Dimitris Mossialos

Posttranslational regulation impacts the fate of duplicated genes

Gene and genome duplications create novel genetic material on which evolution can work and have therefore been recognized as a major source of innovation for many eukaryotic lineages. Following duplication, the most likely fate is gene loss; however, a considerable fraction of duplicated genes survive. Not all genes have the same probability of survival, but it is not fully understood what evolutionary forces determine the pattern of gene retention. Here, we use genome sequence data as well as large-scale phosphoproteomics data from the baker’s yeast Saccharomyces cerevisiae, which underwent a whole-genome duplication ∼100 mya, and show that the number of phosphorylation sites on the proteins they encode is a major determinant of gene retention. Protein phosphorylation motifs are short amino acid sequences that are usually embedded within unstructured and rapidly evolving protein regions. Reciprocal loss of those ancestral sites and the gain of new ones are major drivers in the retention of the two surviving duplicates and in their acquisition of distinct functions. This way, small changes in the sequences of unstructured regions in proteins can contribute to the rapid rewiring and adaptation of regulatory networks.

Bioactive compounds synthesized by non-ribosomal peptide synthetases and type-I polyketide synthases discovered through genome-mining and metagenomics.

Non-ribosomal peptide synthetases (NRPS) and type-I polyketide synthases (PKS-I) are multimodular enzymes involved in biosynthesis of oligopeptide and polyketide secondary metabolites produced by microorganisms such as bacteria and fungi. New findings regarding the mechanisms underlying NRPS and PKS-I evolution illustrate how microorganisms expand their metabolic potential. During the last decade rapid development of bioinformatics tools as well as improved sequencing and annotation of microbial genomes led to discovery of novel bioactive compounds synthesized by NRPS and PKS-I through genome-mining. Taking advantage of these technological developments metagenomics is a fast growing research field which directly studies microbial genomes or specific gene groups and their products. Discovery of novel bioactive compounds synthesized by NRPS and PKS-I will certainly be accelerated through metagenomics, allowing the exploitation of so far untapped microbial resources in biotechnology and medicine.

Evolution and taxonomic distribution of nonribosomal peptide and polyketide synthases

The majority of nonribosomal peptide synthases and type I polyketide synthases are multimodular megasynthases of oligopeptide and polyketide secondary metabolites, respectively. Owing to their multimodular architecture, they synthesize their metabolites in assembly line logic. The ongoing genomic revolution together with the application of computational tools has provided the opportunity to mine the various genomes for these enzymes and identify those organisms that produce many oligopeptide and polyketide metabolites. In addition, scientists have started to comprehend the molecular mechanisms of megasynthase evolution, by duplication, recombination, point mutation and module skipping. This knowledge and computational analyses have been implemented towards predicting the specificity of these megasynthases and the structure of their end products. It is an exciting field, both for gaining deeper insight into their basic molecular mechanisms and exploiting them biotechnologically.

Siderophores in fluorescent pseudomonads: new tricks from an old dog

Iron is an essential nutrient for almost all bacteria; however, at neutral pH its bioavailability is limited. Siderophores are iron-binding compounds of low molecular weight that enable the microorganisms that produce them to obtain the necessary iron from the environment. Fluorescent pseudomonads include those that are plant growth promoting, human and plant pathogens, as well as bacteria involved in the biodegradation of xenobiotics. Although pyoverdine is the main siderophore produced by different fluorescent pseudomonads, other siderophores produced by fluorescent pseudomonads include pyochelin, (thio)quinolobactin and pyridine-2, 6-bis thiocarboxylic acid. Research on siderophores continues to reveal new information on their regulation, biosynthesis, function and properties. In this review, we focus on recent advances in the field, particularly on newly characterized siderophores produced by fluorescent pseudomonads and their biotechnological potential.

Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes.

Considering that 70% of our planet’s surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs) and polyketides (PKs) are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes) and type-I polyketide synthases (PKSes-I), respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS) technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds.

Pepino mosaic virus triple gene block protein 1 (TGBp1) interacts with and increases tomato catalase 1 activity to enhance virus accumulation.

Various plant factors are co-opted by virus elements (RNA, proteins) and have been shown to act in pathways affecting virus accumulation and plant defence. Here, an interaction between Pepino mosaic virus (PepMV) triple gene block protein 1 (TGBp1; p26) and tomato catalase 1 (CAT1), a crucial enzyme in the decomposition of toxic hydrogen peroxide (H₂O₂), was identified using the yeast two-hybrid assay, and confirmed via an in vitro pull-down assay and bimolecular fluorescent complementation (BiFC) in planta. Each protein was independently localized within loci in the cytoplasm and nuclei, sites at which their interaction had been visualized by BiFC. Following PepMV inoculation, CAT mRNA and protein levels in leaves were unaltered at 0, 3 and 6 days (locally) and 8 days (systemically) post-inoculation; however, leaf extracts from the last two time points contained increased CAT activity and lower H₂O₂ evels. Overexpression of PepMV p26 in vitro and in planta conferred the same effect, suggesting an additional involvement of TGBp1 in potexvirus pathogenesis. The accumulation of PepMV genomic and subgenomic RNAs and the expression of viral coat protein in noninoculated (systemic) leaves were reduced significantly in CAT-silenced plants. It is postulated that, during PepMV infection, a p26-CAT1 interaction increases H₂O₂ cavenging, thus acting as a negative regulator of plant defence mechanisms to promote PepMV infections.

