A specialized MreB-dependent complex mediates the formation of stalk-specific peptidoglycan in Caulobacter crescentus

Many bacteria have complex cell shapes, but the mechanisms producing their distinctive morphologies are still poorly understood. Caulobacter crescentus, for instance, exhibits a stalk-like extension that carries an adhesive holdfast mediating surface attachment. This structure forms through zonal peptidoglycan biosynthesis at the old cell pole and elongates extensively under phosphate-limiting conditions. We analyzed the composition of cell body and stalk peptidoglycan and identified significant differences in the nature and proportion of peptide crosslinks, indicating that the stalk represents a distinct subcellular domain with specific mechanical properties. To identify factors that participate in stalk formation, we systematically inactivated and localized predicted components of the cell wall biosynthetic machinery of C. crescentus. Our results show that the biosynthesis of stalk peptidoglycan involves a dedicated peptidoglycan biosynthetic complex that combines specific components of the divisome and elongasome, suggesting that the repurposing of pre-existing machinery provides a straightforward means to evolve new morphological traits.


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The shape of most bacteria is determined by a cell wall made of peptidoglycan (PG), a mesh-like hetero-15 polymer that surrounds the cytoplasmic membrane and provides resistance against the internal osmotic 16 pressure (Typas et al., 2012;. The backbone of PG is formed by strands of alternating 17 N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) subunits. These glycan chains are 18 connected by short peptides that are attached to the MurNAc moieties, giving rise to a single elastic 19 macromolecule known as the PG sacculus (Schleifer and Kandler, 1972). 20 The PG meshwork needs to be continuously remodeled to allow for cell growth and division (den monofunctional GTases (Vollmer and Bertsche, 2008). The majority of TPases are DD-TPases, also known 30 as penicillin-binding proteins (PBPs) (Suginaka et al., 1972). These proteins catalyze the formation of D-31 Ala 4 -meso-DAP 3 (4-3) crosslinks, in a reaction that releases the D-Ala 5 moiety of the donor molecule 32

Phosphate limitation arrests the cell cycle of Caulobacter in G1-phase 123
Although the stimulatory effect of phosphate starvation on Caulobacter stalk elongation has been known 124 for decades (Schmidt and Stanier, 1966), the underlying regulatory mechanisms are still poorly under-125 stood. Prompted by the fact that stalk formation is tightly linked to cell cycle progression, we set out to 126 investigate the effects of phosphate deprivation on central cellular processes such as DNA replication and 127 cell division. First, flow cytometry was used to assess the replicational state of cells after transfer from 128 standard to phosphate-free (M2G -P ) medium. To this end, replication initiation was blocked with 129 rifampicin and ongoing rounds of replication were allowed to finish. Previous work has shown that 130 Caulobacter cells contain a single chromosome that is replicated only once per division cycle (Collier, 2012; 131 Quon et al., 1998). Consistent with this finding, we observed that cells accumulated either one or two 132 chromosome equivalents when grown in standard conditions, indicating that a large fraction of the 133 population was in S-phase ( Figure 1A). However, upon phosphate deprivation, DNA replication gradually 134 ceased, with most cells arrested in G1-phase after 24 h of incubation. These data suggest that the lack of 135 phosphate leads to a block in the cell cycle prior to S-phase, thereby preventing new rounds of 136 chromosome replication. To support this conclusion, we visualized the number and positions of the 137 chromosomal replication origins. In doing so, we made use of a fluorescently (GFP-) tagged derivative of 138 the chromosome partitioning protein ParB, which interacts with specific motifs (parS) in the origin region 139 (Mohl and Gober, 1997;Thanbichler and Shapiro, 2006). The expression of GFP-ParB thus typically results 140 in the detection of either one or two foci, depending on the number of origin copies in the cell. 141 Microscopic analysis revealed that most (~ 85%) cells exhibited a single ParB focus at the stalked pole 142 when subjected to 24 h of phosphate starvation, indicating that they are arrested in G1 phase ( Figure 1B). 