Genomic analysis of an emerging multiresistant Staphylococcus aureus strain rapidly spreading in cystic fibrosis patients revealed the presence of an antibiotic inducible bacteriophage

BACKGROUND: Staphylococcus aureus is a major human pathogen responsible for a variety of nosocomial and community-acquired infections. Recent reports show that the prevalence of Methicillin-Resistant S. aureus (MRSA) infections in cystic fibrosis (CF) patients is increasing. In 2006 in Marseille, France, we have detected an atypical MRSA strain with a specific antibiotic susceptibility profile and a unique growth phenotype. Because of the clinical importance of the spread of such strain among CF patients we decided to sequence the genome of one representative isolate (strain CF-Marseille) to compare this to the published genome sequences. We also conducted a retrospective epidemiological analysis on all S. aureus isolated from 2002 to 2007 in CF patients from our institution. RESULTS: CF-Marseille is multidrug resistant, has a hetero-Glycopeptide-Intermediate resistance S. aureus phenotype, grows on Cepacia agar with intense orange pigmentation and has a thickened cell wall. Phylogenetic analyses using Complete Genome Hybridization and Multi Locus VNTR Assay showed that CF-Marseille was closely related to strain Mu50, representing vancomycin-resistant S. aureus. Analysis of CF-Marseille shows a similar core genome to that of previously sequenced MRSA strains but with a different genomic organization due to the presence of specific mobile genetic elements i.e. a new SCCmec type IV mosaic cassette that has integrated the pUB110 plasmid, and a new phage closely related to phiETA3. Moreover this phage could be seen by electron microscopy when mobilized with several antibiotics commonly used in CF patients including, tobramycin, ciprofloxacin, cotrimoxazole, or imipenem. Phylogenetic analysis of phenotypically similar h-GISA in our study also suggests that CF patients are colonized by polyclonal populations of MRSA that represents an incredible reservoir for lateral gene transfer. CONCLUSION: In conclusion, we demonstrated the emergence and spreading of a new isolate of MRSA in CF patients in Marseille, France, that has probably been selected in the airways by antibiotic pressure. Antibiotic-mediated phage induction may result in high-frequency transfer and the unintended consequence of promoting the spread of virulence and/or antibiotic resistance determinants. The emergence of well-adapted MRSA is worrying in such population chronically colonized and receiving many antibiotics and represents a model for emergence of uncontrollable super bugs in a specific niche. REVIEWERS: This article was reviewed by Eric Bapteste, Pierre Pontarotti, and Igor Zhulin. For the full reviews, please go to the Reviewers' comments section

55.2, a phage T4 ORFan gene, encodes an inhibitor of Escherichia coli topoisomerase I and increases phage fitness

Topoisomerases are enzymes that alter the topological properties of DNA. Phage T4 encodes its own topoisomerase but it can also utilize host-encoded topoisomerases. Here we characterized 55.2, a phage T4 predicted ORF of unknown function. High levels of expression of the cloned 55.2 gene are toxic in E. coli. This toxicity is suppressed either by increased topoisomerase I expression or by partial inactivation of the ATPase subunit of the DNA gyrase. Interestingly, very low-level expression of 55.2, which is non-lethal to wild type E. coli, prevents the growth of a deletion mutant of the topoisomerase I (topA) gene. In vitro, gp55.2 binds DNA and blocks specifically the relaxation of negatively supercoiled DNA by topoisomerase I. In vivo, expression of gp55.2 at low non-toxic levels alters the steady state DNA supercoiling of a reporter plasmid. Although 55.2 is not an essential gene, competition experiments indicate that it is required for optimal phage growth. We propose that the role of gp55.2 is to subtly modulate host topoisomerase I activity during infection to insure optimal T4 phage yield

Fusion of mitochondria in mammalian cells is dependent on the mitochondrial inner membrane potential and independent of microtubules or actin

Mitochondrial fusion is a poorly characterized process which has mainly been studied in yeast and Drosophila but is thought to occur in all eukaryotes. Until now, there was only indirect evidence to support such a process in mammalian cells. In this study, using a cell fusion system, we found that mitochondrial fusion occurs rapidly in mammalian cells and is completed in less than 24 h. We report that the fusion of mitochondria requires an intact mitochondrial inner membrane potential but is independent of a functional cytoskeleton

Gp55.2 binds to DNA, inhibits Topo I relaxation activity but does not affect DNA gyrase supercoiling activity.

