pMGA Gene Research Articles

α-Tocopherol Plays a Role in Photosynthesis and Macronutrient Homeostasis of the Cyanobacterium Synechocystis sp. PCC 6803 That Is Independent of Its Antioxidant Function

alpha-Tocopherol is synthesized exclusively in oxygenic phototrophs and is known to function as a lipid-soluble antioxidant. Here, we report that alpha-tocopherol also has a novel function independent of its antioxidant properties in the cyanobacterium Synechocystis sp. PCC 6803. The photoautotrophic growth rates of wild type and mutants impaired in alpha-tocopherol biosynthesis are identical, but the mutants exhibit elevated photosynthetic activities and glycogen levels. When grown photomixotrophically with glucose (Glc), however, these mutants cease growth within 24 h and exhibit a global macronutrient starvation response associated with nitrogen, sulfur, and carbon, as shown by decreased phycobiliprotein content (35% of the wild-type level) and accumulation of the nblA1-nblA2, sbpA, sigB, sigE, and sigH transcripts. Photosystem II activity and carboxysome synthesis are lost in the tocopherol mutants within 24 h of photomixotrophic growth, and the abundance of carboxysome gene (rbcL, ccmK1, ccmL) and ndhF4 transcripts decreases to undetectable levels. These results suggest that alpha-tocopherol plays an important role in optimizing photosynthetic activity and macronutrient homeostasis in Synechocystis sp. PCC 6803. Several lines of evidence indicate that increased oxidative stress in the tocopherol mutants is unlikely to be the underlying cause of photosystem II inactivation and Glc-induced lethality. Interestingly, insertional inactivation of the pmgA gene, which encodes a putative serine-threonine kinase similar to RsbW and RsbT in Bacillus subtilis, results in a similar increase in glycogen and Glc-induced lethality. Based on these results, we propose that alpha-tocopherol plays a nonantioxidant regulatory role in photosynthesis and macronutrient homeostasis through a signal transduction pathway that also involves PmgA.

Induction of a mycoplasma gallisepticum pMGA gene in the chicken tracheal ring organ culture model.

Genetic and molecular methods to investigate the pathogenesis of the poultry respiratory pathogen Mycoplasma gallisepticum are quite limited. Therefore, the objective of this study was to design and evaluate a functional genomics approach to identify M. gallisepticum genes involved in colonization of the poultry respiratory tract. To serve as a transcriptional reporter, a promoterless lacZ gene from Escherichia coli was cloned into the Tn4001 transposon. The transposon was used to randomly mutagenize the chromosome of the M. gallisepticum S6 strain, and a bank of 1386 transposon mutants containing lacZ fusions to mycoplasma chromosomal DNA was assembled. Each mycoplasma clone containing the lacZ reporter was independently screened in the chicken tracheal ring organ culture (TROC) model system for increased production of beta-galactosidase. A twofold or greater increase in beta-galactosidase was consistently observed for eight mutants. In one of the mutants, the transposon was inserted in a pMGA gene encoding a cell surface adhesin involved in hemagglutination. Therefore, these data indicate that screening of a M. gallisepticum transposon reporter bank with a chicken TROC model is useful for the identification of genes induced during poultry colonization and virulence.

Characterisation of Mycoplasma gallisepticum strains involved in respiratory disease in pheasants and peafowl

Two cases of Mycoplasma gallisepticum infection in different avian species in backyard gamebird operations in Slovenia were investigated. In the first case, M gallisepticum was associated with severe respiratory disease...

