Three Novel Species with Peptidoglycan Cell Walls form the New Genus Lacunisphaera gen. nov. in the Family Opitutaceae of the Verrucomicrobial Subdivision 4.

fmicb-08-00202 February 10, 2017 Time: 15:47 # 1 ORIGINAL RESEARCH published: 13 February 2017 doi: 10.3389/fmicb.2017.00202 Edited by: Damien Paul Devos, Pablo de Olavide University, Spain Reviewed by: Seong Woon Roh, Korea Basic Science Institute, South Korea George Liechti, Uniformed Services University of the Health Sciences, USA *Correspondence: Christian Jogler [email protected] Mareike Jogler [email protected] Specialty section: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology Received: 28 September 2016 Accepted: 27 January 2017 Published: 13 February 2017 Citation: Rast P, Glöckner I, Boedeker C, Jeske O, Wiegand S, Reinhardt R, Schumann P, Rohde M, Spring S, Glöckner FO, Jogler C and Jogler M (2017) Three Novel Species with Peptidoglycan Cell Walls form the New Genus Lacunisphaera gen. nov. in the Family Opitutaceae of the Verrucomicrobial Subdivision 4. Front. Microbiol. 8:202. doi: 10.3389/fmicb.2017.00202 Three Novel Species with Peptidoglycan Cell Walls form the New Genus Lacunisphaera gen. nov. in the Family Opitutaceae of the Verrucomicrobial Subdivision 4 Patrick Rast1, Ines Glöckner2, Christian Boedeker1, Olga Jeske1, Sandra Wiegand1, Richard Reinhardt3, Peter Schumann4, Manfred Rohde5, Stefan Spring6, Frank O. Glöckner7, Christian Jogler1,8*and Mareike Jogler1* 1Microbial Cell Biology and Genetics, Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, 2Institute for Pharmacology, Toxicology and Clinical Pharmacy, University of Technology, Braunschweig, Germany, 3Max Planck Genome Center, Max Planck Institute for Plant Breeding Research, Köln, Germany, 4Department of Central Services, Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, 5Central Facility for Microscopy, Helmholtz Centre for Infection Research, Braunschweig, Germany, 6Department Microorganisms, Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, 7Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, Bremen, Germany, 8Department of Microbiology, Institute for Water and Wetland Research, Faculty of Science, Radboud University, Nijmegen, Netherlands The cell wall of free-living bacteria consists of peptidoglycan (PG) and is critical for maintenance of shape as dissolved solutes cause osmotic pressure and challenge cell integrity. Surprisingly, the subdivision 4 of the phylum Verrucomicrobia appears to be exceptional in this respect. Organisms of this subdivision are described to be devoid of muramic or diaminopimelic acid (DAP), usually found as components of PG in bacterial cell walls. Here we describe three novel bacterial strains from a freshwater lake, IG15T, IG16bT, and IG31T, belonging to a new genus in the subdivision 4 of Verrucomicrobia which we found to possess PG as part of their cell walls. Biochemical analysis revealed the presence of DAP not only in these novel strains, but also in Opitutus terrae PB901T, the closest described relative of strains IG15T, IG16bT, and IG31T. Furthermore, we found that nearly all genes necessary for peptidoglycan synthesis are present in genomes of subdivision 4 members, as well as in the complete genome sequence of strain IG16bT. In addition, we isolated and visualized PG-sacculi for strain IG16bT. Thus, our results challenge the concept of peptidoglycan-less free-living bacteria. Our polyphasic taxonomy approach places the novel strains in a new genus within the family Opitutaceae, for which the name Lacunisphaera gen. nov. is proposed. Strain designations for IG15T, IG16bTand IG31Tare Lacunisphaera parvula sp. nov. (=DSM 26814 =LMG 29468), L. limnophila sp. nov. (=DSM 26815 =LMG 29469) and L. anatis sp. nov. (=DSM 103142 =LMG 29578) respectively, with L. limnophila IG16bTbeing the type species of the genus. Keywords: peptidoglycan, subdivision 4, Verrucomicrobia, Lacunisphaera, ornithine Frontiers in Microbiology | www.frontiersin.org 1February 2017 | Volume 8 | Article 202 fmicb-08-00202 February 10, 2017 Time: 15:47 # 2 Rast et al. Novel Subdivision 4 Verrucomicrobia Possess Peptidoglycan INTRODUCTION In aquatic environments, abiotic factors such as salinity and temperature, but also intrinsic metabolism-related mechanisms challenge the cellular integrity of microorganisms and their ability to proliferate. Protective elements may be of a structural nature, such as S-layers, or the avoidance of osmotic stress by living in dependency of host organisms which provide stable conditions for survival (Miles, 1992;Engelhardt, 2007). Members of the class Mollicutes for example lack a peptidoglycan cell wall (Razin, 2006), are osmotically fragile and exhibit pleomorphism (Miles, 1992). Thus, they depend on an eukaryotic host to provide an osmotically stable environment for living. On the other hand, free-living bacteria usually possess cell wall structures including three dimensionally cross-linked polymeric glycan strands, interconnected by short peptide elements, a structure commonly known as peptidoglycan (PG) to protect cellular integrity. Among bacteria only few exceptions are described while all controversy discussed species belong to the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum (Wagner and Horn, 2006). In many respects, this PVC-superphylum seems to challenge our concept of the prokaryotic cell (Lee et al., 2009;Fuerst and Sagulenko, 2011; Jacquier et al., 