# The oppD Gene and Putative Peptidase Genes May Be Required for Virulence in Mycoplasma gallisepticum.

Chi-Wen Tseng, Chien-Ju Chiu, Anna Kanci, Christine Citti, Renate

Rosengarten, Glenn F Browning, Philip F Markham

► To cite this version:

Chi-Wen Tseng, Chien-Ju Chiu, Anna Kanci, Christine Citti, Renate Rosengarten, et al.. The oppD Gene and Putative Peptidase Genes May Be Required for Virulence in Mycoplasma gallisepticum.. Infection and Immunity, 2017, 85 (6), 10.1128/IAI.00023-17. hal-01591746

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1 The *oppD* gene and putative peptidase genes may be required for virulence in

2 *Mycoplasma gallisepticum*

3

4

5 Chi-Wen Tseng¹, Chien-Ju Chiu¹, Anna Kanci¹, Christine Citti²,³, Renate Rosengarten³,

6 Glenn F. Browning¹* and Philip F. Markham¹

7

8 ¹Asia-Pacific Centre for Animal Health, Faculty of Veterinary Science, The University

9 of Melbourne, Parkville, Victoria 3010, Australia

10 ²INRA, ENVT, UMR 1225, 31076 Toulouse, France

11 ³Institute of Bacteriology, Mycology and Hygiene, University of Veterinary Medicine,

12 Vienna, A-1210, Vienna, Austria.

13

14 *Corresponding author. Mailing address: Veterinary Preclinical Centre, Faculty of

15 Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia.

16 Phone: 61 3 8344 7342. Fax: 61 3 8344 7374 Email: glenfb@unimelb.edu.au

17

18 Running title: Virulence genes in *Mycoplasma gallisepticum*

19
## 20 Abstract

Relatively few virulence genes have been identified in pathogenic mycoplasmas, so we used signature-tagged mutagenesis to identify mutants of the avian pathogen *Mycoplasma gallisepticum* with reduced capacity to persist *in vivo* and compared the virulence of selected mutants in experimentally infected chickens. Four mutants had insertions in one of the two incomplete *opp*ABCDF operons and a further three had insertions in distinct hypothetical genes, two containing peptidase motifs and one a member of a gene family. The three hypothetical gene mutants and the two with insertions in *opp*D₁ were used to infect chickens and all five were shown to have reduced capacity to induce respiratory tract lesions. One *opp*D₁ mutant and the MGA_1102 and MGA_1079 mutants had greatly reduced capacity to persist in the respiratory tract and to induce systemic antibody responses against *M. gallisepticum*. The other *opp*D₁ mutant and the MGA_0588 mutant had less capacity than wild type to persist in the respiratory tract, but did elicit systemic antibody responses. Although *M. gallisepticum* carries two incomplete *opp* operons, one of which has been acquired by horizontal gene transfer, our results suggest that one of the copies of *opp*D may be required for full expression of virulence. We have also shown that three hypothetical genes, two of which encode putative peptidases, may be required for full expression of virulence in *M. gallisepticum*. None of these genes has previously been shown to influence virulence in pathogenic mycoplasmas.

Abbreviations: MB, mycoplasma broth; MA, mycoplasma agar; ST, signature-tagged; RSA, rapid serum agglutination

## 44 Introduction

*Mycoplasma gallisepticum* is the most important mycoplasmal pathogen in
poultry (1, 2) and a particularly useful model of mycoplasmal pathogenesis, in part as
a typical representative of the Pneumoniae phyogenic group, which includes the
important human pathogen *M. pneumoniae*. There have been relatively few studies
attempting to identify virulence genes in mycoplasmas, in part because of the limited
availability of tools for such studies. Signature-tagged mutagenesis (STM) is a
useful technique for identifying genes likely to be involved in virulence, as it enables
rapid comparison of populations of mutants used to inoculate animals with those
recovered from these animals during the course of infection, allowing identification of
mutants with reduced capacity to persist in experimentally infected animals (3, 4).

However the examination of only pools of mutants may result in a failure to
distinguish between a mutation in a gene that is essential *in vivo* and a mutation in a
gene that reduces the capacity of the mutant to compete with other mutants *in vivo*. In
addition, it is not possible to fully assess the virulence of individual mutants within
populations. Therefore STM needs to be complemented with direct comparisons of
isolated mutants for their capacity to infect, persist, and cause disease.

In a previous study we showed that several signature-tagged (ST) mutants of *M. gallisepticum* that were recovered infrequently from chickens inoculated with pools of mutants were significantly attenuated when assessed for their virulence in isolation (5).

These mutants contained insertions in genes encoding the cytadhesin GapA and its
accessory protein CrmA. Three other virulence-associated determinants in *M. gallisepticum*, *lpd*, *malF*, and *mslA*, which has recently been shown to encode a novel
oligonucleotide binding protein, have also been identified previously using transposon
mutagenesis (5-8). Two of these (*malF* and *mslA*) are members of ABC transporter
operons.

The aims of the study reported here were to characterize further ST mutants that
were recovered infrequently from chickens infected with a pool of mutants and to then
72 examine and compare the virulence of these mutants and their capacity to colonize the respiratory tract of experimentally infected chickens, with the aim of identifying additional genes that play a significant role in the pathogenesis of respiratory mycoplasmosis.

76

## 77 Methods and Materials

### 78 Bacterial strain and construction of mutants

79 The virulent *M. gallisepticum* strain Ap3AS was originally isolated from the air sacs of a broiler chicken with airsacculitis (9). It was cultured in modified Frey's broth (MB) containing 10% swine serum (10), as described previously (11).

81

82 Plasmid pISM 2062.2, carrying the transposon Tn4001mod, was used to construct a signature-tagged (ST) library, as described previously (5, 12-15). The transposon contained a gentamicin resistance gene, allowing selective culture. The presence of the different tags in the ST mutants was confirmed by polymerase chain reaction (PCR) using the P2/P4 primer set (Table 1) (5). The insertion site of the transposon in each mutant was determined by genomic DNA sequencing using the IGstmGenmeF3 primer (Table 1) and comparing the sequence to that of the *M. gallisepticum* strain Rlow genome (16).

