YlmD and YlmE are required for correct sporulation-specific cell division in Streptomyces coelicolor A3(2)

Cell division during the reproductive phase of the Streptomyces life-cycle requires tight coordination between synchronous formation of multiple septa and DNA segregation. One remarkable difference with most other bacterial systems is that cell division in Streptomyces is positively controlled by the recruitment of FtsZ by SsgB. Here we show that the genes ylmD (SCO2081) and ylmE (SCO2080), which lie in operon with ftsZ in the dcw cluster of actinomycetes, are required for correct sporulation-specific cell division in Streptomyces coelicolor. Electron and fluorescence microscopy demonstrated that in particular ylmE mutants have a highly aberrant phenotype with defective septum synthesis, and produce very few spores with aberrant spore walls. FtsZ-ring formation was disturbed in ylmD and ylmE mutants. YlmD and YlmE are not part of the divisome, but instead localise diffusely in aerial hyphae, with differential intensity throughout the sporogenic part of the hyphae. Taken together, our work shows that YlmD and YlmE play an important role in (the control of) sporulation-specific cell division, with a more dominant role for YlmE, likely supported by YlmD. This sheds light on the function of the two genes in the direct vicinity of ftsZ whose function had sofar remained unresolved.


INTRODUCTION
In unicellular bacteria, cell division divides a mother cell in two identical daughter cells, each containing a single copy of the chromosome. The control of cell division thereby revolves around finding the mid-cell position, and chromosome segregation and septum synthesis are closely coordinated in time and space to avoid DNA damage by the nascent septum . The cell division scaffold is formed by FtsZ, which is a homologue of tubulin (Bi and Lutkenhaus, 1991) and forms a contractile ring (or Z-ring) that mediates the recruitment of the cell division machinery to the division site (reviewed in (Adams and Errington, 2009;Goehring and Beckwith, 2005)). Septum-site selection and Z-ring stabilization are mediated by proteins like FtsA and ZipA (Hale and de Boer, 1997;Pichoff and Lutkenhaus, 2002;RayChaudhuri, 1999), ZapA (Gueiros-Filho and Losick, 2002) and SepF (Hamoen et al., 2006;Ishikawa et al., 2006). Z-ring (dis-)assembly is thereby actively controlled (reviewed in (Romberg and Levin, 2003)).
Streptomycetes are filamentous Gram-positive bacteria that belong to the phylum of Actinobacteria. These bacteria produce over 60% of all known antibiotics and many other bioactive natural products (Barka et al., 2016;Hopwood, 2007). Exponential growth of the vegetative hyphae is achieved by apical growth and branching. At this stage of the life cycle, cell division does not effect physical separation of the cells, but instead long syncytial cells are formed that are separated by crosswalls (Wildermuth and Hopwood, 1970). Hence, streptomycetes are model organisms for the study of multicellularity and bacterial morphogenesis (Claessen et al., 2014;Flärdh and Buttner, 2009). Most divisome components except FtsZ are not required for vegetative division, presumably reflecting the lack of constriction (McCormick, 2009). Spacing between the cross-walls is highly variable, and little is known of the way septum-site selection is controlled. Recently, using cryo-electron tomography, our lab and others showed that intracellular membrane assemblies or cross-membranes are involved in DNA protection during septum synthesis in young vegetative hyphae, suggesting a novel way of cell-division control (Celler et al., 2016;Yagüe et al., 2016). In addition, these multicellular bacteria have a complex cytoskeleton, which among others plays a role in the organization of the tip growth machinery (Celler et al., 2013;Holmes et al., 2013).
Canonical division resulting in cell fission occurs during sporulation-specific cell division, which requires all components of the divisome (Jakimowicz and van Wezel, 2012;McCormick, 2009). At this stage of the life cycle up to a hundred septa are formed in a short time span, following a highly complex process of coordinated cell division and DNA segregation, and visualized as long ladders of Z-rings (Schwedock et al., 1997). Eventually, chains of unigenomic spores are formed, which have a thick protective spore wall facilitating long-term survival in the environment. While ftsZ null mutants fail to produce septa and hence do not sporulate, cell division is not essential for growth of Streptomyces, which provides a unique system for the study of this process (McCormick et al., 1994;McCormick and Losick, 1996).
Sporulation-specific cell division is controlled by the SsgA-like proteins (SALPs), which are exclusively found in morphologically complex actinobacteria Traag and van Wezel, 2008). The canonical view is that cell division is negatively controlled by the action of the Min system that inhibits division away from midcell, and by nucleoid occlusion (Noc) to avoid septum synthesis near the chromosome to avoid DNA damage, as seen in B. subtilis and E. coli.
In contrast, cell division in streptomycetes is positively controlled by the recruitment of FtsZ to future septum sites by SsgB, in an SsgA-dependent manner (Willemse et al., 2011). As a consequence, both SsgA and SsgB are required for sporulation (Keijser et al., 2003;van Wezel et al., 2000a). We recently showed that SepG (formerly called YlmG) assists in docking of SsgB to the membrane, and also plays a major role in maintaining the nucleoid shape in the spores (Zhang et al., 2016).
Many of the genes for the components of the divisome and the cell-wall biosynthetic machinery are located in the so-called dcw cluster (division and cell-wall synthesis; (Mingorance et al., 2004)). Most of these genes have been studied extensively and their functions have been well characterized. However, little is known of the genes ylmD and ylmE that lie immediately downstream of, and likely in operon with, ftsZ on the genome of streptomycetes and many other bacteria, including firmicutes. In this study we demonstrate that YlmD and YlmE play an important role in cell-wall synthesis during sporulation, whereby in particular ylmE null mutants show severe cell division defects, resulting in a near complete block of sporulation in Streptomyces coelicolor and Streptomyces lividans.

