Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage-resistant M. smegmatis mutants

Cristian Alejandro Suarez; Jorgelina Judith Franceschelli; Sabrina Emilse Tasselli; Héctor Ricardo Morbidoni

doi:10.1371/journal.pone.0231881

. 2020 May 1;15(5):e0231881. doi: 10.1371/journal.pone.0231881

Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage-resistant M. smegmatis mutants

Cristian Alejandro Suarez ¹, Jorgelina Judith Franceschelli ¹, Sabrina Emilse Tasselli ¹, Héctor Ricardo Morbidoni ^1,^*

Editor: Anirudh K Singh²

PMCID: PMC7194413 PMID: 32357186

Abstract

The sequencing and bioinformatics analysis of bacteriophages infecting mycobacteria has yielded a large amount of information on their evolution, including that on their environmental propagation on other genera such as Gordonia, closely related to Mycobacterium. However, little is known on mycobacteriophages cell biology such as the nature of their receptor(s) or their replication cycle. As part of our on-going screening for novel mycobacteriophages, we herein report the isolation and genome bioinformatics analysis of Weirdo19ES, a singleton Siphoviridae temperate mycobacteriophage with a 70.19% GC content. Nucleotide and protein sequence comparison to actinobacteriophage databases revealed that Weirdo19ES shows low homology to Gordonia phage Ruthy and mycobacteriophages falling in clusters Q and G and to singleton DS6A.Weirdo19ES also displays uncommon features such as a very short Lysin A gene (with only one enzymatic domain) and two putative HNH endonucleases. Mycobacterium smegmatis mutants resistant to Weirdo19ES are cross- resistant to I3. In agreement with that phenotype, analysis of cell envelope of those mutants showed that Weirdo19ES shares receptors with the transducing mycobacteriophage I3.This singleton mycobacteriophage adds up to the uncommonness of local mycobacteriophages previously isolated by our group and helps understanding the nature of mycobacteriophage receptors.

Introduction

Bacteriophages infecting the genus Mycobacterium are to this day, the best characterized bacterial viruses with more than 1,700 genomes sequenced and roughly 12,000 phages isolated. Several reports have described the development of the bioinformatics programs needed for mycobacteriophage analysis, as well as the construction of mycobacteriophage based tools made for genetic manipulation of mycobacteria such as the recombineering method to modify mycobacterial and mycobacteriophage genomes [1, 2]. Taken collectively, the information produced through genome sequence analysis gives an ample view of the scenario of mycobacteriophage evolution. As an example, the characterization of mycobacteriophage Patience revealed a low GC content (53%) and codon usage that did not reflect those of its laboratory host, Mycobacterium smegmatis but was more related to Mycobacterium abscessus [3]. Similarly, phages infecting Gordonia species showed homology to mycobacteriophages suggesting propagation across genera [4].

Several mycobacteriophages were isolated and characterized by our group with the aim of exploring their diversity at a geographical location (South America) in which little work on that matter had been carried out [5–7]. Mycobacteriophages defining novel clusters and subclusters were detected by our group in spite of the reduced number of total isolates (less than 30) [8]. One of such phages, Weirdo19ES (initially named 19ES) was isolated in Argentina from soil samples during a previous screening. Preliminary characterization by TEM of Weirdo19ES showed that it has a Siphoviridae morphology with a length of approximately 250 nm and an icosahedral capsid of 70 nm. This phage produced small turbid plaques at 30°C and larger plaques at 37°C but failed to propagate at 42°C, behaved as temperate and failed to infect M. tuberculosis [5]. Surprisingly Weirdo19ES propagated in M. smegmatis without addition of extra calcium or magnesium cations to the growth medium, a common requirement for most mycobacteriophages [5]. Preliminary analysis of Weirdo19ES genome sequence indicated that it was a singleton (Franceschelli, J.J., personal communication). In sum, those features of this mycobacteriophage led us to pursue its full bioinformatics analysis, the characterization of its life cycle and nature of its mycobacterial receptor(s), results that are reported herein.

Materials and methods

Strains, chemicals, culture media and growth conditions

Mycobacterium smegmatis mc²155 (lab stock) and derivatives were grown at 37°C in Middlebrook 7H9 broth supplemented with 0.5% (w/v) glycerol, 10% ADS (albumin-dextrose-NaCl) and 0.2% (w/v) Tween 80 (hereafter designated as 7H9 ADS Gly Tw for short) as liquid culture medium. The same medium devoid of Tween 80 and supplemented with agar 1.5% (w/v) was as solid medium (7H9 ADS Gly agar). Mutant strain M. smegmatis MSMEG_0398::Tn61 (Myc55), -a glycopeptidolipid (GPL) deficient mutant generated through insertional transposon mutagenesis- was the generous gift of Dr. M. Daffé (Institut de Pharmacologie et de Biologie Structurale, Toulouse). Mycobacteriophages I3 and D29, used as controls throughout this study, were lab stocks. All chemicals and solvents were purchased from Sigma (St Louis, Mo) unless otherwise stated.

General bacteriophage techniques

Preparation of mycobacteriophage high titer lysates and their titer determination were done as previously published [5]; briefly, late-log phase culture of M. smegmatis mc²155 grown at 37°C were spun down and washed twice in Phage Buffer (PhB; 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 2 mM CaCl₂, 10 mM MgSO₄) and resuspended in the same buffer to the original volume. Aliquots containing the desired number of Plaque Forming Units (PFU) (usually 10⁵ PFU) of the phages under assay were mixed with 100 μl of the washed M. smegmatis cells for 30 min at room temperature, followed by addition of 4 ml of molten warm top agar (0.4% agar in 7H9 broth containing 0.5% glycerol (w/v) and 2 mM CaCl₂), gentle mixing and finally poured on top of Middlebrook 7H9 0.5% (w/v) glycerol, 2 mM CaCl₂ agar plates (hereafter designated as indicator plates, IP). After 48–72 h incubation at 37°C, phages on those plates showing confluent lysis were eluted with PhB at 4°C for 12–24 h, the liquid collected, centrifuged and the clean supernatant filter sterilized. For phage titer determination, molten warm top agar (4 ml) were mixed with 100 μl of M. smegmatis washed cells and poured onto IP plates. After hardening of the top agar, 10 μl of 1/10 dilutions of each lysate in PhB were spotted on the top agar surface, plates were dried at room temperature and incubated at 37°C for 48–72 h after which titers were calculated by visual inspection. Lysates were kept at 4°C.

The determination of the latency period was carried out as described by McNerney et al [9]; briefly, 100 μl of stationary cultures of M. smegmatis mc²155 were diluted to 10⁸ CFU /ml and infected with phage at a m.o.i ≈ 0.01. The mixtures were maintained in a water bath at 37°C and at regular time intervals following mixing (time zero), 10 μl aliquots were taken for plating on IP; after overnight incubation at 37°C the numbers of plaques produced were counted. The latent period was determined as the time at which an increase in the numbers of PFU was seen on the IP.

Lysogenization frequencies were determined by plating M. smegmatis mc²155 cells (10⁹ CFU) onto plates seeded with aliquots containing 10⁹−10¹⁰ PFU of Weirdo19ES. Clones surviving on those plates were isolated and purified; their lysogenic nature was assayed by UV induction as previously described [5]. Frequency of lysogenization was calculated as the number of clones releasing phage/number total cells in the assay.

Isolation and characterization of mycobacteriophage resistant M. smegmatis mutants

In order to obtain spontaneous mutants resistant to mycobacteriophages, 1 ml aliquots of late-log cultures of M. smegmatis were washed twice in PhB and resuspended in the same buffer to the original volume. Small aliquots (100 μl) were plated on top of 7H9 ADS Gly agar plates which had previously been seeded with 10⁹–10¹⁰ PFU/ml of mycobacteriophages I3 or Weirdo19ES; afterwards plates were incubated at 37°C for 5 days. Ten colonies arising on each plate, representing possible survivors to each mycobacteriophage, were picked and purified twice by streaking on fresh 7H9 ADS Gly agar plates. Resistance to the mycobacteriophage used as selection agent was tested by growing each colony to late-log phase in 5 ml 7H9 ADS Gly Tw, washing the cultures twice with PhB and resuspending them in the same buffer to the original volume. Each clone was challenged against the mycobacteriophage used for its selection by a cross-streaking method, followed by incubation at 37°C for 3 days. Clones that grew without disturbance after contacting the phage were purified on 7H9 Gly plates; to eliminate possible lysogens, each clone was grown again in 7H9 Gly liquid medium and 100 μl aliquots were tested by UV induction as described [5]. Those clones in which UV light induced lysis were considered lysogens and kept for future studies (not included in this report). Finally, the putative spontaneous resistant mutants were used as indicator strains against the mycobacteriophages used for their selection to confirm the resistance phenotype and also tested for cross-resistance to the other mycobacteriophages used in this study.