83

84

85

86

87

88

89

90

### 91 Screening of ST mutants in infected birds

92 The experimental design has been described previously (5). Briefly, in the initial screening, 102 ST mutants labeled with 34 different signature tags were cultured, combined into three pools, each containing 34 different mutants with a distinct tags (Tables 2 and 3), and each pool used to infect 20 specific pathogen free (SPF) chickens by aerosol (17), with an additional 10 uninfected chickens included with each group to assess transmissibility of the mutants. Necropsies were conducted on 10

93

94

95

96

97
98 inoculated birds 14 days after infection and on the remaining 20 birds 28 days after infection. Gross air sac lesions were assessed for severity as described previously (18).
99
100 Swabs of the trachea and air sacs of each bird were assessed by culture in mycoplasma broth and on mycoplasma agar, as described previously (5), and the
101
102 signature tags present amplified by PCR using the P2/P4 primer pair if the broth culture showed signs of growth of M. gallisepticum. The tags present were identified
103
104 by dot blotting, as described previously (5). Blood samples were collected from all
105 birds before inoculation and before euthanasia and the serum assessed for antibody
106 against M. gallisepticum using commercial agglutination antigen (Intervet
107 International) in the rapid serum agglutination (RSA) test (19).
108

108 Dilutions of the ST mutants that were not re-isolated in the initial screening experiment were subjected to confirmatory screening to further reduce the number of candidate ST mutants for definitive testing. The mutants were separated into two pools, with the mutants in each pool distinguishable from each other by their distinct tags. Each pool contained a similar concentration of each mutant and each pool was used to infect 20 birds, as described above. In-contact controls were not included, but all 40 birds were placed into the same positive pressure fibreglass isolator so each group could be used as an in-contact group for the other. Necropsies and sample collection were performed as described above at 14 days after inoculation. Dot blot hybridization and ST mutant specific PCRs were performed to identify the ST mutants recovered.
111
112
113
114
115
116
117
118
119

120 Comparison of the persistence and virulence of selected mutants

121 Several mutants were detected infrequently in the second screening experiment, suggesting that the genes that were interrupted in these mutants may have a significant
122
123 influence on virulence. Two different oppD₁ ST mutants were recovered from birds at
124 distinct rates, suggesting that the different insertion sites may have differing impacts
125 on persistence *in vivo* and potentially on pathogenicity. To assess this, and to assess
126 the persistence and virulence of three mutants with insertions in hypothetical genes,
127 the pathogenicities and rates of colonization of five distinct mutants were compared.
128 Seven groups of 20 four-week-old SPF chickens were housed separately. Group 1 was
129 exposed to an aerosol of mycoplasma broth only, Group 2 to MGA_1102 ST mutant
130 03-1, Group 3 to MGA_0588 ST mutant 18-1, Group 4 to *oppD*<sub>1</sub> ST mutant 20-1,
131 Group 5 to MGA_1079 ST mutant 22-1, Group 6 to *oppD*<sub>1</sub> ST mutant 26-1 and the
132 7th group to wild type Ap3AS. All birds were subjected to necropsies 14 days after
133 infection, with samples collected as described above. Sections of the upper, middle
134 and lower trachea were also taken from each bird.

135 Dot blot hybridization and specific PCRs were used to identify ST mutants
136 reisolated in broth cultures. Because strain Ap3AS did not have a specific sequence
137 tag, dot blot hybridization could not be used for cultures from the positive control
138 group. A pair of PCR primers, STM13-KE-C'-1-Rev and STM13-KF-C, which
139 yielded a 400 bp product, were used to confirm the presence of Ap3AS (Table 1) (5).
140 Sections of the trachea were processed and stained for histological examination,
141 scored for mucosal lesions and the mucosal thickness measured (18). Statistical
142 analyses were conducted using Kruskall-Wallis tests to compare median tracheal
143 lesion scores, with the Dunn post-hoc test used to assess the significance of
144 differences between pairs of groups, and an analysis of variance and Tukey's test to
145 evaluate the mean tracheal mucosal thicknesses. Differences in the proportions of
146 birds with lesions, of birds with serum antibody against *M. gallisepticum*, and of birds
147 positive by culture for *M. gallisepticum* were assessed using Fisher's exact test (20). A
148 probability less than or equal to 0.05 was regarded as significant.
149
150 SDS-PAGE electrophoresis and Western blot analysis of ST mutants

151 The cells in a 1 ml sample of a mid-log phase culture of each of the ST mutants,
152 as well as the Ap3AS strain, were collected by centrifugation at 16,000 g for 5 min,
153 then resuspended in 1 x SDS-PAGE lysis buffer and incubated at 100°C for 5 min
154 before rapid cooling on ice. Total cell proteins were separated in a 12.5%
155 polyacrylamide gel together with molecular mass standards (Marker 12 Wide Range
156 Protein Standard, Novex) and then stained with Coomassie brilliant blue.

157 Whole cell proteins of MGA_0588ST mutant 18, *oppD*₁ mutant 20, *oppD*₁ mutant 26,
158 and *M. gallisepticum* strain Ap3AS were subjected to sodium dodecyl sulphate
159 polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and
160 immunostaining to detect differences in protein expressed in the mutants.

161 Total cell proteins of mutants with protein profiles that differed in Coomassie
162 stained gels were separated in a 10% polyacrylamide gel along with molecular mass
163 standards (PageRuler Prestained Protein Ladder, Thermo Scientific),
164 electrophoretically transferred onto a PVDF membrane (Immobilon™-P Transfer
165 Membrane, Millipore) and antigen-free sites blocked by overnight incubation in 5%
166 skim milk (Devondale) in PBS containing 0.1% Tween 20 (PBS-T) at 4°C. The
167 membrane was then washed three times (10 min each) in PBS-T and then incubated in
168 pooled sera collected from birds infected with Ap3AS (1:2000 in PBS-T), at room
169 temperature for 1 h with gentle rocking, again washed as above and then incubated for
170 1 h at room temperature in anti-chicken horseradish peroxidase conjugate (DAKO) at
171 a dilution of 1:2,000 in PBS-T. After again washing as above, bound conjugate was
172 detected using enhanced chemiluminescence (CD/DAB Substrate Kit, Thermo
173 Scientific) following the manufacturer's recommendations and results recorded
174 digitally using a BioRad Molecular Imager (Bio-Rad) according to the manufacturer's
175 instructions.
176

177 **Results**

178 **Identification of insertion sites in ST mutants**

179 The insertion points of the transposon in ST mutants examined in detail in this
180 study are shown in Table 2. The insertion points in the other ST mutants in the pools
181 are listed in Table 3. In most cases the insertion site for a specific tagged transposon
182 was the same in each of the three initial mutant pools. There were four ST mutants
183 that had a tagged transposon inserted within the *oppAB*₁*C*₁*D*₁F*₁ operon.
184 Transposons were located within *oppB*₁a in ST mutant 36-1 (and in 36-2 and 36-3)
185 and within *oppC*₁ in ST mutant 24-1 (and in 24-2 and 24-3). In ST mutant 20-1 (and
186 20-2 and 20-3) and 26-1 (and 26-2 and 26-3) the transposons were inserted at
187 different sites in *opp*D*₁. No mutants were obtained with insertions in the other copy of
188 the *opp* operon in *M. gallisepticum* (*oppB*₂*C*₂*D*₂F*₂). Three ST mutants, 03-1 (and 03-2
189 and 03-3), 18-1 (and 18-2 and 18-3) and 22-1 (and 22-2 and 22-3), had the transposon
190 inserted within genes encoding distinct hypothetical proteins.

191

192 **Initial screening**

193 **Pathological and serological findings**

194 No anti-*M. gallisepticum* antibodies was detected in the serum of any bird at the
195 time of inoculation. More sera were anti-*M. gallisepticum* antibody-positive at 4
196 weeks than at 2 weeks after infection, but no antibody response was detected in
197 uninoculated in-contact birds in any group (Table 4). Fewer birds had air sac lesions
198 at 4 weeks than at 2 weeks after inoculation and more severe air sac lesions were
199 generally observed at 2 weeks after infection (scores of 0.5 in Group A, 1.0 to 2.0 in
200 Group B and 0.5 to 2.5 in Group C). The severity of the lesions did not correlate
201 closely with the serology results, with some birds having high RSA scores but no
202 detectable air sac lesions.

