1 Characterization of Novel Virulent Broad-Host Phages of Xylella ...

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Characterization of Novel Virulent Broad-Host Phages of Xylella fastidiosa and 1

Xanthomonas 2

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Running title: Novel Virulent Broad-Host Phages of Xylella fastidiosa 4

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Stephen J. Ahern,a,b

Mayukh Das, a,b

Tushar Suvra Bhowmick, a,b

Ry Young, a,c

Carlos F. 6

Gonzalez# a,b

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Center for Phage Technology, Texas A&M University, College Station, Texas 77843-2128a; 9

Department of Plant Pathology and Microbiology, Texas A&M University, College Station, 10

Texas 77843-2132b; Department of Biochemistry and Biophysics, Texas A&M University, 11

College Station, Texas 77843-2128c 12

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#Corresponding author: Carlos F. Gonzalez. E-mail: [email protected] 22

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JB Accepts, published online ahead of print on 8 November 2013J. Bacteriol. doi:10.1128/JB.01080-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 24

The xylem-limited bacterium, Xylella fastidiosa, is the causal agent of several plant 25

diseases, most notably Pierce’s Disease of grape and citrus variegated chlorosis. We report on 26

the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano 27

and Salvo and podophages Prado and Paz, with host range that includes Xanthomonas spp.. 28

Phages propagated on homologous hosts had an observed adsorption rate constant of ~4 抜 10-12

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ml cell-1

min-1

for X. fastidiosa strain Temecula and ~5-7 抜 10-10

ml cell-1

min-1

for Xanthomonas 30

strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions 31

and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding 32

member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-33

like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also 34

spanin gene families that provide insight into the evolution of the lysis pathway for phages of 35

Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each 36

other, are new members of the phiKMV-like phage type, based on the position of the single-37

subunit RNA polymerase gene. The four phages are Type IV pilus-dependent for infection of 38

both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and 39

environmentally responsible strategy for the control of diseases caused by X. fastidiosa. 40

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INTRODUCTION 43

The plant pathogenic bacterium Xylella fastidiosa is the causative agent of a number of 44

economically important diseases, including Pierce’s Disease of grape, phony peach disease, 45

periwinkle wilt, citrus variegated chlorosis, almond leaf scorch, oleander leaf scorch and coffee 46

leaf scorch (1). Existing disease control methods are often only partially successful, and in the 47

case of systemic insecticides used to control vector populations, may be potentially harmful to 48

the environment (2-3). Recently, there has been renewed interest in the application of 49

bacteriophages (phage) as an environmentally acceptable mode for the control of bacterial plant 50

disease (4). 51

To this end, our group isolated and characterized the first temperate (lysogenic) phage of 52

X. fastidiosa, Xfas53 (5). However, temperate phage should not be used as biocontrol agents 53

because of their capacity for lysogenic conversion, superinfection immunity, and risk of 54

generalized transduction. Instead, virulent (lytic) phages are needed for an effective and 55

sustainable phage-based control strategy (6). Here we report on the isolation and characterization 56

of the first virulent siphophages and podophages for X. fastidiosa that are members of Nazgul-57

like phage and the phiKMV-like phage types, respectively. 58

MATERIALS AND METHODS 59

Bacterial strains and culture conditions. Bacterial strains and plasmids used in this study are 60

listed in Table 1. X. fastidiosa strains were cultured at 28°C in PW-M broth (PW-MB) (7) or 61

PW-M agar plates [PW-MA; PW-MB with 20 g/liter plant cell culture tested agar (Sigma)] (5). 62

PW-M soft agar (PW-MSA, PW-MB with 7.5 g/liter plant cell culture tested agar) was used for 63

overlays, with 5-7 day old plate cultures used to make indicator suspensions in PW-M broth 64

(OD600 = ~0.5). PD3 agar (20 g/liter) (8) was used for preparation of electrocompetent cells and 65


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transformations. For X. fastidiosa cultures carrying antibiotic resistance cassettes or plasmids, the 66

medium was supplemented with chloramphenicol (Cm) (5 µg/ml) or kanamycin (Km) (5 µg/ml). 67

Xanthomonas strains were cultured at 28°C in tryptone nutrient broth (TNB) (9) or tryptone 68

nutrient agar (TNA; TNB lacking KNO3 with 20 g/liter agar). TNA soft agar (TNSA, TNB 69

lacking KNO3 with 7.5 g/liter agar) was used for overlays, with 18 h plate cultures used to make 70

indicator suspensions in TNB broth (OD600 = ~0.5). For Xanthomonas cultures harboring 71

plasmids, the medium was supplemented with Km (50 µg/ml). Yeast tryptone broth (YTB) and 72

yeast- tryptone-sucrose agar [YTSA; YTB with 20 g/liter agar and sucrose at a final 73

concentration of 15% (wt/vol)] were used for resolution of mutants (10). Plasmid constructs were 74

generated using Escherichia coli DH5g MCR as host. E. coli strains were cultured at 37°C in LB 75

broth or LB agar (11). For E. coli cultures harboring plasmids, medium was supplemented with 76

Cm (50 µg/ml), Km (50 µg/ml) or ampicillin (Amp) (100 µg/ml), as appropriate. Plant extracts 77

were plated to PW-MA or TNA supplemented with cycloheximide (40 µg/ml), designated PW-78

MC or TNAC, respectively. 79

Bacteriophage isolation and purification. Plant and sewage samples were assayed for 80

the presence of phage able to form plaques on X. fastidiosa strain Temecula. Plant extracts were 81

prepared by macerating 10 g of plant tissue (Oryzae sativa or weeds obtained from rice fields in 82

Jefferson County and Wharton County, Texas) in 50 ml of P-buffer (50 mM Tris-HCl pH 7.5, 83

100 mM NaCl, 8 mM MgSO4) using a mortar and pestle, strained through a sterile double layer 84

of cheese cloth to remove large particles, and filter sterilized. Sewage influent collected from a 85

municipal wastewater treatment plant in Brazos County, TX was centrifuged twice and filter 86

sterilized. Plant and sewage filtrates were directly screened for phage activity using the spot-test 87

and soft agar overlay method (5). Soft agar overlays containing 100 µl of X. fastidiosa strain 88


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Temecula (~108 CFU/ml) as an indicator host were allowed to solidify, spotted with 10 µl drops 89

of serially diluted filtrates in P-buffer, and assayed after incubation at 28ºC for 5-7 days. Filtrates 90

producing either plaques or cleared zones were serially diluted in P-buffer and assayed using the 91

overlay method, where 100 µl of each dilution was directly mixed with the host suspension in 92

tempered soft agar before overlaying. Individual plaques formed on overlays of X. fastidiosa 93

strain Temecula were excised, suspended in P-buffer, filter-sterilized and titered. This procedure 94

was repeated three times to obtain single plaque isolates. Additionally, filtrates were screened for 95

phage activity using Xanthomonas strain EC-12 (isolated from rice, see below), as described 96

above. Plaques were purified three times using Xanthomonas strain EC-12, and purified phages 97

were tested for activity on X. fastidiosa strain Temecula. 98

High-titer phage plate lysates (>1 抜 1010

PFU/ml) were prepared by harvesting overlay 99

plates of X. fastidiosa strain Temecula or Xanthomonas strain EC-12 exhibiting confluent lysis. 100

After being flooded with 5 ml of P-buffer the soft agar overlay was macerated and clarified by 101

centrifugation and filter sterilized. The resulting lysates were stored at 4 ºC. High-titer phage 102

lysates were further purified by isopynic CsCl centrifugation as previously described (5). 103

Bacterial isolation, purification and identification. Samples were tested for the 104

presence of X. fastidiosa using quantitative real time polymerase chain reaction (qRT-PCR) with 105