143 To clarify the reason for this G1 arrest, we analyzed the cellular levels of the replication initiator protein 144 DnaA and the cell cycle master regulator CtrA, which act as positive and negative regulators of 145 chromosome replication, respectively (Collier, 2012). Interestingly, both proteins were rapidly depleted 146 from the cells during phosphate starvation (Figure 1C), indicating that key drivers of the Caulobacter cell 147 cycle are absent under this condition. 148 To correlate changes in cell cycle progression with the growth behavior of cells, we monitored changes in 149 cell mass and number after a shift to phosphate-limiting conditions. Interestingly, the optical density of 150 cultures kept increasing exponentially for more than 10 h and only leveled off after ~ 50 h of incubation 151 ( Figure 1D), suggesting that cells made use of internal phosphate storage compounds to compensate for 152 the lack of an external phosphate source. Consistent with the detection of DNA replication events 153 ( Figure 1A), cells still multiplied during the initial exponential phase. However, after longer starvation 154 periods (> 24 h), the viable-cell count started to decline, whereas the cell mass still increased, likely due 155 to continued elongation of the cell bodies and stalks in the absence of cell division events. Western blot 156 analysis indeed revealed that the essential cell division protein FtsZ was depleted from the cells upon 157 phosphate starvation ( Figure 1C). The same was true for the cell division regulator MipZ, an inhibitor of 158 FtsZ polymerization that limits Z-ring formation to the midcell region (Thanbichler and Shapiro, 2006). In 159 line with these findings, an FtsZ-YFP fusion induced after prolonged phosphate starvation formed multiple 160 foci in the vicinity of the stalk-distal pole instead of a defined midcell band (Figure 1B), indicating the 161 absence of a functional and properly localized Z-ring (Thanbichler and Shapiro, 2006). Notably, FtsZ was 162 never observed at the stalk base, supporting the previous notion that it does not play any role in stalk 163 formation (Thanbichler and Shapiro, 2006). 164 Taken together, our results demonstrate that phosphate starvation arrests the Caulobacter cell cycle in a 165 G1-like phase, thereby stalling DNA replication and cell division until phosphate becomes available again. followed by zonal growth at midcell during the constriction phase (Figure 2-figure supplement 1). 175 Moreover, concurrent with the switch from disperse to zonal growth, an additional intense focus of 176 fluorescence appeared at one of the cell poles, reflecting the establishment and outgrowth of the stalk. 177 This polar signal faded gradually as the cell cycle progressed and was no longer detectable in late pre-178 divisional cells. Thus, HADA reliably detected all known growth zones in Caulobacter cells. 179 Next, we used HADA labeling to determine the pattern of PG synthesis under phosphate-limiting 180 conditions (Figure 2A). After 6 h of incubation in phosphate-free medium, most cells showed a bright 181 fluorescent patch at the stalked pole as well as a faint disperse signal extending throughout the rest of 182 the cell body. Cells longer than ~ 4 µm often displayed an additional bright focus at their center, which 183 supplement 1), PG was isolated from each of the fractions and subjected to muropeptide analysis. 215 Interestingly, stalk PG contained a high proportion of 3-3 crosslinked peptides and non-crosslinked 216 tripeptides (resulting from the cleavage of 3-3 bonds), whereas these muropeptide species were barely 217 detectable in the cell body samples (Figure 3 and Supplementary file 1). Similarly, the total fraction of 218 crosslinked peptide side chains was significantly higher in stalk PG, mostly because of a higher proportion 219 of trimeric muropepides. The glycan chain lengths, by contrast, did not vary between the two 220 compartments. Collectively, these findings indicate that the PG layers of stalks and cell bodies differ in 221 both the type and extent of peptide crosslinks. 