Elimination of Chromosomal Island SpyCIM1 from Streptococcus pyogenes Strain SF370 Reverses the Mutator Phenotype and Alters Global Transcription

This work was made possible by an Oklahoma Center for the Advancement of Science and Technology (OCAST) grant HR11-133 and by NIH Grant Number R15A1072718 to WMM and NIH Grant AI11822 to VAF.Streptococcus pyogenes chromosomal island M1 (SpyCIM1) integrates by site-specific recombination into the 5’ end of DNA mismatch repair (MMR) gene mutL in strain SF370SmR, blocking transcription of it and the downstream operon genes. During exponential growth, SpyCIM1 excises from the chromosome and replicates as an episome, restoring mutL transcription. This process is reversed in stationary phase with SpyCIM1 re-integrating into mutL, returning the cells to a mutator phenotype. Here we show that elimination of SpyCIM1 relieves this mutator phenotype. The downstream MMR operon genes, multidrug efflux pump lmrP, Holliday junction resolution helicase ruvA, and DNA base excision repair glycosylase tag, are also restored to constitutive expression by elimination of SpyCIM1. The presence of SpyCIM1 alters global transcription patterns in SF370SmR. RNA sequencing (RNA-Seq) demonstrated that loss of SpyCIM1 in the SpyCIM1 deletion mutant, CEM1Δ4, impacted the expression of over 100 genes involved in virulence and metabolism both in early exponential phase, when the SpyCIM1 is episomal, as well as at the onset of stationary phase, when SpyCIM1 has reintegrated into mutL. Among these changes, the up-regulation of the genes for the antiphagocytic M protein (emm1), streptolysin O (slo), capsule operon (hasABC), and streptococcal pyrogenic exotoxin (speB), are particularly notable. The expression pattern of the MMR operon confirmed our earlier observations that these genes are transcribed in early exponential phase but silenced as stationary phase is approached. Thus, the direct role of SpyCIM1 in causing the mutator phenotype is confirmed, and further, its influence upon the biology of S. pyogenes was found to impact multiple genes in addition to the MMR operon, which is a novel function for a mobile genetic element. We suggest that such chromosomal islands are a remarkable evolutionary adaptation to promote the survival of its S. pyogenes host cell in changing environments.Yeshttp://www.plosone.org/static/editorial#pee

A. The loss of SPyCIM1 reverses of the mutator phenotype in SF370SmR. The rate of spontaneous mutation to ciprofloxacin resistance for isogenic strains CEM1Δ4 (SpyCIM1^-) and SF370SmR (SpyCIM1⁺) was determined by fluctuation test, and the rate of mutations per generation (μ) was calculated. For each strain, 30 parallel cultures were established with ~1,000 CFU/culture, grown for 24 h at 37°C, and plated individually on selective media. After 48 to 96 h of incubation, colonies were enumerated, and mutation rates with 95% confidence limits were calculated using the maximum likelihood estimation technique as implemented by the program <i>ft</i> [27, 34, 35]. SpyCIM1-free strain CEM1Δ4 showed a 200-fold reduction in the rate of spontaneous mutation as compared to SF370SmR. B. Enhanced resistance to UV irradiation following loss of SpyCIM1.

<p>Ten-fold dilutions of strains SF370SmR and CEM1Δ4, which were growing in either logarithmic or stationary phase, were spotted onto an agar plate and exposed to 254 nm UV light (120 μW/cm²) for 0 to 120 sec. At logarithmic phase, when SpyCIM1 is excised from the chromosome [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145884#pone.0145884.ref015" target="_blank">15</a>], there was little difference in UV sensitivity between the strains. However, at stationary phase when SpyCIM1 is integrated into <i>mutL</i>, SpyCIM1 containing strain SF370SmR showed >100-fold more killing than isogenic strain CEM1Δ4, which lacks this element and is consistent with the restoration of <i>ruvA</i> expression. The protocol was performed in a darkened room to prevent photoreactivation. In the figure, the time in seconds (0–120) and the dilution factor of the cells are shown on the x-axis and y-axis, respectively.</p

Resistance to ethidium bromide in <i>S</i>. <i>pyogenes</i> with and without SpyCIM1.

