Silencing of cryptic prophages in Corynebacterium
ABSTRACT 
DNA of viral origin represents a ubiquitous element of bacterial genomes . 
Its integration into host regulatory circuits is a pivotal driver of microbial evolution but requires the stringent regulation of phage gene activity . 
In this study , we describe the nucleoid-associated protein CgpS , which represents an essential protein functioning as a xenogeneic silencer in the Gram-positive Corynebacterium glutamicum . 
CgpS is encoded by the cryptic prophage CGP3 of the C. glutamicum strain ATCC 13032 and was first identified by DNA affinity chromatography using an early phage promoter of CGP3 . 
Genome-wide profiling of CgpS binding using chromatin affinity purification and sequencing ( ChAP-Seq ) revealed its association with AT-rich DNA elements , including the entire CGP3 prophage region ( 187 kbp ) , as well as several other elements acquired by horizontal gene transfer . 
Countersilencing of CgpS resulted in a significantly increased induction frequency of the CGP3 prophage . 
In contrast , a strain lacking the CGP3 prophage was not affected and displayed stable growth . 
In a bioinformatics approach , cgpS orthologs were identified primarily in actinobacterial genomes as well as several phage and prophage genomes . 
Sequence analysis of 618 orthologous proteins revealed a strong conservation of the secondary structure , supporting an ancient function of these xenogeneic silencers in phage-host interaction . 
1Institute of Bio - und Geosciences , IBG-1 : Biotechnology , Forschungszentrum Jülich , 52425 Jülich , Germany and 2Quantitative and Theoretical Biology , Heinrich-Heine-Universita ̈t Düsseldorf , 40225 , Düsseldorf , Germany 
INTRODUCTION
Viral DNA , in the form of functional prophages or degenerated ( cryptic ) phage elements , is ubiquitously found in bacterial genomes and may constitute up to 20 % of the host genome ( 1 -- 3 ) . 
The mosaic-like structure of bacterial genomes indicates that phage-mediated horizontal gene transfer is a pivotal driver of bacterial evolution ( 4 ) . 
Recent 
Nucleic Acids Research, 2016, Vol. 44, No. 21 10117–10131 doi: 10.1093/nar/gkw692
studies demonstrated that these elements might contribute significantly to the fitness of their respective host by improving stress tolerance , antibiotic resistance , biofilm formation or virulence ( 5,6 ) . 
Phage-mediated gene transfer may provide the cell with novel adaptive traits , improving the fitness of the receptor cell , but this does not occur without risks . 
The integration of selfish replicators , including transposable elements , integrative/conjugative elements ( ICE ) or phages , can lead to high transcriptional and translational costs or even cell death ( 7,8 ) . 
Hence , bacteria possess a number of different systems that confer resistance to foreign genetic elements , e.g. CRISPR/Cas and restriction modification ( RM ) systems ( 9,10 ) . 
However , to harness the adaptive potential of foreign DNA and enable its integration into the host regulatory circuitry , bacteria have evolved a rather mediative mechanism called xenogeneic silencing ( XS ) ( 11 -- 13 ) . 
This mechanism relies on the function of small nucleoid-associated proteins ( NAPs ) to target and inhibit the expression of foreign DNA , which is recognizable by its typically higher AT content in comparison to the host genome ( 1,14 ) . 
The major role of XS proteins is the binding of foreign DNA elements and the inhibition of transcription by a complex formation of AT-rich DNA stretches causing either the occlusion or trapping of the RNA polymerase ( 15,16 ) . 
Currently known XS proteins belong to one of four classes , consisting of H-NS-type proteins found in several proteobacteria ( 12,17 ) , Lsr2-like proteins of the actinomycetes ( 18 ) , MvaT of Pseudomonas species ( 16 ) and Rok of Bacillus subtilis ( 19 ) . 
To date , most studies have focused on host-encoded XS proteins acting as silencers of foreign DNA . 
However , it may also be of benefit for the foreign element to bring its own silencer protein to improve tolerance within the host cell . 
Here , we describe a novel prophage-encoded XS protein of the Lsr2-type in Corynebacterium glutamicum ATCC 13032 . 
The genome of this important industrial amino acid producer contains three cryptic prophages ( 20,21 ) . 
Whereas CGP1 and CGP2 are highly degenerated , CGP3 comprises almost 6 % of the entire genome ( 187 kb ) and is inducible in an SOS-dependent manner ( 22,23 ) . 
Even under non-inducing conditions , spontaneous prophage induction ( SPI ) was observed , preceded by a spontaneous activation of the SOS response in > 60 % of cases ( 20,22,23 ) . 
However , the precise regulatory control of CGP3 induction has not been studied thus far . 
In this study , we demonstrate the essential role of a prophage-encoded NAP , which is a homolog to the mycobacterial Lsr2 protein and functions as a silencer of cryptic phage elements in C. glutamicum ( CgpS , C. glutamicum prophage silencer ) . 
Genome-wide profiling of the CgpS -- DNA interaction revealed its association with AT-rich DNA regions located primarily within prophage regions . 
Countersilencing of CgpS activity via the expression of its truncated oligomerization domain resulted in the induction of CGP3 , causing cell death . 
A bioinformatics analysis revealed homologous proteins mainly in actinomycetes , but , interestingly , also in several phage and prophage genomes . 
These data demonstrate the importance of XS proteins for the tolerance of viral DNA and indicate that this mechanism is exploited by both the host and the virus . 
MATERIALS AND METHODS
Bacterial strains and growth conditions
The bacterial strains and plasmids used in this study are listed in Supplementary Table S1 . 
Corynebacterium glutamicum ATCC 13032 was used as wild-type strain ( 24 ) . 
E. coli DH5 was used as host for cloning procedures and cultivated in Lysogeny Broth ( LB ) medium or on agar plates at 37 ◦ C ( 25 ) . 