X. fastidiosa specific primers (INF2 and INR1) as previously described (12). Additionally, non-106

filtered plant extracts and sewage samples were plated to PW-MC or TNAC, incubated at 28ºC 107

and evaluated for growth (3 days on TNAC and 10 days on PW-MC). Representative single 108

colonies were picked from plates and streak purified three times to obtain stocks on appropriate 109

media. Xanthomonas isolates were identified by sequencing the 16S-23S ITS region (primers 110


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used are listed in Table S1). One Xanthomonas isolate, EC-12, was selected for further use in 111

this study. 112

Phage DNA isolation and genome sequencing. DNA was isolated from CsCl-purified 113

phage suspensions as previously described (13). Phages Sano, Salvo, Prado and Paz were 114

sequenced as part of two pools of phage genomes using the 454 pyrosequencing method (Emory 115

GRA Genomics Core). Phage genomic DNA was prepared from phage isolates as described 116

above and mixed in equimolar amounts to a final concentration of ca. 100 ng/µl. The pooled 117

DNA was sheared, ligated with a multiplex identifier (MID) tag specific for each of the four 118

pools and sequenced by pyrosequencing using a full-plate reaction on a Roche FLX Titanium 119

sequencer according to the manufacturer’s protocols. The pooled phage DNA was present in two 120

sequencing reactions. The trimmed FLX Titanium flowgram outputs corresponding to each of 121

the four pools were assembled individually using the Newbler assembler version 2.5.3 (454 Life 122

Sciences) by adjusting settings to include only reads containing a single MID identifier per 123

assembly. The identity of individual contigs was determined by PCR using primers generated 124

against contig sequences and individual phage genomic DNA preparations as template. The 125

coverages, calculated as assembled reads (in nucleotides) divided by the size of the genome, 126

were 41-, 62-, 140-, and 46–fold for phages Sano, Salvo, Prado and Paz, respectively. 127

Sequencher 4.8 (Gene Codes Corporation) was used for sequence assembly and editing. Phage 128

chromosomal end structures were determined experimentally. Cohesive (cos) ends for phages 129

Sano and Salvo were determined by sequencing off the ends of the phage genome and 130

sequencing PCR products derived by amplification through the ligated junction of circularized 131

genomic DNA, as previously described (13). Direct terminal repeats for phages Prado and Paz 132

were determined by sequencing off the ends of the phage genome with primers designed within 133


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the first and last identified genes. Protein-coding regions were initially predicted using 134

GeneMark.hmm (14), refined by manual analysis in Artemis (15), and analyzed using BLAST 135

(E-value cutoff of 0.005) (16). Proteins of particular interest were additionally analyzed by 136

InterProScan (17), HHpred searches against the pdb70_29jun13 database (18), NCBI's 137

Conserved Domain Database (19), LipoP (20), SignalP 4.1 (21), and TMHMM (22). The 138

sequence of phage Salvo gene 36 was confirmed by PCR and sequencing the resulting PCR 139

product. 140

Nucleotide sequence accession number and nomenclature. In accordance with Texas 141

A&M Center for Phage Technology policy, novel phages were given names for mnemonic 142

purposes. Names were checked for uniqueness by literature searching and were prefixed with 143

Xfa, which is the ReBase species acronym for X. fastidiosa (rebase.neb.com). Completed phage 144

genomes were deposited in GenBank under the following accession numbers: XfaSano, 145

KF626665; XfaSalvo, KF626668; XfaPrado, KF626667; XfaPaz, KF626666. For simplicity, the 146

phage names will be used without the Xfa prefix. Phages Sano, Salvo, Prado and Paz were 147

deposited under ATCC accession numbers PTA-13096, PTA-13095, PTA-13099 and PTA-148

13100, respectively. 149

Transmission electron microscopy. Electron microscopy of CsCl purified 150

bacteriophage (~1 抜1011

PFU/ml) was performed by diluting stock with P-buffer. Phages were 151

applied to thin 400-mesh carbon-coated Formvar grids, stained with 2% (wt/vol) uranyl acetate 152

and air dried. Specimens were observed on a JEOL 1200EX transmission electron microscope 153

operating at an acceleration voltage of 100 kV. Five virions of each phage were measured to 154

calculate mean values and standard deviations for dimensions of capsid and tail, where 155

appropriate. 156


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Host range and efficiency of plating. Host ranges of purified phages (propagated on X. 157

fastidiosa strain Temecula) were determined by the serial dilution spot-test method, as described 158

above, using a panel of X. fastidiosa and Xanthomonas isolates as indicator hosts. Phage 159

sensitivity of Type IV pilus mutants and complements were tested similarly. Efficiency of 160

plating (EOP) was determined by calculating the ratio of phage plaque titer obtained with 161

heterologous (non-propagating) host, to that obtained on homologous (propagating) host. All 162

experiments were done in triplicate. 163

One-step growth curve. One-step growth curves were used to determine burst size and 164

latent period of the phages (23). Liquid cultures of logarithmically growing Xanthomonas strain 165

EC-12 were infected with individual phages at a multiplicity of infection (MOI) of ~3 and 166

allowed to adsorb at 28oC for 5 min. To stop further phage adsorption, cultures were diluted 167

1000-fold in TNB. Infected centers were incubated at 28oC with constant shaking (150 rpm). 168

Samples were taken at 3 min intervals, immediately filter sterilized and plated in soft agar lawns 169

of Xanthomonas strain EC-12. All experiments were done in triplicate. 170

Bacteriophage adsorption. The kinetics of phage adsorption was determined as 171

previously described (5). Liquid cultures of logarithmically growing cells (X. fastidiosa strain 172

Temecula or Xanthomonas strain EC-12) were infected with individual phages (propagated on 173

homologous hosts) at a MOI of ~0.1. The mixture was incubated at room temperature with 174

shaking (150 rpm). Samples were taken (2 h intervals for X. fastidiosa strain Temecula and 2 min 175

intervals for Xanthomonas strain EC-12), immediately filter sterilized, and titered. The rate of 176

phage particle disappearance is defined as dP/dt = –kBP, where B is the concentration of 177

bacteria, P is the concentration of free phage at any time (t), and k is the adsorption rate constant 178

in ml cell-1

min-1

(24). All experiments were done in triplicate. 179


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Construction of X. fastidiosa strain Temecula pilA deletion mutant and in trans 180

complementation. The X. fastidiosa pilA (NP_780105.1) open reading frame (ORF) from 181

nucleotide 2,256,755 to 2,257,201 (X. fastidiosa Temecula1, NC_004556.1) was removed and 182

replaced with a Kmr cassette by site-directed gene disruption as described by Matsumoto et al. 183

(25), except pUC19 served as cloning vector and the downstream-pilA, Kmr cassette (amplified 184

from pXF004), and upstream-pilA regions were cloned sequentially. Primers used are listed in 185

Table S1. The deletion of pilA and replacement with Kmr cassette was confirmed by PCR. 186

Complementation of X. fastidiosa strain Temecula pilA deletion mutant, XF15-1, was 187

accomplished by introducing a wild-type copy of pilA using a chromosome-based 188

complementation system (25). Insertion of pilA and Cmr cassette into the neutral site (NS1) of 189

putative complements was confirmed by PCR amplification using primers pair NS1-f and NS1-r 190

(25), and sequencing of product. 191

Construction of Xanthomonas strain EC-12 pilA deletion mutant and in trans 192

complementation. Deletion of pilA in Xanthomonas strain EC-12 was performed as described 193

by Hamad et al. (10), except cells were transformed using electroporation. Primers used are 194

listed in Table S1. PCR primers were designed based on the annotated pilA sequence of 195

Xanthomonas strain EC-12 draft genome. The deletion of pilA was confirmed by PCR. To 196

complement Xanthomonas strain EC-12-1 (〉pilA), wild-type pilA was introduced in trans (10). 197

The presence of the wild-type pilA was confirmed by PCR and sequenced for confirmation. 198

Twitching motility assay. Twitching motility was examined as previously described 199

(26). Briefly, late log phase liquid cultures of X. fastidiosa strain Temecula (5 day), 200