<p>(A) Electrophoretic mobility shift assays (EMSA). Mixtures containing 300 ng (= 79 fmol) of linear, form I’, or form I pDB29 DNA and the indicated amount of gp55.2-His₆ (95 ng = 6.9 pmol, namely one gp55.2 molecule per 65.6 bp for the amount DNA used in this assay) were incubated and analyzed as described in Materials and Methods. An arrow indicates the migration position of the form II DNA contaminating the form I DNA. (B) Relaxation assays mixtures containing 600 ng (= 158 fmol) form I pDB29 DNA and the indicated units of Topo I (0.47 U = 415 fmol) were incubated in the presence (+) or absence (–) of 855 ng of gp55.2-His₆ (= 63 pmol, one gp55.2 molecule per 14.5 bp of DNA), and the DNA products were analyzed as described in Materials and Methods. The migration positions of form I, form I’ and form II DNA are indicated on the left; linear DNA migration position is indicated by an arrowhead. Results representative of two independent experiments are shown. (C) Supercoiling assays mixtures containing 158 fmol form I’ pDB29 DNA and the indicated units of DNA gyrase (0.42 U = 196 fmol) were incubated in the presence (+) or absence (–) of 855 ng of gp55.2-His₆ and the DNA products were analyzed as described in the Materials and Methods section. Results representative of two independent experiments are shown. (D) Representation of form I, form I’ and form II plasmid DNA. Note that the treatment of form I plasmid DNA by a eukaryotic Topo I to obtain form I’ DNA results, at equilibrium, in a distribution of relaxed DNA topoisomers (A, middle panel) whose maximum corresponds to the fully relaxed form I’ plasmid illustrated in D.</p

hFis1, a novel component of the mammalian mitochondrial fission machinery

The balance between the fission and fusion mechanisms regulate the morphology of mitochondria. In this study we have indentified a mammalian protein that we call hFis1, which is the orthologue of the yeast Fis1p known to participate in yeast mitochondrial division. hFis1, when overexpressed in various cell types, localized to the outer mitochondrial membrane and induced mitochondrial fission. This event was inhibited by a dominant negative mutant of Drp1 (Drp1(K38A)), a major component of the fission apparatus. Fragmentation of the mitochondrial network by hFis1 was followed by the release of cytochrome c and ultimately apoptosis. Bcl-xL was able to block cytochrome c release and apoptosis but failed to prevent mitochondrial fragmentation. Our studies show that hFis1 is part of the mammalian fission machinery and suggest that regulation of the fission processes might be involved in apoptotic mechanisms.</p

<i>55</i>.<i>2</i> expression affects the control of DNA topology and plasmid copy number in <i>E</i>. <i>coli</i>.

<p>(A) Plasmid topoisomers analysis. Left panel: plasmid DNA was extracted from exponentially growing DH5α harboring pYM58 (<i>55</i>.<i>2</i>_IS) or pDB2114 (<i>55</i>.<i>2</i>) plasmids. Plasmid topoisomers were resolved on TBE agarose gels containing the indicated amount of CLQ. The position of migration of relaxed and/or nicked circular DNA is indicated (R). Right panel: Densitometry analysis of the topoisomer distribution on 1.5 μg ml^-1 CLQ gel of four independent samples of pYM58 or pDB2114 plasmid DNA (gel images are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124309#pone.0124309.s004" target="_blank">S4A Fig</a>). Plotted is the average (lines) and standard deviation (shaded area) of relative plasmid density as a function of negative supercoiling. (B) 2D electrophoretic separation of plasmid topoisomers. Left panel: schematic representation of a 2D gel. The migration positions of negatively supercoiled (–), positively supercoiled (+), and form II topoisomers (N) are indicated. Right panel: gel images show the 2D topoisomer distribution of plasmid DNA samples prepared as in A. Chloroquine concentration was 1.5 μg ml^-1 and 25 μg ml^-1 in the first and second dimension, respectively. (C) Plasmid copy number analysis. Linearized plasmid DNA samples from the experiment shown in A were quantified and normalized to the amount (A_600nm) of bacteria used to extract the plasmids. The data represents means and standard error of four independent cultures. (D) Plasmid DNA was extracted from overnight cultures of DHB3, DB503, and DH5α transformed with pBAD101 (<i>55</i>.<i>2</i> –) or pDB2114-101 (<i>55</i>.<i>2</i> +), and a reporter plasmid (pDB868-2). Linearized plasmids were analyzed by agarose gel electrophoresis.</p

The toxicity of <i>55</i>.<i>2</i> in <i>E</i>. <i>coli</i> is suppressed by an increase in the copy number of <i>topA</i>.

<p>(A) Liquid growth assay. Overnight cultures of DB503 cells harboring pBAD101 (ctr) or pDB2114-101 (<i>55</i>.<i>2</i>) plasmids were diluted in fresh LB medium and optical density (A_600nm) was measured at the indicated times. At A_600nm = 0.3, cultures were split in two and one half was induced with 0.2% arabinose (vertical arrow). When A_600nm reached ≈ 1, cultures were diluted 10-fold in prewarmed medium plus or minus arabinose. The graph depicts the data of a representative experiment. (B) Reversibility assay. During a growth curve assay, aliquots of arabinose-induced DB503 cultures, harboring pBAD101 (<i>55</i>.<i>2</i> –) or pDB2114-101 (<i>55</i>.<i>2</i> +) plasmids, were withdrawn at the indicated times, washed in cold media without arabinose, and adjusted to the same A_600nm. Serial 10-fold dilutions were spotted on LB plates without arabinose. (C) DB503 cells transformed with pBAD101 (vector) or pDB2114-101 (<i>55</i>.<i>2</i>) and one of the compatible plasmids, pDB868-2 (vector, 1–2), pDB34-8-4 (<i>topA</i>, 3–4), or pDB34-8 (<i>topA</i>, 5–6) were streaked on LB plates with or without 0.2% arabinose. (D) Plasmid based lethality assay. Overnight cultures of AS1047 (<i>topA</i> +) or AS1050 (<i>topA</i>-) transformed with pBAD33-K (<i>55</i>.<i>2</i> –) or pDB2114-33-K (<i>55</i>.<i>2</i> +) were diluted and outgrown as indicated in the Materials and Methods section. Aliquots were diluted, and plated on M63 plates supplemented with glucose and X-gal. The number of blue and white colonies was scored after 36h at 37°C. Representative photographs are shown in the upper panel (the position of a rare white colony in inset IV is indicated by an arrow). The lower panel depicts percentage of white colonies; average and standard deviation are from three independent experiments.</p

