alignments and of knowledge-based rules that enhance assignments made by sequence similarity. Compared to general domain annotation servers such as PFAM, daTAA captures more domains and provides more sensitive domain detection, as well as integrated and detailed coiled-coil assignments. Availability: The daTAA server is freely accessible at http://toolkit.tuebingen.mpg.de/dataa Contact: andrei.lupas@tuebingen.mpg.de Supplementary information: Supplementary data are available at Bioinformatics online.