Haemagglutinins of pathogenic avian mycoplasmas

The pathogenic avian mycoplasmas, Mycoplasma gallisepticum , Mycoplasma synoviae , Mycoplasma meleagridis , Mycoplasma iowae and Mycoplasma imitans , synthesize haemagglutinins that are immunogenic, variably expressed, surface proteins. The haemagglutinins of M. gallisepticum (pMGA), M. synoviae (VlhA) and M. imitans are lipoproteins, encoded by related multigene families that appear to have arisen by horizontal gene transfer. M. gallisepticum also has genes encoding cytadhesins in its genome but these are present as a single copies, while the pMGA gene family contains 30 to 70 genes. The switch in expression of distinct pMGA genes (e.g. pMGA1.1 to pMGA1.9) generates antigenic variation, which is thought to be important in immune evasion but also has significance in the preparation of M. gallisepticum antigens for serological diagnosis. In the majority of M. synoviae strains, post-translational cleavage of the VlhA protein generates an amino-terminal part (the lipoprotein MSPB) and a carboxyl-terminal part (MSPA), which mediates binding to erythrocytes. The 5' vlhA gene region, which encodes proline-rich repeats in the amino-terminal part of MSPB, is highly polymorphic among M. synoviae strains. Insertions or deletions in the part of vlhA encoding the proline-rich repeats cause MSPB length variation in different M. synoviae strains. Recombination between the 5' vlhA gene and pseudogenes in the genome generates changes in antigenic determinants in the carboxyl two-thirds of the MSPB molecule, and in MSPA, resulting in changes in the domains involved in the binding of M. synoviae to erythrocytes. Variant haemagglutinins of M. gallisepticum (pMGA1.7) and M. synoviae (diverse VlhA forms) share sequences that may be responsible for antigenic cross-reactions between M. gallisepticum and M. synoviae . Shared epitopes have been demonstrated using specific antibodies against MSPB that also recognize proteins of M. gallisepticum and of M. iowae (serotype N). Size and antigenic variants have also been reported for M. meleagridis and M. iowae proteins, but it is not known if these are their haemagglutinins. Advances in the molecular characterization of M. gallisepticum (pMGA, pvpA ) and M. synoviae ( vlhA ) genes and their sequencing in numerous strains is likely to enable significantly improved epidemiological studies and improved tracing of M. gallisepticum and M. synoviae strains in different flocks.

Characterization of pMGA Genes from the F - Strain (Vaccine Strain) of Mycoplasma Gallisepticum

Our long term goal is to determine the role that pMGA hemagglutinins play in Mycoplasma gallisepticum F-strain (termed as F-strain for brevity) persistence in poultry. To do this however, we first had to identify the pMGA genes expressed by an isolate of the F-strain. To identify F-strain pMGA genes, a genomic library was constructed in bacteriophage lambda and screened with polyclonal antisera raised against F-strain surface antigens capable of inhibiting agglutination of chicke n red blood cells. One phage clone was identified, designated λ 9, which remained positive through additional rounds of screening and was sequenced. Sequence data analysis predicted one complete and two partial open reading frames (ORF) arranged in tandem within the 4.5 kb genomic insert, termed ORF 9.1, ORF 9.2 and ORF 9.3, which showed significant homology to the pMGA gene family of M. gallisepticum . The number of GAA trinucleotide repeats in the intergenic region of 9.3 ORF suggested that this ORF is expressed. The pMGA cDNAs amplified from the F-strain showed significant homology to the 9.3 ORF. One set of cDNAs however, differed from the 9.3 ORF sequence by a repeat of the sequence AACCAAA in the 5' end. We conclude that the F-strain vaccine isolate expresses pMGA gene variants, similar to what has been described in field strains of M. gallisepticum.

Trinucleotide GAA repeats dictate pMGA gene expression in Mycoplasma gallisepticum by affecting spacing between flanking regions.

