The Mycoplasma genitalium MG352‐encoded protein is a Holliday junction resolvase that has a non‐functional orthologue in Mycoplasma pneumoniae

Recombination between repeated DNA elements in the genomes of Mycoplasma species appears to lie at the basis of antigenic variation of several essential surface proteins. It is therefore imperative that the DNA recombinatorial pathways in mycoplasmas be unravelled. Here, we describe the proteins encoded by the Mycoplasma genitalium MG352 and Mycoplasma pneumoniae MPN528a genes (RecU_Mge and RecU_Mpn respectively), which share sequence similarity with RecU Holliday junction (HJ) resolvases. RecU_Mge was found to: (i) bind HJ substrates and large double‐stranded DNA molecules and (ii) cleave HJ substrates at the sequence 5′‐^G/_TC↓^C/_TT^A/_GG‐3′ in the presence of Mn²⁺. Interestingly, RecU_Mpn(from M. pneumoniae subtype 2 strains) did not possess obvious DNA binding or cleavage activities, which was found to be caused by the presence of a glutamic acid residue at position 67 of the protein, which is not conserved in RecU_Mge. Additionally, RecU_Mpn appears not to be expressed by subtype 1 M. pneumoniae strains, as these possess a TAA translation termination codon at position 181–183 of MPN528a. We conclude that RecU_Mge is a HJ resolvase that may play a central role in recombination in M. genitalium.     

Functional characterization of the RuvB homologs from Mycoplasma pneumoniae and Mycoplasma genitalium

Homologous recombination between repeated DNA elements in the genomes of Mycoplasma species has been hypothesized to be a crucial causal factor in sequence variation of antigenic proteins at the bacterial surface. To investigate this notion, studies were initiated to identify and characterize the proteins that form part of the homologous DNA recombination machinery in Mycoplasma pneumoniae as well as Mycoplasma genitalium. Among the most likely participants of this machinery are homologs of the Holliday junction migration motor protein RuvB. In both M. pneumoniae and M. genitalium, genes have been identified that have the capacity to encode RuvB homologs (MPN536 and MG359, respectively). Here, the characteristics of the MPN536- and MG359-encoded proteins (the RuvB proteins from M. pneumoniae strain FH [RuvB(FH)] and M. genitalium [RuvB(Mge)], respectively) are described. Both RuvB(FH) and RuvB(Mge) were found to have ATPase activity and to bind DNA. In addition, both proteins displayed divalent cation- and ATP-dependent DNA helicase activity on partially double-stranded DNA substrates. The helicase activity of RuvB(Mge), however, was significantly lower than that of RuvB(FH). Interestingly, we found RuvB(FH) to be expressed exclusively by subtype 2 strains of M. pneumoniae. In strains belonging to the other major subtype (subtype 1), a version of the protein is expressed (the RuvB protein from M. pneumoniae strain M129 [RuvB(M129)]) that differs from RuvB(FH) in a single amino acid residue (at position 140). In contrast to RuvB(FH), RuvB(M129) displayed only marginal levels of DNA-unwinding activity. These results demonstrate that M. pneumoniae strains (as well as closely related Mycoplasma spp.) can differ significantly in the function of components of their DNA recombination and repair machinery.     

Comprehensive Methylome Characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at Single-Base Resolution

In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expression. However, full DNA methylome analyses are scarce due to a lack of a simple methodology for rapid and sensitive detection of common epigenetic marks (ie N⁶-methyladenine (6 mA) and N⁴-methylcytosine (4 mC)), in these organisms. Here, we use Single-Molecule Real-Time (SMRT) sequencing to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129, with single-base resolution. Our analysis identified two new methylation motifs not previously described in bacteria: a widespread 6 mA methylation motif common to both bacteria (5′-CTAT-3′), as well as a more complex Type I m6A sequence motif in M. pneumoniae (5′-GAN₇TAY-3′/3′-CTN₇ ATR-5′). We identify the methyltransferase responsible for the common motif and suggest the one involved in M. pneumoniae only. Analysis of the distribution of methylation sites across the genome of M. pneumoniae suggests a potential role for methylation in regulating the cell cycle, as well as in regulation of gene expression. To our knowledge, this is one of the first direct methylome profiling studies with single-base resolution from a bacterial organism.     

The <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis> MPN229 gene encodes a protein that selectively binds single-stranded DNA and stimulates Recombinase A-mediated DNA strand exchange

Background  

Mycoplasma pneumoniae has previously been characterized as a micro-organism that is genetically highly stable. In spite of this genetic stability, homologous DNA recombination has been hypothesized to lie at the basis of antigenic variation of the major surface protein, P1, of M. pneumoniae. In order to identify the proteins that may be involved in homologous DNA recombination in M. pneumoniae, we set out to characterize the MPN229 open reading frame (ORF), which bears sequence similarity to the gene encoding the single-stranded DNA-binding (SSB) protein of other micro-organisms.     