2015;Rivas-Marín et al., 2016). In particular, the suggested absence of PG in Planctomycetes (König et al., 1984), Chlamydia (Fox et al., 1990) and subdivision 4 Verrucomicrobia (Yoon, 2011) is remarkable. While the assumed lack of PG seems to be associated with the lack of the otherwise universal bacterial cell division protein FtsZ in Planctomycetes (Pilhofer et al., 2008;Jogler et al., 2012) and Chlamydia (Stephens et al., 1998), subdivision 4 Verrucomicrobia encode the tubulin homolog FtsZ (Pilhofer et al., 2008). However, Planctomycetes were recently found to possess a PG cell wall (Jeske et al., 2015;van Teeseling et al., 2015). For Chlamydia, the existence of PG was demonstrated but a canonical PG sacculus was not isolated (Liechti et al., 2014;Packiam et al., 2015). However, for some other members of the phylum Chlamydiae a PG sacculus was identified (Pilhofer et al., 2013). Chlamydia are obligate intracellular pathogens (Jacquier et al., 2015) and thus dwell in an environment isotonic to their cytoplasm, they do not necessarily require a peptidoglycan sacculus to maintain cell shape. Accordingly, recent evidence suggests that PG forms an MreB regulated ring at mid-cell to allow cell division in pathogenic Chlamydia (Liechti et al., 2016). In contrast a typical bacterial sacculus was reported for the free-living Planctomycetes that have to withstand various osmotic challenges in their natural habitats (Jeske et al., 2015;van Teeseling et al., 2015), while free-living bacteria of the verrucomicrobial subdivision 4 are still considered to lack a PG sacculus. This bacterial group belongs to the phylum Verrucomicrobia which is divided into six so-called subdivisions. Thus far, cultured representatives are available for subdivisions 1–4. Recently for subdivision 5 the new Phylum Kiritimatiellaeota was proposed, with one characterized isolate (Spring et al., 2016). Playing a crucial role in environmental nutrient cycles, members of the Verrucomicrobia have not only been found to degrade a variety of complex polymeric compounds in, e.g., soil communities (Wang et al., 2014, 2015), some were also identified as methanotrophs (Sharp et al., 2013; van Teeseling et al., 2014). Increasing efforts to extend the knowledge about this environmentally important phylum have led to the successful isolation and description of several new species in recent years (Lee et al., 2014;Kim et al., 2015). However, the majority of new strains brought into pure culture is affiliated with subdivision 1. Therefore, the scarce data existing to date leaves inconclusive results about the suspected peptidoglycan anomaly of subdivision 4 Verrucomicrobia. Furthermore, thus far only two genomes from validly described species (Opitutus terrae and Coraliomargarita akajimensis) are available. Both genomes were not yet analyzed for PG related genes with state-of-the-art bioinformatic methods (Jeske et al., 2015). Some members of this subdivision have been found to be resistant to various β-lactam antibiotics, indicating either absence of PG or an resistance mechanism such as β-lactamases. For other strains the presence of typical cellular PG building blocks was not investigated at the time of their description (Shieh and Jean, 1998;Choo et al., 2007), leaving open the question whether peptidoglycan exists in verrucomicrobial subdivision 4. Members of this subdivision have been isolated from soil communities and leafs, while most strains originate from aquatic habitats, including freshwater lakes, marine waters and extreme habitats such as hot springs (Shieh and Jean, 1998;Chin et al., 2001;Choo et al., 2007; Yoon et al., 2007c, 2010). Here we describe the targeted isolation of subdivision 4 Verrucomicrobia, using antibiotic agents as selective markers for β-lactam resistant bacteria. Our strategy led to the successful cultivation of three novel strains from surface fresh water samples. By biochemical, microscopic and computational analysis we found that the novel and previously reported members of the verrucomicrobial subdivision 4 possess PG as part of their cell walls. Our findings challenge the proposed absence of peptidoglycan among subdivision 4 Verrucomicrobia, while at the same time extending the scarce pool of cultivated species in this environmentally important phylum. MATERIALS AND METHODS Sample Collection and Preparation Surface freshwater samples were collected in triplicates from a local pond (52◦903800 N, 10◦3204000 E, Wolfenbüttel, Germany) on August 30th, 2012 after the observation of a massive cyanobacterial blooming event. Water was collected in sterile polypropylene bottles, immediately transferred to the laboratory, homogenized and processed within 2 h. Culture Media and Bacterial Isolation Cultivation medium M1H was prepared with double distilled water containing 0.25 g/l peptone (BactoTM), 0.25 g/l yeast extract (BactoTM), 2.38 g/l HEPES (Serva), 20 ml/l mineral salt solution and a pH adjusted to 8.0 with 5 M KOH. After sterilization, the medium was complemented with 10 ml/l of a 2.5% glucose solution, 5 ml/l double concentrated vitamin solution, 1 ml/l of 100 mg/ml carbenicillin and 20 mg/ml cycloheximide stock solutions, respectively. Solid medium was Frontiers in Microbiology | www.frontiersin.org 2February 2017 | Volume 8 | Article 202 fmicb-08-00202 February 10, 2017 Time: 15:47 # 3 Rast et al. Novel Subdivision 4 Verrucomicrobia Possess Peptidoglycan prepared with three times washed 12 