For growth studies and fluorescence assays ( e.g. preparation of cells for fluorescence microscopy ) , C.glutamicum cells were pre-cultivated in BHI ( brain heart infusion , DifcoTM BHI , BD , Heidelberg , Ger - ◦ many ) medium at 30 C for 6 h . 
This first preculture was used to inoculate an overnight culture in CGXII minimal · − medium ( 26 ) containing 2 % ( w/v ) glucose and 30 mg l 1 protocatechuat acid . 
The CGXII culture was finally used to inoculate the main culture in the same medium ( CGXII with 2 % ( w/v ) glucose ) to a start OD600 of 1 , unless specified · − · − otherwise . 
If necessary , 50 g ml 1 ( E. coli ) or 25 g ml 1 · − ( C. glutamicum ) kanamycin and/or 34 g ml 1 ( E. coli ) or · − 10 g ml 1 ( C. glutamicum ) chloramphenicol were added . 
Recombinant DNA work
Plasmids and oligonucleotides used in this study are listed in Supplementary Table S2 , respectively . 
Standard methods including PCR , DNA restriction and ligation , were performed according to established protocols ( 25 ) . 
In some cases , Gibson assembly ( 27 ) was used for the constructions of plasmids . 
DNA sequencing and oligonucleotides synthesis were conducted by Eurofins MWG Operon ( Ebersberg , Germany ) . 
The chromosomal integration of the Strep tagged cgpS gene variant was performed using the two-step homologous recombination method ( 28 ) . 
The 500 bp up and downstream regions of cgpS were amplified using the oligonucleotides LF cgpS pK19 fw and LF cgpS rv and , accordingly , RF cgpS fw and RF cgpS pK19 rv . 
Amplification of the Strep-tagged cgpS gene was done by using the plasmid pAN6-cgpS-strep as template for the oligonucleotide pair cgpS strep fw and cgpS strep rv . 
The three resulting PCR products and the digested pK19mobsacB plasmid ( with BamHI , EcoRI ) were assembled using Gibson assembly ( 27 ) . 
Correct integration into the cgpS locus was confirmed by sequencing of the colony PCR product with the oligonucleotides Cgps indel-fw and CgpS indel rv . 
Cultivation in the BioLector System
Growth experiments were performed predominantly in the BioLector ® microcultivation system of m2p-labs ( Aachen , Germany ) as described by ( 29 ) . 
Cultivation was performed in 48-well FlowerPlates ( m2p labs , Germany ) at 30 ◦ C and a shaking frequency of 1200 rpm . 
The cells were cultivated in 750 l of CGXII minimal media with 2 % ( w/v ) glucose containing different additives ( e.g. Isopropyl - D-1-thiogalactopyranoside ( IPTG ) , MMC , kanamycin ) , as indicated . 
Measurements were taken at 15-min intervals . 
DNA affinity chromatography with the promoter region of alpAC
The promoter region of alpAC was amplified by PCR with the oligonucleotides PalpAC-Biotin-Tag-fw and PalpAC rv ( product size 516 bp ) . 
To flag the amplified product further PCRs were performed but with the Biotin-Primer ( MWG Eurofins , Ebersberg , Germany ) and the PalpAC rv . 
At least 220 pmol of the biotinylated products were purified by size exclusion chromatography with the usage of an 8 ml sepharose s400-HR column from GE Healthcare ( Freiburg , Germany ) . 
A total of 5 mg of the M-280 Streptavidin Dynabeads ® ( Invitrogen , Carlsbad , CA , USA ) were washed twice with the binding and wash ( BW ) buffer ( 10 mM Tris-HCl pH 7.5 , 2 M NaCl ) , subsequently suspended in BW buffer containing biotinylated products and incubated for 1 h at room temperature . 
To eliminate unbound DNA fragments the beads were washed three times with the BW buffer and finally suspended in the binding and storage ( BS ) buffer ( 20 mM Tris-HCl pH 7.5 , 1 mM EDTA , 10 % ( v/v ) glycerin , 0.01 % ( v/v ) Triton-X-100 , 100 mM NaCl , 1 mM DTT ) . 
A total fo 500 ml of cells were grown in CGXII minimal media with glucose as carbon source ( as described in bacterial strains and growth conditions ) to an OD600 of ∼ 5 . 
After the cells were harvested by centrifugation ( 20 min , 5300g and washed once with phosphate buffered saline ( PBS ) buffer ( 137 mM NaCl , 2.7 mM KCl , 20 mM Na2HPO4 , 1.8 mM KH2PO4 ) , cell pellets were suspended in BS buffer supplemented with 1 mM phenylmethylsulfonyl fluoride ( PMSF ) . 
Cell disruption was performed by five passages at 172 MPa through a French pressure cell ( Heinemann , Schwaebisch Gmuend , Germany ) . 
The DNA binding reactions were set up with complete prepared crude extracts , the DNA-coupled beads and 500 g of chromosomal DNA for 45 min at room temperature . 
After the binding reaction , beads were washed once with BS buffer , twice with BS buffer and 400 g chromosomal DNA and , as a final washing step , again with BS buffer . 
The elution was fulfilled in two subsequent steps with BS buffer containing 2 M sodium chloride . 
After TCA precipitations ( 30 ) of the pooled elution fractions the samples were analyzed vi sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( SDS-PAGE ) ( 31 ) . 
Identification of proteins was conducted by MALDI-ToF analysis as described in the section below . 
Preparation of ChAP-Seq samples
Cells of the wild-type strain ATCC 13032 and the variant containing the Strep-tagged CgpS protein ( WT : : cgpS-strep ) were first grown in BHI for 6 h and then 1 ml was used to inoculate minimal media cultures ( CGXII with 2 % ( w/v ) glucose ) . 
