Bacterial strains, plasmids, oligonucleotides, and growth conditions
Bacterial strains and plasmids used in this study are listed in Table 1 and Supplementary Table S2, respectively. The S. venezuelae strains are derivatives of strain NRRL B-65442. E. coli strain DH5α was used for cloning, while strain DY380 was used to carry out λRed-mediated mutagenesis of cosmids. E. coli strain ET12567/pUZ8002 was used for mobilization of oriT-containing cosmids and plasmids into S. venezuelae as described previously [47]. Media, growth conditions and genetic manipulation were generally performed as described previously for E. coli [48] and Streptomyces strains [49], unless otherwise stated. E. coli strains were grown in lysogeny broth (LB) or on LB agar [50] with 10 g l− 1 NaCl, or without NaCl when hygromycin was used for selection. S. venezuelae cells were grown in maltose-yeast extract-malt extract medium (MYM) or MYM agar, as described by Bush et al. [47]. Chitin agar and MOPS glucose minimal agar were prepared as previously described [51, 52]. For monitoring of exploratory growth phenotype, cultures were grown on yeast extract-peptone (YP) agar medium as described previously [53]. Antibiotics were used with the following final concentrations: 50 µg ml− 1 apramycin, 100 µg ml− 1 carbenicillin, 25 µg ml− 1 chloramphenicol, 25 µg ml− 1 hygromycin, 50 µg ml− 1 kanamycin, and 20 µg ml–1 nalidixic acid. Synthesis of oligonucleotides used in the study (Supplementary Table S3) and DNA sequencing was done by Eurofins Genomics.
Table 1
Bacterial strains used in this study
Strains | Genotype or Relevant characteristics | Reference or source |
Escherichia coli |
BTH101 | F– cya-99 araD139 galE15 galK16 rpsL1 (Strr) hsdR2 mcrA1 mcrB1 | [57] |
DH5α | supE44 ΔlacU169 (Φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | [56] |
DY380 | F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZM15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara, leu)7649 galU galK rspL nupG [ λcI857 (cro-bioA) <> tet] | [72] |
ET12567/pUZ8002 | dam-13::Tn9 dcm-6 hsdM, carrying helper plasmid pUZ8002 | [49] |
Streptomyces venezuelae |
NRRL B-65442 | Wild type S. venezuelae strain | |
LUV041 | attBϕBT1::pMS82 | [73] |
LUV052 | attBϕBT1::pKF543(ftsZ-ypet) | [73] |
LUV080 | ΔsepIVA::apra1 | This work |
LUV112 | ΔsepIVA::apra attBϕBT1::pKF652(sepIVA) | This work |
LUV119 | ΔsepIVA::apra attBϕBT1::pMS82 | This work |
LUV125 | ΔsepIVA::apra attBϕBT1::pKF543(ftsZ-ypet) | This work |
LUV169 | attBϕBT1::pKF703(kasOp*-mNeongreen-sepIVA) | This work |
LUV171 | attBϕBT1::pSS76(kasOp*) | This work |
LUV189 | attBϕBT1::pKF733(kasOp*-mNeongreen-sepIVA divIVA-mCherry) | |
LUV339 | ΔsepIVA::apra Δ(scy-filP)::FRT | This work |
LUV340 | ΔsepIVA::apra | This work |
NA1256 | Δ(scy-filP)::FRT | [27] |
1 apra denotes here denotes here a cassette containing both the apramycin resistance gene aac(3)IV and an oriT, derived from plasmid pIJ773.
Isolation of sepIVA mutant strain
S. venezuelae sepIVA deletion strain was constructed essentially following the ‘Redirect’ PCR targeting protocol [54], but employing a different strain and protocol for λRed recombineering of cosmid DNA in E. coli [55]. The apramycin resistance cassette from pIJ773 ([aac(3)IV-oriT], hereafter referred to as apra) was amplified using primer pair KF1568/KF1569 and introduced into E. coli strain DY380 harboring cosmid 3-B07 containing sepIVA (vnz26025). The mutagenized cosmid, named pKF651, was verified for the replacement of sepIVA gene with apra resistance cassette by PCR with primer pairs KF1570/KF435 and KF1570/KF1571. The verified pKF651 was transferred to S. venezuelae by conjugation, as described previously [49] with some modifications. Conjugation mix containing E. coli donor and S. venezuelae recipient cells was plated on Soya Flour-Mannitol (SFM) agar supplemented with 10 mM MgCl2 and was incubated overnight (15–16 hours) at room temperature. Thereafter, the plates were overlaid with nalidixic acid (20 µg ml–1) and apramycin (50 µg ml–1) and were further incubated at 30°C until ex-conjugants appeared (2–4 days). The ex-conjugants were purified by streaking on MYM agar containing nalidixic acid and apramycin to select for S. venezuelae recombinants with the apra cassette integrated into the chromosome. Putative null mutants were identified based on their apramycin resistance and kanamycin sensitivity. Genomic DNA from mutant candidates was isolated and PCR verification was done using primer pairs KF1570/KF1571 flanking the sepIVA locus and KF1570/KF435 detecting the gene replacement. Null mutants were readily isolated in which sepIVA had been replaced with the apra cassette. (Fig. 1A). Four S. venezuelae sepIVA null mutants were isolated and found to be phenotypically indistinguishable, both macroscopically and microscopically, when grown on MYM agar and in liquid MYM. One representative verified sepIVA mutant was named LUV080. In a subsequent experiment, the ΔsepIVA::apra gene replacement was introduced and verified in both the Δ(scy-filP) mutant background and the wild type by the same methods as outlined above, generating strain LUV339 and LUV340, respectively.
