Addressing the functionality of predicted genes remains an enormous challenge in the postgenomic era. A prime example of genes lacking functional assignments are the poorly conserved, early expressed genes of lytic bacteriophages, whose products are involved in the subversion of the host metabolism. In this study, we focused on the composition of important macromolecular complexes of Pseudomonas aeruginosa involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism, and cell division during infection with members of seven distinct clades of lytic phages. Using affinity purifications of these host protein complexes coupled to mass spectrometric analyses, 37 host complex-associated phage proteins could be identified. Importantly, eight of these show an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes. Using complementary protein-protein interaction assays, we further mapped the inhibitory interaction of gp12 of phage 14-1 to the ? subunit of the RNA polymerase. Together, our data demonstrate the powerful use of interactomics to unravel the biological role of hypothetical phage proteins, which constitute an enormous untapped source of novel antibacterial proteins. (Data are available via ProteomeXchange with identifier PXD001199.).
USA300 methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent MRSA in the United States of America (USA) and a global epidemic threat. We investigated the prevalence of USA300 at a tertiary care hospital in Zurich, Switzerland, where all MRSA strains have been collected and PFGE typed since 1992. These strains were retrospectively compared to the PFGE pattern of USA300 strain JE2. Isolates with a respective PFGE pattern were spa-typed and tested for the presence of the arginine catabolic mobile element (ACME) arc gene cluster and Panton-Valentine Leucocidin (PVL) genes. The first MRSA strain with a USA300 PFGE pattern was isolated in 2001 from a patient visiting from the USA. USA300 strains represented between 0% (in 2002) and 9.2% (in 2012) of all MRSA isolates in our hospital. We identified various USA300 subtypes based on either the PFGE pattern, the spa-type or absence of either the PVL genes or ACME arc gene cluster. All the USA300 strains including the variants (n=47) accounted for 5.6% of all MRSA isolates typed between 2001 and 2013 and reached a maximum of 14.5% in 2009. They predominantly caused skin and soft tissue infections (74.4%). In conclusion, even though USA300 has been present in our hospital for over twelve years it has not become the predominant MRSA clone like in the USA. However, in light of the global burden of USA300, care must be taken to further contain the spread of this lineage and of MRSA in general in our hospital.
Extracytoplasmic function (ECF) ? factors control the transcription of genes involved in different cellular functions, such as stress responses, metal homeostasis, virulence-related traits, and cell envelope structure. The genome of Bradyrhizobium japonicum, the nitrogen-fixing soybean endosymbiont, encodes 17 putative ECF ? factors belonging to nine different ECF ? factor families. The genes for two of them, ecfQ (bll1028) and ecfF (blr3038), are highly induced in response to the reactive oxygen species hydrogen peroxide (H(2)O(2)) and singlet oxygen ((1)O(2)). The ecfF gene is followed by the predicted anti-? factor gene osrA (blr3039). Mutants lacking EcfQ, EcfF plus OsrA, OsrA alone, or both ? factors plus OsrA were phenotypically characterized. While the symbiotic properties of all mutants were indistinguishable from the wild type, they showed increased sensitivity to singlet oxygen under free-living conditions. Possible target genes of EcfQ and EcfF were determined by microarray analyses, and candidate genes were compared with the H(2)O(2)-responsive regulon. These experiments disclosed that the two ? factors control rather small and, for the most part, distinct sets of genes, with about half of the genes representing 13% of the members of H(2)O(2)-responsive regulon. To get more insight into transcriptional regulation of both ? factors, the 5 ends of ecfQ and ecfF mRNA were determined. The presence of conserved sequence motifs in the promoter region of ecfQ and genes encoding EcfQ-like ? factors in related ?-proteobacteria suggests regulation via a yet unknown transcription factor. By contrast, we have evidence that ecfF is autoregulated by transcription from an EcfF-dependent consensus promoter, and its product is negatively regulated via protein-protein interaction with OsrA. Conserved cysteine residues 129 and 179 of OsrA are required for normal function of OsrA. Cysteine 179 is essential for release of EcfF from an EcfF-OsrA complex upon H(2)O(2) stress while cysteine 129 is possibly needed for EcfF-OsrA interaction.
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