Gp55.2 binds to DNA, inhibits Topo I relaxation activity but does not affect DNA gyrase supercoiling activity.

<p>(A) Electrophoretic mobility shift assays (EMSA). Mixtures containing 300 ng (= 79 fmol) of linear, form I’, or form I pDB29 DNA and the indicated amount of gp55.2-His₆ (95 ng = 6.9 pmol, namely one gp55.2 molecule per 65.6 bp for the amount DNA used in this assay) were incubated and analyzed as described in Materials and Methods. An arrow indicates the migration position of the form II DNA contaminating the form I DNA. (B) Relaxation assays mixtures containing 600 ng (= 158 fmol) form I pDB29 DNA and the indicated units of Topo I (0.47 U = 415 fmol) were incubated in the presence (+) or absence (–) of 855 ng of gp55.2-His₆ (= 63 pmol, one gp55.2 molecule per 14.5 bp of DNA), and the DNA products were analyzed as described in Materials and Methods. The migration positions of form I, form I’ and form II DNA are indicated on the left; linear DNA migration position is indicated by an arrowhead. Results representative of two independent experiments are shown. (C) Supercoiling assays mixtures containing 158 fmol form I’ pDB29 DNA and the indicated units of DNA gyrase (0.42 U = 196 fmol) were incubated in the presence (+) or absence (–) of 855 ng of gp55.2-His₆ and the DNA products were analyzed as described in the Materials and Methods section. Results representative of two independent experiments are shown. (D) Representation of form I, form I’ and form II plasmid DNA. Note that the treatment of form I plasmid DNA by a eukaryotic Topo I to obtain form I’ DNA results, at equilibrium, in a distribution of relaxed DNA topoisomers (A, middle panel) whose maximum corresponds to the fully relaxed form I’ plasmid illustrated in D.</p

Loss of gene <i>55</i>.<i>2</i> function reduces T4 phage fitness.

<p>(A) CR63 cells grown in M9 medium were infected with T4 K10 (wt) or T4 K10-<i>55</i>.<i>2</i> (<i>55</i>.<i>2</i>) at a moi of 6 at 30°C. Intracellular phage accumulation was followed at the indicated time points; free phages at 25 min represented < 12% of the total infective centers. Data represents mean and ranges of two (wt) and four (<i>55</i>.<i>2</i>) independent experiments. (B) Competition experiment. A mix of T4 K10 and T4 K10-<i>55</i>.<i>2</i> with an initial ratio of 1:9 was grown on <i>E</i>. <i>coli</i> CR63 in M9S medium at 37°C at low moi (< 0.1) over successive growth cycles. The percentage of <i>55</i>.<i>2+</i> phages was determined by PCR and digestion as described in Materials and Methods. Data represent mean and standard deviation of four independent experiments; the dotted line represents the result of a simulation were the <i>55</i>.<i>2</i> mutant has a 16% disadvantage per growth cycle. The intracellular phage accumulation over a single growth cycle in these conditions is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124309#pone.0124309.s005" target="_blank">S5A Fig</a>.</p

55.1, a gene of unknown function of phage T4, impacts on Escherichia coli folate metabolism and blocks DNA repair by the NER.

Phage T4, the archetype of lytic bacterial viruses, needs only 62 genes to propagate under standard laboratory conditions. Interestingly, the T4 genome contains more than 100 putative genes of unknown function, with few detectable homologues in cellular genomes. To characterize this uncharted territory of genetic information, we have identified several T4 genes that prevent bacterial growth when expressed from plasmids under inducible conditions. Here, we report on the various phenotypes and molecular characterization of 55.1, one of the genes of unknown function. High-level expression from the arabinose-inducible P(BAD) promoter is toxic to the bacteria and delays the intracellular accumulation of phage without affecting the final burst size. Low-level expression from T4 promoter(s) renders bacteria highly sensitive to UV irradiation and hypersensitive to trimethoprim, an inhibitor of dihydrofolate reductase. The delay in intracellular phage accumulation requires UvsW, a T4 helicase that is also a suppressor of 55.1-induced toxicity and UV sensitivity. Genetic and biochemical experiments demonstrate that gp55.1 binds to FolD, a key enzyme of the folate metabolism and suppressor of 55.1. Finally, we show that gp55.1 prevents the repair of UV-induced DNA photoproducts by the nucleotide excision repair (NER) pathway through interaction with the UvrA and UvrB proteins