The vlhA loci of Mycoplasma synoviae are confined to a restricted region of the genome

Joanne L. Allen, Amir H. Noormohammadi and Glenn F. Browning

Department of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia

Correspondence
Glenn Browning
glenfb{at}unimelb.edu.au


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mycoplasma synoviae, a major pathogen of poultry, contains a single expressed, full-length vlhA gene encoding its haemagglutinin, and a large number of vlhA pseudogenes that can be recruited by multiple site-specific recombination events to generate chimaeric variants of the expressed gene. The position and distribution of the vlhA pseudogene regions, and their relationship with the expressed gene, have not been investigated. To determine the relationship between these regions, a physical map of the M. synoviae genome was constructed using the restriction endonucleases SmaI, I-CeuI, BsiWI, ApaI and XhoI and radiolabelled probes for rrnA, recA and tufA. A cloned fragment encoding the unique portion of the expressed vlhA gene and two PCR products containing conserved regions of the ORF 3 and ORF 6 vlhA pseudogenes were used to locate the regions containing these genes on the map. The chromosome of M. synoviae was found to be 890·4 kb and the two rRNA operons were in the same orientation. Both the expressed vlhA gene and the vlhA pseudogenes were confined to the same 114 kb region of the chromosome. These findings indicate that, unlike Mycoplasma gallisepticum, in which the vlhA genes are located in several loci around the chromosome and in which antigenic variation is generated by alternating transcription of over 40 translationally competent genes, M. synoviae has all of the vlhA sequences clustered together, suggesting that close proximity is needed to facilitate the site-specific recombinations used to generate diversity in the expressed vlhA gene.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mycoplasma synoviae is a major pathogen of poultry throughout the world, causing chronic respiratory disease and arthritis in infected chickens. As with several other mycoplasmas, the generation of antigenic variants of major surface antigens is thought to be a significant contributor to the chronicity of M. synoviae infections (Behrens et al., 1994; Bhugra & Dybvig, 1992; Glew et al., 2000b; Markham et al., 1993, 1994; Neyrolles et al., 1999; Noormohammadi et al., 1997; Rosengarten & Wise, 1991; Theiss et al., 1993). In M. synoviae two of the major surface antigens are encoded by a single gene, vlhA, the product of which is cleaved post-translationally to yield a lipoprotein, referred to as MSPB, and a haemagglutinin, referred to as MSPA (Noormohammadi et al., 1997, 1998). A single strain of M. synoviae is able to generate a large number of variants of the vlhA gene at high frequency in vitro by site-specific recombination with a large suite of pseudogenes encoding different extents of the coding region of the expressed gene (Noormohammadi et al., 2000). Five different sites appear to be used for these site-specific recombinations, allowing generation of chimaeric expressed genes, with at least four different coding regions capable of being drawn from different pseudogenes.

Homologues of the vlhA gene have only been found in two other Mycoplasma species, M. gallisepticum and M. imitans, both of which are also poultry pathogens (Markham et al., 1999). However, these two species are phylogenically quite distant from M. synoviae and the mechanism used to generate antigenic diversity in vlhA genes of these two species is also quite distinct from that used in M. synoviae. In these species there are large numbers of translationally competent vlhA genes, each with an independent promoter controlled by a trinucleotide repeat element 5' to the –35 box (Glew et al., 1998, 2000a). Mapping studies and the recent determination of the genomic sequence of M. gallisepticum have shown that the vlhA genes are located in five different genomic regions, but to our knowledge there have been no studies examining the location of the vlhA loci in the M. synoviae genome (Baseggio et al., 1996; Papazisi et al., 2003). The differences between the vlhA loci in M. gallisepticum and those in M. synoviae, and the absence of homologues in species more closely related to these two organisms than they are to each other, have led to the suggestion that vlhA loci in at least one, and possibly both species, may have arisen by horizontal gene transfer (Markham et al., 1999).

The aim of the studies reported in this paper was to examine the distribution of the expressed vlhA gene and the vlhA pseudogenes in the genome of M. synoviae to allow further comparison with M. gallisepticum. As a genomic map of M. synoviae has not been deduced, an essential prerequisite was to map the genome of this species and locate some of the key housekeeping genes.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strain and culture conditions.
Mycoplasma synoviae strain WVU1853 was grown to mid-exponential phase in mycoplasma broth (MB) containing 10 % swine serum and 0·01 % NAD as modified by Whithear (1993) from the original formulation of Frey et al. (1968).