222 Previous work has shown that 3-3 crosslinks are generated by LD-TPases, which are characterized by a 223 (now referred to as LdtD and LdtX, respectively). To determine how these factors contribute to the 226 distinctive composition of the stalk cell wall, we generated a strain carrying in-frame deletions in both the 227 ldtD and ldtX gene and analyzed the composition of PG purified from its stalk and cell body compartments. 228 In both samples, 3-3 crosslinked peptides and non-crosslinked tripeptides were virtually undetectable 229 (Figure 3 and Supplementary file 1), indicating that the formation of these muropeptide species is linked 230 to the activity of the two predicted LD-TPases. Notably, however, the total fraction of crosslinked peptides 231 barely changed in either of the compartments, because the loss of 3-3 crosslinks was compensated by a 232 proportional increase in the fraction of 4-3 crosslinks. Thus, LD-TPase activity is not the main factor 233 responsible for the elevated degree of crosslinking in stalk PG. 234 Previous work has shown that the stalk is physiologically separated from the cell body, because it is devoid 235 of cytoplasm and contains crossband complexes that block the exchange of periplasmic and membrane 236 proteins (Schlimpert et al., 2012). It was conceivable that crossbands could help establish the differences 237 in the PG composition observed for the two compartments, for instance by facilitating the establishment 238 of distinct pools of PG biosynthetic enzymes or blocking the diffusion of lipid II into the stalk structure. To 239 test this idea we determined the muropeptide profile of stalk and cell body PG isolated from a crossband-240 less strain (ΔstpAB, SW51). Notably, we still observed a higher content of 3-3 crosslinks and a higher total 241 proportion of crosslinked peptides in stalk PG (Supplementary file 1). Similar to the differences in PG 242 turnover ( Figure 2C), this characteristic thus appear to be independent of the presence of crossbands. 243

Stalk formation involves class A and class B penicillin-binding proteins 244
Stalk formation involves a growth process that is distinct from the disperse and zonal incorporation of PG 245 mediated by the elongasome or division complex, respectively. To determine the composition of the 246 underlying machinery, we systematically analyzed all predicted PG biosynthetic proteins encoded in the 247 Caulobacter genome for their contribution to stalk elongation under phosphate-limiting conditions. In 248 doing so, we initially focused on enzymes with PG synthase activity, including PBPs and LD-TPases. A 249 previous study has shown that inhibition of the monofunctional DD-TPase PBP2 with mecillinam largely 250 abolished the synthesis of stalks under standard conditions, although it concomitantly induced severe 251 morphological defects in the cell body (Seitz and Brun, 1998). To further investigate the role of this 252 protein, we generated a strain producing a fully functional GFP-PBP2 fusion in place of the wild-type Our results confirm that deletion of pbpC led to a moderate reduction in stalk length, whereas the absence 261 of any other PBP, either alone or in combination, did not have any effect ( Figure 4B). However, as 262 observed under standard growth conditions (Strobel et al., 2014;Yakhnina and Gitai, 2013), at least one 263 bifunctional PBP was required for viability during phosphate starvation (Figure 4-figure supplement 1A). 264 In line with the results of the deletion studies, localization analyses revealed that none of the bifunctional 265 PBPs except for PbpC accumulated at the stalked pole, indicating that these proteins may not be 266 specifically associated with the stalk biosynthetic machinery (Figure 4-figure supplement 1B). Notably, 267 however, PbpX appeared enriched in the stalk compartments, but the significance of this observation 268 remains unclear. 269 Finally, we analyzed the role of the two predicted LD-TPases LdtD and LdtX in stalk formation. Although 270 these proteins make a significant contribution to PG crosslinking in the stalk compartment (Figure 3), their 271 inactivation did not have any apparent phenotypic effect ( Figure 4B). LD-TPase activity may thus not 272 contribute to the establishment of the stalk structure per se but rather have an accessory function that 273 serves to modify the biophysical properties of the PG layer. Localization studies indicate that LdtD and 274 LdtX do not accumulate at the stalk base, suggesting that they may act independently of the polar stalk 275 biosynthetic machinery (Figure 4-figure supplement 1C). 276