After cultivation overnight , these precultures were used to inoculate 500 ml of the same minimal medium , were grown to an OD600 5 to 6 , and finally harvested by centrifugation ( 10 min , 11 325g at 4 ◦ C ) . 
After washing the cells with CGXII medium without ( w/o ) MOPS , the cells were resuspended in 10 ml MOPS-free CGXII containing 1 % ( v/v ) formaldehyde . 
The fixation was conducted by incubation at room temperature for 20 min . 
Subsequently , glycine was added to a final concentration of 125 mM and the cells were incubated for further 5 min at room temperature . 
Then , the cells were washed twice with buffer A ( 100 mM Tris-HCl , pH 8.0 , 1 mM EDTA ) and resuspended in 10 ml buffer A supplemented with cOmplete Protease Inhibitor ( Roche , Basel , Switzerland ) and 5 mg RNase A. Cell disruption was performed as described in the DNA affinity chromatography section ( five passages through a French Press cell ) . 
The chromosomal DNA of the lysates were sheared by sonication 3 × 30 s with a Branson sonifier 250 ( Heinemann , Schwaebisch Gmuend , Germany ) using a pulse length of 40 % and an intensity of one to give an average fragment size of 200 -- 1500 bp as confirmed by agarose gel electrophoresis . 
Cell debris was first removed by centrifugation at 5300g for 20 min and then centrifuged for 1 h at 150 000g both steps at 4 ◦ C . 
The supernatant was used for protein -- DNA purification according to the standard Strep-tag ® purification protocol ( see below , protein purification ) . 
The pooled elution fractions were incubated overnight at 65 ◦ C , followed by a treatment with proteinase K ( final concentration 400 mg · ml − 1 ) for 3 h at 55 ◦ C. Finally , the DNA of the samples was purified by phenol -- chloroform extraction ( 32 ) , precipitated with ethanol , washed with 70 % ( v/v ) ethanol , dried and resuspended in 50 -- 100 l ddH2O . 
ChAP-Seq
The obtained DNA fragments of each sample ( 2 g ) were used for library preparation and indexing using the TruSeq DNA PCR-free sample preparation kit according to the manufacturer 's instruction , yet omitting the DNA size selection steps ( Illumina , Chesterford , UK ) . 
The resulting libraries were quantified using the KAPA library quant kit ( Peqlab , Bonn , Germany ) and normalized for pooling . 
Sequencing of pooled libraries was performed on a MiSeq ( Illumina , San Diego , US ) using paired-end sequencing with a read-length of 2 × 150 bases . 
Data analysis and base calling were accomplished with the Illumina instrument software and stored as fastq output files . 
The obtained sequencing data of each sample were imported into CLC Genomics Workbench ( Version 7.5.1 , Qiagen Aarhus A/S ) for trimming and base quality filtering . 
The output was mapped to accession BX927147 as C. glutamicum reference genome 
( 21 ) . 
For peak detection the resulting mapping coverage of each sample was exported and imported into the in-house software Genome Data Viewer ( unpublished ) . 
A peak was automatically annotated if the coverage of a region is above the 3-fold average of the averaged genome coverage . 
All peaks were inspected and confirmed manually . 
The relative amount of circular phage DNA was determined via quantitative PCR ( qPCR ) . 
Therefore , C. glutamicum wild type cells containing empty pAN6 plasmid ( control ) , pAN6-cgpS gene or pAN6-N-cgpS were grown in 48-well FlowerPlates containing CGXII minimal medium at 30 ◦ C and 900 rpm in a microtron ( Infors-HT , Bottmingen , Switzerland ) . 
The overexpression of cgpS and the Nterminal part were induced with 150 M IPTG ( for control samples no IPTG was added ) . 
After 24 h , 750 l of the cells were harvested and the DNA was extracted using the NucleoSpin microbial DNA Kit ( Macherey Nagel , Dueren , Germany ) and DNA concentration was quantified using a nanophotometer ( Implen , München , Germany ) . 
Each sample contained 1 g total DNA as a template . 
For the reaction an innuMIX qPCR MasterMix SyGreen ( Analytic Jena , Jena , Germany ) and a qTOWER 2.2 ( Analytic Jena ) was used . 
The reaction protocol was divided into two parts ( i ) polymerase chain reaction ( PCR ) ( ( a ) 3 min preincubation at 95 ◦ C , ( b ) 5 s denaturation at 95 ◦ C , ( c ) 25 s elongation at 62 ◦ C , 40x repetition of step ( b ) to ( c ) ) and a ( ii ) melting curve analysis ( T = 1 ◦ C/6 s ) . 
The PCR product size using oligonucleotides belonging to the circular phage product is 150 bp ( listed in Supplementary Table S2 ) . 
As reference gene ddh was used with the oligonucleotides listed in Supplementary Table S2 resulting in a 150 bp product . 
For data analysis the qPCR software qPCR 3.1 ( Analytik Jena ) and the Livak method were used ( 33 ) to determine the 2 − Ct based on the measured CT-values . 
DNA microarrays
For a comparative transcriptome analysis of C. glutamicum ATCC 13032/pAN6 with cells carrying the pAN6-N-cgpS - ( used for countersilencing ) were cultivated in CGXII with 2 % ( w/v ) glucose and 100 M IPTG as described in bacterial strains and growth conditions . 
The preparation of labeled cDNA and DNA microarray analysis was performed as described previously ( 34 ) . 
Array data were deposited in the GEO database ( ncbi.nlm.nih.gov / geo ) under accession number GSE80674 . 
Cultivation and perfusion in microfluidic device
For single-cell analysis an in-house developed microfluidic platform was used ( 22,35 -- 37 ) . 
Phase-contrast and fluorescence time-lapse imaging was performed at 6 min intervals . 
Medium was supplied continuously to ensure stable and constant environmental conditions . 