In order to detect differences in colony morphology, each mycobacteriophage resistant clone was plated on 7H9 ADS Gly plates containing Congo Red (100 μg ml-1) and incubated for 3–4 days at 37°C [10]. Colony morphology was assessed by visual inspection and pictures taken at a magnification of 8X with an Olympus MVX10 microscope; differences in morphotypes were recorded.

Aggregation assay

In order to determine the extent of cell-cell interaction in wild-type and mycobacteriophage resistant M. smegmatis mutants, each strain was grown to early stationary phase in 7H9 ADS Gly, followed by spontaneous sedimentation (1xg) for 10 min to separate aggregates as described [11]. The supernatants were gently transferred to new tubes and their OD₆₀₀ was measured. The cell aggregates in the sediments were broken up by briefly shaking them by vortex with 4 mm glass beads and followed by OD₆₀₀ determination. The aggregation index (AI) was calculated as the ratio between OD₆₀₀ supernatant/OD₆₀₀ sediment [11]. Control cultures were grown in 7H9 ADS Gly Tw to reduce aggregation. In each case the aggregation assay was performed in triplicate.

Sliding motility assay

The assay was carried on by spotting 10 μl of stationary phase culture of each mutant under study in the center of 7H9- 0.4% (w/v) agarose plates with no carbon source added; plates were sealed with Parafilm and incubated at 37°C for 7 days, at which time visual inspection of plates and measurement of the diameter of the halo of visible growth were carried out.

Cell envelope analysis of mycobacteriophage resistant M. smegmatis mutants

Extractable lipids were obtained from 100 ml cultures of each mutant under study grown at 150 rpm at 37°C in 7H9 Gly supplemented with dextrose (2% w/v) until saturation (usually 24 h-36 h) following the protocol described by Chiaradia et al [12]. Cultures were centrifuged at room temperature at 4500 xg and the supernatant discarded; the tubes containing the cell pellet were allowed to dry by inverting the tubes on paper, afterwards kept at 4°C until its use. Lipid extraction was performed by solvent extraction, sequentially adding methanol/chloroform mixtures at three different ratios (2:1, 1:1 and 1:2 v/v) in volumes equal to the packed cell volume [12]. Briefly, at the time of each addition cells were manually mixed and the extraction proceeded with gentle stirring for 12 h at room temperature; afterwards cell pellets were collected by centrifugation and the supernatants carefully removed. The three supernatants were afterwards combined and dried. TLC analysis of extracted lipids was done on silica gel plates using chloroform/methanol (90:10 v/v) as solvent system [12]. Elimination of the acetyl/acyl substituents of GPLs, yielding the base-stable GPLs, was performed by an alkali treatment as described by Burguiere et al [13]. Visualization of the bands of the different compounds extracted was carried out by spraying the plates with an ethanol solution of α- naphthol followed by the plates heating at 120°C for 30 min.

Molecular biology techniques

Chromosomal DNA extraction from M. smegmatis mc²155 and derived strains was performed according to Pavelka et al [14]. Amplification by PCR of MSMEG_0392 was carried out using primers MSMEG_0392Fw1 CCACGTCGTCGTCTTTCAG and MSMEG_0392Rev ACGAGTGGCGATGGACGAG under the following conditions: denaturation (94°C, 5 min), followed by 30 cycles of denaturation (94°C, 1 min), annealing (62°C, 40 sec) and extension (68°C, 90 sec); the amplification product of the expected size was gel purified and sequenced at a commercial facility.

Bioinformatics analysis

Genome sequences were analyzed using DNA Master (http://cobamide2.bio.pitt.edu/). Genes were determined using Glimmer11 and GeneMark [15–17] integrated in DNA Master program aided by visual inspection. The presence of tRNA was analyzed by Aragorn [18] and tRNA Scan SE (available at http://cobamide2.bio.pitt.edu). Probable functions were assigned when possible using BLASTP alignments to GenBank and PhageDB database (https://phagesdb.org/), HHPred [19, 20] and HMMER [21]. ClustalW [22] was used to align Lys A domains while visualization and editing of the alignments were performed using Jalview [23]. Comparative genome analyses of Weirdo19ES were performed with Phamerator [24] using our own database which was generated using the Genebank files from 54 mycobacteriophages belonging to different clusters and subclusters (S1 Table). Pairwise comparisons using ClustalW and BLASTP of gene products made by this program yield families of related sequences which are designated as “phamilies” or phams and numbered as pham 1, pham 2, etc. For our analysis we considered that two proteins belonged to the same pham if the percent identity and E-value are higher than 32,5% and lower than 1e^-50, respectively. Genome maps of each bacteriophage under comparison were generated by Phamerator; the genes are color coded automatically according to their pham, with “orphams” (those with only a single gene member) shown in white [24]. Genomic parameters (ORF density, gene length, coding percentage and GC content) were obtained through the use of Artemis version 16.0.0 [25].

Genome pair-wise comparison were performed using a Python program, dRep version 2.2.4 [26], this program compares all genomes using Mash [27] and then executes a secondary algorithm (ANImf) on the genomes having more than 90% Mash. ANImf aligns whole genome fragments and filters the alignment, then calculates the nucleotide identity of aligned regions. We set as default an ANI threshold to form primary (MASH) clusters of 90% with a Mash sketch of 50000.

A phylogenetic network of the 55 mycobacteriophages (inclusing Weirdo19ES) was performed using a fragmented alignment approach using Gegenees 3.1 [28] (50 bp fragment size and 25 bp sliding step size), the results were exported as a Nexus file that was loaded into Splitstree 4 software applying a NeighborNet [29] method. CGView comparison tool was used to perform the circular BLAST analysis [30].

The analysis of gene content dissimilarity (GCD) was performed with the Python scripts available in Github (https://github.com/tmavrich/mavrich_hatfull_nature_micro_2017) written by Mavrich and Hatfull [31]. The data used by the scripts to calculate the GCD values were obtained from our Phamerator local database described above. A distance matrix was calculated with the GCD values for each pair of phages using the DendroUPGMA web server (http://genomes.urv.cat/UPGMA/). The Newick tree of the UPGMA clustering was imported with SplitsTree 4, EqualAngle algorithm was used to construct the tree [29].

Relative synonymous codon usage (RSCU) analysis was performed as described by Esposito et al [32]. To this end RSCU values were calculated using MEGA X for each phage analyzed, and the values for stop codons, methionine and tryptophan were removed. Then a similarity matrix of Pearson coefficient correlation was obtained using the DendroUPGMA web server (http://genomes.urv.cat/UPGMA/) [33]. TreeView3 program (https://bitbucket.org/TreeView3Dev/treeview3) was used to perform a hierarchical clustering by Pearson correlation and complete linkage method; the results were represented as a heatmap. All graphics were edited using Inkscape 0.92 (www.inkscape.org). All the analyses were performed on public databases, last accessed on 06/10/2019.

Nucleotide accession number

The nucleotide sequence data of the bacteriophage Weirdo19ES has been deposited in GenBank (accession number MN103533).

Results

Weirdo19ES genome organization

The genome of Weirdo19ES spans 52581 bps, having a 70.19% GC content with a coding percentage of 96.1% and an average gene length of 568 bps which results in an ORF (open reading frame) density of 1,692 genes per kbp. No tRNA encoding genes were detected by using Aragorn and tRNA Scan SE. The analysis of the genomic sequence of Weirdo19ES predicted 89 ORFs of which more than 50% (48/89) encoded proteins of unknown function. The remaining ORFs (S2 Table) contained readily assignable functions for genes encoding structural proteins of the virion, assembly and lysis proteins, as shown in Fig 1. Interestingly, most of the genes are transcribed in the rightward direction with only a handful of genes (ORFs 33–40, 43, 44, 62 and 77) being transcribed leftward. Clear functions in this group could only be assigned to genes 43 and 44 encoding the putative phage integrase (Int) and the immunity repressor respectively (Fig 1).