Xanthomonas strain EC-12 (18 h) and their derivatives were centrifuged and resuspended to 200 201

たl in liquid medium as appropriate (1 抜 109 CFU/ml). Five microliter cell suspensions were 202


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spotted to appropriate solid medium modified with 1.2% agar, and incubated at 28˚C [X. 203

fastidiosa (5 day) and Xanthomonas (18 h)]. Pseudomonas aeruginosa strain PAO1 grown in a 204

similar manner at 37˚C for 24 h, was used as positive control. Following incubation, colony edge 205

morphology was visualized using an SZ-PT Olympus Microscope equipped with QImaging Go-206

21 CMOS camera system. Colonies with a peripheral fringe were designated as twitch positive. 207

Lysogen formation assay. To assay for phage lysogen-formation, survivors of phage 208

infection were tested for the presence of prophages. For each phage, bacteria were infected with 209

at an input MOI of ~3 and plated in a soft agar overlay. Plates were monitored for colony growth 210

(10-15 days for X. fastidiosa strain Temecula and 2-3 days for Xanthomonas strain EC-12). 211

Individual colonies that emerged were picked, purified (three times) and re-tested for phage 212

sensitivity by spotting dilutions of the same phage in a soft agar overlay. Primer pairs specific to 213

Sano and Salvo primase or Prado and Paz helicase genes (Table S1) were then used to test for the 214

presence of prophage sequences in the phage-insensitive isolates. Wild type bacterial DNA was 215

used as negative control and wild type bacterial DNA spiked with phage DNA served as positive 216

controls. 217

To test whether evidence could be found for abortive lysogeny (i.e., the establishment of 218

repression), we followed the procedure of Gill et al. (27), except reversibly bound phage were 219

removed by three successive washes. Liquid cultures of logarithmically growing Xanthomonas 220

strain EC-12 were cultured to an OD600 of ~ 0.3-0.5. One ml aliquots were pelleted by 221

centrifugation and resuspended in 0.20 ml of phage lysate (harvested in TNB) or sterile TNB. 222

After a 30 min incubation at 25°C cell-phage mixtures were centrifuged and the supernatant 223

removed and titered to determine adsorbed phage. In preliminary experiments it was determined 224

that phage were reversibly bound, which affected the MOIactual (28) calculation. To circumvent 225


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this problem and to obtain an accurate MOIactual, cells were resuspended in sterile TNB, allowed 226

to incubate for 5 min at 25°C, centrifuged and supernatants removed. This procedure was 227

repeated three times to remove unbound phage. Each supernatant was titered to determine PFU. 228

Cell pellets were resuspended in 0.20 ml of sterile TNB, serially diluted and plated to enumerate 229

the bacterial survivors remaining following phage exposure. From these data, the MOIactual, i.e., 230

the ratio of the number of adsorbed phage to the number of CFU in the phage-free controls) was 231

calculated. These MOIactual values were used to calculate the predicted proportion of uninfected 232

cells using the Poisson distribution. This experiment was replicated three times, using both Sano 233

and Prado. 234

RESULTS 235

Phage isolation and characterization. Our initial efforts focused on the screening of rice and 236

weed extracts for phages that formed plaques on lawns of X. fastidiosa strain Temecula. Extracts 237

yielded 10-105 PFU/g of tissue on lawns of X. fastidiosa. X. fastidiosa was not detected in 238

extracts (by direct plating or qRT-PCR to limits of detection). However, Xanthomonas hosts 239

were isolated from extracts, including strain EC-12. Subsequently, samples from both plant 240

tissues and sewage were routinely screened against both X. fastidiosa strain Temecula and 241

Xanthomonas strain EC-12. Of the phages isolated, Sano, Salvo, Prado and Paz are 242

representative. 243

The physical and host-range properties of the four representative phages are summarized 244

in Tables 2 and 3 and in Fig. 1. Phages Sano and Salvo exhibit siphophage morphology with 245

isometric heads and non-contractile tails (Fig. 1A and 1B, respectively), whereas phages Prado 246

and Paz exhibit podophage morphologywith isometric heads and short-stubby tails (Fig. 1C and 247

1D, respectively). All four phages formed plaques on both X. fastidiosa and Xanthomonas hosts, 248


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with differences in host range (Table 3). The siphophages formed small clear plaques, whereas 249

the podophages formed large clear plaques. 250

Phages Salvo and Paz plated efficiently to X. fastidiosa strain Temecula and 251

Xanthomonas strain EC-12 (Table 4). However, there was a ~2- to 3-log reduction in efficiency 252

for phages Sano and Prado on the same hosts (Table 4). Adsorption rate constants for the four 253

phages on X. fastidiosa strain Temecula were on the order of ~4 抜 10-12

ml cell-1

min-1

, which is 254

much lower than most phages previously reported, but similar to phage Xfas53 (5). In contrast, 255

the rate constants for the Xanthomonas strain EC-12 were ~100-fold greater at ~5 抜 10-10

ml cell-

256

1 min

-1. The burst size of all four phages was ~10

2 PFU/cell at ~52 min with Xanthomonas strain 257

EC-12 as host (Table 2). The extremely slow adsorption to X. fastidiosa strain Temecula made it 258

unfeasible to determine burst size in this host. 259

Genomics of siphophages Sano and Salvo. The general characteristics of the phage 260

genomes are summarized in Table 5 and complete annotations with supporting evidence are 261

provided in Table S2. The genomes of Sano and Salvo were found to be 56.1 and 55.6 kb, 262

encoding 77 and 72 genes, respectively. Both have 12 nt 5’-protruding single-stranded cos ends. 263

Having cos ends makes it unlikely that Sano and Salvo are capable of generalized transduction. 264

When analyzed by pairwise DNA MegaBLAST, the genomes of phages Sano and Salvo 265

can be aligned over ~69% of their length, with an average ~80% sequence identity in aligned 266

regions. Sano and Salvo contain 58 homologous genes with amino acid sequence similarities 267

ranging from ~13- 94%, and have retained genome synteny (Table S2, Fig. 2). Despite no direct 268

evidence of transcriptional boundaries or rho-independent terminators, breaks between genes are 269

not large enough to contain promoters. Therefore, the arrangement of genes on the two strands 270

suggests that the genomes are organized into four transcriptional units. Genes found in 271


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transcriptional unit I (starting at the left end, reverse strand) and II (forward strand) primarily 272

encode proteins of unknown function and show very limited similarity to other phage genes in 273

the NCBI database. The lysis cassette, structural module and DNA metabolism module are all 274

encoded in transcriptional unit III (reverse strand), whereas transcriptional unit IV (forward 275

strand) encodes only a few genes, including a primase. 276

When analyzed by BLASTp, Sano and Salvo are most closely related to Burkholderia 277

phage BcepNazgul (NC_005091) (herein referred to as Nazgul); however, the relationship is 278

distant. Of the 77 predicted Sano genes, only 23 show homology to Nazgul, ranging from ~11-279

48% sequence identity at the amino acid level. Homologs are primarily located in the structural 280

and DNA metabolism regions (excluding tail fiber and assembly proteins). However, despite the 281

low observed total homology, phages Sano and Salvo are syntenic with Nazgul (Fig. 2). 282

As defined by Casjens (29), Nazgul does not belong to any existing phage type because 283

of its unique architecture and low homology to previously described phages. We therefore 284

propose that Nazgul is the founder of a new phage type, to which both Sano and Salvo belong. 285

Re-annotation of the genomes of Burkholderia phage AH2 (30) and the closely related 286

Enterobacter phage Enc34 (31), enteric bacteria phage Chi (32) and Providencia phage Redjac 287

(33) indicate they are all members of the Nazgul-like phage type (Fig 2). 288

In the following, except where significant differences exist between Sano and Salvo, only 289

the relationship between the proteins of phage Sano and their homologs will be discussed in 290

detail (Fig. 2, Table S2). 291

Transcriptional units I and II. Most proteins encoded in transcriptional units I and II do 292

not have homologs in the database. Despite this, 24 of the 38 proteins encoded in Sano 293

transcriptional units one and two have homologs in Salvo, with ~13-94% sequence identity at the 294