LlaFI, a Type III Restriction and Modification System in Lactococcus lactis

We describe a type III restriction and modification (R/M) system, LlaFI, in Lactococcus lactis. LlaFI is encoded by a 12-kb native plasmid, pND801, harbored in L. lactis LL42-1. Sequencing revealed two adjacent open reading frames (ORFs). One ORF encodes a 680-amino-acid polypeptide, and this ORF is followed by a second ORF which encodes an 873-amino-acid polypeptide. The two ORFs appear to be organized in an operon. A homology search revealed that the two ORFs exhibited significant similarity to type III restriction (Res) and modification (Mod) subunits. The complete amino acid sequence of the Mod subunit of LlaFI was aligned with the amino acid sequences of four previously described type III methyltransferases. Both the N-terminal regions and the C-terminal regions of the Mod proteins are conserved, while the central regions are more variable. An S-adenosyl methionine (Ado-Met) binding motif (present in all adenine methyltransferases) was found in the N-terminal region of the Mod protein. The seven conserved helicase motifs found in the previously described type III R/M systems were found at the same relative positions in the LlaFI Res sequence. LlaFI has cofactor requirements for activity that are characteristic of the previously described type III enzymes. ATP and Mg²⁺ are required for endonucleolytic activity; however, the activity is not strictly dependent on the presence of Ado-Met but is stimulated by it. To our knowledge, this is the first type III R/M system that has been characterized not just in lactic acid bacteria but also in gram-positive bacteria.     

Defining a minimal cell: essentiality of small ORFs and ncRNAs in a genome-reduced bacterium

Identifying all essential genomic components is critical for the assembly of minimal artificial life. In the genome-reduced bacterium Mycoplasma pneumoniae, we found that small ORFs (smORFs; < 100 residues), accounting for 10% of all ORFs, are the most frequently essential genomic components (53%), followed by conventional ORFs (49%). Essentiality of smORFs may be explained by their function as members of protein and/or DNA/RNA complexes. In larger proteins, essentiality applied to individual domains and not entire proteins, a notion we could confirm by expression of truncated domains. The fraction of essential non-coding RNAs (ncRNAs) non-overlapping with essential genes is 5% higher than of non-transcribed regions (0.9%), pointing to the important functions of the former. We found that the minimal essential genome is comprised of 33% (269,410 bp) of the M. pneumoniae genome. Our data highlight an unexpected hidden layer of smORFs with essential functions, as well as non-coding regions, thus changing the focus when aiming to define the minimal essential genome.     

Identification of 9α-Hydroxy-17-Oxo-1,2,3,4,10,19-Hexanorandrostan-5-Oic Acid in Steroid Degradation by Comamonas testosteroni TA441 and Its Conversion to the Corresponding 6-En-5-Oyl Coenzyme A (CoA) Involving Open Reading Frame 28 (ORF28)- and ORF30-Encoded Acyl-CoA Dehydrogenases

Comamonas testosteroni TA441 degrades steroids via aromatization and meta-cleavage of the A ring, followed by hydrolysis, and produces 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid as an intermediate compound. Herein, we identify a new intermediate compound, 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. Open reading frame 28 (ORF28)- and ORF30-encoded acyl coenzyme A (acyl-CoA) dehydrogenase was shown to convert the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid to the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid. A homology search of the deduced amino acid sequences suggested that the ORF30-encoded protein is a member of the acyl-CoA dehydrogenase_fadE6_17_26 family, whereas the deduced amino acid sequence of ORF28 showed no significant similarity to specific acyl-CoA dehydrogenase family proteins. Possible steroid degradation gene clusters similar to the cluster of TA441 appear in bacterial genome analysis data. In these clusters, ORFs similar to ORFs 28 and 30 are often found side by side and ordered in the same manner as ORFs 28 and 30.     

Sequence divergence in the ORF6 gene of Mycoplasma pneumonia.

The ORF6 gene product of Mycoplasma pneumoniae is involved in a yet-unknown manner in the adhesion of the bacterium to its host cell. Part of the ORF6 gene is a repetitive DNA sequence (RepMP5), about 1,900 bp long. Seven additional similar copies of RepMP5 are dispersed on the genome. In the independently isolated strains M. pneumoniae M129 and FH, the RepMP5 copies residing in the ORF6 gene are not identical. Two conserved regions, ranging from nucleotides 1 to 799 and from nucleotide 1795 to the end of the gene, border a variable region, ranging from nucleotides 800 to 1794. This variable region differs in DNA sequence and by 201 bp. Analysis of RepMP5 copies outside the ORF6 gene showed that both M. pneumoniae M129 and M. pneumoniae FH carry a RepMP5 copy on a 6-kbp EcoRI fragment which has the same DNA sequence as the variable region of RepMP5 in the M. pneumoniae FH ORF6 gene. According to these data, a switch from the M. pneumoniae M129 ORF6 gene to the M. pneumoniae FH ORF6 gene could take place by gene conversion.     

