The p40* adhesin pseudogene of Mycoplasma bovis

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Veterinary Microbiology 104 (2004) 213–217

Short communication

The p40* adhesin pseudogene of Mycoplasma bovis

Anne Thomasa,1, Annick Lindena, Jacques Mainila, Isabelle Diziera,Joel B. Basemanb, Thirumalai R. Kannanb, Benedicte Fleuryc,

Joachim Freyc, Edy M. Vileic,*

aDepartment of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liege,

B43A, Sart Tilman, 4000 Liege, BelgiumbDepartment of Microbiology and Immunology, University of Texas Health Science Center at San Antonio,

San Antonio, TX 78229, USAcInstitute of Veterinary Bacteriology, University of Bern, Langgass-Strasse 122, Postfach, 3001 Bern, Switzerland

Received 6 April 2004; received in revised form 2 September 2004; accepted 16 September 2004

Abstract

An analogue of the adhesin gene p40 of Mycoplasma agalactiae was found in Mycoplasma bovis. Nucleotide sequence

analysis of the p40* gene in M. bovis revealed the presence of a large deletion involving a frameshift that causes premature

truncation of the translated protein, indicating that p40* exists as a pseudogene in M. bovis.

# 2004 Elsevier B.V. All rights reserved.

Keywords: Mycoplasma bovis; p40* gene; Pseudogene; Evolution; Adhesin

1. Introduction

Mycoplasma bovis is the most important myco-

plasma species in cattle in countries free of contagious

bovine pleuropneumonia (Burnens et al., 1999; Brice

et al., 2000; Kusiluka et al., 2000; Byrne et al., 2001;

Thomas et al., 2002). Adherence to host cells is a

prerequisite for colonization and infection (Razin et

al., 1998). Several proteins such as P26 and Vsps

* Corresponding author. Tel.: +41 31 631 2369;

fax: +41 31 631 2634.

E-mail address: [email protected] (E.M. Vilei).1 Present address: Department of Morphology and Pathology,

Faculty of Veterinary Medicine, University of Liege, B43A, Sart

Tilman, 4000 Liege, Belgium.

0378-1135/$ – see front matter # 2004 Elsevier B.V. All rights reserved

doi:10.1016/j.vetmic.2004.09.009

(variable surface proteins) are involved in cytadher-

ence of M. bovis (Sachse et al., 1996, 2000). Recent

results suggest that other proteins could also be

implicated in the first step of infection (Thomas et al.,

2003). M. bovis (previously called Mycoplasma

agalactiae subsp. bovis) was differentiated from

Mycoplasma agalactiae by 16S rRNA and uvrC

genes analysis (Mattsson et al., 1994; Subramaniam et

al., 1998; Thomas et al., 2004). However, these two

mycoplasma species share several antigens (Rasberry

and Rosenbusch, 1995) and genes such as vspA

(Flitman-Tene et al., 1997, 2000) and the tyrosine–

recombinase gene (Ron et al., 2002), and presence of

insertion sequence (IS) elements (Pilo et al., 2003). In

M. agalactiae, functional analysis of the P40 protein

.

A. Thomas et al. / Veterinary Microbiology 104 (2004) 213–217214

by means of monospecific, polyclonal antibodies

directed against mature P40 revealed that it is involved

in M. agalactiae adherence to lamb cells and is strongly

immunogenic (Fleury et al., 2002). In the present study,

we focused our work on studying the presence and the

expression of a p40-like gene in M. bovis.

2. Characterization of the p40-like

gene in M. bovis

Genomic Sau3AI fragments of sizes between 1.0

and 8.0 kb from the M. bovis isolate 2610P7 (Table 1)

were cloned into the BamHI site of pBluescriptII

SK(+). Ligation products were transformed into XL1-

Blue MRF’ Escherichia coli. Colony screening with

the M. agalactiae specific digoxigenin-11-dUTP

(DIG)-labeled p40 probe was performed at 55 8C,

the p40 probe being prepared with oligonucleotide

primers MagaP13a-L2 (50-TGTCAAAAATACAAA-

TCTAGGTG-30) and MagaP13a-R2 (50-CTTTAACT-

TGTGATGAGGTATC-30) (Fleury et al., 2002), using

DNA from M. agalactiae strain 3990.

DNA analysis of one clone (56P7) from three

positive transformants was done by creation of

deletion subclones by exonuclease III digestion and

sequencing with primers complementary to the T3 and

T7 promoters flanking the cloning site of pBluescriptII

SK(+). As shown in Fig. 1, the 1,716-bp insert of

plasmid 56P7 (EMBL/GenBank accession number

AJ579372) contained two adjacent DNA regions that

presented greatest similarity to the p40 gene of M.