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amino acid level (Fig. 2, Table S2). Only one protein (Sano gp28) has a homolog in phage 295

Nazgul (Nazgul gp34). 296

Lysis genes. Phages of Gram-negative hosts have genes coding for three functional 297

classes of lytic proteins; holins, endolysins, and spanins (34-35). In addition, many phages 298

encode an antiholin, a specific inhibitor of the holin. Analysis of the genes of the Nazgul-like 299

phage type suggested all four functional classes are represented, although with unexpected and 300

novel variations. As with many phage genomes, the lysis proteins are clustered in lysis cassettes 301

for the Nazgul-like phage type. Fig. 3 shows the lysis cassettes of Sano, Salvo, Nazgul, AH2 and 302

Chi; Redjac and Enc34 are not shown because the lysis gene arrangements are essentially 303

identical to Chi. In each case, the lysis genes are the last cistrons of transcriptional unit III that 304

includes a long series of morphogenesis genes, except for AH2, which has a single novel gene 305

inserted before the convergence point with unit III (Fig. 2 and 3). Except for Sano, the lysis 306

cassettes have the order most commonly found in phages of Gram-negative hosts, with the genes 307

encoding the holins and antiholins first, followed by the endolysin and then the i-spanin/o-spanin 308

gene pair. In Sano, the endolysin gene is the first of the cassette. Beyond this synteny, however, 309

the Nazgul-like family exhibits extreme diversity and mosaicism in its lysis genes, in that among 310

the 24 lysis genes in the five cassettes shown in Fig. 3, 16 are unrelated to any of the others. 311

Although the spanin genes fall into only two groups (see below), the five endolysin genes lack 312

similarity with each other. Moreover, the nine genes encoding integral membrane proteins 313

encode seven proteins with no detectable sequence similarity, presumably including at least three 314

holins and four antiholins as explained below. 315

Endolysins. The endolysin genes (diagonal hatching, Fig. 3) in these five phages can be 316

identified independently of their proximity to the other lysis genes, although with varying 317


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degrees of confidence. Gp38 of Nazgul is very similar to the P endolysin of the paradigm 318

coliphage 186; P is a transglycosylase of the well-characterized lambda R family. Gp43 of Sano 319

is closely related to the endolysin of a Pseudomonas phage, PaP1, which has been shown to have 320

muralytic activity, although its enzymatic type is not known (36). Gene 42 of AH2 has many 321

related genes in prophage elements, although not to any identified endolysins. However, HHpred 322

analysis reveals weak similarity to eukaryotic and prokaryotic lysozyme (glycosidase) proteins. 323

The Chi/Redjac/Enc34 endolysins were similar to proteins designated as Lysis Protein A in a 324

Salmonella phage iEPS5 (37). Although all of these endolysins are distinct and belong to at least 325

two enzymatic classes, they lack signal sequences or membrane domains and are thus soluble 326

endolysins that require canonical holins for release across the cytoplasmic membrane (see 327

below). Gp36 of phage Salvo stands in stark contrast and represents an endolysin of 328

unprecedented structure. The last ~170 residues of gp36, although lacking any similarity to 329

individual proteins by BLAST analysis, nevertheless are strongly predicted by HHpred analysis 330

to be related to a number of experimentally and structurally characterized phage endolysins, 331

including the classical phage T4 lysozyme. However, the first ~100 residues are unrelated to any 332

sequence in the database. Moreover, the N-terminus has a strongly predicted outer membrane 333

(OM) lipoprotein signal sequence, indicating that this endolysin is exported by the sec translocon 334

and, after processing by signal peptidase II and localization by the Lol system, becomes tethered 335

to the inner leaflet of the OM as a 275 aa mature lipoprotein. In support of this interpretation, 336

gp42 has two pairs of Cys residues in the periplasmic domain; Cys residues in pairs are always 337

found in the catalytic domains of SAR endolysins, presumably to provide stabilizing 338

intramolecular disulfide linkages necessary for the protease-rich environment of the periplasm 339

(38). In contrast, the cytoplasmic endolysins of the other phages of the Nazgul-like family have 340


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no Cys residues, except for the Redjac endolysin, which as a single Cys; this supports the notion 341

that these are all cytoplasmic endolysins. 342

Spanins. Most phages of Gram-negative hosts have two-component spanins, comprised 343

of an integral inner membrane (IM) protein, or i-spanin, and an OM lipoprotein, or o-spanin, 344

that, together, form a complex that spans the periplasm by C-terminal interactions (39). Genes 345

for two spanin proteins can be arranged in three distinct architectures (39). All 7 members of the 346

Nazgul-like phage type encode two-component spanins (Fig. 3). The sequences fall into two 347

families with Sano and Salvo comprising one family and the other five members forming the 348

second, as shown most clearly in Fig. 4 for the mature o-spanin lipoproteins. Surprisingly, in 349

both families, there are representatives of two spanin gene architecture classes. In the Sano/Salvo 350

family, the former has its spanin genes in the separated class whereas in the latter, the spanin 351

genes are overlapped. Similarly, the AH2 genes are entirely separated, whereas the four spanin 352

gene pairs with similarity to the AH2 genes are in the overlapped architecture. These are the first 353

two examples of spanin gene families that include members from different architectural classes 354

(see Discussion). 355

Holins and antiholins. Absent similarity to a known holin, the general practice is to 356

assign holin function to any integral membrane protein encoded by a gene clustered with the 357

more easily identified endolysin and spanin genes, especially if the putative holin shares 358

topology with one of the three established holin topologies (40-41). Among the Nazgul-like 359

phages, the Chi/Enc34/Redjac subset is the simplest for holin annotation, since there is only a 360

single gene encoding an integral membrane protein immediately upstream of the endolysin and 361

spanin genes, the most common arrangement for lysis cassettes. This protein, gp02 in Chi (Fig. 362

3) has a single TMD and is arranged in an N-in, C-out topology, making it the first member of 363


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topology class III that is not related to the phage T4 T holin family. The other four members of 364

the Nazgul-like phage type each have genes for two integral membrane proteins clustered with 365

the lysis genes. Among these eight proteins, seven different membrane topologies are 366

represented. Both of the two membrane proteins encoded by Nazgul have homologs in AH2 367

(yellow and pink, Fig. 3) but otherwise all of these proteins are unrelated and novel. Although 368

Nazgul gp39 has a predicted topology consistent with the most common holin topology, class I 369

(three TMDs with N-out and C-in), its AH2 homolog lacks TMD1. Since it has been shown for 370

two paradigm class I holins, lambda S105 and P2 Y, that the first TMD is required for function, 371

it seems unlikely that these proteins encode holins. In contrast, Nazgul gp40 and its homolog 372

AH2 gp44 both have class III topology, like the Chi holin noted above. The lysis cassettes of 373

Sano and Salvo each encode two novel integral membrane proteins, and because of ambiguity as 374

to the identity of the holin or antiholin we have annotated all four genes (Sano genes 41 and 42 375

and Salvo genes 37 and 38, respectively) as “holin-antiholin”. 376

Structural genes. There is limited amino acid sequence homology between the tail fiber 377

and assembly genes of Sano with Nazgul (Table S2). However, Sano genes 44-52 can be 378

presumptively assigned the functions of tail fiber and assembly proteins based on synteny to 379

similar genes in Nazgul (genes 41-49) (Fig. 2). Sano gp53 is a tail tape measure protein based on 380

the conserved tape measure domain (IPR013491). In most siphophages and myophages, the 381

reading frames upstream of the tape measure protein encode the functional homologs of the 382

lambda tail assembly chaperones, G and G-T. These represent alternate translational products 383

related by programmed -1 frameshifts, and both products have been shown to be required for tail 384

assembly (42). In Sano, the G and G-T equivalent frames are genes 55 and 54; manual inspection 385