g/l agar (BactoTM) and cooled to 55◦C prior to the addition of heat sensitive solutions. Both, mineral salt solution and double concentrated vitamin solution were prepared according to DSMZ medium 621, while metal salts solution consisted of 250 mg/l Na–EDTA, 1095 mg/l ZnSO4.7H2O, 500 mg/l FeSO4.7H2O, 154 mg/l MnSO4.H2O, 39.5 mg/l CuSO4.7H2O, 20.3 mg/l CoCl2.6H2O, and 17.7 mg/l Na2B4O7.10H2O of which 50 ml were added per liter of mineral salt solution. For initial bacterial isolation, solid M1H medium was supplemented with 100 µl of carbenicillin stock solution (100 mg/ml), dried for 30 min and inoculated with 100 µl homogenized sample material per plate in a 10–10−2dilution series and incubated at 20◦C in the dark until colony formation became visible. Single colonies were inoculated on fresh solid medium with respective antibiotics. Pure cultures were cryopreserved in M1H medium supplemented with 50% glycerol or 5% DMSO and stored at −80◦C. Strains isolated and later identified as members of the verrucomicrobial subdivision 4 were designated IG15T, IG16bTand IG31T. Unless otherwise indicated, verrucomicrobial strains were cultivated at 28◦C to ensure reproducibility of cultivation dependent experiments. Cultivation medium of thin layer chromatography (TLC) reference strains, Bacillus subtilis DSM 10 and Escherichia coli DSM 498, was standard LB medium contained 10 g/l tryptone, 10g/l sodium chloride and 5 g/l yeast extract at pH 7.0 (Bertani, 1951). For O. terrae PB90-1Tcultivation was performed following the recommendations of the Leibniz Institute DSMZ (DSMZ medium no. 295). Molecular Identification and Phylogenetic Analysis Novel isolates were identified by direct sequencing of the 16S rRNA gene after amplification with the optimized universal primers 8f (50–AGA GTT TGA TCM TGG CTC AG–30) and 1492r (50–GGY TAC CTT GTT ACG ACT T–30) modified from (Lane, 1991). PCR reactions were performed directly on single colonies for identification or liquid cultures to check for purity, using the Taq DNA Polymerase Kit (Qiagen) with one reaction of 25 µl containing 11 µl PCR–grade H2O, 2.5 µl 10x CoralLoad buffer, 2.5 µl Q-Solution, 0.5 µl dNTPs (10 mM each), 1 µl sterile bovine serum albumin solution (20 mg/ml), 0.5 µl MgCl2 solution (25 mM), 0.125 µlTaq–Polymerase (1 U/µl) and 1 µl of each primer (10 pmol). The employed protocol consisted of two steps, the first step with an initial denaturation at 94◦C, 5 min, 10 cycles of denaturation at 94◦C, 30 s, annealing at 59◦C, 30 s, elongation at 72◦C, 1 min, followed by the second step with 20 cycles denaturation at 94◦C, 30 s, annealing at 54◦C, 30 s, elongation at 72◦C, 1 min and a final elongation step at 72◦C, 7 min. All PCRs were carried out in an Applied BiosystemsR VeritiR thermal cycler (Thermo Fisher Scientific) and PCR products were stored at 4◦C until Sanger sequencing. To generate near full length 16S sequences, additional primers (compare Supplementary Table S1) were used for sequencing and assembly of the resulting sequences was performed with the ContigExpress application of the Vector NTIR Advance 10 software (Thermo Fisher Scientific). Alignment of near full length 16S rRNA sequences was performed using the SINA web aligner (Pruesse et al., 2012), corrected manually and used for phylogenetic tree reconstruction. Tree reconstruction was performed with the ARB software package (Ludwig et al., 2004) using the Maximum Likelihood RAxML module and rate distribution model GTR GAMMA running the rapid bootstrap analysis algorithm, the Neighbor Joining tool with Felsenstein correction for DNA and Maximum Parsimony method employing the Phylip DNAPARS module. Bootstrap values for all three methods were computed with 1,000 resamplings including the E. coli 16S rRNA gene positions 101–1,371. The analysis involved 68 nucleotide sequences of described type strains and uncultured clones, related to the novel strains (compare Supplementary Table S2). 16S rRNA gene identity values of novel isolates and related type strains were calculated using neighbor joining clustering of the ARB package. Characterization of Novel Isolates Morphological, Physiological, and Biochemical Analysis Bacterial cells were immobilized on a 1% agarose–pad in MatTek 35 mm glass-bottom dishes and imaged under phase–contrast illumination using a Nikon Eclipse Ti invers microscope at 100× magnification and the Nikon DS–Ri2 camera. To determine the cell size of the novel strains, 100 individual cells of each strain were measured using the NIS-Elements software V4.3 (Nikon Instruments). For field emission scanning electron microscopy (FESEM) bacteria were fixed in 1% formaldehyde in HEPES buffer (3 mM HEPES, 0.3 mM CaCl2, 0.3 mM MgCl2, 2.7 mM sucrose, pH 6.9) for 1 h on ice and washed one time with HEPES buffer. Cover slips with a diameter of 12 mm were coated with a polyL-lysine solution (Sigma–Aldrich) for 10 min, washed in distilled water and air-dried. 