Use of Real-Time Quantitative PCR for the Analysis of φLC3 Prophage Stability in Lactococci

Lunde, Merete; Blatny, Janet Martha; Lillehaug, Dag; Aastveit, Are H.; Nes, Ingolf F.

doi:10.1128/aem.69.1.41-48.2003

Cited by 29 publications

(27 citation statements)

References 42 publications

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“…2 is the presence of late gene phagespecific transcripts at time zero. This may be attributable to a stationary-phase-dependent decrease in prophage stability, as previously described for LC3 in lactococci and P22 in Salmonella enterica serovar Typhimurium (30,40). Evidence in support of this hypothesis comes from the lack of phage transcripts at time zero in Fig.…”

Section: Discussionsupporting

confidence: 65%

Transcription of the Toxin Genes Present within the Staphylococcal Phage φSa3ms Is Intimately Linked with the Phage's Life Cycle

2003

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Sa3ms, a lysogenic bacteriophage encoding the staphylococcal enterotoxins SEA, SEG, and SEK and the fibrinolytic enzyme staphylokinase (Sak), was identified in the unannotated genome sequence of the hypervirulent community-acquired Staphylococcus aureus strain 476. We found that mitomycin C induction of Sa3ms led to increased transcription of all four virulence factors. The increase in sea and sak transcription was a result of read-through transcription from upstream latent phage promoters and an increase in phage copy number. The majority of the seg2 and sek2 transcripts were shown to initiate from the upstream phage cI promoter and hence were regulated by factors influencing cI transcription. The lysogeny module of Sa3ms was shown to have some -like features with divergent cI and cro genes. Band shift assays were used to identify binding sites for both CI and Cro within the region between these genes, suggesting a mechanism of control for the Sa3ms lytic-lysogenic switch. Our findings suggest that the production of phage-encoded virulence factors in S. aureus may be regulated by processes that govern lysogeny.Staphylococcus aureus is a leading cause of nosocomial and community-acquired infections worldwide (42). S. aureus causes a wide range of diseases that vary in severity from mild skin infections, such as boils and furuncles, to life-threatening diseases, like toxic shock syndrome and endocarditis. The ability of S. aureus to cause such a wide variety of diseases is thought to be due in part to its elaboration of a large number of secreted and cell-surface associated virulence factors (1,5,21,36,48).Much of the variation between S. aureus strains appears to be attributable to mobile genetic elements, such as plasmids, bacteriophages, pathogenicity islands, transposons, insertion sequences, and the staphylococcal chromosomal cassette (2, 27). The bacteriophages and pathogenicity islands of S. aureus encode many virulence factors (35). The known phage-encoded virulence factors include Panton-Valentine leukocidins (24, 33), exfoliative toxin type A (47), and staphylococcal enterotoxins (SEs) (3). The SEs constitute a family of related proteins whose activities are associated with staphylococcal food poisoning, toxic shock syndrome, and possibly several autoimmune disorders (17, 41). SEs are powerful superantigens that activate subsets of T lymphocytes to liberate various cytokines, including gamma interferon and tumor necrosis factor (41). In addition to these characterized phage-encoded virulence factors, a number of putative S. aureus virulence factors, such as staphylokinase (Sak), are also encoded in phage genomes (25). Staphylokinase, a potent plasminogen activator, has been hypothesized to aid in the dissemination of S. aureus from fibrinous clots and abscesses (1).The genome sequences of two S. aureus isolates derived from life-threatening community-acquired infections have been determined (2; unpublished data). The methicillin-resistant strain MW2 (sequenced at Juntendo University, Tokyo, Japan) was i...

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Section: Discussionsupporting

confidence: 65%

Transcription of the Toxin Genes Present within the Staphylococcal Phage φSa3ms Is Intimately Linked with the Phage's Life Cycle

2003

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“…We have previously shown that spontaneous prophage induction frequency is dependent on the growth phase of the lysogenic bacterial host (29). It was shown that the induction frequency increased in the exponential growth phase and reached a maximum in the early stationary phase, both at growth temperatures of 30 and 15°C.…”

Section: Resultsmentioning

confidence: 99%

Effects of Diverse Environmental Conditions on φLC3 Prophage Stability in Lactococcus lactis