Components of the autolytic machinery are critical for stalk formation 277
Apart from PG synthases, stalk formation must also involve autolytic enzymes that cleave the PG sacculus 278 and, thus, enable the insertion of new cell wall material at the stalk base. However, to this point, the 279 nature of the factors involved has remained unknown. To address this issue, we systematically screened 280 mutants lacking one or multiple predicted PG hydrolases for defects in stalk growth under phosphate- To further investigate the functions of the five autolytic factors identified in the mutational screen, we 303 generated fluorescently (mCherry-) tagged derivatives of these proteins and analyzed their localization 304 patterns under conditions of phosphate starvation (Figure 6). Both the DipM and CrbA fusions accu-305 mulated at the stalk base and may, thus, be specifically associated with the polar stalk biosynthetic 306 machinery. The SdpA and SdpB fusions, by contrast, were distributed throughout the cell envelope, sug-307 gesting that the two proteins may either act independently of the polar complex or associate with it in a 308 very transient manner. Unlike the other proteins analyzed (Figure 6-figure supplement 1), LdpA-mCherry 309 was quantitatively cleaved at the junction between the two fusion partners, preventing further analysis 310 (data not shown). 311 In order to determine how the absence of the different autolytic factors influenced the pattern of PG 312 biosynthesis, mutants lacking these proteins were grown in phosphate-limiting conditions and subjected 313 to HADA staining (Figure 7). Consistent with their relatively mild stalk elongation defect, ΔldpA cells still 314 displayed a pattern similar to that of the wild-type strain. In the ΔdipM and ΔcrbA strains, by contrast, the 315 polar signals were much fainter and new cell wall material was often incorporated at non-polar sites. An 316 even more pronounced effect was observed in the ΔsdpAB mutant, which virtually lacked polar foci and 317 instead showed patchy or even HADA fluorescence throughout the cells. Thus, the severity of the stalk 318 elongation defect scales with the loss in polar PG biosynthesis. 319 To obtain more detailed insight into the effects of the different mutations on the structure of the PG layer, 320 we isolated whole-cell sacculi from wild-type and mutant cells after prolonged (24 h) phosphate starva-321 tion and subjected them to muropeptide analysis (Supplementary file 3). For the wild-type strain, whole-322 cell sacculi gave similar results as PG from isolated from a cell body fraction (compare with BacA, which retains its polar position irrespective of changes in the phosphate supply (Figure 9). The 389 MreC fusion, by contrast, formed a broad band at midcell, whereas it was largely excluded from the polar 390 regions (Figure 9). In line with the global morphological defects caused by its depletion, MreC may have a 391 general role in cell wall integrity, but it does not appear to be specifically associated with the polar stalk 392 biosynthetic machinery. 393 To determine the role of the different scaffolds in polar PG biosynthesis, cells lacking these factors were 394 subjected to HADA staining after phosphate deprivation (Figure 10). Interestingly, despite its severe stalk 395 elongation defect (Figure 8) the ΔbacA mutant still displayed intense polar foci, indicating that BacA is an 396 accessory factor that is not critical for the global reorganization of PG biosynthesis induced under 397 phosphate-limiting conditions. Consistent with this idea, muropeptide analysis showed that deletion of 398 bacA did not have any appreciable effects on global PG composition (Supplementary file 4). Depletion of 399 MreB or RodZ, by contrast, strongly decreased the intensity of the polar HADA signals, and frequently led 400 to the insertion of cell wall material at pole-distal sites. In both cases, these defects were accompanied by 401 significant changes in the whole-cell muropeptide profiles. Similar to the ΔsdpAB mutant (compare Sup-402 plementary file 2), the degree of crosslinkage was significantly reduced, mostly due to a decrease in the 403 proportion of highly crosslinked (trimeric and tetrameric) muropeptide species. Moreover, there was a 404 striking increase in the proportion of muropeptides with tripeptide side chains, indicative of high levels of 405 LD-TPase activity. Thus, cell wall stress caused by reduced levels of PBP2-mediated DD-transpeptidation 406 may trigger a fail-safe mechanism that stabilizes the PG meshwork through the formation of abundant 3-407 3 crosslinks. 