50 µl of the fixed bacteria solution was placed on a cover slip and allowed to settle for 10 min. Cover slips were then fixed in 1% glutaraldehyde in TE buffer (20 mM TRIS, 1 mM EDTA, pH 6,9) for 5 min at room temperature and subsequently washed twice with TE–buffer before dehydrating in a graded series of acetone (10, 30, 50, 70, 90, and 100%) on ice for 10 min at each concentration. Samples from the 100% acetone step were brought to room temperature before placing them in fresh 100% acetone. Samples were then subjected to critical-point drying with liquid CO2(CPD 300, Leica). Dried samples were covered with a gold/palladium (80/20) film by sputter coating (SCD 500, Bal–Tec) before examination in a field emission scanning electron microscope (Zeiss Merlin) using the Everhart Thornley HESE2–detector and the inlens SE–detector in a 25:75 ratio at an acceleration voltage of 5 kV. Temperature optima of the novel isolates were determined by optical density measurements of growing cultures at 600 nm (OD600nm). Strains were inoculated 1:10 from early stationary phase cultures in glass tubes with M1H medium and incubated under constant agitation in temperature controlled shakers (for exact temperatures tested, compare Supplementary Figure S1). Frontiers in Microbiology | www.frontiersin.org 3February 2017 | Volume 8 | Article 202 fmicb-08-00202 February 10, 2017 Time: 15:47 # 4 Rast et al. Novel Subdivision 4 Verrucomicrobia Possess Peptidoglycan Measurements were performed in triplicates and each tube served as its own blank prior to inoculation. Resulting growth curves were analyzed by plotting change of OD600nm during exponential growth phases (slope values), of each individual temperature against temperature values in ◦C. To determine the pH optimum, M1H medium was buffered to pH values of 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 10.0 using 10 mM MES, HEPES, HEPPS and CHES buffers, corresponding to their individual buffer range. OD600nm was determined in glass tubes, incubated at 28◦C, with three replicates as measure of growth. Catalase activity was determined by bubble formation with fresh 3% H2O2solution. Cytochrom oxidase activity was determined using BactidentR Oxidase test stripes (Merck Millipore) following the manufacturer’s instructions. Gram properties were determined by reaction of fresh biomass with fresh 3% KOH solution (Suslow et al., 1982). Substrate utilization of the isolated strains was investigated using the Biolog GN2 MicroLogTM test panel for Gram-negative bacteria. Sterile glass tubes were prepared in duplicates with a basic medium mixture containing 15.7 ml IF-0a inoculation fluid (Biolog), 160 µl of 1 M HEPES buffer (pH 8.0) and 80 µl double concentrated vitamin solution. Tubes were inoculated with bacterial colony material from exponentially growing cultures to a turbidity of 56–68%. Two individual plates per strain were evaluated. To enable the comparison of the derived data, the data of each single experiment were normalized to 100. Only values corresponding to >25% utilization were considered as positive. The heat map graphic was obtained in the R environment (R Core Team, 2015) by using the heatmap.2() function of the gplots package. Analysis of Cellular Fatty Acids Biomass of the isolated strains was obtained from liquid cultures grown in M1H medium at 28◦C until stationary phase. The obtained biomasses were stored at −20◦C. For fatty acid analysis, 30 mg of lyophilized biomass was processed according to the standards of the Identification Service of the German Collection of Microorganisms and Cell Cultures (DSMZ) (Miller, 1982; Kuykendall et al., 1988). Determination of Molar G +C Content Strains were grown in liquid culture to stationary phase and biomass was obtained by centrifugation. For strains IG15T and IG31T, the molar G +C content was determined by the service facilities of the DSMZ. In brief, genomic DNA is isolated (Cashion et al., 1977), hydrolyzed, dephosphorylized (Mesbah et al., 1989) and analyzed by HPLC (Tamaoka and Komagata, 1984) in comparison to DNA standards from organisms with published genome sequences and a G +C content range from 43 to 72 mol%. G +C content of strain IG16bTwas determined during genome sequencing with the Pacific Bioscience sequencer. Antibiotic Susceptibility Tolerance of IG15Tand IG16bTtoward β-lactam antibiotic agents was investigated in a treatment assay using carbenicillin. Strains were inoculated as triplicates 1:10 in glass tubes with M1H medium and final concentrations of 0, 500, 1000, or 2000 mg/l carbenicillin were added. Tubes were incubated at 28◦C and growth was measured as change in optical density at 600 nm. After 120 h of incubation, cell viability was investigated by FESEM and cell numbers per ml were calculated by counting with a Neubauer chamber. Genome Sequencing of Strain IG16bT DNA Extraction and Purity Control To obtain high molecular weight DNA of strain IG16bT, nucleic acid was extracted from whole-cells using a tweaked Genomic DNA kit protocol with Genomic tips 100/G (Qiagen). The protocol was performed as recommended by the manufacturer with one exception: incubation time with digestive enzymes was prolonged to an overnight step to ensure complete lysis of bacterial cells. An aliquot of the extracted DNA was used to prepare 16S rRNA clone libraries (Zero BluntR PCR Cloning kit; Invitrogen) and resulting clones were sequenced to ensure purity of the extracted DNA. Sequencing and Gene Content Analysis De novo genome sequencing of strain IG16bTwas performed using a PacBio RS sequencer. Single molecule real-time (SMRT) bellTM libraries (Pacific Bioscience) were prepared using ∼10 µg genomic DNA. Sequencing data was processed and assembled using the SMRT analysis software. The closed and complete chromosome of strain IG16bTwas annotated using the Prokka annotation tool (Seemann, 2014) and subjected to analysis for putative genomic islands and phage regions using IslandViewer3 (Dhillon