Genes involved in translation of Mycoplasma hyopneumoniae and Mycoplasma synoviae

This is a report on the analysis of genes involved in translation of the complete genomes of Mycoplasma hyopneumoniae strain J and 7448 and Mycoplasma synoviae. In both genomes 31 ORFs encoding large ribosomal subunit proteins and 19 ORFs encoding small ribosomal subunit proteins were found. Ten ribosomal protein gene clusters encoding 42 ribosomal proteins were found in M. synoviae, while 8 clusters encoding 39 ribosomal proteins were found in both M. hyopneumoniae strains. The L33 gene of the M. hyopneumoniae strain 7448 presented two copies in different locations. The genes encoding initiation factors (IF-1, IF-2 and IF-3), elongation factors (EF-G, EF-Tu, EF-Ts and EF-P), and the genes encoding the ribosome recycling factor (frr) and one polypeptide release factor (prfA) were present in the genomes of M. hyopneumoniae and M. synoviae. Nineteen aminoacyl-tRNA synthases had been previously identified in both mycoplasmas. In the two strains of M. hyopneumoniae, J and 7448, only one set of 5S, 16S and 23S rRNAs had been identified. Two sets of 16S and 23S rRNA genes and three sets of 5S rRNA genes had been identified in the M. synoviae genome.


Introduction
Mycoplasmas comprise a very large group of prokaryotes widely distributed in nature as parasites and pathogens of humans, animals, plants and insects (Razin, 1992).Because mycoplasmas have an extremely small genome (0.58-2.20 Mb compared to the 4.64 Mb of Escherichia coli), these organisms have limited metabolic options for replication and survival (Maniloff, 1996).
Mycoplasma-induced arthritis of poultry is almost entirely caused by Mycoplasma synoviae infection, which is present on poultry farms all over the world, either sporadically or endemically (Jordan, 1981;Bencina et al., 1999).Infection most frequently occurs as a subclinical respiratory tract infection, which can progress to respiratory disease or to infectious synovitis, in which the synovial membranes of joint and tendon sheaths are affected.Under so far unknown conditions, the M. synoviae disease can become systemic, causing systemic vasculitis and pathological changes in numerous organs.Porcine enzootic pneumonia, with M. hyopneumoniae as the primary agent, is a chronic respiratory disease present in the vast majority of swine farms worldwide, that causes respiratory distress resulting from pneumonia in growing pigs (Kobisch and Friis, 1996;Kobisch, 2000).
Translation is a key step in gene expression, converting the genetic information encoded in messenger RNAs (mRNAs) into contiguous chains of amino acids with structural and/or catalytic properties (Lafontaine and Tollervey, 2001).In this work, we analyzed several aspects of the translation machineries by searching the genes involved in translation of M. hyopneumoniae strain J, M. hyopneumoniae strain 7448, and M. synoviae in the Mycoplasma database and by comparing the results to Mycoplasma and other genomes.

Methods
Genes involved in translation of M. hyopneumoniae strains J and 7448 and M. synoviae where retrieved in the Mycoplasma database.Annotation data were used to compare the gene organization among the three mycoplasmas and to the E. coli and Corynebacterium glutamicum models (Martín et al., 2003).The translated amino acid sequences of L33 ribosomal and gat (A, B and C) proteins were retrieved from the database and used to search for related sequences by using BLAST tools (Altschul et al., 1997).
Seventeen and twenty-two amino acid sequences of L33 and gat proteins, respectively, were used to construct phylogenetic trees by multiple sequence alignments, using the Clustal X program, version 1.8 (Thompson et al., 1997) and the neighbor-joining method (Saitou and Nei, 1987).Robustness of branches was estimated using 100-bootstrap replicates.The amino acid sequences were visualized using the TreeView software.

Genes encoding ribosomal proteins
The amino acid sequences of the ribosomal proteins of M. synoviae, M. hyopneumoniae strain J, and M. hyopneumoniae strain 7448 have been deduced from the genome sequence (Vasconcelos et al., 2005).Thirty-one ORFs (Open Reading Frames) encoding large ribosomal subunit proteins, and 19 ORFs encoding small ribosomal subunit proteins were found in the genomes of M. synoviae, M. hyopneumoniae J and M. hyopneumoniae 7448, as shown in Table 1.
Those ribosomal protein families including L1-L6, L9-L24, L27-L29 and L31-36, as well as S2 to S20, correspond to the stable pool of 50 ribosomal proteins described in bacteria.S1, S21, L25 and L30 are the only four wellestablished bacterial ribosomal proteins which exhibit a disparate distribution (Lecompte et al., 2002).Those four genes were not found in the three mycoplasma genomes sequenced.Among the mycoplasmas, the S1 protein was also absent in the genomes of M. genitalium and M. pneumoniae, but has been identified in M. pulmonis (GenBank accession number: Q98R80).The S21 gene has been identified in the genomes of M. gallisepticum, M. genitalium, M. penetrans, M. pneumoniae and Ureaplasma urealyticum (Lecompte et al., 2002).
The four proteins L7/L12 form a protruding domain of the large subunit that participates in the GTPase activity of ribosomes (Uchiumi et al., 2002).Other proteins, such as S7p, S9p, S12p, S13p, L1p and L5p, are in contact with the tRNA or surround the polypeptide exit channel (L22p, L24p and L29p) (Yusupov et al., 2001).