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reveals a translational slippery sequence 5’-GGGAAAC-3’ near the end of Sano gene 55. This 386

sequence is conserved in Salvo. 387

Genomics of podophages Prado and Paz. The general characteristics of the phage 388

genomes are summarized in Table 5 and complete annotations with supporting evidence are 389

provided in Table S3. The genomes of Prado and Paz were found to be 43.9 and 43.8 kb, 390

encoding 52 and 51 genes, respectively. The genomes of Prado and Paz contain non-permutated 391

direct terminal repeats of 619 and 675 bp, respectively. 392

Prado and Paz are clearly phiKMV-like based on genome organization and protein 393

homology with other phiKMV-like phages. Prado is most similar to Caulobacter phage Cd1 394

(GU393987), with 26 homologous genes ranging from ~8-55% amino acid similarity. Similar to 395

T7-like phages, phiKMV-like phage genomes consist of three functional gene clusters encoded 396

on the forward strand. Class I encodes early genes with functions to overcome host restriction 397

and to convert the metabolism of the host cell to the production of phage proteins. Class II 398

encodes proteins involved in DNA metabolism. Class III encodes structural and assembly 399

proteins, DNA packaging proteins and lysis proteins. The phiKMV-like phages are distinguished 400

from T7-like phages in that they encode a single-subunit RNA polymerase (RNAP) at the end of 401

the class II gene cluster, rather than in the early genomic region (43). Phages Prado and Paz 402

share no significant DNA homology (~4%) when compared by pairwise DNA megaBLAST. 403

Despite divergence of DNA sequence, Prado and Paz contain 43 homologous genes with amino 404

acid similarities ranging from ~17- 79%, and have canonical phiKMV-like gene order (Fig. 6, 405

Table S3). 406


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In the following, except where significant differences exist between Prado and Paz, only 407

the relationship between Prado proteins and database homologs will be discussed in detail (Fig. 408

6, Table S3). 409

Early genes. Early genes are involved in host conversion and have been reported to 410

protect phage from bacterial defense mechanisms or alter host cell mechanisms (44). When 411

comparing phages of the same phage type, phage proteins that interact most intimately with the 412

bacterial host have been reported to be the most diverse (45). Five out of 12 Prado early genes 413

have homologs in Paz. Only two early gene proteins, Prado gp02 and gp11, have homologs in 414

other phiKMV-like phages, namely Cd1 gp01 and gp09, respectively. 415

DNA metabolism. Phage Prado DNA metabolism genes follow the canonical phiKMV-416

like gene order of DNA primase (gene 13), DNA helicase (gene 14), DNA polymerase A (gene 417

17), 5’-3’ exonuclease (gene 19), DNA exonuclease VII (gene 20), ATP-dependent DNA ligase 418

(gene 26), and a single-subunit RNAP (gene 28) (Fig. 6). Prado gp21 and gp22 do not have 419

homologs in Cd1, but do have homologs in other phiKMV-like phages, such as Ralstonia phage 420

RSB1 gp22 and gp24, respectively. The Prado gene encoding single-subunit RNAP is located at 421

the right end of the DNA metabolism gene cluster, a distinguishing characteristic of all reported 422

phiKMV-like phages. However, it has been noted that the position of the DNA ligase gene can 423

vary within phiKMV-like phages (46). In phiKMV, the DNA ligase is encoded at the beginning 424

of the DNA metabolism genes, whereas in Cd1 and Salvo the DNA ligase is encoded directly 425

before the RNA polymerase (Fig. 6). 426

The DNA metabolism region of Prado also shares high homology to several 427

Xanthomonas phages, including CP1 (NC_019933), Xop411 (NC_009543), Xp10 (NC_004902), 428

OP1 (NC_007709), phiL7 (NC_012742) and CP2 (NC_020205). These phages are non-429


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phiKMV-like, yet have homology and preserved gene order characteristic of phiKMV-like DNA 430

metabolism regions, including a single-subunit RNAP located at the end of the region. 431

Structural and lysis genes. As for DNA metabolism genes noted above, Prado’s 432

structural and lysis gene region follows the phiKMV-like gene order. It begins immediately 433

downstream of the single-subunit RNAP, with three small genes for conserved proteins of 434

hypothetical function (genes 29-31) that are homologs to Cd1 genes 27-29. Next in sequence are 435

genes encoding a head-tail connector (gene 32), scaffold protein (gene 33), major capsid protein 436

(gene 34), tail tube A (gene 36), tail tube B (gene 37), and three internal virion proteins (genes 437

38-40). Prado gp40 possesses a C-terminal lysozyme-like domain (IPR023346), identical to that 438

found in Cd1 gp38. However, Prado gp40 is ~300aa larger than any previously reported homolog 439

in phiKMV-like phages. 440

There is diversity within the tail fiber proteins of the phiKMV-like phages, both in terms 441

of sequence homology and components (46). In phiKMV-like phages, tail structural proteins are 442

found immediately downstream of the internal virion proteins. Similarly, genes for the Prado tail 443

fiber proteins (genes 41-44) are encoded in the same location. Prado gp41 contains a T7 tail fiber 444

protein domain (IPR005604) and gp43 and gp44 have homologs to tail fiber proteins found in 445

phiKMV-like phages. It appears, however that Prado genes 41-44 may have been acquired 446

through horizontal gene transfer with a non-phiKMV like phage. The four genes are found as a 447

cluster and are homologs to genes 26-29 in Xanthomonas phage CP2, with ~31-41% identity. 448

The lysis cassette of phage phiKMV has been studied in detail and is organized with 449

genes encoding holin, endolysin, i-spanin (formerly Rz) and o-spanin (formerly Rz1) 450

immediately downstream of TerS and TerL (47). The lysis cassette of Prado is similar to that of 451

phiKMV, except that the predicted holin is found upstream of the endolysin, separated by three 452


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genes encoding for TerS (gene 46), TerL (gene 47) and a hypothetical protein (gene 48) (Fig. 6). 453

This configuration, with lysis and terminase genes intermingled, has been previously observed in 454

phage SP6 and the phiKMV-like phage JG068 (48-49). 455

Prado gene 45 encodes a putative type II holin based on the presence of two TMDs (N-in, 456

C-in). TMD2 (residues 31 to 48) is the most hydrophobic of the two. TMD1 (residues 13 to 29) 457

is not predicted by TMHMM and has characteristics of a SAR domain, with a high percentage of 458

weakly hydrophobic or polar residues. The class II topology and presence of a SAR domain 459

followed by a typical TMD suggests that gp45 is a pinholin, similar to phiKMV gp45 (47). Prado 460

gene 49 encodes an endolysin, based on homology to other phiKMV-like phage endolysins in the 461

database. Prado endolysin gp49 exhibits an N-terminal hydrophobic domain rich in weakly 462

hydrophobic residues that is characteristics of a SAR endolysin (50). A glycoside hydrolase 463

domain (IPR002196) is predicted by InterProScan and contains an E-8aa-D-8aa-T catalytic triad, 464

similar to the E-8aa-D-5aa-T and E-9aa-D-6aa-T catalytic triads found in phage T4 protein E and 465

phiKMV gp45, respectively (38, 47). Prado spanins were identified by characteristic gene 466

orientation and proximity to the endolysin. Prado gp52 is homologous with HslV family proteins 467

of Xanthomonas phages OP1 (gp51), Xp10 (gp51) and Xop411 (gp51), with ~38-42% identity, 468

but does not contain the N-terminal hydrolase domain present in the other phage proteins. 469

Phage receptor site identification. In Gram-negative bacteria, OM proteins, pili, 470

flagella, oligosaccharides or lipopolysaccharides can act as phage receptors (51). Our 471

preliminary results (not shown) demonstrated that two transposon insertion mutants of X. 472

fastidiosa strain Temecula, TM1 (unpiliated) (26) and “tonB1 mutant” (hyperpiliated) (52), were 473

resistant to the four phages, indicating the phages may be Type IV pilus-dependent. To confirm 474

these observations, we generated Type IV pili mutants (in-frame deletions of pilA) in X. 475