408 Collectively, these results demonstrate that MreB and its transmembrane adapter RodZ play a central role 409 in the establishment of the polar PG biosynthetic zone that gives rise to the stalk structure. 410

MreB orchestrates the polar stalk biosynthetic complex 411
Our data demonstrate that several components of the PG biosynthetic machinery localize to the stalked 412 pole in phosphate-starved cells, suggesting that they assemble into a complex mediating the synthesis of 413 stalk PG. To obtain more insight into the factors mediating the recruitment of these proteins, we reanal-414 yzed the localization patterns of DipM-mCherry, CrbA-mCherry, Venus-MreB, CFP-RodZ, and BacA-CFP in 415 all deletion strains that showed defects in stalk elongation (dipM, sdpAB, crbA, ldpA, and bacA). 416 However, in all cases, the positioning of the fusion proteins remained unaffected, indicating that neither 417 lytic factors nor the bactofilin cytoskeleton are required for complex assembly. Given the prevalence of 418 elongasome components among the polarly localized proteins, we then tested the role of MreB in the 419 recruitment process. Treatment of cells with the MreB inhibitor A22 not only led to the delocalization of 420 the known MreB interactor RodZ but also abolished the polar foci of DipM and CrbA (Figure 11). Thus, 421 MreB appears to be a key organizer of the stalk biosynthetic complex. Notably, A22 had no effect on the 422 polar localization of BacA, indicating that the bactofilin scaffold acts independently of MreB. 423 To analyze the dynamics of the polar MreB assembly, we aimed to construct a sandwich fusion in which 424 mCherry was inserted into a surface-exposed loop of the MreB protein (Bendezu et al., 2009) (Figure 12A). Although MreB clearly has a key role in stalk biogenesis, it is not the only scaffolding protein contributing 508 to this process. Previous work has shown that the bactofilin BacA is required for proper stalk length {Kühn, 509 2010 #41}, and our analyses revealed an additional role for this protein in cell body elongation during 510 phosphate starvation (Figure 8). Notably, the bacA gene lies in a putative operon with ldpA, a gene 511 encoding a putative LytM-like endopeptidase that also functions in stalk formation. This genetic context 512 is conserved in a variety of other species, suggesting a functional link between the two gene products 513 Although the functionality and localization of the peptidoglycan biosynthetic machinery changes drasti-534 cally upon transition of Caulobacter cells from phosphate-replete to phosphate-limiting media, the overall 535 composition of their PG layer remains largely unaffected. This finding is unexpected because significant 536 changes in both glycan chain lengths and the degree of cross-linking were observed in other species in 537 response to changes in their growth conditions . However, analyzing the muro-538 peptide profiles of isolated stalk and cell body fractions, we identified clear differences between these 539 two compartments that are likely obscured in whole-cell analyses due to the small contribution of stalks 540 to the total cellular PG content. Most importantly, stalk PG showed a significantly higher degree of 541 crosslinkage, which was mostly due to a higher frequency of 3-3 crosslinks, indicative of elevated LD-TPase 542 activity. The precise reason for this difference remains unclear. It is conceivable that the LD-TPases LdtD 543 and LdtX are part of the polar stalk biosynthetic complex and, thus, preferentially act on newly synthesized 544 PG produced by this machinery. However, localization studies did not give any evidence for an