DNA extraction.
For pulsed-field electrophoresis, M. synoviae cells (1·5 ml) were harvested by centrifugation at 16 000 g, washed and resuspended in 300 µl PBS (140 mM NaCl, 2·7 mM KCl, 1·5 mM KH2PO4, 8·1 mM Na2HPO4). The genomic DNA was prepared in agarose blocks by mixing the cellular suspension with an equal volume of 2 % (w/v) Seaplaque agarose (BioWhittaker Molecular Applications). The mixture was cast into moulds and allowed to set before adding to ESP buffer (0·5 M EDTA, pH 8·0; 1 %, w/v, lauryl sarcosine; proteinase K, 1 mg ml–1). The blocks were incubated at 50 °C for 72 h. They were washed in TE (10 mM Tris/HCl, 1 mM EDTA; pH 8·0) and stored at 4 °C in EDTA (0·25 mM, pH 8·0) until digestion.

For continuous-field gel electrophoresis, used to characterize smaller fragments, and for PCR, DNA was extracted using the method described by Markham et al. (1993).

Restriction endonuclease digestion and electrophoresis.
Slices approximately 1 mm thick were taken from the agarose blocks, equilibrated in TE and incubated for 30 min at room temperature in 100 µl of the appropriate reaction buffer supplied with the restriction endonuclease. Fresh buffer (100 µl) and the restriction endonuclease (20 units) were added to the slices and the mixture was incubated at 4 °C for approximately 1 h. The reactions were then transferred to the appropriate digestion temperature and incubated for a further 18 h. For double digests where reaction conditions varied, the first enzyme used was the enzyme that required the lowest salt concentration. After the first digestion, the reaction buffer was replaced and the protocol repeated for the second enzyme.

The digested genomic DNA was separated by pulsed-field gel electrophoresis using the CHEF DRIII system (Bio-Rad). The agarose slices were loaded in a 1 % (w/v) DNA-grade agarose gel (Progen) in 0·5x TBE (45 mM Tris/HCl, 45 mM borate, 1·0 mM EDTA, pH 8·3). The electrophoresis conditions used to separate fragments of 20–400 kb were an initial switch time of 0·5 s, a final switch time of 40 s, a field strength of 6 V cm–1, 14 °C, and a fixed angle of 120° for 24 h. To separate fragments greater than 400 kb an initial switch time of 60 s, a final switch time of 120 s, a field strength of 6 V cm–1, 14 °C, and a fixed angle of 120° for 24 h were used. The molecular mass markers used for pulsed-field gel electrophoresis were the CHEF DNA size standard lambda DNA ladder (Bio-Rad) and phage lambda DNA digested with HindIII.

The size of fragments less than 20 kb was determined by continuous-field electrophoresis (0·8 % agarose gels, 1 V cm–1, 18 h) using phage lambda DNA digested with HindIII as a molecular mass marker. The gels were stained for 30 min in ethidium bromide solution (0·5 µg ml–1), destained with distilled water and visualized using an ultraviolet light transilluminator.

Southern blotting.
The DNA was transferred onto Hybond-N+ nylon membranes (Amersham Biosciences) for hybridization to radiolabelled probes. The gel was washed twice in HCl (0·25 M) for 10 min, and then washed in NaOH (0·4 M) for 15 min. DNA was adsorbed onto the membrane via capillary action over 24 h using 2 litres of a transfer solution (0·4 M NaOH, 1·5 M NaCl).

Preparation of probes.
The oligonucleotides listed in Table 1 were used to amplify gene sequences of interest from M. synoviae. All PCR reactions were carried out using 200 µM of each dNTP, 1·5 mM MgCl2, 1 µM of each primer and 1·25 units of Taq DNA polymerase (Promega) in a total volume of 25 µl. Reaction mixtures were incubated through 30 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s.