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fastidiosa strain Temecula and Xanthomonas strain EC-12, and tested for phage sensitivity. PilA 476

is the major subunit of Type IV pili, and is required for Type IV pili formation (53). Wild type X. 477

fastidiosa strain Temecula and Xanthomonas strain EC-12 exhibited twitching motility 478

(indicative of pilus retraction) (Fig. 7A and 7D, respectively) and were sensitive to all four 479

phages. The ∆pilA derivatives, XF15-1 and EC-12-1, were devoid in twitching motility (Fig. 7B 480

and 7E, respectively) and were resistant to all four phages. Twitching motility and phage 481

sensitivity were restored in XF15-1and EC-12-1 by complementation of pilA in trans (Fig. 7C 482

and 7F, respectively), providing evidence that the phages are Type IV pilus-dependent. 483

Lysogeny. To examine the potential for lysogeny, 40 phage-insensitive isolates of X. 484

fastidiosa strain Temecula and Xanthomonas strain EC-12 each were recovered following a 485

challenge by phage Sano, Salvo, Prado or Paz. PCR using phage specific primers did not detect 486

the presence of phage lysogens in resistant isolates, indicating resistance was not due to 487

lysogeny. Additionally, we examined the potential for abortive lysogeny, using infection at a 488

high MOI and measuring survival (27). As shown in Table 6, following infection with Sano or 489

Prado, there was no significant difference between predicted and actual survivors, indicating 490

phage infection at a high MOI did not lead to the establishment of repression. Together, these 491

results indicate there is no evidence for lysogeny or repression, supporting the conclusion that 492

the four phages are virulent. 493

494

DISCUSSION 495

We have previously reported on the isolation and propagation of the first temperate phage of X. 496

fastidiosa, Xfas53. Here we report on the isolation and propagation of the first virulent phages 497

able to infect X. fastidiosa and Xanthomonas. Phages were isolated from plant extracts that 498


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contained Xanthomonas spp. but not X. fastidiosa. We have more recently isolated phage directly 499

from the petioles of grapevines that plate to both X. fastidiosa isolates and Xanthomonas. Both 500

genera are members of the Xanthomonadaceae and are associated with a broad range of plant 501

hosts (54-55). Xylella and Xanthomonas share a common origin and have diverged, with X. 502

fastidiosa having a reduced genome that may reflect xylem-limited lifestyle (56-57). Despite 503

their divergence, both have retained Type IV pili (58-62), which have been previously identified 504

as phage receptors (63-70). Phages Sano, Salvo, Prado and Paz are Type IV pilus-dependent. 505

Sano and Salvo are siphophages with isometric heads and non-contractile tails, whereas 506

Prado and Paz are podophages with isometric heads and short-stubby tails (Fig. 1). The 507

adsorption rate of the four phages to X. fastidiosa strain Temecula was similar to that previously 508

observed for Xfas53 (5), but was ~100-fold higher for Xanthomonas strain EC-12. The observed 509

difference in rate constants between hosts may be due to possible differences in affinity to the 510

Type IV pilus of X. fastidiosa and Xanthomonas spp., or potential differences in Type IV pilus 511

retraction speed/frequency that may affect irreversible binding to a secondary receptor site(s). 512

The genomes of Sano, Salvo, Prado and Paz have an average GC content of ~62.1%, which is 513

similar to that of sequenced Xanthomonas spp. (~63.6-65.3%) and Xanthomonas phages (~60%); 514

but significantly higher than that of X. fastidiosa strains (~49-55.6%) (54, 57), suggesting that 515

the primary hosts in the plant environment are Xanthomonas spp.. We speculate that overlapping 516

plant host ranges and shared environmental niches, along with the conservation of Type IV pili, 517

may have led to the selection of phages able to infect both genera. Restriction-modification and 518

CRISPR-cas systems have been identified in some Xanthomonas spp. (71-72), with X. fastidiosa 519

exhibiting only the former (73). The observed weak inhibition of efficiency of plating (Table 4) 520


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suggests that either the above systems are relatively inefficient in both hosts, or the phages are 521

subverting them, as has been demonstrated in phage T7 (74). 522

Phages Prado and Paz are members of the phiKMV-like phage type, a large group of 523

virulent phage, and presumably have diverged from a common phiKMV-like ancestor due to 524

genetic drift. However, the DNA metabolism regions of Prado and Paz share homology to 525

several non phiKMV-like (based on genome architecture) Xanthomonas phages. Additionally, 526

the tail fibers of Prado and Paz share highest homology to the non phiKMV-like phage CP2. The 527

observed homology in individual genes and gene clusters between Prado and Paz to non-528

phiKMV-like Xanthomonas phages is indicative of horizontal gene transfer between these two 529

phage groups, and further demonstrates the mosaic nature of phages. 530

We propose that phage Nazgul is the founder of a new phage type, the Nazgul-like 531

phages. Phages Sano and Salvo are members of this new phage type. Analysis indicates that 532

Burkholderia phage AH2, Enterobacter phage Enc34, enteric bacteria phage Chi and 533

Providencia phage Redjac are Nazgul-like (Fig. 2). It would be expected that closely related 534

phages would share a similar infection lifestyle (i.e., temperate or virulent). However, closely 535

related virulent and temperate mycobacteriophage and siphophage of P. aeruginosa have been 536

reported (75-76). Phage AH2 was not defined by the authors as temperate (30), but there is clear 537

evidence that it may be a temperate phage. AH2 is predicted to encode three excisionase proteins 538

(genes 30, 31 and 69), an integrase (gene 37) and a CI-like repressor (gene 70). Homologs to 539

these genes are not present in the other Nazgul-like phages. In fact, AH2 genes 30, 31 and 37 are 540

encoded in two adjacent transcriptional unites (based on coding strand position; boxed in red in 541

Fig. 2) not present in other Nazgul-like phages. Additionally, there is no clear evidence that the 542

small proteins with helix-turn-helix DNA-binding domains of phages Nazgul, Sano and Salvo 543


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(gp73, gp75 and gp70, respectively) are Cro-like proteins, as have been annotated in the other 544

Nazgul-like phages (30-33). Rather, these proteins may be involved in transcriptional regulation. 545

The lysogeny experiments conducted with phages Sano and Salvo support the conclusion that the 546

phages are virulent. Together, this suggests that the Nazgul-like phages, excepting AH2, have 547

diverged to become virulent, due to possible deletions and modifications of lysogeny-related 548

genes. 549

Despite the rather complete synteny of the Nazgul-like phage type genomes, the lysis 550

cassettes are examples of pronounced mosaicism. Among the seven genomes, there are at least 551

four novel, unrelated holins represented. Two of these (gp02 of Chi, with homologs in Redjac 552

and AH2; gp40 in Nazgul, with gp44 homolog in AH2) can be identified with confidence and 553

represent holins with class III topology. These are the first holins of class III that are not related 554

to the T holin of the paradigm phage T4. Moreover, all of the holins except for Sano must be 555

canonical “large hole” holins, rather than pinholins, since the respective endolysins lack 556

secretory or membrane localization signals and thus require large holin-mediated lesions for 557

escape across the bilayer (34). 558

In the Nazgul-like phage type, the most surprising finding is that the Salvo endolysin 559

gene 36, has a strong lipobox signal (Fig. 3), which would mandate that the enzyme is secreted 560

through the translocon, modified as a lipoprotein and sorted to the inner leaflet of the OM. 561

Heretofore, only two modes of endolysin regulation have been characterized in phages of Gram-562

negative hosts: canonical (cytoplasmic) endolysins, which are simply sequestered fully active in 563

the cytoplasm until liberated by holin-mediated hole formation; and SAR endolysins, which are 564

secreted in a membrane-tethered, inactive state by the sec system and activated by release from 565

the bilayer after pinholin-dependent membrane depolarization. Neither of these strategies would 566