enrichment 545 of these proteins at the stalked pole. An alternative explanation may be provided by the observation that 546 the turnover rate of PG is significantly lower in the stalk than in the cell body. Thus, LD-TPases may act 547 uniformly throughout the entire cell envelope, but most of the 3-3 crosslinks formed in the cell body may 548 be lost as a consequence of PG remodeling, whereas those in the stalk are retained over prolonged periods Collectively, our study shows that, in Caulobacter, multiple cell-wall biosynthetic machineries act in 555 concert to generate stalks of proper size and stability, thereby ensuring optimal performance of this 556 cellular structure in the environmental context. It will be interesting to see how the nature and the 557 regulation of these components have changed during evolution to bring about the large variety of mor-558 phologies found in other stalked members of the alphaproteobacterial lineage. 559 560

Media and growth conditions 562
Caulobacter strains (Evinger and Agabian, 1977) were grown at 28°C in peptone-yeast-extract (PYE) 563 medium (Poindexter, 1964) Copper; Plano GmbH, Germany) and incubated for 1 min at room temperature. Excess liquid was removed 608 with Whatman filter paper. Subsequently, the cells were negatively stained for 5 sec with 5 μl of 1% uracyl 609 acetate. After three washes with H 2 O, the grids were dried, stored in an appropriate grid holder, and 610 analyzed in a 100 kV JEM-1400 Plus transmission electron microscope (JEOL, USA). 611

Western blot analysis 612
Western blot analysis was performed as described (Thanbichler and Shapiro, 2006

Peptidoglycan analysis 642
For whole-cell analyses, cultures were rapidly cooled to 4 °C and harvested by centrifugation at 16,000 643 rpm for 30 min. The cells were resuspended in 6 ml of ice-cold H 2 O and added dropwise to 6 ml of a boiling 644 solution of 8% sodium dodecylsulfate (SDS) that was stirred vigorously. After 30 min of boiling, the 645 suspension was cooled to room temperature. Peptidoglycan was isolated from the cell lysates as 646 described previously (Glauner, 1988) and digested with the muramidase cellosyl (kindly provided by 647 Hoechst, Frankfurt, Germany). The resulting muropeptides were reduced with sodium borohydride and 648 separated by HPLC following an established protocol (Bui et al., 2009;Glauner, 1988). The identity of 649 eluted fragments was assigned based on the retention times of known muropeptides from Caulobacter 650 (Takacs et al., 2013). 651 To prepare stalk and cell body fractions, 100 ml cultures grown in M2G -P medium were rapidly cooled to 652 4 °C and harvested by centrifugation at 16,000 rpm for 30 min. After resuspension in M2G -P medium, the 653 cells were vigorously agitated for 2 min at maximum speed in a kitchen blender. The suspension was sub-654 mitted to three rounds of centrifugation at 9,000 rpm and 4 °C. The supernatants (stalk fraction) and the 655 first pellet (cell body fraction) were collected separately and kept in ice. The stalk fraction was subjected 656 to an additional centrifugation step at 10,000 rpm and 4 °C to remove residual cell bodies and cell debris. 657 Subsequently, stalks were collected by centrifugation at 20,000 rpm and 4 °C for 30 min, resuspended in 658 3 ml ice-cold H 2 O, added dropwise to 3 ml of a boiling 8% SDS solution, and then further processed as 659 described above to isolate stalk PG. The isolation of cell body PG was achieved as described for whole-cell  The full analysis is presented in Figure 1-figure supplement 2C. 701

1160
The data are shown as box plots, with the horizontal line indicating the median, the box the interquartile range and the wiskers 1161 the 2 nd and the 98 th percentile (n=210 per strain). In addition rotated kernel density plots (grey) are depicted for each dataset to 1162 indicate the distribution of the raw data (*** p < 10 -6 ; t-test).  and MT260 (bacA::bacA-cfp). Cells were grown in PYE medium, diluted into M2G -P medium, and incubated for 23 h. Subsequently, 1181 A22 (10 μg/ml) was added to the media, and cultivation was continued for 1 h prior to imaging. Strains AM208, MAB247, and 1182 MT309 were induced for 3 h with 0.3% xylose to induce synthesis of the fusion proteins before analysis (scale bars: 3 μm).   