Fig 1 — This map was generated by Phamerator, the colors of the gene indicate the assigned pham, orphans are shown in white. The pham numbers are above the boxes, whereas the number of the pham members are indicated between brackets.

Analysis of the left side of the genome of Weirdo19ES

The analysis of the left side of the genome of Weirdo19ES reveals genes encoding the small and large terminase subunits, followed by ORFs 3–22 encoding recognizable head, tail and assembly proteins including portal, protease, capsid, head-tail connector proteins, major tail subunit, tail assembly chaperones, tape measure protein (TMP), and minor tail protein genes. This organization is shared by many mycobacteriophages, except for ORF 13—with no known function–which is unique to Weirdo19ES.

The predicted lysis functions (ORFs 26–28) are positioned to the right of the structural genes in Weirdo19ES and include an endolysin gene (ORF26, Lys A) a putative mycolyl-arabinogalactan esterase (gene 27, Lys B) and a holin (ORF28) [34]. A detailed review of mycobacteriophage Lys A enzymes shows that there are at least 25 different module organization arrangements in Lys A (named Org-A to Org-Y) based on the presence of one or two enzymatic domains, N-terminal extensions and one of three C-terminal domains (C1-C3) [35]. In most cases, Lys A proteins contain three domains [35]. Of note, Weirdo19ES Lys A is shorter than in the majority of the mycobacteriophages analyzed until now (283 amino acid residues in length), containing only two domains, an ami2B peptidoglycan hydrolyzing domain and a C2 C-terminal domain (Fig 2A). Weirdo19ES lacks a N-terminal domain, a situation also reported in mycobacteriophages of Org-R (WildCat), Org-S (Barnyard) and Org-X (DS6A and Che9d) [35]. Interestingly, Weirdo19ES Lys A displayed good homology to that of the singleton DS6A (E-value 4e^-99), including homology at the C domain. In spite of that, Weirdo19ES Lys A clearly defines a different domain combination.

Fig 2 — A) Examples of the different LysA domain organization as represented by those for mycobacteriophages Barnyard, Wildcat, DS6A, and Weirdo19ES. B) ClustalW alignment was performed with the C2 domains of different reported Lys A enzymes. In this analysis, the C2 domain of DS6A Lys A displayed the highest homology to that of Weirdo19ES (29%). The alignment was visualized and edited by Jalview program [23].

Analysis of the central region of the genome of Weirdo19ES

Genes encoding nonstructural ORFs 33–40. Weirdo19ES genes 33 to 40 are transcribed to the left; although this arrangement of genes encoding proteins of unknown function is conserved in other mycobacteriophages such as mycobacteriophage Giles (ORFs 39–45), the placement is usually at the right of the integration cluster (integrase-repressor-excisionase) while in Weirdo19ES genome this cluster is placed to the left of the integration cassette (Fig 1). Four of the seven leftwards-transcribed genes have no close relatives in other mycobacteriophages, while three (ORF35, 36 and 38) display homology (E-value e^-11, e^-9 and e^-27, respectively) to genes present in phages Webster (which is still under analysis and has not been assigned a cluster yet) and Milly (member of the cluster K2). Milly ORF39 belongs in pham 59124 which also includes homologous ORFs from other 63 phages belonging to different clusters like K, D, O and N. Until now no function as been demonstrated for those genes in spite of their conserved presence in many phages.

Integration and immunity region

As for the majority of temperate Siphoviridae mycobacteriophages, the genes playing a role in their integration are located at the middle of the genome; in the case of Weirdo19ES ORFs 43–45 encode a tyrosine integrase, an immunity repressor and an excisionase. ORF43 has homology to members of the pham 43705 and it is related to integrases present in mycobacteriophages of cluster G1 (such as BPs, Halo, Annihilator and Avrafan); it is also closely related to the integrase of phage UmaThurman (pham 41717, having 3 members), a Gordonia infecting phage belonging to cluster CV, and a few members of cluster Y [36] (Fig 3A). A representative set of amino acid sequences of repressors (Rep) of temperate phages was used to build a Maximum Likelihood phylogenetic tree, showing that ORF44 of Weirdo19ES is more related to its homologues present in mycobacteriophages of the cluster Y. In this case, the repressors of phages of cluster G1 were more distantly to Weirdo19ES (Fig 3B).

Fig 3 — The multiple sequence alignment of the integrases and repressors were performed using MUSCLE and the evolutionary history was inferred by using Maximum Likelihood method; both analyses were conducted by MEGAX. Circular dendrograms depict the phylogenetic relationships between integrases A), and repressors B). The graphics were colored by host where M. *smegmatis*, G. *terrae* and M. *tuberculosis* phages are shown in green, blue and orange, respectively, and Weirdo19ES is shown in black.

Nucleotide search determined that Weirdo19ES attP is encoded in the repressor gene (ORF44), encompassing bp 33321–33354. A similar nucleotide sequence presumably corresponding to the mycobacterial counterpart attB, is located at MSMEG_6349, encoding a tRNA-Arg which has already been reported as the attachment site for mycobacteriophage BPs [37]. We found a cognate attB in Gordonia terrae 3612 genome, also located at a tRNA-Arg, thus suggesting that Weirdo19ES may also be able to lysogenize that species. A peculiar characteristic of the int-attP organization is the location of the putative attP core which is placed within the coding region of the repressor gene encoded by ORF44, a common trait described for mycobacteriophages Halo and BPs [38]. In agreement with the proposed function, ORF44 contains a pfam01381 motif which is associated with helix–turn–helix DNA-binding motifs and usually present in phage repressors. Finally, ORF45 (a member of the pham 39621 consisting of 54 members) is proposed to be the excisionase protein (Cro) based on its homology (although limited) to several other predicted excision proteins in databases; of those, ORF33 of mycobacteriophage Lemuria (G4 subcluster) is the closest one (e^-13).

The organization of the integration system of Weirdo19ES is similar to that reported for phages BPs, Halo and Brujita [38], in which Int and Rep are transcribed leftward and Cro rightward. The intergenic region between Rep and Cro proteins (151 bp), contains a palindromic region that could function as a regulatory motif for the expression of these genes (Fig 4A). The mechanism of integration-dependent bacteriophage immunity, which was determined in detail for the mycobacteriophages mentioned above, is based on the stability of the integrase and repressor proteins which depends in turn on the action of a host protease (ClpXP) on proteins containing a specific aminoacid tag (ssrA) [38]. The alignment of the C-termini of these proteins and the ssrA tag of M. smegmatis showed a conserved Ala residue in Weirdo19ES, which is known as critical for the action of the protease on the integrases analyzed (Fig 4B). Intriguingly the C-terminal domain of the integrases of phage BPs has a Gly residue and that of phage Bipper has a Pro residue instead of the conserved Ala while the C-term domain of the repressor of phage UmaThurman has a Pro residue instead of the Ala residue (Fig 4B). In all the cases analyzed a second Ala residue is adjacent to the first one (with the exception of phage BPs which contains a Ser residue) (Fig 4B), thus mutational analysis will be required to identify which residue is the critical one.

Fig 4 — A) Graphical representation of the organization of integration system. The integration module of the Weirdo19ES, BPs, UmaThurman and Bipper were extracted from Phamerator, where the color indicated the assigned pham. Rep protein from UmaThurman phage could not be assigned to any pham (white box). The intergenic region between *rep* and *cro* genes was analyzed and a palindromic sequence was identified as indicated by a yellow box. B) The C-terminal sequence of the repressors and integrases were aligned with the *ssr*A proteolytic tag of the M. *smegmatis*, where the conserved alanine residue is colored.

Analysis of the right side of the genome of Weirdo19ES

Finally, the right side of Weirdo19ES contains non-structural genes of which only a few could be assigned function, among them ORF66 (Ruv like resolvase) and ORF67 (WhiB-like transcriptional regulator). ORF86 is only present in the two phages forming the Z cluster, REM711 and 32HC, and in both cases the homology was low.