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apply to gp36, since there is no obvious way that a holin could alter the IM in a way that would 567

directly affect the OM attachment of the gp36 endolysin molecules. It seems unlikely that gp36 568

endolysin accumulates as a fully active, OM-attached endolysin throughout the morphogenesis 569

period without causing premature lysis, so a novel mode of holin-mediated endolysin control 570

may be in place. 571

The second most surprising finding concerns the spanin genes of the Nazgul-like phage 572

type. There are two distinct families of i-spanin/o-spanin gene pairs: the Sano/Salvo family and 573

the Nazgul family, shared by the other four phages (Fig. 4). Unexpectedly, both of these families 574

have members in the overlapped and separated architectural classes (Fig. 2 and 3). The Salvo o-575

spanin gene is overlapped with its i-spanin gene, whereas in Sano, the genes are separated. 576

Similarly, the AH2 genes are separated, but the homologs of the AH2 i-spanin and o-spanin 577

genes in the other phages are all overlapped. Besides this being the first instances where spanin 578

gene families have members that cross an architectural class boundary (39), the alignments of the 579

homologs of differing class may provide a clue to the evolutionary process that underlies the 580

unexpected gene arrangements. When the two gene pairs are aligned, it can be seen that the 581

homologous regions of the i-spanin and o-spanins of Sano and Salvo are separated by ~100 bp in 582

the Sano separated genes (Fig. 5). The most parsimonious interpretation is that a deletion that 583

crossed the gene boundaries flanking this intervening DNA resulted in the overlapped 584

architecture seen in Salvo. Paradoxically, the deletion, which presumably included the DNA 585

encoding the lipobox of the mature lipoprotein, resulted in a fusion to the +1 reading frame of the 586

upstream i-spanin gene and a consequent extension of the o-spanin by 8 N-terminal residues. The 587

structures examined here suggest that the overlapped gene architecture evolved by a process of 588

successive deletion and reading frame fusion and supports our previous hypothesis that the 589


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spanin gene architectures originated with the “normal” separated gene format, in which both 590

proteins could evolve independently (39). 591

In medicine and agriculture, the search for innovative solutions to control disease is an 592

ongoing process. Phage therapy offers a novel and environmentally acceptable treatment for the 593

control of disease caused by X. fastidiosa. This is the first report for the propagation and 594

characterization of virulent phages for X. fastidiosa. The fact that virulent phages for X. 595

fastidiosa can now be easily propagated in a surrogate host (Xanthomonas) that grows in a 596

standard media allows for the efficient isolation, characterization, and propagation of virulent 597

phages that can be implemented as biocontrol agents for prevention or treatment of disease 598

caused by X. fastidiosa. The reported phages are therefore attractive candidates for the 599

development of phage cocktails to control disease caused by X. fastidiosa. 600

Acknowledgements. This project was funded by a grant to the Center for Phage 601

Technology and Texas A&M AgriLife Research from Otsuka Pharmaceutical Co., Ltd. 602

We thank Dr. Thomas J. Burr for kindly providing strains. We thank Dr. Michele Igo for 603

kindly providing plasmids used in this study. We would like to thank Dr. Jason Gill for his 604

critical reading of the manuscript and Guichun Yao for her technical assistance. We thank Dr. 605

Tsutomu Nishida for his support throughout this project. 606

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Kimura ET, Ferro ES, Harakava R, Kuramae EE, Marino CL, Giglioti E, Abreu 821

IL, Alves LM, do Amaral AM, Baia GS, Blanco SR, Brito MS, Cannavan FS, 822

Celestino AV, da Cunha AF, Fenille RC, Ferro JA, Formighieri EF, Kishi LT, Leoni 823

SG, Oliveira AR, Rosa VE, Jr., Sassaki FT, Sena JA, de Souza AA, Truffi D, 824

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sequences of Pierce's Disease and citrus variegated chlorosis strains of Xylella fastidiosa. 827

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80. Bhattacharyya A, Stilwagen S, Reznik G, Feil H, Feil WS, Anderson I, Bernal A, 829

D'Souza M, Ivanova N, Kapatral V, Larsen N, Los T, Lykidis A, Selkov E, Walunas 830

TL, Purcell A, Edwards RA, Hawkins T, Haselkorn R, Overbeek R, Kyrpides NC, 831

Predki PF. 2002. Draft sequencing and comparative genomics of Xylella fastidiosa 832

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81. Yanisch-Perron C, Vieira J, Messing J. 1985. Improved M13 phage cloning vectors 834

and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 835

33:103-119. 836

837

838


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FIGURE CAPTIONS 839

FIG 1 Morphology of phages Sano, Salvo, Prado and Paz. Transmission electron micrographs 840

show (A) Sano, (B) Salvo, (C) Prado and (D) Paz CsCl-purified phage particles. Samples were 841

negatively stained with 2% (wt/vol) aqueous uranyl acetate, as defined in Materials and 842

Methods. Bars, 100 nm. 843

FIG 2 Synteny of siphophages Sano and Salvo as compared to Burkholderia phage Nazgul, 844

Burkholderia phage AH2, Enterobacter phage Enc34, enteric bacteria phage Chi and 845

Providencia phage Redjac. Predicted genes are represented as boxes. Functional modules are 846

represented by different colors (color key in the bottom right). Functionally equivalent genes are 847

connected by solid lines (lysis genes are omitted for clarity). Homologous genes between Sano 848

and Salvo are connected by dotted lines, and similarity at the amino acid level is indicated by 849

color (color key in the bottom left). Apparent transcriptional units are indicated by roman 850

numerals. A distinct divergent transcriptional module in AH2 is indicated by a red box. 851

FIG 3 Lysis cassettes in the Nazgul-like phage type. Color, shade and hatching of each gene 852

indicate the following: (1) black boxes = unrelated genes of unknown function; (2) gray and 853

brown = o-spanin and i-spanin genes. Shades of gray and brown indicate sequence homology; 854

(3) diagonal hatching = endolysin genes. No sequence similarity is present among the five 855

endolysin genes shown. Blue box in Salvo endolysin gene indicates N-terminal lipoprotein 856

domain; (4) vertical hatching = genes encoding proteins with at least one TMD, potentially 857

holins or antiholins. Pink and yellow colors indicate sequence homology. Topological models of 858

Sano, Salvo, Nazgul, AH2 and Chi membrane proteins are shown to right. Genes are drawn to 859

scale. 860


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FIG 4 Alignments of o-spanin proteins from the Sano/Salvo and Nazgul/AH2/Chi/Enc34/Redjac 861

families. Alignments using the Feng–Doolittle (77) progressive algorithm are shown for the (A) 862

Sano/Salvo and (B) Nazgul/AH2/Chi/Enc34/Redjac families of o-spanin proteins. Residues are 863

highlighted with RASMOL (78) colors. Consensus residues were determined based on 100% 864

identity. 865

FIG 5 Spanin gene architecture. Putative deletion (diagonal hatched bar) that could convert the 866

Sano separated spanin gene pair into a Salvo-like overlapped gene pair. Grey connecting green 867

domains shows homologous domains of Salvo and Sano i-spanins (blue bars) and o-spanins 868

(yellow bars). Orange sub-bars: lipoprotein signal sequences in o-spanins. Pink sub-bars: TMD 869

in i-spanin. 870

FIG 6 Synteny of podophages Prado and Paz as compared to Pseudomonas phage phiKMV and 871

Caulobacter phage Cd1. Predicted genes are represented as boxes. Functional modules are 872

represented by different colors (color key in the bottom right). Functionally equivalent genes are 873

connected by solid lines. Homologous genes between Prado and Paz are connected by dotted 874

lines, and similarity at the amino acid level is indicated by color (color key in the bottom left). 875

FIG 7 Bacterial colony morphology as indicator of twitching motility. Colonies of indicated 876

strains were grown on appropriate solid medium modified with 1.2% agar, and incubated at 28˚C 877