The pMGA genes of the avian respiratory pathogen Mycoplasma gallisepticum encode a family of hemagglutinins that are subject to phase variation. A trinucleotide GAA repeat region is located upstream of the pMGA transcription start site. The length of the repeat region varies at a high frequency due to changes in the number of repeat units. Previous studies have shown that pMGA genes are transcribed when 12 GAA repeats are present but are not transcribed when the number of repeats is not 12. To further analyze the mechanism of gene regulation, the pMGA promoter region was modified either by deleting the nucleotides 5" of the GAA repeats or by inserting linkers of 10 or 12 bp at a position 3" of the repeats. The modified promoter region was fused to a promoterless lacZ gene and transformed into M. gallisepticum by using transposon Tn4001 as a vector. Transformants and successive generations of progeny were analyzed with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to monitor beta-galactosidase activity. For the transformants of M. gallisepticum containing the reporter with deletion of nucleotides 5" of the GAA repeats, GAA-dependent pMGA gene regulation was abolished. For the transformants containing the reporter with an addition of 10- or 12-bp linkers, lacZ was expressed only when eight GAA repeats were present. These data indicate that the nucleotides 5" of the GAA repeats as well as the spacing between the GAA repeats and sequences downstream (3") of the repeats are important for pMGA gene expression.

Sequence polymorphisms within the pMGA genes and pMGA antigenic variants in Mycoplasma gallisepticum.

Antigenic variants of Mycoplasma gallisepticum major surface lipoprotein, pMGA, are encoded by a large gene family. In this study sequence analyses of the PCR-amplified pMGA genes showed two types of sequences similar to the pMGA1.2 gene in M. gallisepticum strains. They differed in the sequence encoding a proline-rich region (PRR) at the N-terminus of the pMGA protein. The type A genes had sequences similar to the published pMGA1.2 gene sequence of strain S6, whereas the type B genes lacked the second repetitive segment encoding PTPN sequence within PRR and were similar to the published sequence of PG31 strain. Low in vitro passages of M. gallisepticum strains isolated recently in Slovenia from four avian species showed very different expression patterns of pMGA1.2 and pMGA1.9 genes. Among isogenic populations of S6(B) and IHB1 strains a high frequency of pMGA antigenic variants lacking an epitope for monoclonal antibody (mAb) 71 was found. Strain IHB1 clones, which synthesized pMGA recognized by mAb 71, transcribed pMGA genes whose partial sequence encoded the amino acid sequence (262)TNGDEPRSVS of the mAb 71 epitope. Other IHB1 clones synthesized pMGA variants with different isoelectric points, lacking the epitope for mAb 71, but expressing downstream epitopes for other mAbs. Our study suggests that a molecular basis for pMGA antigenic variation lies in the corresponding changes at the DNA level.

Sequence polymorphisms within the pMGA genes and pMGA antigenic variants in Mycoplasma gallisepticum

Antigenic variants of Mycoplasma gallisepticum major surface lipoprotein, pMGA, are encoded by a large gene family. In this study sequence analyses of the PCR-amplified pMGA genes showed two types of sequences similar to the pMGA1.2 gene in M. gallisepticum strains. They differed in the sequence encoding a proline-rich region (PRR) at the N-terminus of the pMGA protein. The type A genes had sequences similar to the published pMGA1.2 gene sequence of strain S6, whereas the type B genes lacked the second repetitive segment encoding PTPN sequence within PRR and were similar to the published sequence of PG31 strain. Low in vitro passages of M. gallisepticum strains isolated recently in Slovenia from four avian species showed very different expression patterns of pMGA1.2 and pMGA1.9 genes. Among isogenic populations of S6 B and IHB1 strains a high frequency of pMGA antigenic variants lacking an epitope for monoclonal antibody (mAb) 71 was found. Strain IHB1 clones, which synthesized pMGA recognized by mAb 71, transcribed pMGA genes whose partial sequence encoded the amino acid sequence 262TNGDEPRSVS of the mAb 71 epitope. Other IHB1 clones synthesized pMGA variants with different isoelectric points, lacking the epitope for mAb 71, but expressing downstream epitopes for other mAbs. Our study suggests that a molecular basis for pMGA antigenic variation lies in the corresponding changes at the DNA level.

A novel mechanism for control of antigenic variation in the haemagglutinin gene family of mycoplasma synoviae.