Phylogenetic analysis of YlmE (SCO2080) and YlmD (SCO2081)
Many of the genes in the dcw gene cluster have been extensively studied and their functions are well established. However, this is not the case for ylmD and ylmE, which lie immediately downstream of ftsZ in many bacteria. In all Streptomyces genomes analyzed, ftsZ (SCO2082), ylmD (SCO2081) and ylmE (SCO2080) form an operon, an observation that is supported by high-resolution transcript mapping (Romero et al., 2014); moreover there is apparent translational fusion between ftsZ and ylmD (overlapping start and stop codons) and the mere 6 nt spacing between ylmD and ylmE. This transcriptional coupling to ftsZ suggests that these genes may play a prominent role in the cell division process. Transcript levels of ftsZ are similar to those of ylmD and ylmE during vegetative growth on MM agar plates; however, ftsZ is transcribed at significantly higher levels during sporulation (Swiatek et al., 2013). YlmD and YlmE are widespread in Gram-positive bacteria, and particularly in actinobacteria. SCO2081 and SCO2080 share 32% aa identity with YlmD and YlmE of B. subtilis, respectively. Orthologues of these proteins are also found in several genera of Gram-negative bacteria. Phylogenetic analysis of the YlmD and YlmE proteins in actinobacteria shows that while YlmE is widespread, YlmD is often absent in actinobacteria, such as Stackebrandtia, Catenulispora, Salinispora, Micromonospora, Amycolatopsis and Mycobacterium, several of which are spore-forming actinobacteria ( Fig. 1). Remarkably, no examples of ylmD being present in the absence of ylmE and thus it would appear that loss of YlmD (SCO2081) has occurred on multiple occasions, given that there is wide but patchy distribution of this gene across distinct actinobacteria lineages and the sequences clade tightly within the accepted actinomycete phylogenies. This would suggest that there is potentially overlapping function in these two genes.
Analysis of ylmD using the EMBL String engine (Szklarczyk et al., 2011) shows functional linkage in two groups to cell division associated genes (sepF2, SCO2085 and ftsZ) along with a group of mainly hypothetical proteins (STRING Data link: http://bit.ly/2gc5kCB). YlmD has a domain that has homology to the multiple-copper polyphenol oxidoreductase laccases, which are oxidoreductases that are widely distributed in both prokaryotes and eukaryotes (Claus, 2003). A similar analysis of YlmE also indicates a functional linkage to cell-wall biosynthesis and cell division (STRING Data link: http://bit.ly/2gbPyHK). YlmE is a YBL036c-like protein which generally contain pyridoxal 5-phosphate dependent enzymes. The structure of YBL036c from Saccharomyces cerevisiae was resolved at 2.0 Å resolution (PDB 1CT5; (Eswaramoorthy et al., 2002). The protein has homology to the N-terminal domains of alanine racemases but lacks of the β-sandwich domain which would likely limit the activity of YBL036c as alanine or nonspecific amino acid racemase (Percudani and Peracchi, 2003). To test the hypothesis that YlmE may have alanine racemase activity and thus may play a role in determining the amino acid composition of the peptidoglycan, we tested purified YlmE for alanine racemase assays as described previously (Tassoni et al., 2017). YlmE failed to catalyze the conversion of L-alanine to D-alanine in vitro, and over-expression of the enzyme also failed to restore a D-Ala auxotrophy to alr null mutants (data not shown). These observations coupled with protein structure homology data make it highly unlikely that YlmE functions as an alanine racemase in vivo.