Free standing HNH endonucleases

The genome of Weirdo19ES contains an ORF (ORF89) which encodes a protein with a length of 114 amino acids in which a HNH endonuclease domain was predicted by InterProScan. A BlastP search of the Actinobacteriophage database (available in www.phagesdb.org) using this putative HNH shows that it is similar to gp54 of the actinophage KaiHaiDragon (E-value 4e^-16), which infects Microbacterium foliorum. This protein is member of the pham 36880, having 12 members. ORF61 is also proposed to be HNH; using HMMER it was recognized as a member of the HNHc_6 family (PF16784.59), although showing a low similarity to the family domain.

Comparative genomics of Weirdo19ES

In order to gain insight on the relationship of Weirdo19ES to other mycobacteriophages we first compared its genome using BLASTN against the Actinobacteriophage database. Phages belonging into clusters G, K, CV and DI (this last two containing Gordonia phages) showed homology to Weirdo19ES. Interestingly, the most significant alignments to Weirdo19ES were those of two singletons; Ruthy which infects Gordonia spp. and DS6A which is the only mycobacteriophage directly isolated using M. tuberculosis as host (E-value of 2e^-52 and 6e^-47, respectively). Further comparison was performed against a set of 54 phages (chosen randomly from each cluster and subcluster of phages displaying the highest homology to Weirdo19ES) belonging to mycobacteriophage subclusters G1-4, Q, K2, X, Z, 5 mycobacteriophage singletons and 17 phages that infect Gordonia spp. belonging into the clusters CV and DI. The results represented in CGView BLASTN show that there is little sequence homology between Weirdo19ES and the 54 phages under comparison (S1A Fig).

Interestingly, although displaying a very low nucleotide sequence similarity, the genes present in Weirdo19ES have homologues to phages belonging to cluster Q (11%), Z and G (8% each one). However, when the comparison was made on the basis of the encoded proteins, we detected higher homology between the phages on proteins encoded on the left half of their genomes. This result is expectable as structural proteins are encoded in that part of the phages genomes; each of those proteins very frequently have similar folds and domains dictated by conserved structures of virion particle components. Nevertheless, the proteins encoded on the right half of the genomes are much less homologous or completely unrelated (S1B Fig).

A phylogenetic network of the 54 phages mentioned above plus Weirdo19ES was built using Gegenees and Splitstree; the results obtained again located Weirdo19ES as a unique phage in a separated branch (Fig 5). A distant relationship with phages of cluster Q was observed when the area of the center of the network was enlarged and analyzed; mycobacteriophages DS6A (a singleton) and the two members of cluster Z (REM711 and 32HC) were the closest ones to Weirdo19ES (Fig 5).

Fig 5 — Gegenees was used to calculate the distance between bacteriophages and the phylogenetic network was build using the Neighbor-Net algorithm in Splitstree 4. The different clusters of bacteriophages were represented in different colors. The center of the network was zoomed in to analyze the most distant relationships.

A pair-wise comparison of all genome was performed using dRep [26], and a primary cluster was made using Mash, with ANImf used to perform a secondary, more sensitive clustering (Fig 6). This analysis also showed that Weirdo19ES is a singleton with a distant relation to bacteriophages members of the cluster Q. To confirm these results, we compared the sequence of TMPs, conserved proteins that have been used to cluster mycobacteriophages [39]; again our results showed that the Weirdo19ES TMP was close to homologues belonging to cluster Q (S2 Fig).

Fig 6 — Primary clustering was performed using the pair-wise Mash distance with a 90% of ANI (dotted line) in dRep program [22]. The different mycobacteriophages clusters were colored and singletons are shown in dotted boxes.

Finally, we sought to determine whether Weirdo19ES was originally hosted by Gordonia, switching genus barriers and beginning to infect a yet undetermined mycobacterial species by the time we isolated it. In order to accomplish that goal, we first utilized a novel measure recently developed by Hatfull´s group, termed GCD which has been applied to study how Gordonia phages are related to other actinobacteriophages by comparing phams [4]. GCD correlates shared and unshared phams, and its values range from 1 (no genes are shared) to 0 (identical gene content). Comparison of Weirdo19ES versus representatives of each cluster and subcluster under study and singleton mycobacteriophages was carried out. In this way, we obtained a representation in which mycobacteriophages from cluster Q and Z were the most similar to Weirdo19ES (average GCD = 0.74 and 0.804 respectively), while phages infecting Gordonia and belonging into cluster DI had a GCD value of 0.87 (Fig 7A). Thus this analysis did not clarify whether Weirdo19ES was originally a Gordonia infecting phage as the mycobacteriophages under analysis displayed GCD values larger or smaller than those displayed by phages infecting Gordonia. Importantly all the singleton mycobacteriophages displayed the highest level of dissimilarity indicating that the analysis was valid (Fig 7A, for the sake of clarity, a partial scale of GCD values is shown). When all the phages including Weirdo19ES were compared against each other, the results also showed that phages from cluster Q were the most similar ones as assessed by GCD values. Interestingly, Weirdo19ES and phages of cluster Q and Z were more related to Gordonia phages like Ruthy, Ghobes and members of clusters CV and DI, than to mycobacteriophages of clusters G, K2 or X (Fig 7B).

Fig 7 — A) Boxplot of the GCD values of 54 bacteriophages against Weirdo19ES. As shown in picture, cluster Q are sharing more genes with Weirdo19ES, represented by the lower values of GCD. B) EqualAngle algorithm was used to build a phylogenetic network in Splitstree 4 with the GCD values of all bacteriophages in the analysis. The different clusters were colored whereas singletons are in dotted boxes.

A different approach to assess the role of Gordonia as a host for Weirdo19ES was undertaken by analyzing the RSCU which measures the relative frequency of codon usage in bacterial hosts and bacteriophages [8]. As shown in Fig 8, in the resulting dendrogram (left side of the heatmap) all phages analyzed are assigned to four different clusters (1 to 4) as defined by RSCU. Weirdo19ES is a member of cluster 3, in close relation with the cluster K2 mycobacteriophages ZoeJ and Dismalfunk. Bacteriophages members of the cluster DI of Gordonia as well as a singleton were positioned on a separate branch on the same cluster. Interestingly, two phages previously isolated by our group (Jolie2 and 32HC, belonging into cluster G4 and cluster Z respectively), also clustered with Weirdo19ES, in spite of having phylogenetically distant genomes.

Fig 8 — RSCU values obtained by MEGA X where used to construct a similarity matrix. Hierarchical clustering and heatmap representation was performed in TreeView3 program. The four main clusters are depicted in the dendrogram at the left of the heatmap.

Biology cycle of Weirdo19ES

To add information of mycobacteriophage Weirdo19ES, we determined some of its biological features such as infection cycle parameters and phage-bacterial interactions (see Materials and methods). Our analysis showed that Weirdo19ES was able to lysogenize the mycobacterial host at a frequency of 12% (an average of three independent experiments) which is intermediate between the frequency displayed by L5 (22%) or Giles (2%) as previously reported [2]. This singleton also had an unusual lytic cycle characterized by an extremely long latency time of ≈300 min which largely exceeds those reported for other mycobacteriophages such as Kampy which showed a latency time of 100 min [40] or D29 (100–120 min) [41, 42].

There are very few reports on the biology behind M. smegmatis resistance to mycobacteriophages or the nature of receptors for mycobacteriophages, being the mutants resistant to the transducing phage I3 the best characterized till now [43, 44]. In order to gain knowledge on the subject we obtained several spontaneous M. smegmatis mutants resistant to Weirdo19ES (see Materials and methods), one of which, designated 1R-Weirdo19ES, was kept for further analysis. Simultaneously we isolated spontaneous M. smegmatis mutants resistant to mycobacteriophage I3 following the same protocols, keeping one clone which we designated as 3R-I3. Importantly, successive passages on fresh medium showed that morphotypes were stable although revertants with colony morphology similar to the parental strain arose with a frequency of 10⁻⁶–10⁻⁷. In order to characterize those phage resistant mutants, aliquots of each one of them, along with the parental strain were plated onto solid medium in the presence of the hydrophobic dye Congo Red; observation of the plates at low magnification revealed that I3 and Weirdo19ES M. smegmatis resistant mutants displayed a rough colony phenotype being approximately half the size of that of the wild-type M. smegmatis mc²155 (Fig 9 panel A). M. smegmatis Myc55, a Tn611 transposon insertional mutant affecting GPL synthesis [45] that was added as control, also displayed a highly similar colony morphology in agreement with what was described in the literature (Fig 9 Panel A). The behavior of the phage resistant mutants on sliding motility was also tested finding that while the parental strain displayed the well-known growth pattern the three mutants (1R-Weirdo19ES, 3R-I3 and Myc55) failed to move from the point where the cultures were spotted onto the plate (Fig 9 Panel B). In concordance with the loss of the sliding motility, the aggregation index showed that the three mutants were much more prone to aggregation (4–5 fold) than the wild-type strain (Fig 9).

