Deciphering the Rules Underlying Xenogeneic Silencing and Counter-Silencing of Lsr2-like Proteins Using CgpS of Corynebacterium glutamicum as a Model.

In actinobacteria, Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XS) of horizontally acquired genomic regions, including viral elements, virulence gene clusters in Mycobacterium tuberculosis, and genes involved in cryptic specialized metabolism in Streptomyces species. Consequently, a detailed mechanistic understanding of Lsr2 binding in vivo is relevant as a potential drug target and for the identification of novel bioactive compounds. Here, we followed an in vivo approach to investigate the rules underlying xenogeneic silencing and counter-silencing of the Lsr2-like XS CgpS from Corynebacterium glutamicum. Our results demonstrated that CgpS distinguishes between self and foreign by recognizing a distinct drop in GC profile in combination with a short, sequence-specific motif at the nucleation site. Following a synthetic counter-silencer approach, we studied the potential and constraints of transcription factors to counteract CgpS silencing, thereby facilitating the integration of new genetic traits into host regulatory networks.

microcultivation system (m2p-labs, Aachen, Germany) (2). Therefore, 750 µl of the main 23 culture (see Growth conditions) were cultivated in 24 Aachen, Germany) at 30°C and 1200 rpm. Biomass production was measured as 25 The temporal dynamics of the genetic toggle switch were analyzed on single-cell level by 37 cultivating C. glutamicum wild type cells harboring the plasmid-based construct in an in-38 house developed microfluidic platform (4, 5). The chip design and the experimental setup 39 was performed as described before (6). Phase contrast as well as Venus and E2-Crimson 40 fluorescence were imaged at 20 min intervals by fully motorized inverted Nikon Eclipse Ti 41 microscope (Nikon GmbH, Düsseldorf, Germany) as described previously (4, 5, 7). The 42 exposure times for phase contrast was 100 ms, for Venus 200 ms and for E2-crimson 300 43 ms. Cells were cultivated in the microfluidic chip system in CGXII medium supplemented 44 3 with 25 µg/ml kanamycin and either 100 mM gluconate or 111 mM glucose. Continuous 45 medium supply with a flow rate of 200 nl/min and waste removal was achieved by a high-46 precision syringe pump system (neMESYS, Cetoni GmbH, Korbussen, Germany) using 47 disposable syringes (Omnifix-F Tuberculin, 1 ml; B. Braun Melsungen AG, Melsungen, 48 Germany). After 17 hours of cultivation, the carbon source supply was switched from 49 gluconate to glucose or vice versa by changing the syringes and the connecting tubing to 50 ensure an immediate medium change. The temperature was set to 30°C during the 51 complete cultivation using an incubator system (PeCon GmbH, Erbach, Germany). Data 52 analysis was performed using the image-processing package Fiji (8)  plasmid. This plasmid contains the cgpS gene fused to its native promoter, which was 62 flanked by 500 bp upstream and downstream regions of the integration site (intergenic 63 region of cg1199-cg1201). Two step homologous recombination and selection was 64 performed as described previously (10). Successful integration was verified by 65 sequencing using the oligonucleotides cg1199_1201_seq_fw and cg1199_1201_seq_rv 66 (Table S2G). 67 4 68

Design of disruptive counter-silencing constructs 69
Reporter studies were performed based on plasmid pJC1 (approximately 30 copies per 70 cell) (11, 12). All counter-silencing constructs were based on the same design scheme. 71 First, the native promoter region was amplified from C. glutamicum genomic DNA, fused 72 to the gene venus via a consistent linker, which contains a ribosomal binding site, and 73 inserted into the pJC1 plasmid using Gibson assembly (13). To ensure that all regulatory 74 promoter elements are present, forward primers were designed so that the 5´-sequence 75 end coincided with the upstream end of the CgpS binding peak (14). Reverse primers 76 were chosen so that the promoter constructs contain the first 30 bp of the coding 77 sequence. The resulting plasmids serve as template for the counter-silencer constructs. 78 Overlap PCR was performed for the insertion of the GntR binding site (BS: 79 TATGATAGTACCAAT) (15) at different positions. All constructed plasmids are listed in 80 Table S2C and oligonucleotides are listed in Table S2D. 81 82