Organization of gene clusters encoding ribosomal proteins
Most genes encoding ribosomal proteins are clustered forming operons.In the three sequenced genomes, we found 10 ribosomal protein gene clusters encoding 42 ribo-somal proteins in M. synoviae, and 8 clusters encoding 39 ribosomal proteins in both M. hyopneumoniae strains, as shown in Figure 1.In the E. coli and C. glutamicum models (Martín et al., 2003), there are 11 gene clusters encoding 41 and 42 ribosomal proteins, respectively.In E. coli, the S10 operon encodes 11 ribosomal proteins in the following order: S10, L3, L2, L4, L23, S19, L22, S3, L16, L29 and S17, and is located close to the spc operon that includes 12 genes which encode the proteins L14, L24, L5, S14, S8, L6, L18, S5, L30, L15, Sec Y and L36 (Cerretti et al., 1983).These two operons correspond in organization to the big cluster in M. hyopneumoniae and M. synoviae with minor differences, as shown in Figure 1A.In M. hyopneumoniae, the large block of approximately 15 kb contains 29 genes, 24 of which encode ribosomal proteins.This block of genes also contains those encoding the SecY subunit of the preprotein translocase (secY), adenylate kinase (adk), methionine aminopeptidase (map), translation initiation factor IF-1 (infA), and the RNA polymerase subunit alpha (rpoA).In M. synoviae, there is a cluster of 22 genes encoding ribo- somal proteins in a region of about 10 kb.The block of genes secY-adk-map-infA-L36-S13-S11-rpoA-L17 is located in another region of the genome when compared to M. hyopneumoniae (Figure 1C).In addition, the L11 and L1 genes are not grouped in a separate cluster as in M. hyopneumoniae, E. coli and C. glutamicum (Figure 1C).The clusters "L10-L7/L2", "L13-S9" and "S12-S7-EF-G" of M. hyopneumoniae and M. synoviae are organized as in E. coli and C. glutamicum (Figure 1B).Figures 1C and 1D show particularities of the analyzed mycoplasma ribosomal protein clusters compared to E. coli and C. glutamicum.For example, the "L21-L27" gene cluster is absent in M. hyopneumoniae and present in E. coli and C. glutamicum in inverted position, as compared to M. synoviae (Figure 1C).

Organization and phylogenetic relationships among L33 ribosomal proteins
Comparing the two M. hyopneumoniae strains, the organization of the ribosomal genes was identical, except for the L33 gene, of which strain 7448 presented two ORFs (MHP0638 and MHP0658) in different locations, while strain J presented only one (MHJ0658) (Table 1).The extra ORF in strain 7488 encodes a predicted protein of 46 amino acid residues, while the conserved ORF encodes a protein with 50 amino acid residues.
The phylogenetic relationships of the L33 ribosomal proteins were visualized by grouping into two big clades, A and B (Figure 2).Some mycoplasmas (M.pulmonis, M. pneumoniae M129, M. genitalium G37 and MP12668) presented two L33 genes.When present in duplicates, the orthologues were clustered in different clades, suggesting interphylum horizontal transfer and a possible specialized function to those proteins.

Ribosomal RNAs
In the M. synoviae genome, the 16S rDNA is separated from the 23S rDNA by 259 bp, while in the M. hyopneumoniae strains the distance is about 490 bp (data not shown).Downstream of the 23S rDNA, there is another spacer region followed by the 5S rDNA gene.This region is located at a greater distance (about 100 kb) than usual from the 16S-23S cluster.In M. pulmonis, for example, the 5S gene is located 350 bp distant from the genes encoding 16S and 23S rRNAs (Chambaud et al., 2001).Those data confirm the previous observation that these sets of genes are localized in different regions of the M. hyopneumoniae genome (Taschke et al., 1986;Stemke et al., 1994).All mycoplasmas presented intervening tRNA genes, which seems to be common to all Mollicutes, except the phytoplasmas (Chambaud et al., 2001).In both M. hyopneumoniae strains only one set of 5S, 16S and 23S rRNAs was identified.Two sets of 16S and 23S rRNA genes and three sets of 5S rRNA genes were identified in the M. synoviae genome.