et al., 2015) and PHAST (Zhou et al., 2011), respectively. The verrucomicrobial genomes for the gene content analysis were derived from NCBI and IMG (Markowitz et al., 2012) in April 2016 and had to match the following criteria upon CheckM analysis (Parks et al., 2015): completeness >90, contamination <5 and strain heterogeneity <20. Orthologs were detected by Proteinortho (Lechner et al., 2011), a tool that identifies the reciprocal best hits from the given protein sequences. The genome plot was then generated with BRIG (Alikhan et al., 2011). Peptidoglycan Analysis Identification of Peptidopglycan Synthesis Genes and β-lactamase Protein Homologs The presence of peptidoglycan synthesis genes was analyzed using blastp (Altschul et al., 1997), while protein sequences of Phycisphaera mikurensis FYK2301M01Tor Gimesia maris 53430Tserved as query and were compared with protein sequences encoded in the genomes of O. terrae PB90-1T,C. akajimensis 04OKA010-24Tand strain IG16bT.β-lactamase encoding genes were detected in IG16, O. terrae and C. akajimensis as previosly described (Bush, 2013;Jeske et al., 2015). For both analysis, homologous proteins required an identity >30%, an e-value lower than 1e−6and a conserved domain architecture. Lysozyme Assay Susceptibility to lysozyme was investigated by incubation of the novel strains in M1H medium. Since strain IG31Tshowed no lysis after 24 h in M1H medium, osmotic stress was Frontiers in Microbiology | www.frontiersin.org 4February 2017 | Volume 8 | Article 202 fmicb-08-00202 February 10, 2017 Time: 15:47 # 5 Rast et al. Novel Subdivision 4 Verrucomicrobia Possess Peptidoglycan increased by incubation of cells in ddH2O (negative controls as well as lysozyme treated cells). Lysozyme was added to a final concentration of 10 mg/ml and cells were incubated for up to 24 h at 37◦C under constant agitation at 300 rpm. Bacterial cells were immobilized on a 1% agarose–pad in MatTek 35 mm glass–bottom dishes and imaged under phase–contrast illumination using a Nikon Eclipse Ti invers microscope at 100×magnification and the Nikon DS–Ri2 camera (Nikon Instruments). Cell viability was checked by microscopy after 1, 3, 6, and 24 h of incubation in M1H medium or ddH2O until cell lysis was observed. Biochemical Analysis of Peptidoglycan Building Blocks The presence of diaminopimelic acid (DAP) was investigated employing thin-layer chromatography and gas chromatography/mass spectrometry (GC/MS). Thin-layer chromatography of whole-cell hydrolysates of strains IG15T, IG16bT, IG31Tas well as reference strains B. subtilis DSM 10 and E. coli DSM 498 was performed as previously described (Staneck and Roberts, 1974). Novel isolates were grown in M1H medium at 28◦C to stationary phase and cells were harvested by centrifugation. B. subtilis and E. coli served as organismic controls, grown in 50 ml LB medium at 37◦C overnight and harvested by centrifugation, while a mixture of purified DAP isomers (Sigma) was used as detection standard. Whole-cell hydrolysates of strains IG15T, IG16bT, IG31T as well as of O. terrae PB90-1T(DSM 11246) were analyzed using a gas chromatography/mass spectrometry (GS/MS)-based method (Schumann, 2011), previously employed to quantify the peptidoglycan marker DAP and in addition ornithine in a new proposed Verrucomicrobia related phylum (Spring et al., 2016). In brief, cell pellets were obtained from liquid cultures (grown as described above) and biomass was lyophilized. Samples were standardized for the quantification of diagnostic diamino acids by supplementing lyophilized biomass with 2 µmol of norleucine as internal standard. The hydrolysates (200 µl 4N HCl, 100◦C, 16 h) of the samples were dried in a vacuum desiccator. Amino acids derivatized to N-heptafluorobutyryl isobutylesters and were resolved in ethyl acetate and analyzed by GC/MS (Singlequad 320, Varian; electron impact ionization, scan range 60 to 800 m/z). The DAP derivative was detected in extracted ion chromatograms using the characteristic fragment ion set 380, 324, 306, and 278 m/z at a retention time of 22.17 min. A fragment ion of 266 m/z with a retention time of 15.13 min was indicative of the presence of ornithine. Preparation of IG16bTSacculi Cells of IG16bTwere harvested from 2 l of stationary phase cultures grown in M1H medium at 28◦C, by centrifugation at room temperature following a protocol established by van Teeseling et al. (2015). In brief, cells were boiled at 100◦C for 1 h with 4% SDS, while being gently mixed by inverting the reaction tube several times in 15 min intervals. Lysates were transferred to Float-a-LyzerR dialysis tubes (SpectrumLabs, DG Breda, Netherlands) and dialyzed against deionized water in a five-liter beaker over the course of 3 days (water was exchanged two times). Dialyzed samples were stored at RT until analysis by transmission electron microscopy (TEM). Negative Staining of IG16bTSacculi Thin carbon support films were prepared by sublimation of a carbon thread onto a freshly cleaved mica surface. Lysate containing the sacculi was adsorbed onto a carbon film for 1 min and negatively stained with 1% (w/v) aqueous uranyl acetate, pH 5.0 (Valentine et al., 1968). After air-drying, samples were examined in a TEM 910 transmission electron microscope (Carl Zeiss, Oberkochen, Germany) at an acceleration voltage of 80 kV and calibrated magnifications using a line replica. Images were recorded digitally with a Slow-Scan CCD-Camera (ProScan, 1024x1024, Scheuring, Germany) with ITEM-Software (Olympus Soft Imaging Solutions, Münster, Germany). Nucleotide Sequence Accession Numbers Near full-length sequences of the 16S ribosomal RNA genes as well as the complete genome sequence of strain IG16bT were deposited with the National Center for Biotechnology Information (NCBI) and are available under KX058881 (IG15T), KX058882 (IG16bT), KX058883 (IG31T) and CP016094 (IG16bT whole genome). RESULTS Novel Species of the Verrucomicrobial Subdivision 4 Isolation and Identification Surface water samples from a local duck pond were used for the targeted isolation of novel subdivision 4 Verrucomicrobia. Given that members of subdivision 4 were thought to lack peptidoglycan, β-lactam antibiotics were used as selection pressure to enrich target bacteria. Obtained colonies of β-lactam resistant bacteria were screened by 16S rRNA gene sequencing analysis and three isolates were identified as members of the verrucomicrobial subdivision 4. Phylogenetic tree reconstruction based on near full-length 16S rRNA gene sequences (Figure 1) revealed that strains IG15T, IG16bT, and IG31Tbelong to the family of Opitutaceae, sharing 92.21, 92.39, and 92.90% sequence identity with the closest related species O. terrae PB90-1T, respectively (Table 1). Based on recent threshold values for 16S rRNA gene sequence comparison (Rosselló-Móra and Amann, 2015), the novel strains represent three distinct species that form a novel genus within the family Opitutaceae, with IG15T, IG16bT, and IG31Tbeing the type strains. Morphological, Physiological, and Biochemical Characterization of Novel Strains Cells of strains IG15T, IG16bT, and IG31Twere investigated using light microscopic and electron microscopic techniques, revealing a coccoid cell shape with cells present as monoor diplococci (Figures 2 and 3). No chain or rosette formation was observed. IG15Tcells were the smallest of the three strains in average, measuring 0.6 ±0.1 µm (diameter of single cocci Frontiers in Microbiology | www.frontiersin.org 5February 2017 | Volume 8 | Article 202 fmicb-08-00202 February 10, 2017 Time: 15:47 # 6 Rast et al. Novel Subdivision 4 Verrucomicrobia Possess Peptidoglycan FIGURE 1 | Maximum likelihood 16S rRNA gene-based phylogenetic tree. All three strains cluster within the subdivision 4 of Verrucomicrobia with Opitutus terrae as the closest representative with validly published name. Related sequences of uncultured bacteria are shown for comparison and pronounce the distinct phylogenetic position of strains IG15T, IG16bT, and IG31T. Bootstrap values based on three different tree building methods (Maximum Likelihood: ML; Neighbor Joining: NJ; Maximum Parsimony: MP). Black dots indicate support values above 70% for all three methods while gray dots show support values of more than 50% for all three methods and less than 70%, at least for one method. Branches that were not supported by all three methods show no dot. Scale bar indicates 10% estimated sequence divergence. with standard deviation; n=100 cells) while cells of IG16bT and IG31Tmeasured 0.9 ±0.2 and 0.6 ±0.1 µm in diameter, respectively (Figure 2D). In wide-field microscopy experiments, cell size variability of all three strains (compare Figure 2) became more evident than in scanning electron microscopy, where cells appeared smaller in size (compare Figure 3) due to osmotic stress during fixation. During exponential growth, cells of strain IG15T and IG31Twere highly motile, while IG16bTshowed only very few motile cells. While culture agitation was not necessary for growth, cells of strain IG15Tproduced an extracellular matrix when grown under constant agitation (90 rpm) (Figures 3A,B) with cells embedded in loose aggregates. No extracellular matrix formation was observed for strain IG16bTand IG31T (Figures 3C–F). All strains grow aerobically. Temperature and pH optima measurements revealed a mesophilic growth profile with growth temperatures from 13–38, 13–36, and 20–36◦C for strains IG15T, IG16bT, and IG31T, respectively. Optical density changes during exponential growth pointed to optimum growth temperatures of 33, 32, and 30◦C, respectively (Supplementary Figure S1). IG15Tand IG16bTwere able to grow in pH ranges from 6.0 to 9.0, with an optimum between 7.5 and 8.0. The pH optimum for strain IG31Twas not determined, since its pH growth properties are likely to be similar to strains IG15Tand IG16bT. Additionally, results of the oxidase assays were positive and determination of catalase activity showed negative results for all three strains. Strains were found to be Gram-negative by reaction with 3% KOH solution (Suslow et al., 1982). Substrate TABLE 1 | 16S rRNA gene sequence identity matrix indicating similarity between the three verrucomicrobial isolates and the next relative Opitutus terrae PB90-1. 