pNPTS138 derivative for in-frame deletion of ldtD a) amplification of the CCNA_01579 flanking regions from CB15N chromosomal DNA using primers oAZ70+oAZ71 (upstream) and oAZ72+oAZ73 (downstream), b) restriction of the upstream fragment with PstI and EcoRI, restriction of the downstream fragment with EcoRI and NheI. c) triple ligation with pNPTS138 cut with PstI and NheI pAZ30 pNPTS138 derivative for in-frame deletion of ldtX a) amplification of the CCNA_03860 flanking regions from CB15N chromosomal DNA using primers oAZ81+oAZ82 (upstream) and oAZ83+oAZ84 (downstream), b) restriction of the upstream fragment with HindIII and EcoRI, restriction of the downstream fragment with EcoRI and NheI. c) triple ligation with pNPTS138 cut with HindIII and NheI pMT719 pXCHYC-2 derivative bearing crbA-mCherry a) amplification of CCNA_02243 from CB15N chromosomal DNA using primers oMT657+oMT658, restriction with NdeI and SacI b) ligation with pXCHYC-2 cut with NdeI and SacI pMT1003 pXVENN-1 derivative bearing venus-mreB a) amplification of CCNA_01612 from CB15N chromosomal DNA using primers oMT1107+oMT1112, restriction with BglII and NheI b) ligation with pXVENN-1 cut with BglII and NheI pMAB60 pNPTS138 derivative for in-frame deletion of CCNA_03856 a) amplification of the CCNA_03856 flanking regions from CB15N chromosomal DNA using primers oMAB235+oMAB218 (upstream) and oMAB219+oMAB236 (downstream) b) double-joint PCR with oMAB235 and oMAB236 and restriction of the PCR product with EcoRI and HindIII, c) ligation with pNPTS138 cut with EcoRI and HindIII pMAB62 pNPTS138 derivative for in-frame deletion of CCNA_03031 a) amplification of the CCNA_03031 flanking regions from CB15N chromosomal DNA using primers oMAB245+oMAB246 (upstream) and oMAB247+oMAB248 (downstream) b) double-joint PCR with oMAB245 and oMAB248 and restriction of the PCR product with EcoRI and HindIII, c) ligation with pNPTS138 cut with EcoRI and HindIII pMAB64 pNPTS138 derivative for in-frame replacement of mreB with mreB sw a) amplification of the CCNA_01612 coding regions from CB15N chromosomal DNA using primers oMAB231+oMAB206 (upstream coding region until 227 codon) and oMAB209+oMAB233 (downstream coding region from 228 codon) b) amplification of the mCherry coding region from pXCHYC-2 using primers oMAB207+oMAB208 bearing appropriate linkers and compatible regions with the CCNA_02243 fragments c) triple-joint PCR with oMAB231 and oMAB233 of the upstream, doawnstream and mCherry fragments and restriction of the PCR product with EcoRI and HindIII, d) ligation with pNPTS138 cut with EcoRI and HindIII pMAB65 pXGFPN-4 derivative bearing gfp-pbp2 a) amplification of CCNA_01615 from CB15N chromosomal DNA using primers oMAB221+oMAB222, restriction with KpnI and NheI b) ligation with pXGFPN-2 cut with KpnI and NheI pMAB66 pXCHYC-2 derivative bearing mreC-mCherry a) amplification of CCNA_01613 from CB15N chromosomal DNA using primers oMAB185+oMAB186, restriction with NdeI and KpnI b) ligation with pXCHYC-2 cut with NdeI and KpnI pMAB67 pNPTS138 derivative for in-frame replacement of pbp2 with gfp-pbp2 a) amplification of the CCNA_01615 upstream flanking region from CB15N chromosomal DNA using primers oMAB241+oMAB242 and the GFP with the first 685bp of CCNA_01615 coding region from pMAB65 with primers oMAB243+oMAB244 b) double-joint PCR with oMAB241 and oMAB244 of the upstream and downstream fragments and restriction of the PCR product with EcoRI and HindIII, d) ligation with pNPTS138 cut with EcoRI and HindIII pMAB70 pNPTS derivative for inframe deletion of CCNA_03431 a) PCR amplification of the CCNA_03431 flanking regions from chromosomal DNA using primers oMAB215+oAMAB202 (upstream) and oMAB203+oMAB204 (downstream) b) double-joint PCR with oMAB215 and oMAB204 of the upstream and downstream fragments and restriction of the PCR product with PstI and EcoRI, d) ligation with pNPTS138 cut with PstI and EcoRI pMAB133 pXCHYC-2 derivative bearing ldtD-mCherry a) amplification of CCNA_01579 from CB15N chromosomal DNA using primers oMAB383+oMAB384 b) Gibson assembly with pXCHYC-2 cut with NdeI and KpnI pMAB134 pXCHYC-2 derivative bearing ldtX-mCherry a) amplification of CCNA_03860 from CB15N chromosomal DNA using primers oMAB385+oMAB386 b) Gibson assembly with pXCHYC-2 cut with NdeI and KpnI Oligonucleotides used in this work.