CGXII minimal medium with 2 % ( w/v ) glucose and 25 g · ml − 1 kanamycin was infused at a rate of 300 nl · min − 1 using a high-precision syringe pump ( neMESYS , Cetoni GmbH , Korbussen , Germany ) . 
For the expression of the N-terminal part of Cgp 
150 M IPTG were added to the medium . 
A constant cultivation temperature of 30 ◦ C was ensured ( PeCon GmbH , Erbach , Germany ) . 
The cells were cultivated for 16 h. 
Fluorescence microscopy
The cultivations were done as described in bacterial strains and growth conditions . 
After 6 h of cultivation , 1 -- 3 l were pipetted on a microscope slide coated with a thin 1 % ( w/v ) agarose layer that was based on tris-acetate buffer . 
To stain the DNA with the Hoechst Dye , 33 342 1 ml cells were harvested ( 5300g , 5 min ) , subsequently resuspended − in PBS buffer containing 100 ng · ml 1 Hoechst 33342 and incubated at room temperature for 20 min . 
Images were taken on an AxioImager M2 ( Zeiss , Oberkochen , Germany ) equipped with a Zeiss AxioCam MRm camera . 
Fluorescence was monitored with the filter set 46 HE YFP for eYFP , 63 HE filter was used for mCherry fluorescence and Hoechst fluorescence was examined with the filter set 49 . 
An EC Plan-Neofluar 100x/1 .3 Oil Ph3 objective was used . 
Images were acquired and analyzed with the AxioVision 4.8 software ( Carl Zeiss ) . 
Protein purification
CgpS tagged C-terminal with a Strep-tag ® was heterologously produced in E. coli BL21 ( DE3 ) . 
Cells were grown to an OD600 of 0.4 at 37 ◦ C. Upon induction with 50 M IPTG the cultivation was continued at 16 ◦ C overnight . 
Cells were harvested by centrifugation at 5300g and 4 ◦ C for 10 min and resuspended in buffer B ( 250 mM NaCl , 50 mM Tris-HCl , pH 7.5 ) . 
Cell disruption was performed by two passages through a French pressure cell at 172 MPa . 
Cell debris was removed by centrifugation at 20 min , 5300g and 4 ◦ C , followed by an ultracentrifugation ( 60 min , 229 000g , 4 ◦ C ) . 
The supernatant was applied to an equilibrated 1 ml Strep-Tactin ® - Sepharose ® ( IBA , Göttingen , Germany ) column . 
It was subsequently washed with 10 ml buffer B and the protein was eluted with 10 ml buffer B containing 1 mM d-desthiobiotin ( Sigma Aldrich ) . 
Electrophoretic mobility shift assays (EMSA)
EMSA studies of CgpS and selected DNA regions identified by ChAP-Seq were performed with selected regions ( 500 bp fragments , for oligo sequences see Supplementary Table S3 ) . 
The corresponding regions were amplified by PCR and purified by using the PCR clean-up Kit of Macherey Nagel ( Dueren , Germany ) . 
The promoter region of gntK was used as control fragment ( 560 bp ) . 
A total of 90 ng DNA per lane were incubated with different concentrations ( 1 M and 2 M ) of purified CgpS protein for 20 min in EMSA buffer ( 250 mM Tris-HCl pH 7.5 , 25 mM MgCl , 2 200 mM KCl , 25 % ( v/v ) glycerol ) . 
Subsequently , samples were loaded onto a native 10 % polyacrylamide gel ( TBEbased , TBE ( 89 mM Tris base , 89 mM boric acid , 2 mM Na2EDTA , loading dye : 0.01 % ( w/v ) xylene cyanol dye , 0.01 % ( w/v ) bromophenol blue dye , 20 % ( v/v ) glycerol , 1x TBE ) . 
The DNA was stained with SYBR Green I ( Sigma Aldrich , St. Louis , MO , USA ) . 
Protein pull down and MALDI-TOF analysis
C. glutamicum cells containing the plasmids pAN6 , pAN6-cgpS-strep or pAN6-N-cgpS-strep were cultivated as described in bacterial strains and growth conditions . 
The cultures were grown in 500 ml CGXII with 2 % ( w/v ) glucose to an OD600 of 5 and subsequently induced with 150 M IPTG for further 4 h . 
The cells were harvested ( 5300g , 20 min , 4 ◦ C ) , washed in buffer B ( see protein purification ) and disrupted as descripted in the DNA affinity chromatography section . 
Purification was performed as described in the section above . 
The eluted fractions were analyzed by SDS-PAGE ( 31 ) using a 4 -- 20 % Mini-PROTEAN ® gradient gel ( Bio Rad , Munich , Germany ) . 
The gels were stained with a Coomassie dye based RAPIDstain solution ( GBiosciences , St. Louis , MO , USA ) . 
MALDI-TOF-MS measurements were performed with an Ultraflex III TOF/TOF mass spectrometer ( Bruker Daltonics , Bremen , Germany ) for the identification of the proteins as described ( 38 ) . 
Homology search
BLAST ` nr ' database ( ver . 
February 2015 ) was downloaded from NCBI ( http://www.ncbi.nlm.nih.gov/ ) . 
CgpS amino acid sequence was extracted from the GenBank file Corynebacterium glutamicum ATCC 13032 , accession : NC 006958.1 and locus tag : cg1966 . 
A PSI-BLAST ( ( 39 ) ) search with CgpS sequence as the query was executed against the ncbi nr database . 
The e-value threshold was set to 0.005 , the number of iteration was not limited and the search iteration was performed until it converged . 
A total of 5230 ( 1920 unique ) homologous hits were achieved from which 618 could be allocated to a particular bacterial species or a phage . 
Sequence global identity was calculated by pairwise comparison between the CgpS sequence with all 618 PSI-BLAST hits using the Needleman -- Wunsch algorithm ( 40 ) implemented in the EMBOSS package ( 41 ) needle . 