16S rRNA gene sequence similarity [%] Opitutus terrae PB90-1T IG15TIG16bTIG31T Opitutus terrae PB90-1T 100 92.21 92.39 92.90 IG15T92.21 100 97.07 97.55 IG16bT92.39 97.07 100 97.71 IG31T92.90 97.55 97.71 100 Similarity values were calculated using neighbor joining clustering. utilization profiles of strain IG15Tand IG16bTshowed similar patterns in terms of sugar and sugar acid utilization, while strain IG31Twas clearly distinct, utilizing substrates such as glycylL-glutamic acid, L-rhamnose and succinic acid mono-methyl ester (Figure 4). Cellular fatty acid analysis identified iso-C15:0 as major component of IG15Tand IG16bTcell walls with 33.3 and 48.6%, respectively, while IG31Tonly contained 9.1% of this particular fatty acid (Supplementary Table S3). Furthermore, IG31Tpossessed iso-C14:0as major component (15.4%). Antibiotic Susceptibility of Strains IG15Tand IG16bT Antibiotic susceptibility of strains IG15Tand IG16bTtoward β-lactams was investigated by treatment with carbenicillin. Optical density (OD600nm) measurements indicated growth at Frontiers in Microbiology | www.frontiersin.org 6February 2017 | Volume 8 | Article 202 fmicb-08-00202 February 10, 2017 Time: 15:47 # 7 Rast et al. Novel Subdivision 4 Verrucomicrobia Possess Peptidoglycan FIGURE 2 | Investigation of cell morphology and size by light microscopy. The morphology and average cell size of IG15T(A), IG16bT(B), and IG31T(C) was investigated by light microscopy under phase-contrast illumination. Cells of strain IG15T(A), IG16bT(B), and IG31T(C) are of coccoid morphology and grow as monoor diplococci. Cell size was determined by measuring 100 individual cells per strain (D) and average cell size with standard deviation differed from 0.6 ±0.1 µm (IG15T), 0.9 ±0.2 µm (IG16bT) to 0.6 ±0.1 µm (IG31T). Scale bar indicates 2 µm. all tested antibiotic concentrations for both strains, as values increased over time (Supplementary Figures S2A,B). However, size measurements based on SEM micrographs revealed that treated cells of both strains were significantly increased in size when compared to untreated samples (Figure 5;p=0.0001). Furthermore, the number of cells per ml was significantly lower (about 10-fold) in treated samples (Supplementary Figure S2C; p=0.001). Thus, the increase of OD600nm was rather caused by swelling of the cells, than by multiplication after cell division. Genome Sequencing and Gene Content Analysis of IG16bT The genome of strain IG16bTwas obtained solely with single molecule real-time sequencing (PacBio). Sequencing read length was 3823 bp in average and yielded 616 mega bp of sequencing data from 6 SMRT cells with a coverage of ∼80×per base. Chromosome size was determined at 4,199,284 bp in length and bear a GC content of 66.5 mol%. Annotation with Prokka revealed the presence of 3575 coding sequences, 3 rRNA and 50 tRNA entries (Table 2). In Figure 6 the results of gene content analysis based on reciprocal blast are shown in a circular plot. Known genomes of subdivision 4 Verrucomicrobia are compared to the IG16bTchromosome, thereby revealing its unique genomic regions (Figure 6, gray boxes). Some of these regions were also predicted to be genomic islands (Figure 6, gray zones, outer rim), originating from horizontal gene transfer, and mainly hold hypothetical proteins or proteins with domains of unknown function. All predicted prophage regions (Figure 6, yellow zones, outer rim) were incomplete (Supplementary Table S4), thus no intact prophage exists in the chromosome of strain IG16bT. Peptidoglycan in the Verrucomicrobial Subdivision 4 Bioinformatic Analysis of Peptidoglycan Synthesis Genes and β-lactamase Homologs Using comparative genomics, we analyzed the genomes of strain IG16bT,O. terrae PB90-1Tand C. akajimensis 04OKA010-24T (compare Table 2) with respect to genes required for the synthesis of peptidoglycan (PG). Results of our blast-based approach led to the conclusion that all investigated organisms harbor almost all genes essential for the synthesis of PG (Supplementary Table S5). Interestingly, for the penicillin binding proteins only ftsI was identified above threshold. Gene products of murB and murC were encoded polycistronic in IG16bT,O. terrae and C. akajimensis (compare Supplementary Table S5, orange boxes) leading to the identification of the same protein when investigated with the query protein sequences for MurB and MurC. Frontiers in Microbiology | www.frontiersin.org 7February 2017 | Volume 8 | Article 202 fmicb-08-00202 February 10, 2017 Time: 15:47 # 8 Rast et al. Novel Subdivision 4 Verrucomicrobia Possess Peptidoglycan FIGURE 3 | Field emission scanning electron microscopy. Strains show coccoid morphology with organization as monoor diplococci. Micrographs of IG15Tcells (A, overview; B, close up) illustrate the formation of multicellular aggregates embedded in an extracellular matrix substance (white arrowheads). In contrast, cells of IG16bT(C, overview; D, close up) or IG31T (E, overview; F, close up) did not produce an extracellular matrix. Scale bar indicates 2 µm. Tolerance of β-lactam-derived antibiotic agents in bacteria is often related to one of several modes of resistance, including efflux or exclusion mechanisms, alterations in target proteins or the most common cause being the presence of β-lactamases to degrade the antibiotic compound (Poole, 2004). Growth of strains IG15T, IG16bT, and IG31Ton solid media supplemented with the β-lactam carbenicillin gave rise to the assumptions that these strains possess a mode of tolerance against β-lactams. Employing comparative genomics, we analyzed the presence of β-lactamase genes in the genomes of strain IG16bT,O. terrae and C. akajimensis (see Supplementary Tables S6 and S7). For IG16bTand O. terrae, three β-lactamases were identified, while for C. akajimensis no β-lactamase was found with the tested criteria. Our findings suggest that a tolerance mechanism against carbenicillin exists in strain IG16bTand is at least partially due to the presence of β-lactamases, leading to the survival of the organism until the