ylmD and ylmE are required for proper sporulation
To analyze the role of ylmD and ylmE, deletion mutants were created in S. coelicolor M145 as detailed in the Materials and Methods section. The +25 to +696 region of ylmE (SCO2080) or the +25 to +705 region of ylmD (SCO2081) were replaced by the apramycin resistance cassette, followed by deletion of the cassette using the Cre recombinase so as to avoid polar effects. For each mutant, four independent mutants were selected and all had the same phenotype. Therefore, one was selected for more detailed analysis, designated GAL47 (S. coelicolor M145 ΔylmD) and GAL48 (S. coelicolor M145 ΔylmE).
The ylmD null mutant GAL47 had a light grey appearance, while ylmE null mutant GAL48 hardly produced any grey pigment after 6 days incubation. The lack of grey-pigmented spores is indicative of a failure to complete full development ( Fig. 2A). Phase-contrast light microscopy of impression prints of the top of the colonies demonstrated that while the parent produced typical long spore chains, the ylmD null mutant produced many aberrantly sized (and in particular large) spores, while the ylmE null mutant produced fewer spores and those produced were unusually large. The same defect in sporulation was observed on R5 and MM mannitol agar plates. Antibiotic production was not affected, and the mutants produced normal levels of the pigmented antibiotics actinorhodin and undecylprodigiosin. To further ascertain that the sporulation defects were indeed solely due to the deletion of the respective genes, we introduced plasmids expressing ylmD or ylmD-egfp in the in the ylmD null mutant and ylmE or ylmE-egfp in the ylmE mutant, in all cases with transcription directed from the native ftsZ promoter region. While the majority of the spores of the complemented ylmD mutant had a regular appearance, those of the complemented ylmE mutants still showed variable lengths (Fig.   2B). This partial complementation suggests that the deletion of ylmE may have polar effect on the expression of its downstream genes. This is supported by the fact that introduction of a plasmid harbouring the entire region from ftsZ to divIVA fully restored sporulation to ylmE mutants (Fig. S1).
To study the spores of ylmD and ylmE null mutants at high resolution, cryo-scanning electron microscopy (SEM) was performed, which again demonstrated the sporulation defects.
The parental strain S. coelicolor M145 produced abundant spore chains, with nearly all hyphae fully developed into mature spore chains (Fig. 3AB). The ylmD null mutant GAL47 frequently produced aberrantly sized spores (Fig. 3CD), while in ylmE null mutant GAL48, precious few and often aberrant spores were identified (Fig. 3EF). Mutants were also created in Streptomyces lividans 66, and these again showed the same morphology, strongly supporting the notion that the observed phenotypes were solely due to the mutations in ylmD or ylmE (data not shown).
The highly similar phenotypes of the S. coelicolor and S. lividans mutants support the notion that the genes play a key role in sporulation-specific cell division and are consistent with the conserved function of the genes in sporulation-specific cell division in Streptomyces.