Fig 9 — The mutant strains (1R-Weirdo19ES, resistant to mycobacteriophage Weirdo19ES; 3R-I3, resistant to mycobacteriophage I3 or Myc 55 share colony morphology (Panel A), sliding motility behavior (Panel B) and cell aggregation (Panel C). Panel A: Aliquots containing 10² CFU of each strain were spread onto solid medium plates containing Congo Red (100 μg ml-1). Growth was examined under microscope at 8X magnification after 3 days of incubation at 37°C. Pictures are representative of ten fields observed for each plate. Panel B: 5μl drops of a stationary culture of each strain were placed in the center of 0.3% (w/v) agar plates, the plates were sealed with Parafilm and incubated for 7 days at 37°C. Panel C: Aggregation Index for each strain was determined in the absence or presence of 0.5% (w/w) Tween 80 as described in Materials and Methods.

Determination of the efficiency of plating of each mycobacteriophage against the corresponding resistant mutant revealed a decrease in the efficiency of plating of 10⁵-fold or more confirming the resistance phenotype (Fig 10). Importantly, I3 failed to propagate normally in M. smegmatis mutant 1R-Weirdo19ES; conversely Weirdo19ES propagated at very low efficiency of plating in 3R-I3 (Fig 9). A similar behavior was displayed by M. smegmatis Myc55 and thus the three strains showed cross-resistance to both Weirdo19ES and I3; opposite to that, D29 -used as a control phage- propagated with no substantial differences in the wild-type and phage resistant strains (Fig 10).

Fig 10 — Aliquots (5 μl) of ten-fold dilutions of Weirdo19ES, I3 or D29 (used as control) were spotted on Indicator Plates containing either the wild-type strain, 1R-19WeirdoES, 3R-I3 or Myc 55 on solid medium. Lysis was determined after 24–48 h of incubation at 30°C.

Colony morphology of 1R-Weirdo19ES and 3R-I3 M. smegmatis resistant mutants suggested cell envelope alterations, hypothesis supported by the colony size and morphology shared by these two mutants and Myc55. A previous report on transposon insertions causing resistance to I3 and yielding a rough colony phenotype and loss of GPL synthesis led us to extract and analyze cell envelope lipids of 1R-Weirdo19ES as described in Materials and Methods [12, 45]. Extraction and chromatographic analysis of lipids of the cell envelope of M. smegmatis mc²155, 1R-Weirdo19ES, 3R-I3 and Myc55 supported our hypothesis as no polar GPLs were discernible by TLC analysis in the three mutants while the parental strain displayed the normal content of polar and apolar lipids (Fig 11). Using Tn5730 insertional mutagenesis Chen and co-workers isolated M. smegmatis I3 resistant mutants which upon analysis showed insertions in four genes [43]. We selected one of them, MSMEG_0392, encoding the glycosyltransferase gtf, as an initial candidate possibly underlying the phenotype of 1R-Weirdo19ES. PCR amplification and sequencing of this gene showed a single bp insertion at position 575 of the gene, generating a frameshift that created a premature protein termination. Thus, although the involvement of other genes was not addressed (see Discussion), MSMEG_0392 is affected in independent mutants resistant to phages I3 or Weirdo19ES, giving support to our hypothesis and being consistent with GPL analysis.

Fig 11 — Extractable lipids from strains M. *smegmatis* mc²155, 1R –Weirdo19ES, 3R-I3 and Myc55 were loaded and separated by HPLTC using chloroform: methanol (9:1 v/v) as mobile phase followed by detection using α-naphtol and charring (Panel A). The extracted lipids were also deacylated by mild alkali treatment as described by Burguiere et al. [13], separated on HPTLC plates using chloroform/methanol/water (90:10:1 v/v) and revealed as above; the major GLP species (1–4) are indicated (Panel B). In both panels the origin of the solvent run is shown by an arrow, lanes 1 through 4 indicate the lipids extracted from M. *smegmatis* mc²155, 1R-19WeirdoES, 3R-I3 and Myc 55 respectively.

In conclusion, Weirdo19ES uses one or more of the M. smegmatis GPLs as a receptor, as proven by the defect in GPL synthesis in mutants resistant to this phage.

Discussion

Weirdo19ES is a singleton mycobacteriophage

There are currently over 1,700 mycobacteriophage genomes that have been sequenced and analyzed (information compiled in www.phagesdb.org), puzzlingly, among those phages, only ten are singletons according to the latest actinobacteriophage database release (last accessed Nov. 1, 2019). We report herein the characterization of another singleton, Weirdo19ES, our first one among a small handful (less than 30) of phages isolated and studied in Argentina [5, 6]. Of note, Weirdo19ES was isolated in 2009 and since then still remains a singleton in spite of the large number of mycobacteriophages that were sequenced since then. This mycobacteriophage has very low nucleotide and protein homology to other actinobacteriophages as judged by BLASTN and BLASTP and graphically depicted in S1 Fig. The analysis of GCD showed that all the singleton phages reported and of Weirdo19ES have very little content of common genes when compared to mycobacterial and Gordonia phages in clusters, moreover most of the singletons differ in gene content among themselves (Fig 7A and 7B).

Weirdo19ES has several very interesting features when a specific protein or set of proteins is compared to the homologues present in other actinobacteriophages, one of such features is TMP, the protein that controls the length of the phage tail. Weirdo19ES TMP is related to TMPs of several mycobacteriophages belonging to cluster Q -of which mycobacteriophage Giles is the one more thoroughly studied [2, 46, 47], mycobacteriophage Gaia (one of the two currently known cluster X mycobacteriophages), the singletons MooMoo and Eyre, and cluster Z mycobacteriophages Rem711 and 32HC. However, while all the cluster Q phages have very similar TMPs suggesting a close evolution, Weirdo19ES, as well as the remaining phages mentioned above, is more distantly related. This is more compelling when looking at TMPs from phages Weirdo19ES, Gaia and MooMoo (S2 Fig). Due to that relatedness to singletons or phages infecting G. terrae it is possible that Weirdo19ES has been circulating between Gordonia and mycobacteria although other hosts cannot be excluded.

Recently, Payne and Hatfull analyzed 224 Lys A enzymes encoded by sequenced mycobacteriophages, pointing out the remarkable diversity of those enzymes both in size and in the encoded activities [35]. Most of the Lys A enzymes analyzed in that report contain three conserved domains, namely a C-terminal domain that is possibly involved in the binding to the mycobacterial cell wall, a central domain that contains a sequence motif associated with peptidoglycan hydrolysis, and a peptidase encoding N-terminal domain [34]. The central domain typically consists of amidases (Ami), muramidases or transglycosylhydrolases (GH) which can cleave different linkages in the peptidoglycan structure [35]. The domains encoding the amidases can be grouped in two families, Ami-2A and Ami-2B according to sequence similarities [35]. Importantly, only the singletons Weirdo19ES, DS6A and the V cluster phage Wildcat lack the N terminal domain encoded peptidase. In both DS6A and Wildcat Ami-2B and GH19 domains are present however Weirdo19ES contains only a central Ami2-B domain (Fig 2). Thus, although several combinations of domains are present in the mycobacteriophage endolysins analyzed by Payne and Hatfull [35], Lys A from Weirdo19ES is still an oddity. There are very few reports on the specific activity of Lys A in mycobacteriophages [42], thus, considering the extremely long latency time (300 min) displayed by Weirdo19ES, a possibility worth exploring is its correlation to a less than optimal Lys A activity.