High-frequency phase and antigenic variation of homologous lipoprotein haemagglutinins has been seen in both the major avian mycoplasma pathogens, Mycoplasma synoviae and Mycoplasma gallisepticum. The expression and, hence, antigenic variation of the pMGA gene family (encoding these lipoproteins in M. gallisepticum) is controlled by variation in the length of a trinucleotide repeat motif 5' to the promoter of each gene. However, such a mechanism was not detected in preliminary observations on M. synoviae. Thus, the basis for control of variation in the vlhA gene family (which encodes the homologous haemagglutinin in M. synoviae) was investigated to enable comparison with its homologue in M. gallisepticum and with other lipoprotein gene families in mycoplasmas. The start point of transcription was identified 119 bp upstream of the initiation codon, but features associated with control of transcription in other mycoplasma lipoprotein genes were not seen. Comparison of three copies of vlhA revealed considerable sequence divergence at the 3' end of the gene, but conservation of the 5' end. Southern blot analysis of M. synoviae genomic DNA revealed that the promoter region and part of the conserved 5' coding sequence occurred as a single copy, whereas the remainder of the coding sequence occurred as multiple copies. A 9.7 kb fragment of the genome was found to contain eight tandemly repeated regions partially homologous to vlhA, all lacking the putative promoter region and the single-copy 5' end of vlhA, but extending over one of four distinct overlapping regions of the 3' coding sequence. Examination of sequential clones of M. synoviae established that unidirectional recombination occurs between the pseudogenes and the expressed vlhA, with duplication of pseudogene sequence and loss of the corresponding region previously seen in the expressed gene. Expression of the 5' end of two variants of the vlhA gene showed that they differed in their reaction with monoclonal antibodies specific for this region. These data suggest that the control of vlhA antigenic variation in M. synoviae is achieved by multiple gene conversion events using a repertoire of coding sequences to generate a chimeric expressed gene, with the greatest potential for variation generated in the region encoding the haemagglutinin. Thus, completely distinct mechanisms have been adopted to control antigenic variation in homologous gene families.

A gene family in Mycoplasma imitans closely related to the pMGA family of Mycoplasma gallisepticum.

The avian pathogen Mycoplasma gallisepticum possesses a large gene family encoding lipoproteins which function as haemagglutinins. Representative species of the pneumoniae phylogenetic group of mycoplasmas were examined for the presence of genes homologous to members of this multigene family. Antisera against the pMGA1.1 lipoprotein recognized a 35 kDa protein in Mycoplasma imitans, but did not recognize proteins of Mycoplasma genitalium, Mycoplasma pneumoniae, Mycoplasma pirum, Mycoplasma penetrans of Mycoplasma iowae in Western blots. A fragment of the pMGA 1.2 gene and oligonucleotide probes complementary to highly conserved coding and non-coding regions of pMGA genes bound to fragments of genomic DNA of M. imitans, but not to the genomes of M. genitalium, M. pneumoniae, M. pirum or M. penetrans, and only one probe bound to a fragment of the M. iowae genome. One homologue of the pMGA genes was amplified from the M. imitans genome by PCR and used as a probe to clone a 3.1 kbp DNA fragment from a library of HindIII-digested M. imitans genomic DNA. The contiguous DNA sequence of the PCR and HindIII clones was predicted to encode one complete and one partial ORF which shared some peptide sequence identity with the pMGA genes, including the signal peptidase II cleavage site and the proline-rich amino-terminal region. Like the pMGA genes, the M. imitans genes were found to be members of a large gene family, with an association with GAA trinucleotide repeats, a feature which distinguishes these two families from the homologous vlhA gene family in Mycoplasma synoviae. The identification of these gene families in three phylogenetically distinct avian mycoplasma species, but not in human mycoplasmas, suggests their horizontal transfer between species infecting the same host.