Deletion of ylmD and ylmE results in aberrant morphology and reduced viability of spores
Viability of the spores was tested by plating around 1000 spores -counted in a haemocytometer -of S. coelicolor M145 and its mutants GAL47 (ΔylmD) and GAL48 (ΔylmE) onto SFM agar plates.
While the wild-type strain had close to 100% viability, spores of the ylmD and ylmE mutants had reduced viability, with 50% and 60% viable spores, respectively. Spores of in particular the ylmE mutant were also more heat sensitive, with 24% viability after 5 min incubation at 60 0 C, as compared to 44% survival for ylmD null mutant spores and 50% for the wild-type strain.
To analyze the possible cell wall defects in more detail, all strains were grown on SFM agar plates for 5 days and analysed by Transmission Electron Microscopy (TEM) (Fig. 4). The parent produced typical spore chains and thick-walled spores. Conversely, ylmD and ylmE mutant spores were deformed and highly irregular. In particular the spores of the ylmE mutant had a thin wall similar to that of hyphae, suggesting that spore-wall synthesis was compromised.

YlmD and YlmE are required for peptidoglycan synthesis at the septum
To analyze cell wall and membrane distribution in ylmD and ylmE mutants, fluorescence microscopy was performed on two-days old SFM surface-grown cultures of mutants GAL47 (M145 ΔylmD) and GAL48 (M145 ΔylmE) and compared to the parental strain M145.
Peptidoglycan precursors were stained with FITC-WGA or Oregon-WGA and membranes visualized by staining with FM5-95. In wild-type pre-division aerial hyphae, long symmetrical septal ladders were observed when stained for cell wall or membranes (Fig. 5A). In contrast, aerial hyphae of the mutants showed highly disturbed cell wall and membrane distribution, which was more pronounced in ylmE than in ylmD mutants (Fig. 5AB). The ylmE mutants were mostly devoid of septation, which could be complemented via re-introduction of ylmE; similarly, septation was restored to the ylmE mutant by re-introduction of ylmE, although peptidoglycan synthesis and septum spacing was still irregular (Fig. 5C). These data suggest an important role for YlmD and in particular YlmE in cell-wall synthesis during sporulation.

Cellular localization of YlmD and YlmE
Given the impact of ylmDE on sporulation, we analysed how YlmD and YlmE were localized in the hyphae of S. coelicolor. To this end, constructs based on the low-copy number vector pHJL401 were prepared to allow the expression of YlmD-eGFP and YlmE-eGFP fusion proteins, which were expressed from the natural ftsZ promoter region (see Materials and Methods section for details). These constructs were then introduced into S. coelicolor M145 and (as a control for functionality) in the mutants. The constructs partially restored development to the respective mutants, with significant restoration of the sporulation defects in ylmD mutants, and partial restoration of sporulation to ylmE mutants (Fig. 2B). Both YlmD-eGFP and YlmE-eGFP mostly showed diffuse localization along the wall of the aerial hyphae and in spores of S. coelicolor M145 (Fig. 6). During early stages of sporulation, fluorescence became more intense, indicative of increased expression of the proteins (Fig. 6). During later stages of sporulation, YlmD-eGFP and YlmE-eGFP showed an irregular pattern of varying intensity, with some spores showing bright fluorescence indicative of high concentrations of YlmD or YlmE, while others hardly showed any fluorescence (Fig 6). This suggests that YlmD and YlmE are differentially expressed throughout nascent spore chains, and do not exclusively co-localise with FtsZ and the divisome.