Construction of plasmids
For in trans complementation tests, the sepIVA gene was PCR amplified with primer KF1574 containing NdeI restriction site and primer KF1575 with KpnI restriction site. The amplified PCR product was cloned into NdeI and KpnI sites of plasmid pIJ10770, generating plasmid pKF652. The inserted sepIVA gene was verified by PCR using primer pair KF1272/KF1246 and by DNA sequencing. The plasmid was introduced into S. venezuelae strains by conjugation and integrated at the фBT1 attachment site.
For the study of subcellular localization of SepIVA, PCR amplification of sepIVA was done using primers KF1646/KF1647 with AflII and HindIII restriction sites. The PCR product was digested and ligated into pKF699 (kasOp*-mNeongreen-cvnD2) resulting in the plasmid pKF703 (kasOp*-mNeongreen-sepIVA). Verification of pKF703 was done by restriction mapping and DNA sequencing using primer pairs KF1646/KF1647. Plasmid pKF703 was conjugated into S. venezuelae wild type as described earlier, resulting in the strain LUV169. The kasOp*-mNeongreen-sepIVA fragment was also excised using XbaI and AvrII and ligated in the SpeI site of pSS204, resulting in plasmid pKF733, which allows studies of co-localisation of mNeongreen-SepIVA and DivIVA-mCherry.
In order to construct plasmids for bacterial two-hybrid assay [57], sepIVA was PCR amplified using primer pairs KF1656/KF1657 and S. venezuelae genomic DNA as a template. The PCR product was digested using XbaI and KpnI and ligated into pUT18, pKT25 and pKNT25. The resulting plasmids were named pKF705, pKF707 and pKF708, respectively. The divIVA plasmids were created by amplifying divIVA from chromosomal DNA of S. venezuelae with primers KF1370/KF1371, digesting PCR products with XbaI and KpnI and ligating to the corresponding sites in the BACTH vectors. Plasmid inserts were verified by DNA sequencing.
For creating pKF756, sepIVA was amplified with KF1646 and KF1579, digested with AflII and XhoI and ligated with similarly digested pKF748 to create kasO*p driven expression of sepIVA. pKF748 was derived from pSS76 by overlapping fragments with partial FLAG-tag sequence (created from pSS76 as template with primer sets KF1727/KF1752 and KF1753/KF1751), digesting pSS76 and the overlapping product with KpnI and HindIII and ligation.
Microscopy
For phase-contrast microscopy of aerial hyphae and spores, colonies were grown at 30°C on MYM agar for 4–5 days. Aerial hyphae and spores were sampled by pressing a cover slip against the colony surface and then mounting it on a slide coated with 1% agarose in phosphate-buffered saline (PBS). To observe growth during vegetative stage, spores were diluted and inoculated onto cellophane membranes placed on the top of MYM agar or MOPS glucose minimal agar. Plates were incubated at 30°C for 17 hours and membranes were carefully transferred to agarose-coated (1% in PBS) slides. Prepared slides were imaged using phase-contrast microscopy. Cell wall and nucleoid staining of cultures with Wheat germ agglutinin-Oregon Green (WGA-Oregon Green; Molecular Probes) and 7-Aminoactinomycin D (7-AAD; Molecular Probes) was done as described previously [58]. To observe subcellular localization of fusion proteins by fluorescence microscopy, cells were grown in liquid MYM and transferred to agarose-coated slides. To study FtsZ dynamics and SepIVA localization by time-lapse imaging, bacteria were cultivated in the CellASIC ONIX2 microfluidic system and B04A-03 microfluidic plates (Merck Millipore), as described previously [59]. Imaging was performed on a Zeiss AxioObserver.Z1 microscope with Illuminator HXP 120 V lamp (Zeiss), appropriate fluorescence filters, Zeiss Plan-Apochromat 63×/1.4 Oil Ph3 or 100×/1.4 Oil Ph3 objective, ZEN software (Zeiss), and an ORCA Flash 4.0 LT camera (Hamamatsu). ImageJ/Fiji [60] was used to generate images and movies, as previously described [59]. Fluorescence profiles for SepIVA localization were obtained using ZEN and a width of 5 pixels. Prior to analysis, profiles were aligned at the hyphal apex guided by intensity profiles from the phase contrast channel.