VARIABLE DNA SEQUENCE OF THE pMGA GENE IN Mycoplasma gallisepticum

Specific fragments of Mycoplasma gallisepticum genomic DNA, representing different regions of pMGA1.1 and pMGA1.2 genes, were amplified by Polymerase Chain Reaction (PCR). Using sequence analysis of pMGA terminal domains, differences in nucleotide sequences among strains of M. gallisepticum were identified. Restriction analysis revealed polymorphism at the 5? -end of the pMGA coding region. By means of sequence analysis it was established that the most variable part of pMGA sequence was the 5? -end of the pMGA gene, the region coding amino-terminal part of pMGA protein, while the 3? -end of the gene was conserved among various M. gallisepticum strains analysed. In some strains insertions or deletions were found that indicate the diversity of the pMGA gene family and might be related to the variable expression of the pMGA gene family. Changes in nucleotide sequences of the pMGA genes were also found within isogenic lineages. Genotypic variability is also confirmed by results of the study of the gene product expression.

Expression of the pMGA genes of Mycoplasma gallisepticum is controlled by variation in the GAA trinucleotide repeat lengths within the 5' noncoding regions.

We analyzed the segment of DNA which contains the expressed pMGA gene from one strain of Mycoplasma gallisepticum in normal (strain S6) cells and in cells in which pMGA1.1 gene expression had ceased as a consequence of in vitro culture in the presence of pMGA1. 1-specific antibodies. Sequence analysis of isolates lacking pMGA1.1 expression revealed that this gene, which is typically expressed, exhibited sequence changes within a region 5' to its promoter. Specifically, pMGA1.1(+) cells contained a (GAA)12 motif upstream of the promoter, whereas in pMGA1.1(-) cells the corresponding region contained a (GAA)10 motif; when such cells were grown in medium no longer containing pMGA-specific antibodies, pMGA1.1 was reexpressed and the 5' (GAA)12 motif was restored. Two other genes, pMGA1.9 and pMGA1.2, were also shown to acquire a (GAA)12 motif in clones which expressed these genes. The results imply the evolution by the pMGA genes of M. gallisepticum of a novel transcriptional requirement which facilitates rapid and reversible switches in the pMGA expression pattern.

A protein (M9) associated with monoclonal antibody-mediated agglutination of Mycoplasma gallisepticum is a member of the pMGA family.

A 62-kDa cell surface antigen (M9) of Mycoplasma gallisepticum PG31 that mediates antibody-induced agglutination of the organism was purified and subjected to N-terminal amino-acid sequencing. A 999-bp region of the cDNA encoding the M9 protein was generated by reverse transcription-PCR, and its nucleotide sequence was determined. PCR primers based on this sequence were used to screen a genomic DNA library of PG31. A full-length M9 protein-encoding gene was isolated and sequenced, revealing 96% nucleotide identity with the pMGA1.1 gene of M. gallisepticum S6. Sequence analyses of the M9 gene and flanking open reading frames that encode other pMGA family members suggest that a tandemly repeated GAA sequence may influence pMGA gene expression.

Size and genomic location of the pMGA multigene family of Mycoplasma gallisepticum

The pMGA multigene family encodes variant copies of the cell surface haemagglutinin of Mycoplasma gallisepticum. Quantitative Southern blotting, using an oligonucleotide probe complementary to a region conserved in the leader sequence of all known pMGA genes, was used to estimate the number of members of the family in the genome of seven strains of M. gallisepticum. The number of copies estimated to be present in the genome varied from 32 in strain F to 70 in strain R, indicating that the pMGA gene family may be second in size only to the tRNA family among prokaryotes. If all members of the pMGA family are of similar length to those which have been characterized, a minimum of 79 kb (7.7%) of the genome of strain S6, 82 kb (8.2%) of PG31 and 168 kb (16%) of the genome of strain R is dedicated to encoding variants of the same haemagglutinin. The GAA repeat motif identified in the intergenic region between all characterized pMGA genes appeared to be a feature common to most, if not all, pMGA genes, and furthermore probably exclusive to them. The genomic locations of members of the pMGA family were determined by PFGE and Southern blot hybridization of M. gallisepticum strain S6. The hybridizing regions were localized to four separate regions on the chromosome. The pMGA genes are likely to be predominantly arranged as tandem repeats within these regions, similar to the restricted regions for which the genomic sequence has been determined.