Localization of FtsZ is disturbed in ylmD and ylmE null mutants
To investigate the localization of FtsZ in the mutants, integrative plasmid pKF41 that expresses FtsZ-eGFP from the native ftsZ promoter region (Grantcharova et al., 2005) was introduced into the ylmD and ylmE mutants, to generate strain GAL52 and GAL53, respectively. Prior to sporulation, typical ladders of Z-rings were observed in the parental strain S. coelicolor M145, while ylmD and ylmE null mutants showed abnormal Z-ladders (Fig. 7). In the absence of YlmD, ladders were still observed, but the intensity varied and spacing between the individual rings was far less regular, with many neighboring Z-rings either close together or widely spaced.
Consistent with the sporulation defect, ylmE null mutants produced very few Z-rings, and the few Z-ladders that where formed were highly irregular or unfinished, and significantly shorter than in the parental strain. Thus, FtsZ localization is irregular in ylmD mutants and highly compromised in ylmE mutants, consistent with a role for YlmE and to a lesser extent YlmD in (the control of) sporulation-specific cell division.

CONCLUSIONS
Phylogenetic analysis shows strong linkage between ylmD, ylmE and ftsZ, and this is further validated by the transcriptional organization, with the three genes forming a single transcription unit in streptomycetes. All bacteria that have ylmD also have ylmE, while conversely, ylmD is often lost in strains that have retained ylmE. This suggests that is unlikely that YlmD is solely required for the proper function of YlmE. In line with this, ylmE plays a key role in sporulationspecific cell division, while ylmD appears less important. Mutants lacking ylmE have a severe phenotype, with highly compromised cell-wall synthesis, and aberrant division. Mutants that lacked ylmD produced abundant spores, but with aberrant shapes and variable lengths. FtsZladders were seen in sporogenic aerial hyphae of ylmD mutants, although with imperfect symmetry, while FtsZ failed to localise properly in ylmE mutants. Taken together, the racemaselike protein YlmE plays an important role in (the control of) sporulation-specific cell division in streptomycetes, supported by YlmD. We are currently performing detailed structural and functional analysis of these proteins, to elucidate their precise role in bacterial cell division.

Phylogenetic analysis of ylmD and ylmE
The amino acid sequence of YlmD and YlmE were extracted from StreptDB (http://strepdb.streptomyces.org.uk) and used to search the NCBI database (www.ncbi.nlm.nih.gov) using BLASTP against the non-redundant protein sequence database.
Alignment of YlmD and YlmE was generated using ClustalW (Thompson et al., 1994) followed by manual editing in MEGA v. 4.0. The neighbour-joining trees (Saitou and Nei, 1987) were generated with default parameters settings as implemented in MEGA v. 4.0 (Tamura et al., 2007). The maximum-likelihood trees were made using the best fit models predicted by MEGA.
Tree reliability was estimated by bootstrapping with 1000 replicates. Trees were drawn with either N-J or ML algorithms gave trees of similar topologies indicating that the phylogenies are likely to reliable.

Plasmids and constructs
All plasmids and constructs described in this study are summarized in Table S2. The oligonucleotides used for PCR are listed in Table S3. PCR reactions were performed using Pfu DNA polymerase as described (Colson et al., 2007).