Secondary structure prediction
The amino acid sequence of the CgpS protein and the sequences of the 618 homologous hits were used to predict the secondary structure by psipred ( 42 ) . 
The visualization of the psipred output was done in R ( 43 ) . 
All statistical analysis and data visualization from formatic section was performed in R (43).
RESULTS
A small nucleoid-associated protein encoded by a cryptic prophage element
To decipher the control of prophage induction and activation of cryptic elements in C. glutamicum ATCC 13032 , we performed DNA affinity chromatography with the promoter of the early phage operon alpAC using the crude extract of log-phase cells grown in glucose minimal medium ( ( 34 ) , Figure 1A ) . 
SDS-Page analysis of the proteins boun to the alpAC promoter revealed a prominent band corresponding to the 13.4 kDa protein Cg1966 encoded within the CGP3 prophage region ( Figure 1B ) . 
In particular , the C-terminal domain of Cg1966 shares significant sequence similarity with the nucleoid-associated protein Lsr2 of Mycobacterium tuberculosis ( Supplementary Figure S1 ) . 
This domain corresponds to the DNA binding domain of Lsr2 ( IPR024412 ) , which was previously found to bind AT-rich DNA via an AT-hook motif and functions as a silencer of xenogeneic DNA ( 44,45 ) . 
Based on the data described in the following sections , we renamed Cg1966 as CgpS ( Corynebacterium glutamicum prophage silencer ) . 
Secondary structure predictions of CgpS as well as of CgpS homologs suggest a significant structural similarity with Lsr2 and reveal the presence of an AT-hook-like motif ` RGI ' between the two predicted C-terminal alpha helices ( Figure 1C ) ( 18,45 ) . 
CgpS functions as a silencer of CGP3 activity
To study the impact of cgpS expression on the activity of the CGP3 prophage , we overexpressed cgpS in a strain carrying a reporter construct ( WT-Plys-eyfp ) indicative for the activation of CGP3 by the production of the yellow fluorescent protein eYFP under the control of a phage promoter ( 22 ) . 
Upon induction with mitomycin C , the control strain carrying the empty plasmid displayed increased reporter activity . 
Consistent with our assumption , overexpression of cgpS reduced the reporter output to nearly the background level ( Figure 2A ) . 
To study the intracellular localization of CgpS in C. glutamicum cells , we C-terminally fused this protein to mCherry and analyzed its distribution via fluorescence microscopy . 
As shown by Hoechst staining , this NAP appeared associated with the nucleoid but formed distinct foci in the cell ( Figure 2B and C ) . 
Remarkably , CgpS-mCherry foci co-localized with foci of an AlpA-eYFP fusion that was previously described as a CGP3 DNA adaptor protein binding to the alpAC promoter region ( 34 ) ( Figure 2C ) . 
Th functionality of this CgpS-mCherry fusion was confirmed by the counteraction of CGP3 activation upon addition of MMC ( Supplementary Figure S2 ) . 
Genome-wide binding profile of CgpS
The data of the co-localization experiments suggest binding of CgpS to the CGP3 prophage region . 
In the following , the genome-wide binding profile was analyzed by combining affinity chromatography purification of crosslinked CgpS -- DNA complexes followed by sequencing of associated DNA ( ChAP-Seq ) . 
For this purpose , we replaced the native cgpS gene in the genome of ATCC 13032 with cgpS-Strep encoding a C-terminal Strep-tagged CgpS variant . 
This analysis revealed that CgpS associates with 1.5 % of the ATCC 13032 genome and with ∼ 20.5 % of the cryptic CGP3 prophage region ( Supplementary Figure S3 ) . 
In total , 90 peaks were detected , 58 of which were within and 32 were located outside the CGP3 prophage ( Figure 3A , Supplementary Table S4 ) . 
The majority of the peak maxima were located within promoter regions ( 60 % ) , but CgpS binding was also observed within genes ( 31 % ) or intergenic regions ( 9 % ) ( Supplementary Figure S4B and C ) . 
To deduce a binding motif of CgpS , sequences of the 90 peaks ( Supplementary Table S4 ) were extracted and analyzed using the MEME-ChIP software platform ( 46 ) . 
A 21-bp long AT-rich motif was predicted , which was present in 87 of 90 sequences ( Figure 3B ) . 
The occurrences of the found DNA binding sites were validated using a FIMO search ( Find Individual Motif Occurrences , ( 47 ) ) in the ATCC 13032 genome , which revealed significant matches ( > 75 % ) of the predicted and experimentally identified CgpS binding sites ( Supplementary Figure S5 ) . 
Remarkably , the % GC content of the 90 peak sequences is considerably lower than the average GC content of the ATCC 13032 strain , indicating the preferred binding of CgpS to AT-rich DNA ( Figure 3C ) . 
Moreover , the GC contents of the CgpS bound regions within the prophage revealed no significant differences from that of the regions bound outside the prophage ( Figure 3C ) . 
Most of the identified CgpS targets were located within the CGP3 prophage and code for hypothetical proteins . 
The two strongest signals were found within transposase-encoding genes ( cg1950-cg1951 ) and in the promoter region of cgpS itself , indicating a negative autoregulation similar to that of H-NS ( 48 ) . 
Other potential target genes encode the actin-like protein and the corresponding adaptor protein ( alpAC , cg1890 and cg1891 ( 34 ) ) , a resolvase ( cg1929 ) , a prophage primase ( cg1959 ) , a putative phage lysin ( cg1974 ) and a phage integrase ( cg2071 ) , which are spread across the cryptic prophage element . 
In addition to regions within CGP3 , CgpS target sites are located in the low GC island 1 ( LCG1 ) , in the cryptic phage element CGP1 , or proximal to transposases encoding genes . 