The complex evolutionary history of aminoacyl-tRNA synthetases

Abstract Aminoacyl-tRNA synthetases (AARSs) are a superfamily of enzymes responsible for the faithful translation of the genetic code and have lately become a prominent target for synthetic biologists. Our large-scale analysis of >2500 prokaryotic genomes reveals the complex evolutionary history of these enzymes and their paralogs, in which horizontal gene transfer played an important role. These results show that a widespread belief in the evolutionary stability of this superfamily is misconceived. Although AlaRS, GlyRS, LeuRS, IleRS, ValRS are the most stable members of the family, GluRS, LysRS and CysRS often have paralogs, whereas AsnRS, GlnRS, PylRS and SepRS are often absent from many genomes. In the course of this analysis, highly conserved protein motifs and domains within each of the AARS loci were identified and used to build a web-based computational tool for the genome-wide detection of AARS coding sequences. This is based on hidden Markov models (HMMs) and is available together with a cognate database that may be used for specific analyses. The bioinformatics tools that we have developed may also help to identify new antibiotic agents and targets using these essential enzymes. These tools also may help to identify organisms with alternative pathways that are involved in maintaining the fidelity of the genetic code.

Intra- and inter-serotypic recombinations in the 5΄ UTR-VP4 region of Echovirus 30 strains

Recombination has been recognized as a major mechanism of evolution in enteroviruses. The Echovirus 30 (E-30) strain Gior was sequenced and phylogenetically compared to all available E-30 sequences to detect recombination events between the 5΄UTR and VP1 genomic regions. The comparison of phylogenetic trees of the 5΄UTR and VP1 revealed incongruences concerning strains, lineages and sub-lineages. Comparative analysis of 62 E-30 sub-genomic sequences revealed six different recombination events that almost all occurred in the same region, having a start point in the 3΄end of the 5΄ UTR and end point in VP4. The only exception was the sub-lineage of Gior for which both borders of recombination were located in the 5΄UTR. These results describe for the first time recombination events in this region in circulating EV-B strains, revealing the exact points of these recombination events, highlighting the impact of such events on the evolution and epidemiology of enteroviruses.

NAT/NCS2-hound: A webserver for the detection and evolutionary classification of prokaryotic and eukaryotic nucleobase-cation symporters of the NAT/NCS2 family.

Nucleobase transporters are important for supplying the cell with purines and/or pyrimidines, for controlling the intracellular pool of nucleotides and for obtaining exogenous nitrogen/carbon sources for the metabolism. Nucleobase transporters are also evaluated as potential targets for antimicrobial therapies, since several pathogenic microorganisms rely on purine/pyrimidine salvage from their hosts. The majority of known nucleobase transporters belong to the evolutionarily conserved and ubiquitous NAT/NCS2 protein family. Based on a large-scale phylogenetic analysis that we performed on thousands of prokaryotic proteomes, we have developed a webserver that can detect and distinguish this family of transporters from other homologous families that recognize different substrates. We can further categorize these transporters to certain evolutionary groups with distinct substrate preferences. The webserver scans whole proteomes and graphically displays which proteins are identified as NAT/NCS2, to which evolutionary groups and subgroups they belong to and which conserved motifs they have. For key subgroups and motifs, the server displays annotated information from published crystal-structures and mutational studies pointing to key functional amino acids that may help experts assess the transport capability of the target sequences. The server is 100% accurate in detecting NAT/NCS2 family members. We also used the server to analyze 9109 prokaryotic proteomes and identified Clostridia, Bacilli, β- and γ-Proteobacteria, Actinobacteria and Fusobacteria as the taxa with the largest number of NAT/NCS2 transporters per proteome. An analysis of 120 representative eukaryotic proteomes also demonstrates the server’s capability of correctly analyzing this major lineage, with plants emerging as the group with the highest number of NAT/NCS2 members per proteome.

Microbial diversity in biodeteriorated Greek historical documents dating back to the 19th and 20th century: A case study

Paper documents in archives, libraries, and museums often undergo biodeterioration by microorganisms. Fungi and less often bacteria have been described to advance paper staining, so called “foxing” and degradation of paper substrates. In this study, for the first time, the fungal and bacterial diversity in biodeteriorated paper documents of Hellenic General State Archives dating back to the 19th and 20th century has been assessed by culture‐dependent and independent methods. The internally transcribed spacer (ITS) region and 16S rRNA gene were amplified by PCR from fungal and bacterial isolates and amplicons were sequenced. Sequence analysis and phylogeny revealed fungal phylotypes like Penicillium sp., Cladosporium sp., Penicillium citrinum, Alternaria infectoria, Alternaria alternata, Epicoccum nigrum, and Penicillium chrysogenum which are often implicated in paper deterioration. Bacterial phylotypes closely related to known biodeteriogenic bacteria such as Bacillus spp., Micrococcus spp., Kocuria sp. in accordance with previous studies were characterized. Among the fungal phylotypes described in this study are included well‐known allergens such as Penicillium spp., Alternaria spp., and Cladosporium spp. that impose a serious health threat on staff members and scholars. Furthermore, fungal isolates such as Chalastospora gossypii and Trametes ochracea have been identified and implicated in biodeterioration of historical paper manuscripts in this study for the first time. Certain new or less known fungi and bacteria implicated in paper degradation were retrieved, indicating that particular ambient conditions, substrate chemistry, or even location might influence the composition of colonizing microbiota.