Lunde¹,

Aastveit²,

Blatny³

et al. 2005

Appl Environ Microbiol

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The effects of various growth conditions on spontaneous LC3 prophage induction in Lactococcus lactis subsp. cremoris IMN-C1814 was analyzed with a half fraction of a 4 4 factorial experimental design. The four factors included in the study were nutrient availability, acidity, osmolarity, and temperature, each applied at four levels. These environmental factors are related to the fermentation processes in the dairy industry, in which bacteriophage attacks on sensitive starter strains are a constant threat to successful fermentation processes. The frequency of spontaneous LC3 induction was determined by quantitative analyses of restored DNA attachment sites (attB) on the bacterial chromosomes in a population of lysogenic cells. Statistical analysis revealed that all four environmental factors tested affected LC3 prophage stability and that the environmental factors were involved in interactions (interactions exist when the effect of one factor depends on the level of another factor). The spontaneous LC3 induction frequency varied from 0.08 to 1.76%. In general, the induction frequency remained at the same rate or decreased when level 1 to 3 of the four environmental factors was applied. At level 4, which generally gave the least favorable growth conditions, the induction frequency was either unchanged, decreased, or increased, depending on the type of stress. It appeared that the spontaneous induction frequency was independent of the growth behavior of the host. It was the environmental growth conditions that were the decisive factor in induction frequency.

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“…After the phage is excised from the bacterial chromosome, the 13 genome recircularizes and the attP site is restored. Therefore, this site can be used as a marker for phage replication in the host cell (16). For this purpose, primers that span the attP site of 13 were designed.…”

Section: Resultsmentioning

confidence: 99%

Ciprofloxacin and Trimethoprim Cause Phage Induction and Virulence Modulation in Staphylococcus aureus

Goerke

Köller

Wolz

2006

Antimicrob Agents Chemother

201

184

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Staphylococcus aureus asymptomatically colonizes the anterior nares of humans but also causes a wide spectrum of acute and chronic diseases. The ability of S. aureus to cause such a wide variety of diseases is probably due to the fact that it produces a large number of cell-bound and secreted virulence factors (15). Much of the dissimilarity between S. aureus strains is dependent on the presence of mobile genetic elements, such as plasmids, bacteriophages, pathogenicity islands, transposons, and insertions sequences (2, 9, 13). Many virulence factors are encoded on such mobile elements (3,5,12,18,19,26). Phages which integrate into the chromosomal gene coding for ␤-hemolysin (hlb) are widely distributed in S. aureus strains of human origin. Most of them encode accessory virulence determinants such as staphylokinase (sak) and enterotoxins (5, 10). Staphylokinase is a plasminogen activator and has thus been postulated to aid the bacteria in their dissemination from clots and abscesses (1). Additionally, it was shown that S. aureus can resist human defensins by the production of staphylokinase (11).Many prophages are induced by environmental conditions that lead to DNA damage, including exposure to reactive oxygen species generated by leukocytes or exposure to exogenous agents such as antibiotics (24). We could show that phages are mobilized during chronic infection of the lungs of patients with cystic fibrosis (CF), possibly due to the strong selective pressure exercised on the pathogen by the specific host response and/or by the regular exposure to antibiotics (8). Quinolones (for instance, ciprofloxacin) or trimethoprim are frequently used for the treatment of lung infections in patients with CF. Ciprofloxacin acts as an antibacterial agent by trapping DNA gyrase on DNA and thus blocking the replication fork movement. Trimethoprim prevents the incorporation of thymine into bacterial DNA by inhibiting the dihydrofolate reductase. Blockage of the replication fork by these drugs may trigger the DNA repair system. For Escherichia coli it was shown that damaged bacterial DNA interacts with and activates the multifunctional RecA protein (25). Stimulated RecA promotes the autoproteolysis of the repressor of DNA repair functions, LexA, and also that of the phage repressor CI (20, 25). The decline in active CI levels permits derepression of phage lytic genes and the resumption of lytic growth.We analyzed the effects of subinhibitory concentrations of ciprofloxacin and trimethoprim on phage induction and the expression of phage-related virulence factors in comparison to those of the classical phage-inducing agent mitomycin C in S. aureus. CF isolates for which induction of hlb-converting phages has been demonstrated in vivo (8) as well as a phage 13 lysogen of prototypic S. aureus strain 8325-4 were selected for this study. We showed that ciprofloxacin and trimethoprim trigger phage induction and lead to enhanced expression of phage-encoded virulence factors. MATERIALS AND METHODSBacterial strains. S. aureus isolates from...

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Use of Real-Time Quantitative PCR for the Analysis of φLC3 Prophage Stability in Lactococci

Cited by 29 publications

References 42 publications

Transcription of the Toxin Genes Present within the Staphylococcal Phage φSa3ms Is Intimately Linked with the Phage's Life Cycle

Transcription of the Toxin Genes Present within the Staphylococcal Phage φSa3ms Is Intimately Linked with the Phage's Life Cycle

Effects of Diverse Environmental Conditions on φLC3 Prophage Stability in Lactococcus lactis

Ciprofloxacin and Trimethoprim Cause Phage Induction and Virulence Modulation in Staphylococcus aureus

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