Furthermore , promoter regions of genes coding for R-M systems ( Pcg1028 and PcglIM , ( Pcg1996 ) ) are also bound by CgpS , which in several studies were shown to be transferred horizontally ( 49 -- 52 ) . 
A considerably high peak was observed for the promoter region of cg0150 that encodes a putative regulatory protein or toxin possessing a predicted fido domain ( IPR003812 ) . 
The binding profile obtained by the ChAP-Seq analysis was validated by EMSAs ( Supplementary Figure S6 ) . 
For this purpose , CgpS was purified as a C-terminal Streptag fusion and incubated with DNA fragments covering selected putative CgpS binding sites as identified by ChAP-Seq ( Figure 3D and Supplementary Figure S6 ) . 
This in vitro approach confirmed the binding of CgpS for all selected target regions ( including the promoters of cg0150 , alpAC and cgpS itself ) in comparison to the control fragment ( gntK promoter ) ( Figure 3D ) . 
Overall , these data are consistent with CgpS acting as a xenogeneic silencer by targeting AT-rich DNA regions , several of which have likely been acquired by HGT . 
Countersilencing of CgpS activity
Several independent efforts to inactivate the cgpS gene failed ( data not shown ) , suggesting that cgpS represents an essential gene for C. glutamicum ATCC 13032 . 
However , previous studies revealed that deletions of all three cryptic phage elements , including the cgpS gene , are possible and do not lead to a significant growth defect of the particular strain ( 53 ) . 
In fact , trials to construct an in-frame deletion of cgpS resulted in the isolation of strains lacking large parts of the CGP3 prophage , indicating that the essentiality of cgpS is a consequence of the de-repression of toxic phage genes in the absence of CgpS . 
For the conditional inactivation of CgpS , we adapted a countersilencing approach similar to the H-NST system described by Williamson and Free ( 54 ) . 
This protein was reported as a truncated H-NS derivative that antagonizes H-NS function by interfering with the multimerization of H-NS . 
Co-purification assays with the N-terminal domain of CgpS confirmed the interaction of this truncated varian with the full-length protein ( Figure 4A ) . 
Based on previous data and the H-NST mechanism ( Figure 4B ) , we constructed the pAN6-N-cgpS plasmid to overproduce a truncated variant of CgpS ( amino acids 1 -- 65 ) under the control of Ptac . 
Homology studies indicated that amino acids 1 -- 65 cover the domain of CgpS required for the oligomerization of this NAP . 
Remarkably , production of the truncated CgpS-N domain in the wild-type strain resulted in a significant growth defect , whereas no impact on growth was observed in a strain lacking the CGP3 prophage ( Figure 5A ) . 
This finding was supported by single-cell analysis of a strain containing a prophage reporter construct ( Plys-eyfp ) ( 22 ) and the countersilencing construct pAN6-N-cgpS . 
Production of the N-terminal domain of CgpS led to a strong increase in fluorescence accompanied by growth arrest and a branched cell morphology ( Figure 5C , Video S1 and S2 ) . 
Quantitative real-time PCR revealed a 3-fold increase in the level of circular CGP3 DNA in comparison to uninduced cells , which is consistent with the induction of this cryptic prophage ( Figure 5B ) ( 20 ) . 
To monitor the impact of countersilencing CgpS activity on gene expression , we performed a comparative transcriptome analysis ( Figure 5D , Supplementary Table S5 ) . 
More than 194 genes were affected , 12 of which exhibited a reduced mRNA level ( mRNA ratio ≤ 0.5 , P-value < 0.05 ) , and 182 genes were upregulated ( mRNA ratio ≥ 2 , P-value < 0.05 ) . 
The majority of upregulated genes ( 148 ) were genes of the prophage CGP3 . 
Additional genes that displayed an increased mRNA level were the ferritin gene ( ftn , cg2782 ) and cg1517 of the CGP1 prophage ( Supplementary Table S5 ) , both of which were also identified as putative CgpS targets by ChAP-Seq . 
Together , these data demonstrate that CgpS is an essential NAP due to its function as a silencer of cryptic phage elements inC. glutamicum . 
CgpS homologs are found in actinomycetes and their phages
Our data support a function for CgpS as a xenogeneic silencer that binds to AT-rich DNA similar to the Lsr2 of M. tuberculosis as well as the H-NS of E. coli . 
This is underlined by the fact that both proteins , Lsr2 and CgpS , are able to complement the phenotype of an hns mutant strain ( ( 18 ) , Supplementary Figure S7 ) . 
These findings highlight the conserved mechanism of a highly diverse set of proteins . 
In the following , we overexpressed the Nterminal oligomerization domains of CgpS orthologs from Corynebacterium amycolatum DSM 44737 ( CORAM0001 2081 ) and Corynebacterium diphtheria DSM 44123 ( CDC7B 2240 ) and the Lsr2 from M. tuberculosis H37R ( Rv3597c ; Lsr2 ) ( Figure 6A and B ) . 
Whereas the production of the oligomerization domain strongly affected cellular growth in all cases ( Figure 6A ) , only the N-terminal domain of the ortholog of C. amycolatum ( DSM 44737 ) led to a significant induction of CGP3 ( Figure 6B ) . 
No significant reporter output was observed with production of the truncated orthologs of C. diphtheria or M. tuberculosis , suggesting a high level of plasticity within this family of xenogeneic silencers ( Figure 6B ) . 
Furthermore , we used a bioinformatics approach to obtain a more general overview of the distribution of CgpS orthologous proteins . 
For this purpose , a PSI-BLAST 
( Position-Specific Iterated BLAST ) search was performed on CgpS and resulted in 5230 hits , of which 1920 protein sequences were unique ( threshold e-value ≤ 0.005 ) . 
Of these , 98.3 % were found in the domain of bacteria and 1.7 % in phages , mostly belonging to the Siphoviridae ( Figure 7A , Supplementary Table S6 ) . 