Bacterial two-hybrid assay
The ‘T25’ and ‘T18’ fusion plasmids were used to co-transform chemically competent E. coli BTH101 cells. Transformants were selected on LB agar containing carbenicillin and kanamycin. Detection of b-galactosidase activity on agar plates or in microtiter trays was done essentially as recommended previously [61]. Three individual transformants per combination were grown overnight at 30°C in LB with antibiotics and 500 µg ml− 1 isopropyl β-D-1-thiogalactopyranoside (IPTG). The resulting cultures were spotted (3 µl) onto LB agar with 40 µg ml− 1 X-gal) and 500 µg ml− 1 IPTG and appropriate antibiotics. Plates were incubated at 30°C in dark conditions for 24–48 hours before photographs were taken. For quantitative assays, three co-transformants were grown overnight in liquid LB with ampicillin, kanamycin and IPTG at 30°C. OD600 was measured in a microplate reader on the next day. b-galactosidase assays were done as described [61] and OD405 was measured every 2 minutes in a FLUOstar OPTIMA Microplate Reader (BMG Labtech).
Analyses of peptidoglycan and cell wall structure
Cultures were grown in liquid MYM until OD600 reached 1.0. Mycelium was harvested by centrifugation and washed 3 times in PBS. Peptidoglycan (PG) samples were analysed following the 24 h protocol as described previously, with some modifications [62, 63]. In brief, samples were boiled in SDS 5% for 2h and sacculi were repeatedly washed with MilliQ water by ultracentrifugation (541.000 × g, 10 min, 20ºC), toughly washed, sonicated, and treated with a-amylase, DNase, RNase, and trypsin, and enzymes were inactivated by boiling. Wall teichoic acids were removed with 1M HCl. The samples were finally treated with muramidase (100 µg ml− 1) for 15 hours at 37ºC. Muramidase digestion was stopped by boiling and, coagulated proteins were removed by centrifugation (10 min, 18.800 × g ). The supernatants were first adjusted to pH 8.5-9.0 with sodium borate buffer and then sodium borohydride was added to a final concentration of 10 mg ml− 1. After reduction for 30 min at room temperature, the pH was adjusted to pH 3.5 with orthophosphoric acid.
UPLC analyses of muropeptides were performed on a Waters UPLC system (Waters Corporation, USA) equipped with an ACQUITY UPLC BEH C18 Column, 130Å, 1.7 µm, 2.1mm X 150mm (Waters, USA) and a dual wavelength absorbance detector. Elution of muropeptides was detected at 204 nm. Muropeptides were separated at 45ºC using a linear gradient from buffer A (formic acid 0.1% in water) to buffer B (formic acid 0.1% in acetonitrile) in a 30-minute run, under a 0.50 ml min− 1 flow.
Relative total PG amounts were calculated by comparison of the total intensities of the chromatograms (total area) from three biological replicas normalized to the same initial biomass and extracted with the same volumes. Quantification of muropeptides was based on their relative abundances (relative area of the corresponding peak) normalized to their molar ratio.
For analyses of overall appearance and thickness of cell walls in vegetatively growing hyphae, cultures were grown in liquid culture in MYM medium for 12 hours. Hyphae were harvested by centrifugation. The supernatant was removed and replaced with freshly prepared 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and incubated for 12 h at 7°C. After fixation the fixative solution was removed, and the pellets were washed with 0.1 M sodium cacodylate buffer (pH 7.4). The pellets were postfixed in 2% osmium tetroxide in distilled water at 7°C for 1 hour. The specimens were then dehydrated in a graded ethanol series (70% 2x10 min, 96% 2x10 min, 100% 2x15 min) and embedded in Pelco Eponate 12 resin (Ted Pella) via acetone. Ultrathin sections (50 nm) were cut with a Leica UC7 with a diamond knife. The sections were stained with uranyl acetate (2%, 30 min) and Reynolds lead citrate (3 min){Reynolds, 1963 #2657}, mounted on copper grids, and viewed with a JEOL 1400 Plus Transmission Electron Microscope at 100 kV. The thickness of the peptidoglycan cell wall layer was measured at multiple points in hyphae that appeared to have been cross-sectioned perpendicularly to the hyphal length axis.
GraphPad PRISM Software (Inc., San Diego CA, www.graphpad.com) was used for statistical analysis.
Sequence analyses
To find putative orthologues of SepIVA in the different suborders of Actinomycetales, M. tuberculosis SepIVA was used to search for homologous proteins with BLAST in genomes of each Actinomycetales suborder in the NCBI Microbial Genomes resource (https://www.ncbi.nlm.nih.gov/genome/microbes/). A few representatives of putative SepIVA homologues were selected from each suborder, and were then subjected to analysis and comparison of the genomic context with the tool webFlaGs [64]. Proteins encoded by genes from an obviously different genomic context were removed and only those originating form a locus with conserved flanking genes similar to those of known sepIVA genes were kept.
Predictions of the structure of homodimers of specific proteins were performed using Alphafold multimer via the LU-fold facility at Lund University (https://www.medicine.lu.se/research-and-research-studies/house-infrastructure/list-research-infrastructures/lu-fold). Five models were obtained per sequence and ranked according to the iptm + ptm score and the pDockQ score [65, 66]. The highest ranking models are displayed.