A set of 8 leftward transcribed genes (ORFs 33 to 40) is also of interest, in first place in the cases of phages where such a set is found -such as mycobacteriophage Giles [48], it is located at the right side of the integration cassette while in Weirdo19ES it is to the left as is also located in phage Tweety. In second place, in the case of Giles, the set is formed by ten genes transcribed leftward, spanning form ORF38 to ORF47: of those 7 are “orphams”. The analysis of essentiality of those genes in phage Giles indicated that only ORF40 and ORF42 encoded essential proteins that were also strictly required for obtaining lysogens while ORF41, although not essential, reduced lysogenization in 70% [2]. Importantly deletion of ORF47 (Cro repressor) reduced the number of phages per plaque as well as abrogated the lysogenization. RNA-seq studies showed a low level of expression of the leftward transcribed genes (with the exception of ORF47 during the lysogenic state) regardless of which phage replication stage (early, late or lysogeny) was analyzed [2]. Thus although we cannot assign roles to the Weirdo19ES leftward transcribed set of genes, it is reasonable to postulate that they may perform roles similar to those of phage Giles.

Temperate bacteriophages encode transcription repressors required to maintain lysogeny and confer superinfection immunity, thus regulation of those repressors is crucial for the choice of lysogenic or lytic growth. Temperate mycobacteriophages contain two types of integrases classified depending on their active site, of which tyrosine integrases (Y-Int) are favored against Serine integrases (S-Int). In recent years a new subclass of Y- Ints has been described which lacks an N-terminal arm-type binding domain [38]; a remarkable feature of those systems is that the attP site is located within the gene encoding the phage repressor, such that integration results in loss of the 3′ end of the gene and removal of a C-terminal degradation tag that would otherwise inactivate the virally-encoded form of the repressor [38]. Mycobacteriophage Brujita, that contains such a Y-Int enzyme, has recently been characterized in detail as a simple system in which the rate of integration and excision could be dictated by Int instability or concentration [49]. The lack of N-terminal Int domains, arm-type sites, and recombination directionality factors make the integration system of Brujita very simple [49]. In a similar way, our analysis of mycobacteriophage Weirdo19ES revealed the presence of a Y- Int encoding gene (ORF43) with homology to that of phage Brujita (Fig 3); however, a detailed inspection of the relationships between the Y-Int available in the databases indicates that Weirdo19ES Int is related to integrases of phages belonging to cluster G as well as to the one present in Gordonia phage UmaThurman (Fig 3), all of which are included in pham 41717, while Brujita´s Int belongs to pham 44980.

M. smegmatis resistance to Weirdo19ES highlights the role of gycopeptidolipids as a key mycobacteriophage receptor

Early work from different groups contributed information on the chemical nature of possible mycobacteriophage receptors extracted by solvent treatment of mycobacterial cells; this fraction was composed of amino acids, sugars modified by oxalic acid or pyruvic acid and fatty acids and was capable of interfering with the adsorption of a phage on Mycobacterium phlei [50]. Thus, it was clear on the basis of the interference of bacteriophage adsorption to mycobacterial cells that one or more GPL species are able to act as part of the bacteriophage receptor. In spite of that, it was only in 2009 that specific genes were linked to resistance to mycobacteriophage I3 through transposon insertions leading to the lack of production of GPLs [43]. The insertions yielded four I3 resistant clones and were mapped into genes MSMEG_0392 and MSMEG_0399-MSMEG_0401 encoding a glycosyltransferase (gtf2), a MbtH-like protein, and two Non-ribosomal peptide synthases 1 and 2 (mps1 and mps2) respectively. Although colony morphology alterations were not reported by the authors, Myc55, carrying a Tn611 insertion in a polyketide synthase gene (MSMEG_0398, pks) showed the same rough colony morphology and loss of GPLs [45]. In our hands all the isolated M. smegmatis clones spontaneously resistant to Weirdo19ES displayed the same rough colony morphology as strain Myc55 and M. smegmatis I3 resistant mutants; additionally, the three strains displayed cross- resistance to I3 and Weirdo19ES. Moreover, the genetic lesion in 1R-Weirdo19ES affected MSMEG_0392, in agreement with its loss leading to resistance to I3; finally the GPL profile on TLC plates correlated with those genetic defects.

As a whole, our isolation genome annotation and biological characterization of the singleton mycobacteriophage Weirdo19ES adds new information to the field and opens up new possibilities of research on the role of the mycobacterial GPLs as receptors for mycobacteriophages.

Supporting information

S1 Fig. Comparative analysis using CGView comparison tools.

Circular maps comparing Weirdo19ES with a set of bacteriophages of different clusters infecting either Gordonia or Mycobacterium hosts are displayed. The outermost ring corresponds to the reference genome and the internal rings shows the regions of BLASTN and BLASTP homology, A and B, respectively. The inner ring displays the GC content of Weirdo19ES.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

S2 Fig. Phylogenetic analysis of the tape measure proteins.

The amino acid sequences of the TMPs were aligned with MUSCLE and the dendrogram was constructed using Maximum Likelihood method in MEGAX: mycobacteriophage clusters are indicated in colored boxes.

(TIF)

Click here for additional data file.^{(424.1KB, tif)}

S1 Table. List of mycobacteriophages used throughout this study.

Corresponding accession numbers, host and clusters classification are listed.

(DOCX)

Click here for additional data file.^{(16.4KB, docx)}

S2 Table. List of predicted gene of mycobacteriophage Weirdo19ES.

The function were predicted by BLASTP analysis using phagedb database. ⁽¹⁾ The highest homologue to each gene product are listed together with the phage cluster and BLASTP E-value ⁽²⁾.

(DOCX)

Click here for additional data file.^{(19.6KB, docx)}

Acknowledgments

We gratefully acknowledge the assistance of Dr. Enrique Morales in microscopy and imaging. We also acknowledge the use of the Microscopy facility at IBR (Instituto de Biología Molecular y Celular de Rosario), Argentina. HRM acknowledges Mr. A. Fornasari for secretarial assistance.

Data Availability

We declare that the data that support the findings reported in this manuscript are available in NCBI at https://www.ncbi.nlm.nih.gov/, Accesion number MN103533. These data were derived from the following resources available in the public domain: The Actinobacteriophage database at https://phagesdb.org/phages/Webster/.

Funding Statement

HRM has received grant ANPCyT PICT 3795 (MinCyT, Argentina) https://www.argentina.gob.ar/ciencia/agencia/fondo-para-la-investigacion-cientifica-y-tecnologica-foncyt The funder played no role in the study, the preparation of the article or the decision to publish.

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PLoS One. doi: 10.1371/journal.pone.0231881.r001

Decision Letter 0

Anirudh K Singh

16 Jan 2020

PONE-D-19-31407

Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage resistant Mycobacterium smegmatis mutants

PLOS ONE

Dear Dr Morbidoni,

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Reviewer #1: Partly

Reviewer #2: Yes

**********

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Reviewer #1: N/A

Reviewer #2: N/A

**********

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

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Reviewer #1: Dear authors,

Please find detailed review comments for the manuscript in attached folder. It need to be rewritten for better understanding of audience with correction in english. The approach is good but scientific finding need to be stated clearly. Figures need to be improved, its hard to read or visualise.

Reviewer #2: In this manuscript titled “Weirdo19ES is a ……………………………Mycobacterium smegmatis mutants” Suarez and co-workers describe the genomic features of a singleton Mycobacteriophage Weirdo19ES by using various bioinformatics tools. They further described the possible receptor for this phage on M. smegmatis surface and demonstrated that cell surface polar GLPs act as receptor. I must admit that authors have done a very thorough analysis of this new mycobacteriophage and their study is well designed and executed. However, there are few issues with the manuscripts;

Major issues:

1. While the M. smegmatis GLP deficient mutants are resistant to Weirdo19ES the experiments performed here do not establish direct interaction of Phage to GLPs on bacterial cells. It will be advisable to do an experiment where Phage is adsorbed on purified GLPs before being used in the plaque assay. In the absence of sufficient quantity of purified GLPs same experiment can be performed using heat killed 1R-Weirdo19ES and wild type MC2155.

2. The manuscript needs careful proofreading for correcting grammatical and typographical mistakes. I have highlighted some of the examples in the minor issues section in the attachment. However, this is not the comprehensive list of language related mistakes. I strongly encourage the authors to have the manuscript proofread for language from their English proficient colleagues.

Minor issues:

Please refer to the attachment.