Of 302 bacterial genomes containing prophage regions predicted by PhiSpy ( 55 ) , 22 contain cgpS orthologs ( Supplementary Table S6 ) . 
The remaining 280 hits were found outside of any predicted prophage region . 
Moreover , secondary structure predictions were performed for 618 unique sequences , which were clearly assigned to bacterial or phage species , exhibiting high resemblances . 
The structural similarity suggests a common function , although the identity of the amino acid sequences is low ( ∼ 23 % ) ( Figure 7B , C and Supplementary Figure S10 ) . 
XS exclusion hypothesis
A recent bioinformatics study on the distribution of XS genes revealed that members of the same family can appear within a particular species but that members of different families are never found together ( 56 ) . 
To test the proposed exclusion mechanism , we expressed the hns gene from E. coli MG1655 in a C. glutamicum ATCC 13032 strain containing the prophage reporter ( : : Plys-eyfp ) . 
As expected , the overexpression of hns caused a severe growth defect , coinciding with a highly increased output of the prophage reporter ( Figure 6C and D ) . 
The effect of hns overexpression was comparable to the countersilencing of CgpS activity with the production of a truncated CgpS variant ( Figure 6E ) . 
When hns was expressed in a CGP3 background the effect on growth was only moderate ( Supplementary Figure S8 ) . 
However , hns expression still negatively affected the growth of the CGP3 mutant strain which can likely be explained by unspecific binding and interference of H-NS at other genomic regions . 
These findings are in agreement with the hypothesis that different XS proteins interfere at AT-rich DNA regions , leading to a disruption of silencing complexes and thereby to an activation of foreign DNA elements . 
Nevertheless , in some cases the scenario is clearly more complex , as illustrated by the finding that the expression of cgpS in the E. coli wild-type strain was not able to counteract H-NS expression at the bgl operon ( Supplementary Figure S9 ) . 
DISCUSSION
CgpS functions as a silencer of cryptic phage elements
In this study , we identified the prophage-encoded XS protein CgpS that inherits an essential role as a silencer of cryptic prophages in C. glutamicum . 
Genome-wide profiling of CgpS binding sites reveals an association of this protein to AT-rich DNA stretches primarily located within horizontally acquired genomic islands and shows a remarkable accumulation of binding sites within the large and cryptic CGP3 prophage . 
Countersilencing of CgpS activity by overproduction of its N-terminal oligomerization domain resulted in a strong increase in CGP3 activity leading to cell death . 
Furthermore , several CgpS binding sites were identified outside the CGP3 region , and the essentiality of the cgpS gene was attributed to the presence of the CGP prophage . 
This is consistent with the finding that the cgpS gene is located on the CGP3 island , suggesting that evolution favored a physical association between this XS and its main target . 
Sequence analysis of CgpS revealed a low sequence identity ( 27 % , Supplementary Figure S1 ) with the mycobacterial Lsr2 protein that was described in previous studies as an H-NS-like protein targeting AT-rich sequences in M. tuberculosis ( 18 ) . 
Both XS proteins , Lsr2 and CgpS , complemented the bgl-based phenotype ( 57 ) of an Escherichia coli hns strain , supporting the overall analogous functions of these XS proteins ( Supplementary Figure S7 ) ( 18 ) . 
Whereas both lsr2 and cgpS are essential for viability in their native hosts , E. coli hns mutant strains are viable although exhibiting severe growth defects ( 58 ) . 
Salmonella Typhimurium null mutants of hns are not viable unless mutations in rpoS ( general stress response ) or phoP ( virulence gene regulator ) counteract this deletion ( 12 ) . 
Because the presence and diversity of phage elements contributes to major strain-specific differences within a bacterial species , our study illustrates that the essentiality of XS genes is highly dependent on the particular strain background . 
The C. glutamicum strain MB001 , cured of all prophage regions as well as the cgpS gene located on prophage CGP3 , displays wild-type-like growth behavior ( 53 ) . 
CgpS binds AT-rich xenogeneic DNA regions
Secondary structure predictions of CgpS-related proteins evince two - helices flanking an ` RGI ' motif ( Figures 1C and 7C ) . 
This motif resembles the prokaryotic AT-hook motif ` Q RGR ' found in H-NS and Lsr2 and may also be / responsible for the binding of AT-rich DNA as a general rule for XS functioning ( 44,59 ) . 
A certain plasticity of the AT-hook motif is supported by experiments with AT-hook muteins of H-NS and Lsr2 , showing that the exchange of a single arginine residue to an alanine reduces DNA binding but does not completely abolish it ( 59 ) . 
Moreover , another member of the H-NS family , the Ler protein , has a hydrophobic amino acid ( ` VGR ' motif ) instead of an arginine at this position ( 60 ) . 
However , significant differences were observed for the number of target genes affected by the binding of the particular XS proteins . 
ChIP-on-Chip analysis revealed a direct influence of S. Typhimurium H-NS on the expression of more than 740 ORFs ( 12,61 ) , and the binding of Lsr2 affected more than 800 regions within the M. tuberculosis genome and > 900 in Mycobacterium smegmatis ( 45 ) . 
ChAP-Seq profiling of CgpS binding , however , yielded only 90 potential target regions . 
Typical for XS function , an AT-rich DNA motif was derived from the ChAP-Seq results , which clusters at a high density within the CGP3 prophage region ( Supplementary Figure S5 ) . 
In general , promoter regions are more often bound by CgpS than genes or intergenic regions ( Supplementary Figure S4 ) , which is not surprising because promoter regions usually possess a higher AT content ( 62,63 ) . 
CgpS targets outside the CGP3 region show a similar or lower GC content ( Figure 3C ) but less altered expression levels , and this may suggest the importance of motif density for XS function . 