Determination of transcriptional start sites (TSS) 83
For the determination of the TSS, C. glutamicum wild type cells were cultivated in CGXII 84 supplemented with 111 mM glucose starting at an OD600 of 1. After one-hour cultivation 85 at 30°C, the SOS response was induced by adding 600 nM MMC leading to prophage 86 induction (16). 50 ml cultures were harvested on ice after one, three and six hours of 87 cultivation at 30°C, respectively. Pellets after centrifugation (5300 g, 4°C, 15 min) were 88 snap-frozen in liquid nitrogen and stored at -80°C until use. Total RNA was prepared using 89 the RNeasy Mini Kit (QIAGEN, Venlo, Netherlands) according to the manufacturer 90 5 protocol. Subsequently, all three RNA samples were pooled in approximately equal 91 amounts. The determination of the TSS and data analysis was performed by Vertis 92 Biotechnology AG (Vertis Biotechnology AG, Freising, Germany) using the Cappable-seq 93 method developed by Ettwiller and Schildkraut (17). Obtained reads were mapped against 94 the reference C. glutamicum genome BX927147 (18) (Table S1). 104 105
AT-rich 109 stretches were determined as the longest possible sequences with at least 70% 110 adenosine/thymidine (AT)-content and a number of guanosines (G) and cytidines (C) 111 below a particular threshold. The scan was performed multiple times with incrementing 112 the limit for maximal allowed G/C interruptions inside AT-rich sequences. The results of 113 6 these multiple scans were then pulled together without further collapsing, meaning that 114 the discovered AT-rich sequences with different G/C numbers may overlap each other. 115 AT-rich regions were then grouped based on their lengths and G/C numbers (or number 116 of AT steps). The grouping was done in a way to fulfil two requirements: the groups should 117 be roughly equal and cover the whole dynamic range of the selected parameters. To remove cell debris, cell lysate was centrifuged at 5300 g, 4°C for 15 min and 141 subsequently ultracentrifuged for one hour (229000 g, 4°C). His-tagged GntR protein from 142 the supernatant was purified with gravity-flow chromatography using Ni-NTA agarose 143 columns (QIAGEN, Venlo, Netherlands). Columns were washed with TNI20 buffer 144 followed by protein elution with TNI200 buffer (20 mM Tris/HCl pH 7.9, 300 mM NaCl and 145 200 mM imidazole). Protein fractions were pooled and buffer was exchanged against TG 146 buffer (30 mM Tris/HCl pH 7.5, 10% (v/v) glycerol) using a PD-10 Desalting Column (GE 147 Healthcare, Chicago, IL, USA). Subsequently, GntR was snap-frozen in liquid nitrogen 148 and stored at -80°C before it was used for EMSAs. 149 150

Surface plasmon resonance (SPR) spectroscopy 151
Binding of Strep-tagged CgpS to native or synthetic target promoters was analyzed by 152 SPR analysis in a Biacore T200 and a Biacore 3000 device (GE Healthcare,Freiburg,153 Germany) using carboxy-methyl dextran sensor chips pre-coated with streptavidin 154 (XanTec SAD500L (XanTec Bioanalytics GmbH, Düsseldorf, Germany) and Sensor Chip 155 SA (GE Healthcare, Freiburg, Germany). As first step, DNA-fragments covering the 156 respective promoters or the promoter region of the gene cg3336 (negative control) were 157 amplified by using biotinylated primer via two-step PCR as described in Table S2E Binding of GntR to the native Plys promoter and the corresponding counter-silencer 184 construct Plys_CS_0 was analyzed by electrophoretic mobility shift assays (EMSAs). 185 Primers and templates used for the PCR amplification of DNA fragments are listed in 186   Table S2F. The PCR products were purified from an agarose gel with the PCR clean-up 187 and gel extraction kit of Macherey Nagel (Düren, Germany). 14 nM DNA fragments 188 covering the promoter regions (Plys: 518 bp; Plys_CS_0: 533 bp) were incubated for 15 min 189 at room temperature with varying amounts (0,28,70,140,180