**********

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Reviewer #1: Yes: Vandana Singh

Reviewer #2: No

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Attachment

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PLoS One. 2020 May 1;15(5):e0231881. doi: 10.1371/journal.pone.0231881.r002

Author response to Decision Letter 0

10 Mar 2020

PONE-D-19-31407

Reply to Reviewer 1

Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage resistant Mycobacterium smegmatis mutants

This is a nice attempt by Cristian Alejandro Suarez and coauthors to characterize singleton mycobacteriophage that selects for glycolipid deficient phage resistant Mycobacterium smegmatis mutants. The authors have compiled large set of bioinformatics data for mycobacteriophage. This characterized mycobacteriophage Weirdo19ES is adding new information and opening research possibilities on the role of mycobacterial GPLs as receptors. On through review of the manuscript, I found several errors which need to be corrected before acceptance for publication in the journal.

Comments:

� The english is very poor, and grammatically incorrect. It’s hard to understand the statements made by authors, as those are too long and complex. Please be consistent in using nomenclature (Use Weirdo19ES or 19WeirdoES, similarly Maximum Likelyhood method or Maximum Likelihood method). Through rewriting with grammar corrections is required for possible acceptance.

Authors reply: We deeply apologize as the reviewer was right, there were many mistakes that went undetected. We have improved the writing by making shorter sentences where possible. We have corrected the discrepancies on the nomenclature.

• Figures are with compromised resolutions, hardly readable and difficult to understand the message. Fonts and figures are not appropriate for publication.

Authors reply: Regarding resolutions, all figures have passed the test requested by the journal submission platform (PACE), and after careful review, we found that they have the fonts required by the Journal. However, we do agree that due to the amount of information some figures may be hard to read. Several of the figures presented in this manuscript are similar (due to the common bioinformatics programs used) to others we previously published in this journal. Comparable figures are often presented by other research groups working on mycobacteriophages. We will address the nature of the problems with fonts and resolution with staff members of PlosOne if needed.

� In line 226, 70,19% GC content need to be corrected with 70.19%. Similarly, in line 227, 96,1% should be 96.1%.

Authors’ reply: We apologize for the mistake which has been corrected.

� Analysis and homology of the LysA needs to be revalidated. In Fig 2A, both Che9C and DS6A are similar in domain organization, as they belong to same group Org-X. But in Fig. 2B, homology has been studied only with DS6A and other genomes? Why not Che9C is included in this study? If it was included, what is the homology with Weirdo19ES? If it has homology, then why not to say that Weirdo19ES is similar to Org-X group. In Fig. 2B, it looks like SG4 and some other genomes also have homology with Weirdo19ES. Please include these other homology.

Authors’ reply: We apologyze as we included Che9c instead of Che9d by mistake. That point has been fixed. Fig 2A represents a depiction of some of the most frequent domain combinations found in mycobacteriophage Lys A enzymes. Homology analysis shown in Fig 2B is made on the C-end of those Lys A enzymes that showed highest homology to Weirdo19ES Lys A C-terminus. The reviewer mentions that other genomes have homology to Weirdo19ES however we were only comparing a fragment of LysA and not whole genomes.

� In line 269, what is the significance of “Nonstructural genes ORFs 33-40”?

Authors’ reply: This line has been corrected to ensure proper understanding. The sentence refers to genes encoding ORFs that do not bear homology to those playing structural roles in virion structure. The designation is widely used and we feel that we are using the appropriate nomenclature.

� In genome organization (line 234), ORF 33-40, 43, 44, 62 and 77 are mentioned to be transcribed leftward. There is no information about gene 45 in this part, but in integration and immunity region (line 283), gene 45 is included as a part of ORF 43-45. What is the orientation of gene 45, it is leftward or rightward?

Author’s reply: As mentioned in the text and in this revised versions genes 43 and 44 are oriented leftwards and gene 45 rightward.

� Statement in line 235 and 236 is that gene 43 (int) and gene 44 (rep) are transcribed leftward, while in line 314, 315 and 316, statement tells that int and rep are transcribed rightward. This kind of statements are due to shear negligence and must be taken care prior submission.

Authors reply: Fixed, the correct orientation is shown also in Fig 1 originated through Phamerator.

In line 285, phages BPs, Halo, Annihilator and Avrafan are mentioned having relationship with Weirdo19ES, but in Fig.3A, Annihilator is missing.

Authors reply: Mycobacteriophages of G1 cluster are numerous and display extensive homology. We have chosen a few of them to illustrate the analysis of integrase and excisionase genes. Once again, the authors would like to mention that a set of phages were included in each analysis to avoid a heavy crowding of sequences and names in the figures. In each case, the set of phages was randomly chosen but representative of a major group displaying homology at the level of genomes or any specific gene under analysis.

� In Fig. 3A on the basis of integrase, homology is reported with BPs, Halo, Annihilator, Avrafan, UmaThurman, cluster CVs and Y. In Fig. 4, only BPs, UmaThurman and Bipper are included for intervening region and ssrA tag study. What was the basis to select these members only?

Authors’ reply: Only one phage from each cluster was picked on the basis of the highest homology for the integrase or the repressor to those of phage Weirdo19ES. In the case of repressor analysis, that of phage UmaThurman was included in spite of being the less homologous one to those of phage Weirdo19ES as it is a Gordonia infecting phage and was included in the previous analysis of integrases.

� Similarly, on the basis of repressor, homology/ similarity was with cluster Y, not with G1. Bipper is from cluster Y, but in Fig.4B, BPs also have that conserved alanine. What is the conclusion of this observation?

Authors’ reply: For the analysis of repressor and integrases we picked only one phage of each cluster displaying the highest homology to repressor and integrase of Weirdo19ES. It is not clear to us what is the question raised by the reviewer.

� In Fig. 4B, colour of highlight is not visible.

Authors’ reply: Fixed

� Heading in line no 334 has error.

� In line 340, ORF 89 is predicted to be HNH using InterProScan. In line 345, ORF 61 is proposed to be HNH, using HMMER. It is not clear that both ORFs (89 and 61) are HNH? Why authors used two different platforms and predicted two different HNH.

Authors´ reply: The use of more than one program to pinpoint specific features, in this case HNHs domains, helps to increase the possibility of detection. Nonetheless, the confirmation of the identity of HNHs will require biological confirmation.

� In comparative genomics study, why group Y (significantly similar in repressor based study) was not included and why K2, Q, D1, G2, G3 and G4 were included as these were not included or mentioned in any other study of the article.

Authors’ reply: Comparative genomics has been carried out with the closest related whole bacteriophage genomes while Rep studies were carried out only based on those temperate phages displaying homology to ours.

� In line 411, authors stated that Weirdo19ES, Q and Z are more related to CV & D1. But what about intermediate values of G1, G4, G2, G3?

According to authors G is distant, it is surprising as it has lower values than CV.

Authors’ reply: Fig 7A displays the GCD of each cluster of phages vs Weirdo19ES while Fig 7B shows GCD contents of all vs all phages (including Weirdo19ES). We looked into GCD content of mycobacteriophages and phages infecting Gordonia in an attempt to find out whether Weirdo19ES would be more related to Gordonia, however, as GCD values for Gordonia phages or mycobacteriophages are not clustered when compared to Weirdo19ES (Fig 7A) it is not possible identify a whether our phage was originally infecting either Gordonia or mycobacteria.

So, intermediate values of other G subclusters are not relevant for our purpose.

� In line 465, it is 102 CFU or 102 CFU?

Authors’ reply: 102. The mistake has been corrected

� Legend of Fig.11 says GLP, while Figure denotes GPL.

Authors´reply: Fixed

� Pictures are not clear in Fig.11 A and B. What was the loading control?

Authors’ reply: Fig. 11 is a typical picture of lipid bands after charring, blurring is unavoidable. If by loading control the reviewer refers to mass, comparable volumes of lipid extracts obtained from comparable culture cell pellets were loaded, thus all lanes should be comparable in terms of contents.

� Conclusion portion must be summarised.

Authors’ reply: We have rewritten most of the text trying to be concise but not at the expense of a reasonable presentation and discussion of results. We hope this new manuscript will be acceptable for the Reviewers.

Reply to Reviewer 2

In this manuscript titled “Weirdo19ES is a ……………………………Mycobacterium smegmatis mutants” Suarez and co-workers describe the genomic features of a singleton Mycobacteriophage Weirdo19ES by using various bioinformatics tools. They further described the possible receptor for this phage on M. smegmatis surface and demonstrated that cell surface polar GLPs act as receptor. I must admit that authors have done a very thorough analysis of this new mycobacteriophage and their study is well designed and executed. However, there are few issues with the manuscripts;

Major issues:

1. While the M. smegmatis GLP deficient mutants are resistant to Weirdo19ES the experiments performed here do not establish direct interaction of Phage to GPLs on bacterial cells. It will be advisable to do an experiment where Phage is adsorbed on purified GLPs before being used in the plaque assay. In the absence of sufficient quantity of purified GLPs same experiment can be performed using heat killed 1R-Weirdo19ES and wild type MC2155.