Table 1

Characteristics of M. bovis strains and isolates used in this study

Strain designation Passage number Years/period of isolation

PG45b unknown (>15) 1962

ML1c 7 before 1999

221/89 7 1980–1990

86p 7 1990–2000

39G 7 1990–2000

2610P7 7 1990–2000

2610P116 116 1990–2000

0435P7 7 1990–2000

0435P80 80 1990–2000

9585P7 7 1990–2000

9585P98 98 1990–2000

a BAL, bronchoalveolar lavage.b PG45, type strain of M. bovis.c ML1, rabbit isolate; all other strains are of bovine origin.

agalactiae strain 4212 (sequence AJ344231) (Fleury

et al., 2002). The first region (positions 439–523 of

sequence AJ579372) had an identity of 79.8% over 85

nucleotides with positions 19–102 of sequence

AJ344231, while the second region (positions 532–

1127) had an identity of 76.5% over 596 nucleotides

with positions 341–915 of AJ344231. The sequence

homology between the peptide coded by this 596-bp

DNA portion (in a single frame) and the P40 adhesin

of M. agalactiae was calculated as 65% identical and

77% similar amino acids. A 238-bp fragment harbored

in the nucleotide region 103–340 of the M. agalactiae

p40 sequence appeared to be completely deleted from

the p40 gene of M. bovis (Fig. 1). This deletion

introduced a frameshift leading to a premature stop

codon (position 557 of sequence AJ579372) imme-

diately downstream of the ATG start codon (position

503), resulting in a truncated protein of 18 amino

acids. Moreover, all three forward frames contained

several TAA stop codons, ruling out the possibility of

having any adhesin production. Thus, the p40-like

gene was designated as p40* pseudogene.

3. Distribution and sequencing of the p40*

pseudogene in M. bovis strains

A DIG-labeled p40* specific probe was constructed

using DNA from M. bovis strain PG45 and the primers

MBO-P40-L (50-ATGAAAACAAATAGAAAAATA-

AGTC-30) and MBO-P40-R (50-GTAGCTTTTTC-

CAATAATTTTCC-30). The 11 M. bovis strains/

Country of origin Sourcea Disease

USA Milk Mastitis

France Lung Bronchopneumonia

Germany Milk Without symptoms

Belgium Milk Mastitis

Belgium BAL Bronchopneumonia

UK Joint fluid Arthritis

UK Joint fluid Arthritis





A. Thomas et al. / Veterinary Microbiology 104 (2004) 213–217 215

Fig. 1. Genetic map of the p40 locus in M. agalactiae and M. bovis. The physical map of the 1716-bp insert in clone 56P7 obtained from M. bovis

isolate 2610P7 is shown together with the homologous locus in M. agalactiae strain 4212. The two partial ORFs in the 1716-bp insert of M. bovis

are indicated with interrupted arrows at both extremities showing the direction of translation. A grey, orientated pentagon represents the p40*

pseudogene. Two segments showing similarity with p40 of M. agalactiae are depicted, whereby thin dotted lines align homologous genomic

sections between the two mycoplasmal species. Percentages of identity are indicated. Sau3AI sites for cloning of the 1716-bp insert are

represented at both extremities. The positions of the oligonucleotide primers derived from M. bovis used in this work are depicted as small

arrowheads.

isolates tested (Table 1) (Ball et al., 1994; Sub-

ramaniam et al., 1998; Thomas et al., 2003) were

grown in modified Hayflick broth medium for 24–48 h

(Ball et al., 1994). M. agalactiae strains 3990 and

4021, and Mycoplasma mycoides subsp. mycoides SC

type strain PG1 were used as controls. Mycoplasmal

genomic DNA was extracted by the phenol/chloro-

form method as described previously (Su et al., 1990)

and digested by EcoRV, a restriction enzyme not

Fig. 2. Detection of the p40* pseudogene in the EcoRV-digested genomic D

SC type strain PG1 and M. agalactiae strain 3990. Std., molecular mass

cutting within the probe sequence. Southern blotting

was performed at 68 8C with the p40* probe following

standard protocols (Ausubel et al., 1999). The results

revealed the presence of a single chromosomal copy of

the p40* pseudogene among the 11 M. bovis strains/

isolates. Nine isolates presented a same pattern with a

reacting band at around 4.4 kb, whereas PG45 and

2610P116 showed larger DNA fragments (Fig. 2). No

hybridization of the p40* probe with the DNA from M.