<p>Strains SF370SmR (lower panel) and its SpyCIM1 cured derivative CEM1Δ4 (upper panel) were grown at 37°C in THY broth with increasing concentrations of ethidium bromide (0, 1, 1.5, 2.5, and 5 μM). Each culture was grown in ten replicates, and growth was monitored every 15 minutes for 24 hours by the absorbance of the cultures at 600 nm. The data is presented as percentage of maximum growth observed for each strain without EtBr. Error bars, which were uniformly very small, are not shown for clarity of presentation. The numbers by each line indicate the concentration (μM) of EtBr in each culture.</p

Elimination of SpyCIM1 alters global transcription patterns.

<p>RNA was isolated from SF370SmR or CEM1Δ4 at the onset of logarithmic growth (Early Log) or immediately before the cells entered stationary phase (Late Log); the cells were either grown at 37°C (<b>panel A</b>) or 39°C (<b>panel B</b>). Samples were then analyzed by RNA sequencing (RNA-Seq). In the ratio of gene expression from SF370SmR compared to CEM1Δ4 for values greater than 3 or less than -3 are plotted against the corresponding gene identification number from the SF370SmR annotation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145884#pone.0145884.ref001" target="_blank">1</a>]. The MMR operon showed that no difference in expression was observed between the strains during early log phase but that the transcription of these genes was inhibited in SF370SmR at late log phase, in agreement with previous studies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145884#pone.0145884.ref015" target="_blank">15</a>]; an expanded view of this region is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145884#pone.0145884.s003" target="_blank">S3 Fig</a>. Notable genes whose expression was altered in SF370SmR or CEM1Δ4 are identified. The ticks on the X-axis mark every 500 genes. Legend: Act–acetate CoA-transferase operon; <i>act</i>–acetyltransferase Spy154; <i>adhA</i>–alcohol dehydrogenase; <i>citM</i>—Mg2+/citrate complex transporter; <i>cspR</i> - 23S rRNA methyltransferase; CT- conserved transmembrane protein Spy0169; <i>dap</i>—diaminopimelate epimerase; <i>emm1</i> –M protein; <i>glfP</i>—glycerol uptake facilitator; <i>grab</i>—protein G-like alpha2-macroglobulin-binding protein; <i>has</i>–hyaluronic acid capsule operon; His–histidine catabolism operon; Hyp–gene encoding a protein of unknown function; <i>lctO</i>–lactate oxidase; MFS–uncharacterized major facilitator family protein (Spy1392); MMR–MMR operon; ϕMTP–phage major tail protein; <i>norA</i>—antibiotic resistance protein NorA; Nuc–nucleotide interconversions operon; <i>pbuG</i>—guanine-hypoxanthine permease; <i>pncA</i>—pyrazinamidase/nicotinamidase; <i>prtS</i>—cell envelope proteinase PrtS; PTS—mannose/fructose PTS system operon; <i>pur</i>–purine biosynthesis operon; sclA—collagen-like surface protein SclA; <i>slo</i>–streptolysin O operon; SpyCIM1 –phage-like CI. The identification of the SpyCIM1 genes is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145884#pone.0145884.s001" target="_blank">S1 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145884#pone.0145884.s005" target="_blank">S1 Table</a>.</p

Elimination of SpyCIM1 (ϕ370.4) from its host strain.

<p><b>A.</b> The prophage-free <i>attB</i> region can be amplified from a SpyCI-free M1 strain MGAS5005 and from SpyCIM1 cured strain CEM1Δ4, but not from the parental SF370SmR strain, where this region is interrupted by the presence of the 13.5 kb chromosomal island in the stationary phase cells. The arrow indicates the size of the expected 1361 bp PCR product following the loss of SpyCIM1. Lane 1 is a no template PCR control. <b>B.</b> Probing of a Southern blot of <i>Hin</i>dIII digested chromosomal DNA with a <i>mutS-mutL</i> probe showed SF370SmR has an altered pattern due to the presence of the integrated SpyCIM1 chromosome (indicated by the arrow), compared to CEM1Δ4, which has an identical hybridization pattern with SpyCIM1-free strains MGAS5005 and K56. MW: Kilobase molecular weight marker (Life Technologies, Grand Island, NY).</p