Here , a variation of the AT-hook motif likely represents a mechanism to adjust the binding behavior of the XS protein to meet the needs of a particular host species . 
In addition to CGP3 as a main CgpS target , further targets were identified which were also likely acquired by horizontal gene transfer , such as the LCG1 island , the cryptic prophage CGP1 ( 21 ) , R-M systems , transposases and also regulatory proteins such as putative transcriptional regulators ( Cg0725 , Cg1340 , Cg2426 ) , the gluconate-responsive repressor GntR1 ( Cg2783 ) ( 64 ) and an operon encoding the two-component system CgtSR6 ( Cg3060 ) ( Supplementary Table S4 ) . 
Several previous studies reported similar tar get genes or regions for H-NS , Lsr2 and MvaT , demonstrating the convergent evolution of XS in bacterial species ( 12,47,61,65 ) . 
Overall , more that 80 % of CgpS-bound regions also exhibited a more than 2-fold altered expression level under countersilencing conditions ( Figure 5D ) confirming the postulated silencing effect of CgpS . 
Several potential targets outside of the CGP3 region , however , showed only a moderate impact on the expression level suggesting a more complex regulatory scheme at the corresponding promoter regions . 
Therefore , the role of CgpS for the control of these potential targets , including , e.g. the gntR1 gene or the cgtSR6 operon , remains to be elucidated in further studies . 
How to overcome CgpS silencing?
Several different mechanisms were described to counteract H-NS-mediated silencing , including structural interference with H-NS-bound nucleoids by transcription factors , temperature or osmolarity effects , and the binding of alternative sigma factors or other NAPs preventing multimerization of the XS protein ( 11,66,67 ) . 
To interfere with CgpS XS activity , we produced a truncated part of the native protei covering the N-terminal oligomerization domain of CgpS ( Figure 5 ) . 
This overcomes the problem of cgpS being essential in the presence of CGP3 and was inspired by the study of Williamson and Free , who described the antagonistic function of a truncated H-NS variant found in an enteropathogenic E. coli strain ( 54 ) . 
As expected , production of the N-terminal CgpS domain resulted in strong activation of CGP3 , leading to cell death . 
In recent studies we described the spontaneous induction of the CGP3 prophage occuring in the absence of an external trigger ( 20,22,23 ) . 
Single-cell analysis demonstrated that a considerable fraction of this SPI is preceded by an activation of the SOS response , which is likely the result of spontaneous DNA damage during replication ( 68,69 ) . 
However , these studies also highlighted a certain ( > 30 % ) fraction of SOS-independent SPI , suggesting that other factors influence this common phenomenon of bacterial populations ( 5 ) . 
The present study shows the sensitive reaction of C. glutamicum cells to the downregulation of CgpS activity ( Video S2 ) . 
It is therefore interesting to determine whether cells can adjust the level of XS proteins to manipulate the frequency of SPI according to their particular requirements . 
Sequence analysis revealed the presence of CgpS/Lsr2 homologs in phage and prophage genomes displaying a low sequence identity but highly conserved secondary structure prediction ( Figure 7 ) . 
This finding is not surprising because bacterial evolution has been shaped by a tight interaction with bacteriophages . 
For the integration of viral DNA into the host genome , both the bacterium and phage benefit from tolerance and a smooth integration into the host genetic circuitry . 
Because the activation of silent prophages or mobile elements often causes serious detrimental effects to host cells ( 11,70,71 ) , the stringent control of xenogeneic elements is required . 
Several examples of XS proteins involved in the control of mobile elements or phages have been described in the recent literature , including H-NS of S. Typhimurium ( 12 ) , Rok from B. subtilis ( 19 ) and MvaT from P. aeruginosa ( 72 ) . 
Their corresponding genes , however , are all located on the host chromosome and are characterized as a type of immunity system protecting hosts against foreign DNA ( 11,66 ) . 
A PSI-BLAST search of CgpS-related proteins revealed that the majority ( > 98 % of all hits , > 92 % of prophage containing strains ) are found in bacterial genomes ( Supplementary Table S6 ) . 
However , several examples located in phages or prophage regions were identified . 
The functions of these phage-encoded XS-like proteins remain to be studied , but their presence suggests the following : ( i ) like CgpS , they may be required to secure tolerance of their carrier DNA within the respective host ; ( ii ) they may , however , also function as antagonistic proteins , interfering with the host XS protein similar to the situation described for H-NST ( 54 ) ; or ( iii ) they may interfere with the function of another class of XS proteins . 
This hypothesis is based on the exclusion theory suggested by Perez-Rueda and Ibarra , who postulated that XS from different families do not appear in the same bacterial organism ( 56 ) . 
Consistent with this bioinformatics study , our data show that the expression of E. coli hns results in strong activation of the cryptic prophage CGP3 and consequently cell death . 
The finding that expression of the C. glutamicum cgpS gene in E. coli MG1655 does not counteract H-NS-mediated silencing at the bgl operon shows , however , that the scenario is more complex and strongly depends on the particular strain and its regulatory equipment . 
However , our data on prophage activation in C. glutamicum provide evidence for an interference of analogous XS proteins at AT-rich DNA regions . 
Here , likely the incompatibility of the oligomerization domains inhibits the formation of XS multimeric structures required for silencing . 
Considering the presence of XS encoding genes in phage and prophage genomes , this principle is likely to be harnessed by any phage predator by encoding an interfering XS . 
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENT
The authors thank Karin Schnetz ( University of Cologne ) for helpful advice and for providing us with the E. coli hns mutant strain . 
FUNDING
Deutsche Forschungsgemeinschaft priority program SPP1617 [ FR 2759/2 -2 and KO 4537/1 -2 ] ; Helmholtz Association [ VH-NG-716 ] . 
Funding for open access charge : Helmholtz Association [ VH-NG-716 ] . 
Conflict of interest statement . 
None declared .