203 *Identification of recoverable ST mutants*

204 The recovery rates of ST mutants from birds at 14 and 28 days after inoculation are summarized in Tables 4 and 5. The *oppD*₁ ST mutant 20 (-1/-2/-3) was the mutant most frequently recovered from the air sacs, as well as from the tracheas, and was isolated at high frequency from all three groups. It was also recovered from the air sacs of one in-contact control bird in Group A 4 weeks after inoculation. In contrast, *oppD*₁ ST mutant 26 (-1/-2/-3) was only recovered from the trachea of one bird in Group A. Six and five isolations of *oppC*₁ ST mutant 24 (-1/-3) were made from the tracheas of birds in Groups A and C, respectively, whilst only one isolation of *oppB*₁a ST mutant 36 (-3) was made, from the trachea of a bird in Group C. MGA_0588 ST mutant 18 (-3) was only re-isolated from the air sacs of one bird in Group C, and from the trachea of one bird each in Groups A and C (-1/-3). Sixteen ST mutants, including ST mutants 03 (-1/-2/-3) (MGA_1102 interrupted) and 22 (-1/-2/-3) (MGA_1079 interrupted), were unable to be recovered from any bird in any of the groups (Table 4A).

218

219 *Confirmatory screening*

220 *Pathological and serological assessments*

221 No anti-*M. gallisepticum* antibody was detected prior to inoculation in any bird (Table 4B). Severe air sac lesions (score of 2.5) were seen in one bird, and mild lesions (0.5 and 1.0) in another two chickens in Group A. One bird had mild air sac lesions (1.0) in Group B. At 2 weeks after inoculation, 15/19 chickens in Group A and 14/19 birds in Group B had detectable antibody against *M. gallisepticum* (Table 4B).

226 *Re-isolation of ST mutants*

227 Birds in Group A were infected with a pool containing MGA_1102 ST mutant
228 03-1, and a pool that contained MGA_1079 ST mutant 22-1 was used to inoculate the
229 birds in Group B. A total of eleven ST mutants, including ST mutant 03-1, were
230 re-isolated from sixteen chickens in Group A and ten, including ST mutants 22-1 and
231 03-1, were re-isolated from eighteen birds in Group B (Table 4B).

232

233 *Infectivity and virulence analysis of selected ST mutants*

234 *Clinical signs and post mortem examination*

235 The prevalence and severity of lesions in chickens infected with individual
236 mutants are shown in Table 6. Air sac lesions were not seen in the uninfected control
237 birds (Group 1), or in birds exposed to aerosols of MGA_1079 ST mutant 22-1
238 (Group 5). Mild lesions (score of 0.25) were observed in one bird inoculated with
239 MGA_1102 ST mutant 03-1 (Group 2). Of the 20 birds infected with *oppD*₁ ST
240 mutant 26-1 (Group 6), four had mild lesions (0.5 to 1.0), whilst lesions were only
241 seen in the abdominal air sacs of five birds exposed to MGA_0588 ST mutant 18-1
242 (Group 3). Six of 20 birds had mild to severe lesions (0.5 to 2.5) in the group
243 infected with *oppD*₁ ST mutant 20-1 (Group 4), while mild to severe lesions (0.5 to
244 3.0) were seen in 11/18 birds infected with the virulent Ap3AS strain (Group 7).

245 *Antibody responses and re-isolation of ST mutants*

246 No anti-*M. gallisepticum* antibody was detected at the time of infection in the
247 serum of any birds (Table 6). Two weeks after exposure, antibody responses were
248 not detected in any of the birds in Groups 5 (MGA_1079 mutant 22-1 infected) or 6
249 (*oppD*₁ mutant 26-1 infected), whilst a response was detectable in only one bird in
250 Group 2 (MGA_1102 mutant 03-1 infected). In contrast, strong antibody responses
251 against *M. gallisepticum* were detectable in all the birds in Groups 3 (MGA_0588
252 mutant 18-1 infected) and 4 (*oppD*₁ mutant 20-1 infected). The uninfected control
253 birds (Group 1) did not develop any antibody against *M. gallisepticum* over the course
254 of the experiment, while all birds infected with the wild type Ap3AS strain (Group 7)
255 had strong antibody responses against *M. gallisepticum*.

256 *M. gallisepticum* was not isolated on MA plates inoculated with swabs of the air
257 sacs of any birds in Groups 2 (MGA_1102 mutant 03-1 infected) or 5 (MGA_1079
258 mutant 22-1 infected), but they were isolated from the trachea of two birds in Group 2
259 (MGA_1102 mutant 03-1 infected) and one bird in Group 5 (MGA_1079 mutant 22-1
260 infected). In Group 6 (*oppD*₁ mutant 26-1 infected), *M. gallisepticum* were isolated
261 from the air sacs of one bird and the trachea of 6 birds. *M. gallisepticum* were also
262 isolated from the trachea of 17/20 birds in Group 3 (MGA_0588 mutant 18-1 infected)
263 and of all the birds in Group 4 (*oppD*₁ ST mutant 20-1 infected), and also from swabs
264 of the air sacs of 5/20 birds in Group 3 and 7/20 birds in Group 4. In the positive
265 control group (Group 7), *M. gallisepticum* were isolated from the air sacs of 9/18
266 birds and the trachea of 17/18 birds (Table 6).

267 *M. gallisepticum* was recovered in MB inoculated with swabs of the air sacs of
268 one bird in Group 6 (*oppD*₁ mutant 26-1 infected), 7 birds in Group 3 (MGA_0588
269 mutant 18-1 infected) and 8 birds in Group 4 (*oppD*₁ mutant 20-1 infected).
270 *M. gallisepticum* were recovered from the trachea of birds in all six of the groups
271 exposed to ST mutants, with the number of colonized birds ranging from 3 in Group 5
272 (MGA_1079 mutant 22-1 infected) to 20 in Group 4 (*oppD*₁ mutant 20-1 infected).
273 The identity of the recovered *M. gallisepticum* ST mutants was confirmed using the
274 unique signature tags they carried (Table 6).

275 *Tracheal lesions and mucosal thicknesses*

276 Median tracheal lesion scores are shown in Table 7. The scores of birds in all
277 the mutant infected groups differed significantly from those of birds in the wild type
278 infected group (Group 7) (P < 0.0001). There was no significant difference between
279 the lesions scores for uninfected group (Group 1) and the mutant infected groups, with
280 exception of those for the upper tracheas of the birds infected with MGA_1102
281 mutant 03-1. The upper tracheal lesion scores of the birds infected with MGA_1102
282 mutant 03-1 (Group 2) did not differ from those of birds in Groups 3, 5 and 6, but did
283 differ significantly from those of birds in Group 4 (*oppD*₁ mutant 20-1 infected).