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Table 1. Oligonucleotides used to generate hybridization probes for this study

 
Plasmid pUC18 containing the entire vlhA gene, as described by Noormohammadi et al. (1998), was used as template for PCR of the MSPA and MSPB regions of the vlhA gene. The probe used to detect vlhA pseudogenes, which contained ORFs 3 and 6, was derived by PCR using oligonucleotide primers C32FB and C92POF from a 9·7 kb M. synoviae AflIII fragment of the M. synoviae genome inserted into pUC18 (Noormohammadi et al., 2000).

An 11 kb fragment containing part of the vlhA region of M. synoviae, derived from an MluNI restriction endonuclease digestion of genomic DNA, was cloned into pUC18 (C132) and used as a probe to refine the location of vlhA.

PCR products of the expected size were purified from gels using the Qiaex II gel extraction kit (Qiagen) and used as a template for random primed DNA labelling with [32P]dATP (Roche Diagnostics).

Southern blot hybridization.
Membranes were prehybridized in Church buffer (1 % BSA, 7 % SDS, 0·5 M Na2HPO4, 1 mM EDTA, pH 8·0) at 56 °C. The denatured radioactively labelled probe was added to the solution and the blot incubated for a further 18 h. The blot was then washed three times in 2x SSC, 0·1 % SDS (1x SSC contains 0·15 M NaCl, 15 mM trisodium citrate, pH 7·2) at 56 °C and exposed to radiographic film.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The size of the M. synoviae WVU1853 genome, 890·4±17·5 kb, was estimated by calculation of the fragment sizes after digestion with SmaI, as the size of all fragments could be estimated from the same electrophoretic gel. The sizes were estimated from four independent gels. The size of fragments yielded by other endonucleases was determined from a minimum of three gels (Table 2, Fig. 1).


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Table 2. Fragment sizes from digestion of the M. synoviae genome with single restriction endonucleases

 


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Fig. 1. A typical pulsed-field electrophoretic gel showing the fragments of M. synoviae after digestion with a panel of restriction endonucleases. The electrophoretic conditions used were an initial switch time of 0·5 s, a final switch time of 40 s, and a field strength of 6 V cm–1, with a fixed angle of 120° between the alternating fields. Separation was performed at 14 °C for 24 h.

 
The rrnA probe bound to both fragments of the I-CeuI digest, the largest fragments of the ApaI, BsiWI and XhoI digests, and fragments C and E of the SmaI digest. The close proximity of the SmaI sites to the I-CeuI sites enabled confirmation of the location of the key features of the map, namely the location of the rrnA genes and two SmaI fragments.

The tufA gene was found to be located between the two rrnA genes, a region of approximately 118 kb. The tufA probe bound to SmaI D, ApaI A, XhoI A, BsiWI A and I-CeuI B fragments. A second housekeeping gene, recA, was located within a region of 29 kb, adjacent to the fragments containing tufA and rrnA. The probe hybridized to the SmaI B, ApaI B, XhoI A, BsiWI B and I-CeuI A fragments.

The two regions of the expressed vlhA gene, MSPA and MSPB, were mapped to the same region of the genome, initially 280 kb. Both probes bound to the largest fragments of all digests. The PCR products containing the vlhA pseudogenes, ORFs 3 and 6, bound to the same area of the genome as the expressed vlhA gene probes. The conditions used for hybridization have been shown previously to allow binding of this probe to a large number of pseudogene sequences (Noormohammadi et al., 1998, 2000).

A series of restriction endonucleases, including BglI, EclXI, KpnI, PmeI, SacI and SalI, were used to further refine the location of vlhA. All of the enzymes except EclXI were able to cut the largest SmaI fragment. Double digestion with KpnI and SmaI generated a 114 kb fragment that hybridized to the expressed vlhA gene probes and also the C132 probe.

The C132 fragment was also shown to contain a single KpnI restriction endonuclease site. The presence of the vlhA gene in the C132 fragment was confirmed by Southern blot hybridization to the MSPA probe. Placement of the 114 kb fragment on the map, at one end of the SmaI A fragment, was also confirmed by the absence of a XhoI site within it.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This study has shown that the genome of M. synoviae, at 890·4±17·5 kb, is among the smaller of the mycoplasma genomes. The estimate was based on the five fragments yielded by SmaI digestion of the genomic DNA, which ranged from 344·3 to 19·8 kb. Estimation of the size of larger fragments from other digests was found to be less accurate with the pulsed-field electrophoretic conditions used to resolve the smaller fragments. Previously, Pyle et al. (1988) estimated the M. synoviae genome to be 900 kb, based on digestion of the genome using XhoI and ApaI and separation using a pulse time of 200 s. The electrophoretic conditions used by Pyle et al. (1988) to separate the fragments of the ApaI restriction digest were optimal for visualization of the ApaI A and hence the smaller fragments of ApaI B and C would not be resolved.