NA from 11 M. bovis strains/isolates, M. mycoides subsp. mycoides

standard.

A. Thomas et al. / Veterinary Microbiology 104 (2004) 213–217216

Fig. 3. Assessment of p40* expression in three M. bovis strains.

Total antigen of M. agalactiae strain 4021 and approximately 1 mg

of recombinant P40-His (Fleury et al., 2002) were used as controls.

mycoides subsp. mycoides SC strain PG1 and from M.

agalactiae strain 3990 was observed.

The sequences from eight M. bovis isolates were

obtained with primers MBO-P40-L and MBO-P40-R.

Sequence comparison of the resulting 797-bp segments

revealed that isolates 2610P7, 2610P116 and 9585P7

(sequences AJ810388, AJ810389 and AJ810386, resp-

ectively) shared an identical sequence to that of the

1716-bp insert (AJ579372) despite their size differ-

ence in the EcoRV-digested genomic fragments. The

four strains PG45, ML1, 221/89 and 86p (seque-

nces AJ810390, AJ810392, AJ810391 and AJ810387,

respectively) presented a unique C–T substitution

corresponding to position 677 of AJ579372, while

isolate 0435P7 (sequence AJ810385) presented a

C–T substitution corresponding to position 703 of

AJ579372. The different band size observed for PG45

and 2610P116 could thus be related to insertion/

deletion event(s) outside of or flanking the p40* gene.

There were no further differences that could allow any

M. bovis strain to express a P40-like protein.

4. Assessment of an eventual expression of

a P40 analogue in M. bovis

Total antigen (10 mg) from mycoplasmas was

treated as already described (Fleury et al., 2001).

Immunoblotting was carried out with the rabbit

monospecific serum anti-P40 of M. agalactiae

(1:1000) (Fleury et al., 2002). The serum was reacted

with whole-cell proteins of three M. bovis strains but

no proteins could be detected (Fig. 3). This was

consistent with the finding that adherence to embryo-

nic bovine lung cells (Thomas et al., 2003) was

not reduced by pre-incubation of M. bovis with the

anti-P40 serum (data not shown). As expected, the

M. agalactiae strain 4021 reacted with the mono-

specific P40 antibody by showing a band at approxi-

mately 40 kDa.

5. Concluding remarks

Pseudogenes are abundant in most organisms, but

their function is still unclear, and they are thought to

be simple molecular fossils (Lee, 2003). Normally, a

pseudogene shows some differences if compared to its

functional equivalent (Feavers and Maiden, 1998). In

fact, silent genes can undergo mutations more

frequently than expressed genes since they do not

undergo any phenotypic selective pressure (Ophir and

Graur, 1997; Petrov and Hartl, 2000; Fleury et al.,

2001). This is the case for p40*, whereby the similarity

over the whole p40 gene between M. agalactiae and

M. bovis (p40* pseudogene) is below 65%. As

comparisons, the nucleotide identity between the

two species for uvrC is 83%, for the tyrosine

recombinase gene is 81% and for IS elements is

93%. Moreover, the conserved 50 part of vspA exhibits

92% identity between the two species, whereas p40

and p40* both display two conserved regions with only

76–80% identity (Fig. 1). An unusual feature of the

p40* pseudogene in M. bovis is the high number of

intragenic stop codons in all three forward frames,

suggesting a target selection for inactivation of p40*

that has not been deleted from the genome. The

presence of p40* in M. bovis as pseudogene, in

contrast to the ovine pathogen M. agalagtiae where

the P40 adhesin is expressed, could lead to the

speculation that the bovine host may lack suitable

receptors for a P40 homologue. In further studies it

would certainly be interesting to identify such P40

receptor in ovine cells and check its presence or

absence in bovine cells.

A. Thomas et al. / Veterinary Microbiology 104 (2004) 213–217 217

Acknowledgements

The authors are grateful to Dr. Ayling (Veterinary

Laboratories Agency, Weybridge, Surrey, UK), Dr.

Ball (DARDNI, Belfast, UK), Dr. Poumarat (AFSSA,

Lyon, France), Dr. Sachse (BGVV, Jena, Germany)

and Dr. Blanchard (INRA, Bordeaux, France) for

providing M. bovis strains. This study was possible

thanks to the grant from the Belgian ‘Ministere de

l’Agriculture’ (convention 6039).

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The p40* adhesin pseudogene of Mycoplasma bovis

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