284 Mean tracheal mucosal thicknesses are shown in Table 7. The mean tracheal
285 mucosal thicknesses of the wild type infected birds were significantly different from
286 those of both the uninfected and the mutant infected birds (P < 0.0001). The mean
287 mucosal thicknesses in the middle and lower trachea did not differ significantly
288 between the uninfected group and any of the mutant infected groups (Groups 2 to 6).
289 However, they were significantly in the wild type infected group (Group 7). While
290 there was no significant difference between the tracheal mucosal thickness in the
291 upper trachea of the uninfected controls and Groups 3, 4, 5 and 6, the mean upper
292 tracheal mucosal thickness in the group infected with MGA_1102 mutant 03-1 was
293 significantly greater than that of the uninfected birds and those infected with *oppD*1
294 mutant 20-1. In summary, the wild type infected controls had significantly more
295 severe tracheal lesions, as assessed by histological lesion score or mucosal thickness,
296 than any of the groups infected with the ST mutants. Group 2 (MGA_1102 mutant
297 03-1 infected) was the most severely affected among the groups infected with the ST
298 mutants.

299

300 SDS-PAGE electrophoresis

301 Total cell proteins of the *M. gallisepticum* strains used in this study were
302 separated by SDS-PAGE and stained with Coomassie brilliant blue (Figure 1).
303 There were no detectable differences in the protein profiles of ST mutants 03-1
304 (MGA_1102 interrupted), 20-1 (*oppD*<sub>1</sub> interrupted), 22-1 (MGA_1079 interrupted),
305 24-1 (*oppC*<sub>1</sub> interrupted), 36-1 (*oppB*<sub>1a</sub> interrupted) and wild-type Ap3AS. However,
306 one protein band of about 40 kDa was absent in MGA_0588 mutant 18-1, whilst
307 *oppD*₁ mutant 26-1 contained an additional band of about 33 kDa compared to the
308 other mutants and wild-type Ap3AS.

309 Two protein bands, of 38 and 30 kDa, were more prominently detected in
310 MGA_0588 mutant 18-1 than in wild-type Ap3AS by sera from birds infected with
311 the parent strain Ap3AS (Figure 2, Figure S1). One protein of 43 kDa was not
312 detected in *oppD*₁ mutant 20-1 by sera from birds infected with Ap3AS and two
313 proteins of 40 kDa and 43 kDa were more prominently detected in *oppD*₁ mutant 26-1
314 than in wild-type Ap3AS, while proteins of 20 kDa were less prominent in the *oppD*₁
315 mutant 26-1 than in the Ap3AS strain (Figure 2, Figure S1).

316

317 Discussion

318 ABC transporters are a family of multi-domain membrane proteins that use
319 energy from hydrolysis of ATP to translocate solutes across cellular membranes (21,
320 22). The four domains within ABC transporters form a conserved core structure
321 composed of two transmembrane domains (TMDs) and two nucleotide-binding
322 domains (NBDs) (23-26).

323 There are two adjacent copies of the *opp* ABC transporter operon (*oppB*₁*C*₁*D*₁F*₁
324 and *oppAB*₂*C*₂*D*₂F*₂) in *M. gallisepticum* strain R (16). *OppB* and *OppC* are predicted
325 to be transmembrane proteins, while *oppD* and *oppF* are predicted to encode
326 ATP-binding proteins. The *oppB*₁ gene contains a frameshift mutation, *oppF*₂ appears
327 to have been truncated, and there is only one gene encoding *OppA*, the
328 substrate-binding lipoprotein (27), in *M. gallisepticum*. Thus it appears that *M.
329 gallisepticum* may contain two complementary partial copies of the *opp* operon, with
330 only a single full-length copy of *oppA*, *oppB* and *oppF*, but two full length copies of
331 *oppCD*.
One of these *opp* operons (MGA_0237, MGA_0235, MGA_0234, MGA_0232, and MGA_0230, or *opp*AB~2~C~2~D~2~F~2~) is most similar to that of *M. synoviae* strain 53 (GenBank accession number: AE017245), suggesting that the operon has been relatively recently acquired from *M. synoviae* or a closely related species within the Hominis phylogenetic group (which includes *M. hyopneumoniae*, *M. synoviae* and *M. pulmonis*) (Figure 3). Phylogenetic analysis of these genes in a number of fully sequenced genomes suggested that this operon was duplicated in a member of the Hominis group, and that these duplicated operons then diverged, with one subsequently transferred horizontally to an ancestor of *M. gallisepticum* and *Ureaplasma urealyticum*. *Ureaplasma urealyticum* appears to have lost its original copy of the operon and retained only the horizontally transferred copy, whilst *M. gallisepticum* has retained most of both copies of the operon (Figure 3). Studies in *M. hominis* have shown that OppA is a cytadhesin, and also that it binds oligopeptides, suggesting that this operon encodes a transporter responsible for import of oligopeptides (27-29). It is notable that duplication of the *opp*ABCDF operon by horizontal gene transfer has also occurred in the ruminant pathogens *Mycoplasma agalactiae* and *Mycoplasma bovis*, although this horizontal gene transfer event was clearly independent of that in *M. gallisepticum* (30-32).

In the studies presented here, four mutants had transposon insertions in genes in the same *opp* operon (in *opp*B~1~, *opp*C~1~, and *opp*D~1~), suggesting that mutations in some of the original *opp* genes may be tolerated (except possibly for the only full length *opp*F, *opp*F~1~), but that the *opp*B~2~C~2~D~2~ genes may be required for survival in *M. gallisepticum*. The transposon was inserted into the *opp*D~1~ gene in mutants 20-1 (-2/-3) and 26-1 (-2/-3), but in different positions (Table 2). Mutant 20-1 induced seroconversion in more birds than mutant 26-1, and also colonized a greater proportion of birds, even though they were both much less pathogenic than the wild
type strain. The insertion site in mutant 20-1 was close to the start of the gene, whilst in mutant 26-1 the first third of the gene may be able to be expressed. It is possible that the two different OppD proteins form heterodimers in the transport complex, and that, in the absence of expression of OppD₁ in mutant 20-1, OppD₂ could form functional homodimers. The partial copy of OppD₁ in mutant 26-1 may retain a capacity to form a heterodimer, but one that is less functional, resulting in less efficient transportation and thus the greater reduction in colonization and virulence seen in this mutant. Further work will be needed to establish whether the two copies of *oppD* in the genome can partially complement each other and whether they form heterodimers.

In ST mutant 36-1 (-2/-3), the transposon was inserted into the middle of the *oppB*₁a gene (MGA_0223). As there is a frame shift mutation within the *oppB*₁ gene, the two proteins predicted to be derived from it, OppB₁a and OppB₁b (MGA_0223 and MGA_0224), may not be fully functional, and thus *oppB*₂ may be the only fully functional copy in the genome. Although the *oppB*₁a mutant 36-1 was isolated very infrequently in the initial screening experiment, it was only assessed as a member of a pool, so the impact of mutation of the *oppB*₁a gene on infectivity and virulence requires further investigation. ST mutant 24-1 (-2/-3) had a transposon inserted into the *oppC*₁ gene (MGA_0221) about two-thirds along the length of the gene and this mutant could be recovered frequently from birds in the initial screening, suggesting that either the essential region of OppC may be within the first two-thirds of the gene, or that *oppC*₂ can complement the loss of *oppC*₁.