It was possible to pinpoint the exact location and orientation of the rrnA genes as I-CeuI cuts within the 23S rRNA gene. As both fragments of the I-CeuI digest hybridized to the rrnA probe, it was evident that the rRNA operons have the same orientation on the genome.

M. synoviae was found to have a SmaI cleavage site in close proximity to each of the I-CeuI sites, as digestion with these two enzymes concurrently did not result in any obvious differences in the fragment patterns compared to digests obtained using SmaI alone (Tables 2 and 3). The published sequence of the 23S rRNA gene of the phylogenically close species Mycoplasma pulmonis has a SmaI site approximately 20 bp upstream of the I-CeuI site, a difference too small to be observed on an agarose gel (Chambaud et al., 2001). The SmaI site found in the rrnB gene is also found in the phylogenically related Mycoplasma species M. hominis, M. pulmonis, M. fermentans, M. hyopneumoniae and M. flocculare. However these species, with the exception of M. hominis and M. fermentans, only contain a single copy of the rRNA genes. In M. fermentans the rrnAB operons are inverted with respect to each other, but in most strains of M. hominis that have been mapped they are organized as direct repeats, as in M. synoviae (Blanchard et al., 1996; Furneri et al., 2000; Huang et al., 1995; Ladefoged & Christiansen, 1992; Stemke et al., 1994).


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Table 3. Fragment sizes of the M. synoviae genome after digestion with two restriction endonucleases

 
The tufA, recA and rrnA genes were confined to an estimated 173 kb region of the M. synoviae genome (Fig. 2). In contrast, the 964 kb M. pulmonis genome contains a single set of rRNA genes, and the 16S rRNA and 23S rRNA genes (at 813 kb on the genome) are distant from the tufA gene (at 481 kb on the genome) and the recA gene (296 kb on the genome) (Chambaud et al., 2001).



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Fig. 2. Physical map of the M. synoviae genome (890±17·5 kb) showing the ApaI, BsiWI, I-CeuI, SmaI and XhoI restriction endonuclease cleavage sites and the location of the rrnA genes and the housekeeping genes, tufA and recA. The location of the vlhA region was more finely mapped by determining the placement of the KpnI site. The designation of the fragments (A, B, C, D or E) corresponds to those shown in Table 2.

 
Fragments of the two pseudogenes, ORFs 3 and 6, were used as a probe to detect the vlhA pseudogene loci. It seems likely that these probes detected all the vlhA loci in the genome because previous studies have shown that, using the same hybridization conditions as were employed here, this probe can bind to approximately 50 kb of the M. synoviae genome (Noormohammadi et al., 1998). Furthermore, sequencing of a 9·7 kb fragment of the genome, which is entirely composed of vlhA pseudogenes and includes ORFs 3 and 6, has shown that the highly conserved sequences within each pseudogene are contained within the ORF 3 and 6 regions. It can be assumed that these highly conserved sequences would enable detection of all the vlhA pseudogenes in the genome (Noormohammadi et al., 2000).

The confinement of both the expressed vlhA genes and the vlhA pseudogene family to a region of approximately 114 kb of the M. synoviae genome is in marked contrast to the arrangement of the M. gallisepticum vlhA (pMGA) multigene family (Baseggio et al., 1996; Papazisi et al., 2003). M. gallisepticum has been shown to have multiple copies of the genes distributed into five distinct loci throughout the genome. This difference in distribution reflects the different method used by M. synoviae to achieve genetic variation in its haemagglutinin gene. While control of expression of vlhA genes in M. gallisepticum clearly does not require the genes to be in close proximity, in M. synoviae, where variation depends on site-specific recombination, the entire gene family apparently needs to be located within the one genomic region, possibly within a single locus.

The derivation of the genomic map of M. synoviae and the location of the vlhA locus on this map are important steps in further understanding the mechanisms involved in genetic variation within this organism.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 26 November 2004; accepted 1 December 2004.



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