antibiotic agent is decayed from the cultivation medium. Lysozyme Susceptibility Assay Treatment with lysozyme leads to the disruption of the cell envelope by hydrolytic cleavage of β-1,4-linkages in the peptidoglycan complex (Johnson et al., 1968). Untreated cells of strains IG15T, IG16bT, and IG31Tmaintained typical coccoid cell morphology, while all three strains displayed a loss of mobility during incubation at 37◦C (Figures 7A–C, respectively). Cells that were treated with lysozyme for up to 24 h at 37◦C in either culture medium (IG15Tand IG16bT) or ddH2O (IG31T) showed different susceptibility levels toward the lysozyme treatment. Cells of strain IG15Tshowed no lysis in M1H medium after 1, 3, or 6 h, but were lysed after 24 h of incubation (Figure 7D; white arrowheads). Cells of strain IG16bTwere destroyed after 3 h incubation in M1H medium (Figure 7E; white arrowheads). Since strain IG31Tshowed no lysis after 24 h in M1H medium, osmotic stress was increased by incubation of cells in ddH2O and cells were disrupted in ddH2O after 24 h (Figures 7D,F; white arrowheads). Biochemical Evidence for the Presence of Peptidoglycan Building Blocks First, the presence of DAP was investigated for strains IG15T, IG16bT, and IG31Tby TLC and no DL-DAP was detected. In contrast, Gram-negative and Gram-positive reference strains, E. coli DSM498 and B. subtilis DSM10, respectively, showed signals for DAP (Supplementary Figure S3), with E. coli giving only a weak signal. However, we analyzed whole-cell hydrolysates of IG15T, IG16bT, and IG31Tusing a more sensitive GC/MS method that previously revealed DAP in Planctomycetes. Despite negative results in TLC, we found the specific ion peaks, characteristic for DAP (compare Figure 8B), indicating the presence of peptidoglycan in IG15T, IG16bT, and IG31Tand O. terrae PB90-1T. The same ion peaks were previously detected for E. coli DSM 498 (Spring et al., 2016), the identical E. coli strain we here used in our TLC experiment. In addition, ornithine was detected in the whole cell hydrolysates of all three novel strains and the closest related type strain, O. terrae (Figure 8A). A quantitative estimation, based on the internal standard used, revealed that DAP and ornithine occurred in nearly equivalent, albeit low amounts in strains IG15Tand IG16bT while ornithine was the dominant substance detected for O. terrae and strain IG31T(Table 3). However, quantities of DAP for strains IG15T(7 nmol), IG16bT(6 nmol), IG31T (3 nmol) and O. terrae (4 nmol) were nearly 10-fold lower than those detected for the control E. coli strain (63 nmol), investigated in the study of Spring et al. (2016), which explains why no signal of DAP was visible in TLC experiments for strains IG15T, IG16bT, and IG31T, but a weak signal for E. coli (compare Supplementary Figure S3). Furthermore, proteins essential for DAP biosynthesis via the aminotransferase pathway are present in the genome of strain IG16bT(LysC :WP_069962807.1, WP_069963418.1; Asd: WP_069963129.1; DapA: WP_069962952.1; DapB: WP_069962953.1; DapL: WP_069960938.1; DapF: WP_069963382.1) as well as a alanine racemase (WP_069962553.1). Thus, we conclude despite negative results in TLC, that all analyzed strains contain DAP as diagnostic diamino acid of peptidoglycan. Additionally, ornithine was detected which is a part of the peptidoglycan backbone of certain gram-negative bacteria (Yanagihara et al., 1984;Spring et al., 2016). Cell Sacculi of IG16bT To give the ultimate proof that PG exists in the novel strains isolated in this study cell sacculi were extracted from strain Frontiers in Microbiology | www.frontiersin.org 8February 2017 | Volume 8 | Article 202 fmicb-08-00202 February 10, 2017 Time: 15:47 # 9 Rast et al. Novel Subdivision 4 Verrucomicrobia Possess Peptidoglycan FIGURE 4 | Heatmap illustration of substrate utilization. Substrate utilization was tested using the GN2 MicrologTM plate system. Substrate spectrum of IG15T was more similar to IG16bT, while some substrates such as succinic acid and α-cyclodextrin were almost solely degraded by IG15T. IG16bTin contrast was able to utilize D-cellobiose and α-D-lactose, distinguishing it from strains IG15Tand IG31T. The utilization pattern of IG31Twas less broad, encompassing eight of the 95 tested substrates, but included for example glycyl-L-glutamic acid, which was not utilized by IG15Tor IG16bT. Frontiers in Microbiology | www.frontiersin.org 9February 2017 | Volume 8 | Article 202 fmicb-08-00202 February 10, 2017 Time: 15:47 # 16 Rast et al. Novel Subdivision 4 Verrucomicrobia Possess Peptidoglycan toward ornithin in PG layers. FG analyzed sequencing data and was involved in genome assembly of IG16b’s genome sequence. CJ is PI and together with MJ functions as corresponding author. MJ and CJ, along with PR designed the study and helped with experimental setups and design. FUNDING This work was kindly funded by the Deutsche Forschungsgemeinschaft (JO 893/3-1). We thank Pacific Bioscience for genome sequencing. ACKNOWLEDGMENTS We thank Anja Heuer for versatile and skillful technical assistance. 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Nucleic Acids Res. 39, W347–W352. doi: 10.1093/nar/ gkr485 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2017 Rast, Glöckner, Boedeker, Jeske, Wiegand, Reinhardt, Schumann, Rohde, Spring, Glöckner, Jogler and Jogler. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 18 February 2017 | Volume 8 | Article 202