ST mutant 18-1, with an insertion in one of multiple paralogous genes for a hypothetical protein (MGA_0588), was able to infect birds, but was significantly less virulent than wild type Ap3AS. This suggests that the paralog disrupted in this mutant may have a significant influence on virulence, although not on colonization.
384 Mutant 22-1 (MGA_1079 interrupted) was relatively apathogenic and had a reduced capacity to colonize. ST mutant 03-1 (-2/-3) (MGA_1102 interrupted) was not recovered from any bird in the initial screen, but was able to be re-isolated in the confirmatory screen, albeit at a relatively low frequency, and was also able to spread, as it was recovered from birds in both groups. Although only one bird infected with this mutant developed detectable serum antibodies, it was the most virulent of all the mutants assessed in isolation, suggesting that mutants need to be assessed individually in hosts to examine the severity and extent of the lesions they induce if their virulence is to be fully assessed, and that capacity to induce seroconversion is not always a reliable indicator of virulence.

394 The *oppD*₁ mutant 20-1, which caused mild air sac lesions and minor damage in the trachea, induced a detectable anti-mycoplasma antibody response. The site of insertion of the transposon would allow expression of only the N-terminal 7.9% of the protein. In contrast, the less virulent *oppD*₁ mutant 26-1, could express the N-terminal 34% of the protein, and did not induce detectable anti-mycoplasma antibody. Western blotting detected differences between this mutant and wild-type Ap3AS strain, suggesting that the transposon insertion did result in changes in protein expression.

401 However, the protein profiles of the mutants did not provide clear evidence of the loss of expression of the proteins encoded by the genes that had been interrupted in each mutant, although some differences in the protein profiles were detected. The lack of a clear correlation between the site of insertion of the transposon and the changes in protein profiles may be attributable to the prevalence of post-translational cleavage in mycoplasmas, which complicates direct attribution of a protein band to a specific gene. In addition, assuming *oppD*₁ was expressed at sufficiently high levels to be detected in these assays, the presence of the product of the paralogous *oppD*₂ gene would be expected to obscure detection of changes in expression of *OppD*₁. In other bacteria
410 signals imported through the Opp system can have diverse effects on expression of
411 other genes, so the changes detected in these mutants could reflect similar effects in *M. gallisepticum*. We would predict that the two *oppD* mutants might have differing
412 patterns of protein expression as a result of the differing extents of their effects on the
413 function of the Opp transport system.
414

415 As it is possible that the mutants described here could have other mutations in
416 addition to the transposon insertion, complementation would be required to
417 definitively establish the role of the genes interrupted by the transposon in virulence
418 in *M. gallisepticum*. Unfortunately, stable extrachromosomal expression of introduced
419 genes, which would be needed to perform experimental infection studies, is difficult
420 to achieve with our current tools for genetic manipulation of *M. gallisepticum*. In
421 addition, the *oppD* insertions may have polar effects on expression of other genes in
422 the operon. However, the attenuation of virulence, albeit to different levels, by two
423 independent insertions in the *oppD* gene strongly suggests that this gene in particular
424 does play a role in virulence. Furthermore, recent studies have shown that this operon
425 can play a role in virulence in both Gram negative and Gram positive bacteria (33,
426 34).
427

427 Notably, genes MGA_1102 and MGA_1079, which, based on the studies
428 described here, may be required for full pathogenicity in *M. gallisepticum*, appear to
429 have been transferred horizontally from *M. synoviae*, as all their most closely related
430 homologs are found in mycoplasmas in the Hominis phylogenetic group. The protein
431 encoded by MGA_1102 has a zinc peptidase-like motif at its carboxyl end, while the
432 protein encoded by MGA_1079 has a trypsin-like peptidase motif at its carboxyl end.
433

433 Thus, in the work presented here, we used STM to screen pools of mutants of *M. gallisepticum* and identified a number of genes that may have a role in establishing
434 infection in the natural host of this pathogen, and on pathogenicity. Our results
435
436 suggested that genes that appear to have been acquired by horizontal gene transfer
437 from *M. synoviae*, MGA_1102 and MGA_1079, as well as the *oppD2* gene, which has
438 been complemented by horizontal gene transfer from *M. synoviae*, may play a
439 significant role in pathogenicity in this important pathogen, even though they are not
440 essential for colonisation and persistence.

441

442 Acknowledgments

443 This work was partially supported by funding from the Australian Egg
444 Corporation Ltd and the Australian Poultry CRC. We would like to thank Professor
445 Chris Minion, Professor Amir Hadji Noormohammadi, Ms Cheryl Colson and Mr
446 Tony Belfiore for their assistance with these studies.

447

448

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566

567
<table><caption>568 Table 1. Oligonucleotides used in this study</caption><thead><tr><td>Name</td><td>Use</td><td>5'-3' sequence (size in bp)</td></tr></thead><tbody><tr><td>P2</td><td>Signature tag region PCR</td><td>ATCCTACAAACCTCAAGCT (18)</td></tr><tr><td>P4</td><td>Signature tag region PCR</td><td>ATCCCATTCTAACCAAGC (18)</td></tr><tr><td>STM13-KE-C'-1-Rev</td><td>Wild-type Ap3AS PCR</td><td>CCACAGAGAACTTGAAG (17)</td></tr><tr><td>STM13-KF-C'</td><td>Wild type Ap3AS PCR</td><td>TATAAACCTGGTACGG (16)</td></tr><tr><td>IGstmGenmeF3</td><td>DNA sequencing and ST mutant PCR</td><td>GGACTGTTATATGGCCTTTTTGGATC (26)</td></tr><tr><td>STM03-B-Rev</td><td>ST mutant PCR</td><td>ACAGCTTGACGTTTTCCA (18)</td></tr><tr><td>STM18-Rev</td><td>ST mutant PCR</td><td>AGCAAAATTTCCACCCAAGA (20)</td></tr><tr><td>STM20-Rev</td><td>ST mutant PCR</td><td>GAGACATCAATCGCGGTTTT (20)</td></tr><tr><td>JZ-STM22</td><td>ST mutant PCR</td><td>CTCAATTGAAGAATTATATGATG (23)</td></tr><tr><td>STM26-Rev</td><td>ST mutant PCR</td><td>CATCCCACCTGAAAACTCGT (20)</td></tr></tbody></table>