[X. fastidiosa (5 day) and Xanthomonas (18 h)], as described in Materials and Methods. (A) 878

Wild-type X. fastidiosa strain Temecula; (B) Temecula ǻpilA; (C) Temecula ǻpilA 879

complemented chromosomally in trans with pilA; (D) wild-type Xanthomonas strain EC-12; (E) 880

EC-12 〉pilA; and (F) EC-12 〉pilA complemented with pilA containing plasmid, pSJA107. Bars, 881

0.1 mm. 882

883


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TABLE 1 Bacterial strains and plasmids used in this study 884

Strain or

plasmid

Genotype and relevant features a

Reference or source

X. fastidiosa

Temecula1 PD strain (subsp. fastidiosa) (ATCC 700964) (79)

Ann-1 Oleander isolate (subsp. sandyi) (ATCC 700598) (80)

Dixon Almond isolate (subsp. multiplex) (ATCC 700965) (80)

XF53 Grape isolate (subsp. fastidiosa) (5)

XF54 Grape isolate (subsp. fastidiosa) (5)

XF95 Oleander isolate (subsp. sandyi) (5)

XF134 Grape isolate (subsp. fastidiosa) This study




XF15-1 Temecula1; 〉pilA::Kmr This study

XF15-1-1 XF15-1, NS1::Cmr pilA This study

TM1 Temecula1; PD1693::Tn5 (26)

tonB1 Temecula; PD0843::Tn5 (52)

Xanthomonas

EC-12 Xanthomonas sp., rice isolate (ATCC PTA-13101) This study

North 40 X. axonopodis pv. citri, sweet orange isolate (N. Wang, U. Florida)

Ft. Basinger X. axonopodis pv. citri, sweet orange isolate (N. Wang, U. Florida)

Block 22 X. axonopodis pv. citri, sweet orange isolate (N. Wang, U. Florida)

Jal-4 X. euvesicatoria, jalapeno isolate Laboratory stock

Presidio-4 Xanthomonas sp., rice isolate Laboratory stock

EC-12-1 EC-12, unmarked deletion of pilA This study


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EC-12-1-1 EC-121, pMo168::pilA This study

E. coli

DH5gMCR F— Ɏ80lacZ〉M15 〉(lacZYA-argF) recA1 endA1gyrA96

thi-1hsdR17supE44 relAl deoR U169

Life Technologies, Inc.

(Gaithersburg, Md.)

Plasmids

pUC19 pMB1 replicon, cloning vector, Apr (81)

pXF004 RSF1010::Kmr, broad-host-range vector (25)

pSJA101 Isogenic to pUC19; Temecula pilA-downstream flanking

region cloned into XmaI and XbaI site

This study

pSJA102 Isogenic to pSJA101; Kmr cassette cloned into KpnI and

XmaI site

This study

pSJA103 Isogenic to pSJA102; Temecula pilA-upstream flanking

region cloned into SacI and KpnI site

This study

pAX1Cm Cmr cassette and multiple-cloning site on pAX1

backbone

(7)

pSJA104 Isogenic to pAX1Cm; Temecula pilA cloned into XbaI

and XhoI site

This study

pMo130 Suicide vector for allelic exchange; ColE1 ori, RK2 oriT,

xylE, sacB, Kmr

(10)

pSJA105 Isogenic to pMo130; EC-12 pilA-upstream flanking

region cloned into NheI and BglII site

This study

pSJA106 pSJA105 with EC-12 pilA-downstream flanking region

cloned into BglII and HindIII site

This study

pMo168 Replicative vector; ori pBBR1, mob+, xylE, Kmr (10)

pSJA107 pMo168::EC-12 pilA This study

a Ap, ampicillin; Cm, chloramphenicol; Km, kanamyacin. 885

886

887

888

889


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TABLE 2 General, physiological and structural characteristics of phages Sano, Salvo, Prado and 890

Paz 891

Phage

Feature Sano Salvo Prado Paz

Isolation host Temecula EC-12 Temecula Temecula

Capsid width (nm)a 64 (±2.1) 64 (±0.8) 69 (±1.4) 68 (±1.1)

Tail length (nm)a 204 (±2.3) 207 (±4.2) n/a n/a

Adsorption rate k (ml

cell-1 min-1)bc

Temecula (4.33 ± 0.28) 抜

10-12

(4.33 ± 0.28) 抜 10-

12

(4.33 ± 0.28) 抜

10-12

(4.33 ± 0.28) 抜

10-12

EC-12 (5.48 ± 0.23) 抜

10-10

(4.58 ± 0.30) 抜 10-

10

(7.26 ± 0.42) 抜

10-10

(5.98 ± 0.37) 抜

10-10

Burst size (PFU/cell)d 100 (±10.1) 112 (±8.3) 99 (±13.2) 104 (±12.5)

a Phage physical dimensions are the means of measurements of five virions, bracketed values 892

after each dimension indicate standard deviations. 893

b Phage adsorption rate constants k are the means of three independent experiments, bracketed 894

values indicate standard deviations. 895

c Phage stocks propagated on homologous host. 896

d Phage burst are the means of three independent experiments, bracketed values indicate standard 897

deviations. Xanthomonas strain EC-12 was used as host. 898

899

900

901


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TABLE 3 Phage host range 902

Strain Phage

Sano Salvo Prado Paz

X. fastidiosa

Temecula1 +a + + +

Ann-1 + + + +

Dixon + + + +

XF53 + + + +

XF54 + + + +

XF95 + + + +

XF134 - + + +

XF136 - + + -

XF140 + + + -

XF141 - + + -

Xanthomonas

EC-12 + + + +

Jal-4 - - + +

Presidio-4 - - + +

North 40 - - + -

Ft. Basinger - - + -

Block 22 - - + -

a Ability to form individual plaques. 903

904

905


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TABLE 4 Influence of bacterial host on efficiency of plating a 906

Phage

Production

host

Indicator

host

Sano Salvo Prado Paz

Temecula Temecula 1.0 1.0 1.0 1.0

Temecula EC-12 (1.2 ± 0.2) 抜

10-2

(1.4 ± 0.5) 抜

10-1

(2.3 ± 0.2) 抜

10-3

(3.1 ± 0.3) 抜 10-1

EC-12 EC-12 1.0 1.0 1.0 1.0

EC-12 Temecula (1.4 ± 0.7) 抜

10-3

(2.0 ± 0.2) 抜

10-1

(9.5 ± 0.5) 抜

10-2

(3.2 ± 0.3) 抜

10-1

a Data shown are the means of triplicate independent experiments ± SD. 907

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TABLE 5 General features of Sano, Salvo, Prado and Paz phage genomes 910

Phage

Feature Sano Salvo Prado Paz

Genome size (bp) 56,147 55,601 43,940 43,869

GC content (%) 62.3 63.0 63.0 60.2

Predicted No. of

genes

77 72 52 51

Coding density (%) 95.4 96.9 96.0 93.9

Genomic termini 12 bp 5’-overhang 12 bp 5’-overhang 619 bp DTRa 675 bp DTR

a DTR, Direct terminal repeats. 911

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TABLE 6 Predicted bacterial survivors based on MOIactual compared to measured bacterial 914

survivors of Xanthomonas strain EC-12 following exposure to phage Sano or Pradoa 915

Replicate

No.

MOIactual Predicted %

surviving cells

Measured %

surviving cells

Fold difference of

survivors vs. prediction

Sano 1 6.51 0.15 0.12 0.80

2 5.57 0.38 0.25 0.65

3 5.99 0 .30 0.24 0.80

Prado 1 5.40 0.45 0.38 0.80

2 5.39 0 .45 0.49 1.08

3 5.52 0.40 0.37 0.92

a Predicted survivors were calculated from the Poisson distribution for the measured MOIactual. 916

Data shown are from three independent replicate experiments. 917

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1 Characterization of Novel Virulent Broad-Host Phages of Xylella ...

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