معن محسن نعمة نعمة النقيب

Staphylococcus aureus subsp. anaerobius is a microaerophilic, catalase-negative bacterium responsible for abscess pathology (Morel’s disease) in small ruminants. We performed whole-genome sequencing to a collection of isolates taken in Europe and Africa over the last 30 years, and carried out an evolutionary genomic analysis to understand the molecular bases of its host adaptation and restricted metabolism. Phylodynamic analyses showed that S. anaerobius emerged from a Staphylococcus aureus progenitor about 1000 years ago (716–1184), with an evolutionary rate of ∼1.2 SNPs/year (approximately 10-fold slower than S. aureus clones), before differentiating into two distinct lineages separating African and European isolates. The S. anaerobius genome displays signatures of extreme adaptation to a highly specific niche, with 205 pseudogenes that together represent over 10 % of the genome and affect many metabolic and pathogenic pathways. In addition, S. anaerobius contains 87 highly similar insertion sequences (IS) located in intergenic regions. Our functional analysis suggests that the IS transcription could affect the expression of their flanking genes, using at least two complementary strategies: antisense RNA or modification of promoter regions. We propose that the IS-mediated control of gene expression underpins an orchestrated mechanism of host adaptation. Remarkably, we also identified 6 large genomic transversions (size range: 70–346 kb) flanked by IS, presumably the result of homologous recombination. In summary, S. anaerobius evolved from S. aureus undergoing restrictive host specialisation, which shaped its genome through widespread pseudogenisation, accumulation of IS that modulate gene expression, and large chromosomal rearrangements.

DNA-single strand annealing proteins (SSAPs) are recombinases frequently encoded in the genome of many bacteriophages. As SSAPs can promote homologous recombination among DNA substrates with an important degree of divergence, these en- zymes are involved both in DNA repair and in the gen- eration of phage mosaicisms. Here, analysing Sak and Sak4 as representatives of two different fami- lies of SSAPs present in phages infecting the clini- cally relevant bacterium Staphylococcus aureus, we demonstrate for the first time that these enzymes are absolutely required for phage reproduction. Dele- tion of the genes encoding these enzymes signifi- cantly reduced phage replication and the generation of infectious particles. Complementation studies re- vealed that these enzymes are required both in the donor (after prophage induction) and in the recipient strain (for infection). Moreover, our results indicated that to perform their function SSAPs require the ac- tivity of their cognate single strand binding (Ssb) pro- teins. Mutational studies demonstrated that the Ssb proteins are also required for phage replication, both in the donor and recipient strain. In summary, our re- sults expand the functions attributed to the Sak and Sak4 proteins, and demonstrate that both SSAPs and Ssb proteins are essential for the life cycle of temper- ate staphylococcal phages.

Targeting conserved and essential processes is a successful strategy to combat enemies. Remarkably, the clinically important Staphylococcus aureus pathogenicity islands (SaPIs) use this tactic to spread in nature. SaPIs reside passively in the host chromosome, under the control of the SaPI-encoded master repressor, Stl. It has been assumed that SaPI de-repression is effected by specific phage proteins that bind to Stl, initiating the SaPI cycle. Different SaPIs encode different Stl repressors, so each targets a specific phage protein for its de-repression. Broadening this narrow vision, we report here that SaPIs ensure their promiscuous transfer by targeting conserved phage mechanisms. This is accomplished because the SaPI Stl repressors have acquired different domains to interact with unrelated proteins, encoded by different phages, but in all cases performing the same conserved function. This elegant strategy allows intra- and inter-generic SaPI transfer, highlighting these elements as one of nature’s most fascinating subcellular parasites.

The dUTPase (Dut) enzymes prevent the misincorporation of uracil into the DNA and are encoded by almost all free-living organisms and some viruses. We have previously showed that phage-encoded trimeric Duts mediates the Staphylococcus aureus pathogenicity island (SaPIs) transfer by interacting to the SaPI-encoded repressor Stl, proposing that these Duts are regulatory proteins. Some S. aureus phages encode structurally unrelated dimeric Duts instead trimeric Duts. Surprisingly, a recent work, has involved one of these predicted dimeric Duts in the transfer of SaPIs by interacting with the same Stl repressor. With the aim of decipher the molecular basis of SaPI induction by dimeric Duts and to compare with the mechanism reported for trimeric Duts, we examined the SaPI mobilization capacity of dimeric Duts in vivo as well as its binding capacity to Stl repressor in vitro. We analyzed the effect of the dUTP in the Stl-Dut interaction, and finally, we solved the 3-D structure by X-ray crystallography of several dimeric Duts in different activation states. Our results show that dimeric and trimeric Duts from S. aureus phages present a striking parallelism in the mechanism of SaPI mobilization. These similarities would confirm the role of dUTP as new nucleotide with second messenger function. However, some differences suggest peculiarities in the molecular mechanism of Stl recognition and binding for each type of Duts. Our results support the idea that the signalling role of the Dut proteins is an important force driving evolution and speciation.