Expression studies on four members of the pMGA multigene family in Mycoplasma gallisepticum 56

A large family of related genes known as pMGA exists in the avian pathogen Mycoplasma gallisepticum but only a single member of this family was previously found to be expressed in one strain of this bacterium. In this work two unrelated strains of M. gallisepticum were also shown by amino-terminal sequencing to express a unique pMGA polypeptide in both cases. To investigate pMGA gene selection in M. gallisepticum, mRNA expression was analysed in M. gallisepticum strain 56 using reverse transcription-PCR (RT-PCR) and Northern blot techniques with probes for several members of the pMGA multigene family. It was shown that the pMGA message is 2.2 kb in size and is monocistronic. RT-PCR detected four different pMGA mRNA molecules but their relative yields were significantly affected by magnesium concentration. By quantitative Northern analysis, the relative abundances of the four pMGA mRNAs in M. gallisepticum S6 total RNA was determined: the pMGA1.1 mRNA predominated [1.88 ng (micrograms total RNA)-1] but at least three other pMGA genes were found to be transcribed but at much lower levels (20 to 40-fold lower). The pMGA1.1 mRNA is expressed at a level five times higher than the tuf gene, known to be one of the most abundantly expressed proteins in the prokaryotic cell. The start point of transcription for pMGA1.1 was determined and probable promoter assigned. From these data it appears likely that transcriptional control plays a major role in the selection of pMGA gene expression in the M. gallisepticum cell.

The organisation of the multigene family which encodes the major cell surface protein, pMGA, of Mycoplasma gallisepticum

The genome of the avian pathogen Mycoplasma gallisepticum contains a number of related genes for putative adhesion molecules (pMGA). Cloning and sequence analysis of several pMGA genes suggested that all of them might be transcriptionally and translationally functional. Analysis of the gene sequence encoding the sole pMGA variant expressed in vitro in the S6 strain (pMGA1.1) revealed no unambiguous feature that could account for its unique expression. It is estimated that the pMGA gene family may contain up to 50 members, and its possible role is discussed herein.

Molecular cloning of a member of the gene family that encodes pMGA, a hemagglutinin of Mycoplasma gallisepticum.

A hemagglutinin with an M(r) of 67,000 (pMGA) from Mycoplasma gallisepticum S6 was purified by using monoclonal antibody affinity chromatography. Purified pMGA was treated with a number of enzymes, the resultant peptides were purified, and their amino acid sequence was determined by using an Applied Biosystems (model 471A) protein sequencer. The DNA sequence encoding two peptides was used to dictate the sequences of synthetic oligonucleotides which were used to screen a library of EcoRI-cut M. gallisepticum DNA in pUC18. A clone reactive to both probes was isolated and found to contain a recombinant insert of 10 kb. The clone was mapped by using restriction endonucleases and fragments subcloned into pUC18 for DNA sequencing. Analysis of part of the DNA sequence revealed an open reading frame containing 1,941 nucleotides which encoded 647 amino acids. The amino terminus was preceded by a putative leader sequence of 25 amino acids. A promoter region preceding the putative start codon GUG was also located. This gene would encode a mature protein of 67,660 Da. There were a number of differences between the predicted amino acid sequence and that determined by direct peptide sequencing. Also, two tryptic peptides of pMGA were not found in the DNA sequence. This suggested that the cloned gene did not encode pMGA but did encode a homolog (pMGA1.2). Furthermore, downstream of pMGA1.2 was a region of DNA encoding a leader sequence followed by an amino acid sequence with high homology to that encoded by the pMGA1.2 gene. The presence within M. gallisepticum of a family of pMGA genes is inferred from the DNA sequence and Southern transfer data. A possible role for this gene family in immune evasion is discussed.