Constructs for the deletion of ylmD and ylmE
The strategy for creating knock-out mutants is based on the unstable multi-copy vector pWHM3 (Vara et al., 1989) as described previously (Świątek et al., 2012). For each knock-out construct roughly 1.5 kb of upstream and downstream region of the respective genes were amplified by PCR from cosmid St4A10 that contains the dcw cluster of S. coelicolor. The upstream region was thereby cloned as an EcoRI-XbaI fragment, and the downstream part as an XbaI-BamHI fragment , and these were ligated into EcoRI-BamHI-digested pWHM3 (for the precise location of the oligonucleotides see Table S3). In this way, an XbaI site was engineered in-between the flanking regions of the gene of interest. This was then used to insert the apramycin resistance cassette aac(3)IV flanked by loxP sites, using engineered XbaI sites. The presence of the loxP recognition sites allows the efficient removal of the apramycin resistance cassette following the introduction of a plasmid pUWL-Cre expressing the Cre recombinase (Fedoryshyn et al., 2008;Khodakaramian et al., 2006). Knock-out plasmids pGWS728 and pGWS729 were created for the deletion of nucleotide positions +25/+696 of ylmE (SCO2080) and +25/+705 of ylmD (SCO2081), whereby +1 refers to the translational start site of the respective genes. This allowed first the replacement by the apramycin resistance cassette. Subsequently the apramycin resistance cassette was removed using expression of pUWLCre.
For complementation of the ylmE and ylmD null mutants, the entire coding regions of SCO2080 and SCO2081 (with stop codons) were amplified from the S. coelicolor M145 chromosome using primer pairs ylmE_F+1 and ylmE_R+723 and ylmD_F+1 and ylmD_R+732, respectively. The PCR products were digested with StuI/XbaI, and inserted downstream of the native ftsZ promoter region in pHJL401, respectively. Thus constructs pGWS1042 and pGWS1043 were generated that express ylmE and ylmD, respectively, under control of the S. coelicolor ftsZ promoter region.

Constructs for the localization of YlmD and YlmE
The entire coding regions of SCO2080 and SCO2081 (without stop codons) were amplified from the S. coelicolor M145 chromosome using primer pairs ylmE_F+1 and ylmE_R+717 and ylmD_F+1 and ylmD_R+726, respectively. The PCR products were digested with StuI/BamHI, and inserted downstream of the native ftsZ promoter region and immediately upstream of egfp in pHJL401. The latter is a highly stable vector with low copy number that generally results in wildtype transcription levels and is well suited for among others complementation experiments (van Wezel et al., 2000b). Thus constructs pGWS757 and pGWS758 were generated that express YlmE-eGFP and YlmD-eGFP, respectively, from the native S. coelicolor ftsZ promoter region.
Constructs pGWS757 and pGWS758 were also used to complement ylmE and ylmD null mutants, respectively.

Microscopy
Fluorescence and light microscopy were performed as described previously (Willemse and van Wezel, 2009). For peptidoglycan staining we used FITC-labeled wheat germ agglutinin (FITC-WGA) or Oregon Green 488 conjugated WGA (Oregon-WGA); for membrane staining, we used FM5-95 (all obtained from Molecular Probes). All images were background-corrected, setting the signal outside the hyphae to 0 to obtain a sufficiently dark background. These corrections were made using Adobe Photoshop CS4. Cryo-scanning electron microscopy (cryo-SEM) and transmission electron microscopy (TEM) were performed as described (Piette et al., 2005).

Computer analysis
DNA and protein database searches were completed by using StrepDB page (http://strepdb.streptomyces.org.uk/). Phylogenetic analysis was done using the STRING engine at EMBL (www.string.EMBL).    spores of the ylmD (B) and ylmE (C) null mutants have an irregular appearance. Note the lighter appearance of the spore wall in the ylmD null mutant and the lack of the typical thick spore wall in ylmE mutants. Cultures were grown on SFM agar plates for 5 days at 30 0 C. Bar, 500 nm.  YlmD-eGFP and YlmE-eGFP. Stages were: early aerial development, late aerial development and sporulation maturation. During sporulation, YlmD had a 'patchy' localization, while YlmE was frequently seen to localize in a ring-like fashion inside the spores, suggesting that at this stage YlmD and YlmE may co-localize with the cell-wall synthetic machinery. Bar, 5 µm.