Authors’ reply: Although the reviewer raises a reasonable point, the fact that mutants deficient in GPLs both by transposon insertion (and thus bearing genetically identified lesions) and spontaneous resistant mutants, become resistant to the phages under analysis strongly suggest that GPLs are part of the natural receptors for the phages. However, as commented in the Discussion section, an approach comparable to that suggested by the reviewer has been undertaken at early times when there were no tools available for genetic manipulation of mycobacteria. Since this is a microbiology lab we were able to show differences in GPL contents in wild-type and phage resistant mutants, however, GPL extraction and species purification in amounts enough to perform the requested experiment is not within our capabilities. GPLs are the outermost components of the mycobacterial cell envelope and thus the first logical choice to be a receptor.

The use of heat killed phage resistant cells will not show any interference with phage binding to the wild type strain. Conversely, heat killed wild-type cells may interfere. However, cell envelope components (mainly GPLs and fatty acids) of M. smegmatis are usually shed to the culture medium specially when Tween is used to achieve smooth growth. Tween inhibits phage adsorption, thus we should need to wash cells and with that loose possible phage receptors in the wash step. Thus, although we do appreciate the insightful suggestion, we preferred to use a genetic approach as shown in the manuscript.

2-The manuscript needs careful proofreading for correcting grammatical and typographical mistakes. I have highlighted some of the examples in the minor issues section below. However, this is not the comprehensive list of language related mistakes. I strongly encourage the authors to have the manuscript proofread for language from their English proficient colleagues.

Authors reply: We deeply apologize, there were a number of grammar mistakes and typos that went undetected. The manuscript has been thoroughly checked by the senior author and an English public translator. An intriguing issue is the fact that figures were processed according to PlosOne specific guidelines using the PACE software and they were approved before uploading the manuscript. If the problem persists we will ask the editorial staff to help us out.

We hope this version will be acceptable and again thank you for your suggestions

Minor issues:

Please remember this is a small list of typographical and grammatical mistakes and the entire manuscript needs to be carefully read and mistakes need to be corrected.

1. Throughout the manuscript punctuations after the reference are added after space, remove them.

2. At several places a hyphen (-) is added before a word for no reason, remove them.

3. At some places a comma (,) is used instead of the decimal (.)

4. Line 41: mycobacteriophage Patience from patient

5. Lines 43-44: In the same scenario, analysis of phages infecting Gordonia species showed similarities to mycobacteriophages [4] (not adding anything)

6. Line 47: It called caught our attention the fact that uncommon

7. Line 79: Preparation and of

8. Line 80: briefly late log fresh phase

9. Line 133: Is it true that the images for colony morphology were taken at 0.8X magnification? Or should it be 8X?

10. Line 152: Was 0.5% glycerol in the medium was taken by weight (w/v) or is it a typographical mistake?

11. Line 168: Provide reference for Jacobs and Hatfull protocol.

12. Line 212: The To this

13. Line 334: genome if of Weirdo19ES

14. Change the title of the last section from Conclusion to Discussion and remove Discussion from Results and Discussion title.

15. Title for Fig. 9 should be “Phage resistant mutants of Mycobacterium smegmatis”

16. Line 468: 5 ml drop seems do big to be used for this experiment. It must be a typographical mistake, correct it.

17. In the legends of Fig. 9 and Fig. 10 the phage is written as 19WeirdoES, match it to nomenclature used in the rest of the manuscript i.e. Weirdo19ES.

18. Include a color key of pham in Fig. 1.

19. Include the schematic of typical LysA with all the domain as a reference.

20. Images attached are blurred, provide good quality images for review.

Authors’ reply: We believe we have corrected the manuscript. Of note , in Line 41 of the original submission, Patience is the name of the phage so it was mantained as such.

18: pham color assignation is by default

19. As stated in the text there is no typical LysA

Attachment

Submitted filename: PONE-D-19-31407 Reply to Reviewers.docx

Click here for additional data file.^{(22.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0231881.r003

Decision Letter 1

Anirudh K Singh

3 Apr 2020

Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage-resistant Mycobacterium smegmatis mutants

PONE-D-19-31407R1

Dear Dr. Morbidoni,

This mail should cheer you up in this time COVID-19 distress. We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Anirudh K. Singh, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: Please take care of grammar and do needful revision before final submission. Have a critical look on Fig.3 B about clustering analysis of repressors.

Reviewer #2: The manuscript has improved improved significantly after the revision. The authors have addressed all the reasonable queries.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Vandana Singh

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0231881.r004

Acceptance letter

Anirudh K Singh

6 Apr 2020

PONE-D-19-31407R1

Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage-resistant M. smegmatis mutants

Dear Dr. Morbidoni:

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Comparative analysis using CGView comparison tools.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

S2 Fig. Phylogenetic analysis of the tape measure proteins.

The amino acid sequences of the TMPs were aligned with MUSCLE and the dendrogram was constructed using Maximum Likelihood method in MEGAX: mycobacteriophage clusters are indicated in colored boxes.

(TIF)

Click here for additional data file.^{(424.1KB, tif)}

S1 Table. List of mycobacteriophages used throughout this study.

Corresponding accession numbers, host and clusters classification are listed.

(DOCX)

Click here for additional data file.^{(16.4KB, docx)}

S2 Table. List of predicted gene of mycobacteriophage Weirdo19ES.

The function were predicted by BLASTP analysis using phagedb database. ⁽¹⁾ The highest homologue to each gene product are listed together with the phage cluster and BLASTP E-value ⁽²⁾.

(DOCX)

Click here for additional data file.^{(19.6KB, docx)}

Attachment

Submitted filename: PONE-D-19-31407.docx

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Attachment

Submitted filename: PONE-D-19-31407_Comments.docx

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Attachment

Submitted filename: PONE-D-19-31407 Reply to Reviewers.docx

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Data Availability Statement

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PERMALINK

Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage-resistant M. smegmatis mutants

Cristian Alejandro Suarez

Jorgelina Judith Franceschelli

Sabrina Emilse Tasselli

Héctor Ricardo Morbidoni

Roles

Abstract

Introduction

Materials and methods

Strains, chemicals, culture media and growth conditions

General bacteriophage techniques

Isolation and characterization of mycobacteriophage resistant M. smegmatis mutants

Aggregation assay

Sliding motility assay

Cell envelope analysis of mycobacteriophage resistant M. smegmatis mutants

Molecular biology techniques

Bioinformatics analysis

Nucleotide accession number

Results

Weirdo19ES genome organization

Fig 1. Weirdo19ES genome map.

Analysis of the left side of the genome of Weirdo19ES

Fig 2. Schematic representation of the Lys A domains.

Analysis of the central region of the genome of Weirdo19ES

Integration and immunity region

Fig 3. Phylogenetic analysis of the integrases and repressors.

Fig 4. Analysis of the integration system.

Analysis of the right side of the genome of Weirdo19ES

Free standing HNH endonucleases

Comparative genomics of Weirdo19ES

Fig 5. Phylogenetic network of mycobacteriophages.

Fig 6. Mash distance dendrogram.

Fig 7. Gene content dissimilarity (GCD) analysis.

Fig 8. Clustering analysis of the relative synonymous codon usage values.

Biology cycle of Weirdo19ES

Fig 9. Phage resistant mutants of Mycobacterium smegmatis.

Fig 10. Cross-resistance of mutants 1R-Weirdo19ES, 3R-I3 and Myc55 to mycobacteriophages Weirdo19ES and I3.

Fig 11. Mutants resistant to mycobacteriophages I3 or 19WeirdoES lack polar GPLs.

Discussion

Weirdo19ES is a singleton mycobacteriophage

M. smegmatis resistance to Weirdo19ES highlights the role of gycopeptidolipids as a key mycobacteriophage receptor

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Anirudh K Singh

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Author response to Decision Letter 0

Decision Letter 1

Anirudh K Singh

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Acceptance letter

Anirudh K Singh

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Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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