569
<table><caption>Table 2. Transposon insertion sites in ST mutants used in this study (inocula A, B and C)</caption><thead><tr><td>ST mutant</td><td>Insertion site in genome (% of gene to insertion point)</td><td>Gene Name</td><td>Function of disrupted gene</td></tr></thead><tbody><tr><td>03-1/2/3</td><td>380999-381000 (16.0)</td><td>MGA_1102</td><td>Hypothetical membrane protein, zinc peptidase-like motif</td></tr><tr><td>18-1/2/3</td><td>980912-980913 (25.1)</td><td>MGA_0588</td><td>Hypothetical membrane protein, multiple paralogs</td></tr><tr><td>20-1/2/3</td><td>710237-710238 (7.9)</td><td>MGA_0220</td><td>Oligopeptide transporter ATP-binding protein OppD<sub>1</sub></td></tr><tr><td>22-1/2/3</td><td>365386-365387 (22.3)</td><td>MGA_1079</td><td>Hypothetical lipoprotein, trypsin-like peptidase motif</td></tr><tr><td>24-1/2/3</td><td>712236-712237 (67.8)</td><td>MGA_0221</td><td>Oligopeptide transporter permease OppC<sub>1</sub></td></tr><tr><td>26-1/2/3</td><td>710587-719588 (34.2)</td><td>MGA_0220</td><td>Oligopeptide transporter ATP-binding protein OppD<sub>1</sub></td></tr><tr><td>36-1/2/3</td><td>712897-712898 (45.4)</td><td>MGA_0223</td><td>Oligopeptide transporter permease (OppB<sub>1a</sub>)</td></tr></tbody></table>
<table><caption>571 Table 3 Classification of transposon insertion sites in ST mutants in this study</caption><thead><tr><th>ST mutant</th><th>Transposon insertion site</th><th>Function</th></tr></thead><tbody><tr><td colspan="3"><strong>Cell envelope – Membranes, lipoproteins</strong></td></tr><tr><td>11-1/-2/-3</td><td>MGA_0964</td><td>VlhA 4.02 (Hemagglutinin)</td></tr><tr><td>17-1/-2</td><td>MGA 0379</td><td>VlhA 3.02 (Hemagglutinin)</td></tr><tr><td>13-1/-2/-3</td><td>No match in strain R<sub>low</sub></td><td>Match to vlhA gene in draft Ap3AS sequence</td></tr><tr><td colspan="3"><strong>Cellular processes</strong></td></tr><tr><td>01-1/-2/-3</td><td>MGA 1142</td><td>OsmC-like stress-induced protein</td></tr><tr><td colspan="3"><strong>Translation – Protein synthesis</strong></td></tr><tr><td>19-1/-2/-3</td><td>MGA 0216</td><td>Elongation factor P (EF-P)</td></tr><tr><td colspan="3"><strong>Transport and binding proteins – ABC transport</strong></td></tr><tr><td>20-1/-2/-3</td><td>MGA 0220</td><td>ATP-binding protein OppD<sub>1</sub></td></tr><tr><td>24-1/-2/-3</td><td>MGA 0221</td><td>Permease protein OppC<sub>1</sub></td></tr><tr><td>26-1/-2/-3</td><td>MGA 0220</td><td>ATP-binding protein OppD<sub>1</sub></td></tr><tr><td>36-1/-2/-3</td><td>MGA 0223</td><td>Permease protein OppB<sub>1a</sub></td></tr><tr><td colspan="3"><strong>Intergenic regions</strong></td></tr><tr><td>10-1/-2/-3</td><td>MGA_0537 – MGA_0539</td><td>HsdM endonuclease – <i>hsd1</i> locus</td></tr><tr><td>16-1/-2/-3</td><td>MGA 0395 – MGA 0398</td><td>vlhA 3.09 – <i>malP</i></td></tr><tr><td>23-1/-2/-3</td><td>MGA 0379 – MGA 0380</td><td>vlhA 4.04 – vlhA 4.05</td></tr><tr><td>27-1/-2/-3</td><td>MGA 0226 – MGA 0230</td><td>Conserved hypothetical protein – <i>oppF</i> (OppF)</td></tr><tr><td>31-1/-2/-3</td><td>MGA 0071 – MGA 0073</td><td>vlhA 1.05 – Putative transposase</td></tr><tr><td>38-1/-2/-3</td><td>MGA_0518 – MGA_0519</td><td>Unique hypothetical protein – conserved hypothetical protein</td></tr><tr><td colspan="3"><strong>Others</strong></td></tr><tr><td>02-2</td><td>MGA 0549</td><td>Unique hypothetical protein</td></tr><tr><td>02-3</td><td>MGA 0145</td><td>Putative transposase</td></tr><tr><td>03-1/-2/-3</td><td>MGA 1102</td><td>Hypothetical membrane protein, zinc peptidase motif</td></tr><tr><td>04-3</td><td>MGA 0073</td><td>Putative transposase</td></tr><tr><td>07-1/-2/-3</td><td>MGA 0554</td><td>Hypothetical lipoprotein</td></tr><tr><td>09-1/-2</td><td>MGA_0662</td><td>Unique hypothetical membrane protein</td></tr><tr><td>12-1/-2/-3</td><td>16S rRNA</td><td>16S rRNA</td></tr><tr><td>14-1/-2/-3</td><td>MGA 0549</td><td>Hypothetical membrane protein unique to <i>M. gallisepticum</i> and <i>M.</i></td></tr><tr><td>18-1/-2/-3</td><td>MGA 0588</td><td>Hypothetical membrane protein, multiple paralogs</td></tr><tr><td>21-1/-2/-3</td><td>MGA 0554</td><td>Hypothetical lipoprotein</td></tr><tr><td>22-1/-2/-3</td><td>MGA 1079</td><td>Hypothetical lipoprotein</td></tr><tr><td>32-1/-2/-3</td><td>MGA 0817b</td><td>Unique hypothetical protein</td></tr><tr><td>35-1/-2/-3</td><td>MGA 0758</td><td>Putative hemoxygenase</td></tr><tr><td colspan="3"><strong>Not determined</strong></td></tr><tr><td>09-3</td><td>Multiple insertions</td><td></td></tr><tr><td>15-1/-2/-3</td><td>Multiple insertions</td><td></td></tr><tr><td>17-3</td><td>Multiple insertions</td><td></td></tr><tr><td>25-1/-2/-3</td><td>Multiple insertions</td><td></td></tr><tr><td>34-1/-2/-3</td><td>Multiple insertions</td><td></td></tr></tbody></table>

572 Mutants 02-1, 04-1, 06-1/2/3, 28-1/2/3, 33-1/2/3, and 39-1/2/3 have been described previously (5).

573
<table><caption>Table 4. Serology, air sac lesions and mutants detected in each group in the initial (A) and confirmatory (B) screens</caption><caption>A.</caption><thead><tr><th rowspan="2">Group</th><th colspan="4">Antibody against <i>M. gallisepticum</i>*</th><th colspan="3">Air sac lesions*</th><th colspan="2">Mutant-tag detected (number of times detected)</th></tr><tr><th>Week 0</th><th>Week 2</th><th>Week 4</th><th>Control</th><th>Week 2</th><th>Week 4</th><th>Control</th><th>Air sacs</th><th>Trachea</th></tr></thead><tbody><tr><td>A<sup>#</sup></td><td>0/30</td><td>7/9</td><td>8/9</td><td>0/8</td><td>2/10</td><td>0/9</td><td>0/8</td><td>oppD<sub>1</sub>-20 (2)<sup>§</sup></td><td>oppD<sub>1</sub>-20 (6), oppC<sub>1</sub>-24 (6), oppD<sub>1</sub>-26 (1)<br>MGA_0588-18(1)</td></tr><tr><td>B</td><td>0/31</td><td>6/10</td><td>9/11</td><td>0/10</td><td>2/10</td><td>1/11</td><td>0/10</td><td>oppD<sub>1</sub>-20 (1)</td><td>oppD<sub>1</sub>-20 (6)</td></tr><tr><td>C<sup>#</sup></td><td>0/29</td><td>5/10</td><td>9/10</td><td>0/7</td><td>4/10</td><td>2/10</td><td>0/7</td><td>oppD<sub>1</sub>-20 (7)</td><td>oppD<sub>1</sub>-20 (10), oppC<sub>2</sub>-24 (5), oppB<sub>1a</sub>-36 (1)</td></tr></tbody></table>