Translation initiation, elongation and termination
The genes encoding initiation factors (IF-1, IF-2 and IF-3) are present in the genomes of M. synoviae and M. hyopmeumoniae J and 7448.IF-1 is part of a cluster containing the genes which encode L36, S13, S11, rpoA and L17, among others (Figures 1A and 1C).In the two M. hyopneumoniae genomes, the gene encoding IF-2 is located between the genes coding for the ribosome-binding factor A (rbfA) and the N utilization substance protein A (nusA).In M. synoviae, the gene is located between the genes encoding adenine phosphoribosyltransferase (apt) and nusA (data not shown).The gene encoding IF-3 is part of a block of genes which encode the ribosomal proteins L35, L-20 and L28 in the genomes of both M. hyopneumoniae strains (Figure 1C).In M. synoviae, this gene is part of a block containing the genes which encode L35 and L20 (Figure 1C).The elongation factors EF-Ts, EF-Tu, EF-G and EF-P are present in the genomes of M. synoviae, M. hyopmeumoniae J, and M. hyopneumoniae 7448.As mentioned before, the gene encoding EF-G is part of the operon S12-S7-EF-G (Figure 1B).The gene encoding EF-Ts is located close to the gene encoding S2 in the genomes of both M. hyopneumoniae strains and of M. synoviae (data not shown).
The gene encoding the ribosome recycling factor (frr), which is responsible for the dissociation of the ribosomes from the mRNA after termination of translation, and is essential for bacterial growth (Janosi et al., 1996), is also present in the genomes of M. synoviae, M. hyopmeumoniae J, and M. hyopneumoniae 7448.Only one polypeptide release factor (prfA) was identified in both M. hyopneumoniae strains and in M. synoviae.This finding is consistent with the use of UAA and UAG as stop codons (Fraser et al., 1995;Himmelreich et al., 1996;Inagaki et al., 1996).Similar to the situation in other mycoplasma genomes, the UGA codon encodes tryptophan instead of stop codon.As a consequence, the peptide-chain release factor 2 (RF2), which recognizes the stop codons UGA and UAA, has become obsolete and was deleted, as proved experimentally in M. capricolum (Inagaki et al., 1993).

Phylogenetic relationships among aminoacyl-tRNA synthetases
Aminoacyl-tRNA synthetases catalyze the ligation of specific amino acids to their cognate tRNAs, which is the initial step in protein synthesis (Kim et al., 1993;Park et al., 2005).In the three sequenced genomes, M. hyopmeumoniae strains J and 7448 and M. synoviae, nineteen aminoacyl-tRNA synthetases were identified.Of the 20 standard tRNA-synthetases, glutaminyl-tRNA synthetase is the only one not detected in M. genitalium (Fraser et al., 1995) and M. pneumoniae (Himmelreich et al., 1996).In Bacillus subtilis, this problem is solved by charging the tRNA Gln first with glutamate, which is subsequently converted to glutamine by an amidotransferase (Strauch et al., 1988).It has been shown that Glu-tRNA Gln amidotransferase is a heterotrimeric enzyme encoded by the genes gatA, gatB, and gatC, which are part of an operon in B. subtilis (Curnow et al., 1997).In M. hyopneumonaie strain J, the genes gatC (MH12539), gatA (MH15039), and gatB (MH21623) are organized in a cluster.The same kind of organization is observed in M. hyopneumoniae strain 7448 (gatC -MP09881, gatA -MP18574, gatB -MP12574).The organization of those genes in M. synoviae follows the order gatA -MS05589, gatB -MS05595, gatC -MS10482.
In order to analyze the grouping of the gat protein subunits, a phylogenetic tree was constructed and visualized, as shown in Figure 3.The deduced amino acid sequences of the ORFs encoding gatA, gatB and gatC proteins of M. synoviae, M. hyopneumoniae J, M. hyopneumoniae 7448 and complete sequences of other mycoplasma proteins from databases were aligned.The bootstrap values (98%) among gatA, gatB and gatC indicate the high identity among the gat groups of the mycoplasmas.In Figure 3, separated clades comprising gatA, gatB and gatC can be observed.Only one gat protein subunit has been described for each mycoplasma.A higher identity was found between gatA (97%), gatB (94%), and gatC (96%) of M. hyopneumoniae 7448 and M. hyopneumoniae J (data not shown) when compared to other mycoplasmas and formed single groups inside the clades.This observation suggests a common ancestral.

232Figure 1 -
Figure 1 -Gene clusters of ribosomal proteins in Mycoplasma synoviae, Mycoplasma hyopneumoniae, Escherichia coli and Corynebacterium glutamicum.Arrows represent genes with their relative orientation in the genomes.Contiguous arrows represent adjacent genes, while interrupted lines indicate loss of proximity.The solid bar indicates the limit of the S10 and spc operons in E. coli and C. glutamicum.The names of the genes were obtained from M. hyopneumoniae ( §) or from M. synoviae ( ‡) (when the name was not defined for M. hyopneumoniae).(A) A big cluster showing differences between the four organisms; (B) Clusters with identical organization in the four organisms; (C) Another cluster showing differences between the organisms; (D) Clusters presenting particularities in M. synoviae and M. hyopneumoniae.Abbreviations used for gene names: SecY (preprotein translocase secY subunit), Adk (adenylate kinase), Map (methionyl aminopeptidase), IF-1 (translation initiation factor IF-1), α (DNA-directed RNA polymerase subunit alpha), EF-G (elongation factor EF-G), Ssb (single-stranded DNA-binding protein), IF-3 (translation initiation factor IF-3), TrmD (tRNA (guanine-N1)-methyltransferase).Data for E. coli and C. glutamicum were retrieved from NCBI's complete genome section and from Martín et al. (2003).

Table 1 -
Genes encoding ribosomal proteins identified in the genomes of M. synoviae, M. hyopmeumoniae J, and M. hyopneumoniae 7448.