<table><caption>B.</caption><thead><tr><th rowspan="2">Group</th><th colspan="2">Antibody against <i>M. gallisepticum</i>*</th><th>Air sac lesions*</th><th colspan="2">Mutant-tag detected (number of times detected)</th></tr><tr><th>Week 0</th><th>Week 2</th><th>Week 2</th><th>Air sacs</th><th>Trachea</th></tr></thead><tbody><tr><td>A<sup>†</sup></td><td>0/20</td><td>15/19</td><td>3/19</td><td>MGA_1102-03 (1)</td><td>MGA_1102-03 (2)</td></tr><tr><td>B<sup>†</sup></td><td>0/20</td><td>14/19</td><td>1/19</td><td></td><td>MGA_1102-03 (1), MGA_1079-22 (2)</td></tr></tbody></table>

<footnote>*Number positive/number examined.</footnote>

<footnote>§Two inoculated and two in-contact birds died in Group A and two in-contact birds died in Group C prior to necropsy.</footnote>

<footnote>†including recovery from one bird in the in-contact group at 4 weeks after inoculation.</footnote>

<footnote>One bird died in Groups A and B before necropsy. Pool A contained mutant 03-1 and pool B contained mutant 22-1.</footnote>
<table><caption>582 **Table 5** Mutants detected in each experimental group in the initial screening and confirmatory screening</caption><thead><tr><th rowspan="2">Group</th><th colspan="2">Signature tags detected (number of times detected)</th></tr><tr><th>Air sacs</th><th>Trachea</th></tr></thead><tbody><tr><td>Initial - A*</td><td>16-1 (1)§, 20-1 (2)§</td><td>07-1 (3), 11-1 (1), 12-1 (4), 14-1 (1), 18-1 (1), 19-1 (2), 20-1 (6), 21-1 (1), 24-1 (6),<br>26-1 (1), 27-1 (2), 31-1 (1), 32-1 (1), 35-1 (1), 38-1 (1)</td></tr><tr><td>Initial - B</td><td>12-2 (1), 20-2 (1)</td><td>07-2 (1), 11-2 (1), 12-2 (3), 14-2 (1), 16-2 (1), 20-2 (6), 27-2 (1)</td></tr><tr><td>Initial - C*</td><td>11-2 (1), 12-2 (1), 16-2 (3), 18-2 (1), 20-2 (7),<br>27-2 (1)</td><td>01-3 (1), 02-3 (8), 11-3 (1), 12-3 (5), 13-3 (1), 14-3 (1), 16-3 (3), 18-3 (1), 20-3 (10),<br>24-3 (5), 27-3 (6), 36-3 (1), 38-3 (2)</td></tr><tr><td>Confirmatory - A<sup>#</sup></td><td>03-1 (1), 09-1 (1), 10-1 (2), 15-1 (1), 23-1 (2)</td><td>02-2 (4), 03-1 (2), 04-3 (1), 09-1 (3), 10-1 (10), 15-1 (1), 17-2 (12), 23-1 (2), 25-1 (1),<br>34-1 (1)</td></tr><tr><td>Confirmatory - B<sup>#</sup></td><td>09-3 (2), 17-3 (1), 23-1 (2)</td><td>02-2 (1), 03-1 (1), 09-3 (14), 10-1 (5), 15-1 (2), 17-3 (10), 23-1 (15), 25-1(3), 34-1 (2)</td></tr></tbody></table>

583 *One inoculated and two in-contact birds died in Group A and two in-contact birds died in Group C prior to necropsy.

584 §including recovery from one bird in the in-contact group at 4 weeks after inoculation.

585 The following tags were not detected in the initial screen: 02-2, 03, 04-3, 09, 10, 15, 17, 22, 23, 25 and 34.

586 <sup>#</sup>One bird each died in Groups A and B before necropsy.

587 Birds in Group A were inoculated with a pool containing ST mutants 02-2, 03-1, 04-1, 06-1, 09-1, 10-1, 15-1 and 17-2; birds in Group B were inoculated with

588 a pool containing ST mutants 04-3, 09-3, 17-3, 22-1, 23-1, 25-1, 33-1 and 34-1.

589 Mutants 02-1, 04-1, 06-1/2/3, 28-1/2/3, 33-1/2/3, and 39-1/2/3 have been described previously (5).

590

26
<table><caption>591 Table 6. Serology, air sac lesion scores and re-isolation rates in the infectivity and virulence study</caption><thead><tr><th rowspan="2">Group</th><th rowspan="2">Inoculum</th><th rowspan="2">Antibody against<br>M. gallisepticum*</th><th rowspan="2">Air sac lesions*<br>(range of scores)</th><th colspan="2">Positive by agar culture</th><th colspan="2">Positive by broth culture†</th></tr><tr><th>Air sacs</th><th>Trachea</th><th>Air sacs</th><th>Trachea</th></tr></thead><tbody><tr><td>1</td><td>Medium</td><td>0/20<sup>a</sup></td><td>0/20<sup>a</sup> (0 – 0)</td><td>0/20<sup>a</sup></td><td>0/20<sup>a</sup></td><td>0/20<sup>a</sup></td><td>0/20<sup>a</sup></td></tr><tr><td>2</td><td>Mutant 03-1</td><td>1/20<sup>a</sup></td><td>1/20<sup>a,b</sup> (0–0.25)</td><td>0/20<sup>a</sup></td><td>2/20<sup>a,b</sup></td><td>0/20<sup>a</sup></td><td>5/20<sup>b,c</sup></td></tr><tr><td>3</td><td>Mutant 18-1</td><td>20/20<sup>b</sup></td><td>5/20<sup>b</sup> (0.5–3.5)</td><td>5/20<sup>b,c</sup></td><td>17/20<sup>c</sup></td><td>7/20<sup>b</sup></td><td>19/20<sup>d</sup></td></tr><tr><td>4</td><td>Mutant 20-1</td><td>20/20<sup>b</sup></td><td>6/20<sup>b</sup> (0.5–4.0)</td><td>7/20<sup>c</sup></td><td>20/20<sup>c</sup></td><td>8/20<sup>b</sup></td><td>20/20<sup>d</sup></td></tr><tr><td>5</td><td>Mutant 22-1</td><td>0/19<sup>a</sup></td><td>0/19<sup>a</sup> (0–0)</td><td>0/19<sup>a</sup></td><td>1/19<sup>a,b</sup></td><td>0/19<sup>a</sup></td><td>3/19<sup>a,b</sup></td></tr><tr><td>6</td><td>Mutant 26-1</td><td>0/20<sup>a</sup></td><td>4/20<sup>a,b</sup> (0.5–1.0)</td><td>1/20<sup>a,b</sup></td><td>6/20<sup>b</sup></td><td>1/20<sup>a</sup></td><td>10/20<sup>c</sup></td></tr><tr><td>7</td><td>Wild type Ap3AS</td><td>18/18<sup>b</sup></td><td>11/18<sup>c</sup> (0.5–8.5)</td><td>9/18<sup>c</sup></td><td>18/18<sup>c</sup></td><td>10/18<sup>b</sup></td><td>15/18<sup>d</sup></td></tr></tbody></table>

<footnote n="592">592 *Number positive/number examined. One bird died in Group 5 and two birds died in Group 7 before necropsy.</footnote>

<footnote n="593">593 †The only signature tag detected in each group was that carried by the mutant used to infect that group. No signature tags were detected in cultures from</footnote>

<footnote n="594">594 groups 1 and 7.</footnote>

<footnote n="595">595 Data in the same column marked with the same superscript letter were not significantly different.</footnote>

<footnote>596</footnote>
<table><caption>597 Table 7 Tracheal lesion scores and mucosal thicknesses in birds in the infectivity and virulence study</caption><thead><tr><th rowspan="2">Group</th><th rowspan="2">Inoculum</th><th colspan="3">Median tracheal lesion score (minimum, maximum)</th><th colspan="3">Mean tracheal mucosal thickness ± SD* (µm)</th></tr><tr><th>Upper</th><th>Middle</th><th>Lower</th><th>Upper</th><th>Middle</th><th>Lower</th></tr></thead><tbody><tr><td>1</td><td>Medium</td><td>0.25 (0, 0.5)<sup>a</sup></td><td>0.25 (0, 0.5)<sup>a</sup></td><td>0 (0, 0.25)<sup>a</sup></td><td>50 ± 11<sup>a</sup></td><td>41 ± 8<sup>a</sup></td><td>37 ± 6<sup>a</sup></td></tr><tr><td>2</td><td>Mutant 03-1</td><td>1 (0.25, 3)<sup>b</sup></td><td>0.375 (0, 1.5)<sup>a</sup></td><td>0.25 (0, 1.5)<sup>a</sup></td><td>89 ± 45<sup>b</sup></td><td>54 ± 12<sup>a</sup></td><td>46 ± 22<sup>a</sup></td></tr><tr><td>3</td><td>Mutant 18-1</td><td>0.25 (0, 10)<sup>a,b</sup></td><td>0.25 (0, 1.5)<sup>a</sup></td><td>0.25 (0, 1.5)<sup>a</sup></td><td>62 ± 15<sup>a,b</sup></td><td>53 ± 15<sup>a</sup></td><td>49 ± 15<sup>ab</sup></td></tr><tr><td>4</td><td>Mutant 20-1</td><td>0.25 (0, 1.5)<sup>a</sup></td><td>0.125 (0, 1.5)<sup>a</sup></td><td>0.125 (0, 1.0)<sup>a</sup></td><td>56 ± 22<sup>a</sup></td><td>49 ± 23<sup>a</sup></td><td>51 ± 19<sup>a</sup></td></tr><tr><td>5</td><td>Mutant 22-1</td><td>0.5 (0, 1)<sup>a,b</sup></td><td>0.25 (0, 1)<sup>a</sup></td><td>0 (0, 0.25)<sup>a</sup></td><td>64 ± 13<sup>a,b</sup></td><td>49 ± 8<sup>a</sup></td><td>39 ± 8<sup>a</sup></td></tr><tr><td>6</td><td>Mutant 26-1</td><td>0.5 (0, 2)<sup>a,b</sup></td><td>0.25 (0, 1)<sup>a</sup></td><td>0 (0, 2)<sup>a</sup></td><td>64 ± 24<sup>a,b</sup></td><td>49 ± 14<sup>a</sup></td><td>48 ± 22<sup>a</sup></td></tr><tr><td>7</td><td>Wild type Ap3AS</td><td>1.5 (1, 3)<sup>c</sup></td><td>1.50 (0.5, 3)<sup>b</sup></td><td>1.50 (0.25, 3)<sup>b</sup></td><td>168 ± 70<sup>c</sup></td><td>153 ± 84<sup>b</sup></td><td>120 ± 56<sup>b</sup></td></tr></tbody></table>

598 *SD : standard deviation

599 Data in the same column marked with the same superscript letter were not significantly different.
FIGURE LEGENDS

600

Figure 1 Protein profiles of ST mutants. Total cell proteins were separated in a 12.5% polyacrylamide gel together with molecular mass standards and then stained with Coomassie brilliant blue. The position of the main differences in the protein profiles (in lanes 2 and 6) are indicated by arrows on the left. Lane 1, ST mutant 03-1 (MGA_1102 interrupted); lane 2, ST mutant 18-1 (MGA_0588 interrupted); lane 3, *oppD*₁ mutant 20-1; lane 4, ST mutant 22-1 (MGA_1079 interrupted); lane 5, *oppC*₁ mutant 24-1; lane 6, *oppD*₁ mutant 26-1; lane 7, *oppB*₁α mutant 36-1; Lane 8, wild-type strain Ap3AS. The positions of the broad range protein markers (Mark12™ Wide Range Protein Standard, Novex) are indicated on the left.

608

609

Figure 2. Western blot analysis of selected ST mutants. Whole cell proteins of ST mutants, and the parent strain Ap3AS were separated in 12.5% polyacrylamide gels, then transferred onto a PVDF membrane. The membranes were then probed with pooled sera from birds infected with Ap3AS diluted 1:2,000 (C). Lanes 1 and 5, MGA_0588 ST mutant 18-1; Lanes 3 and 6, *oppD*₁ ST mutant 20-1; Lanes 2, 4 and 8, Ap3AS; Lane 7, *oppD*₁ ST mutant 26-1. The positions of the broad range protein markers (PageRuler Prestained Protein Ladder, Thermo Scientific) are indicated on the left.

615

616

Figure 3. Phylogenetic analysis of *opp*ABCDF operons of *Mollicutes*. Trees were inferred using DNAmI (35) and then drawn using MEGA version 3.1 (36). Clustal W (37) aligned sequence data were analyzed using global rearrangement and randomized input orders to find the most likely tree. The optimal transition/transversion ratio was found to be 0.8 when analyzing the *oppB* genes, so this ratio was used for analysis of all other genes. A single mutation rate was assumed. MG, *Mycoplasma genitalium*; MGA, *Mycoplasma gallisepticum*; MMOB, *Mycoplasma mobile* 163K; mph, *Mycoplasma hyopneumoniae*; MPN, *Mycoplasma pneumoniae*; MS, *Mycoplasma synoviae*; MSC, *Mycoplasma mycoides* subspecies *mycoides* small colony type; MYPE, *Mycoplasma penetrans*; MYPU, *Mycoplasma pulmonis*; UU, *Ureaplasma urealyticum/parvum*.
<figure><img src="image_2.png" /><figcaption>Fig. 1</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Fig. 2</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Fig. 3</figcaption></figure>