Expression of Mycoplasma Proteins Carrying an Affinity Tag in M. pneumoniae Allows Rapid Purification and Circumvents Problems Related to the Aberrant Genetic Code

In Mycoplasma pneumoniae and several other mollicutes, the UGA opal codon specifies tryptophan rather than a translation stop. This often makes it difficult to express Mycoplasma proteins in heterologous hosts. In this work, we demonstrate that mollicute proteins can be fused to an affinity tag and be expressed directly in M. pneumoniae. The protein can then be purified by affinity chromatography and be used for biochemical or any other desired analysis.     

Identification and characterization of hitherto unknown Mycoplasma pneumoniae proteins

Eleven hitherto unknown Mycoplasma pneumoniae proteins were identified and characterized with respect to their size and subcellular location. This was carried out through the construction of in vitro gene fusions between a modified mouse dehydrofolate reductase (dhfr) gene and selected regions (cosmid clones) of the M. pneumoniae genome and expressing them in Escherichia coli. Positive clones were identified using antibodies against specific fractions of M. pneumoniae. The deduced protein sequences of 11 out of 30 clones did not show significant homologies to known proteins in protein databank searches. Monospecific antibodies against these 11 fusion proteins were used to determine the size and cellular location of the corresponding M. pneumoniae proteins by immunoscreening Western blots of SDS-acrylamide gels from M. pneumoniae cell extracts.     

Genomic repeats, genome plasticity and the dynamics of Mycoplasma evolution

Mycoplasmas evolved by a drastic reduction in genome size, but their genomes contain numerous repeated sequences with important roles in their evolution. We have established a bioinformatic strategy to detect the major recombination hot-spots in the genomes of Mycoplasma pneumoniae, Mycoplasma genitalium, Ureaplasma urealyticum and Mycoplasma pulmonis. This allowed the identification of large numbers of potentially variable regions, as well as a comparison of the relative recombination potentials of different genomic regions. Different trends are perceptible among mycoplasmas, probably due to different functional and structural constraints. The largest potential for illegitimate recombination in M.pulmonis is found at the vsa locus and its comparison in two different strains reveals numerous changes since divergence. On the other hand, the main M.pneumoniae and M.genitalium adhesins rely on large distant repeats and, hence, homologous recombination for variation. However, the relation between the existence of repeats and antigenic variation is not necessarily straightforward, since repeats of P1 adhesin were found to be anti-correlated with epitopes recognized by patient antibodies. These different strategies have important consequences for the structures of genomes, since large distant repeats correlate well with the major chromosomal rearrangements. Probably to avoid such events, mycoplasmas strongly avoid inverse repeats, in comparison to co-oriented repeats.     

Identification of gene products of the P1 operon of Mycoplasma pneumoniae

Gene P1 of Mycoplasma pneumoniae, which codes for a major adhesin, is flanked by two sequences with open reading frames designated ORF4 and ORF6 (Inamine et al., 1988b). In order to identify proteins translated from those ORFs, gene fusions between the N-terminus of the RNA replicase of the Escherichia coli bacteriophage MS2 and selected regions of ORF4 and ORF6 were constructed. The corresponding fusion proteins synthesized in Escherichia coli were used to immunize mice. Antisera directed against ORF4-related sequences did not recognize M. pneumoniae antigens in Western blot analysis, but antisera directed against ORF-6-derived fusion proteins reacted with two M. pneumoniae proteins of 40 kDa and 90 kDa. In addition, some of the antisera also recognized proteins that formed in a sodium dodecyl sulphate/polyacrylamide gel a protein ladder between 115 and 145 kDa.     

Glycerol Metabolism Is Important for Cytotoxicity of Mycoplasma pneumoniae

Glycerol is one of the few carbon sources that can be utilized by Mycoplasma pneumoniae. Glycerol metabolism involves uptake by facilitated diffusion, phosphorylation, and the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate, a glycolytic intermediate. We have analyzed the expression of the genes involved in glycerol metabolism and observed constitutive expression irrespective of the presence of glycerol or preferred carbon sources. Similarly, the enzymatic activity of glycerol kinase is not modulated by HPr-dependent phosphorylation. This lack of regulation is unique among the bacteria for which glycerol metabolism has been studied so far. Two types of enzymes catalyze the oxidation of glycerol 3-phosphate: oxidases and dehydrogenases. Here, we demonstrate that the enzyme encoded by the M. pneumoniae glpD gene is a glycerol 3-phosphate oxidase that forms hydrogen peroxide rather than NADH₂. The formation of hydrogen peroxide by GlpD is crucial for cytotoxic effects of M. pneumoniae. A glpD mutant exhibited a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. Attempts to isolate mutants affected in the genes of glycerol metabolism revealed that only the glpD gene, encoding the glycerol 3-phosphate oxidase, is dispensable. In contrast, the glpF and glpK genes, encoding the glycerol facilitator and the glycerol kinase, respectively, are essential in M. pneumoniae. Thus, the enzymes of glycerol metabolism are crucial for the pathogenicity of M. pneumoniae but also for other essential, yet-to-be-identified functions in the M. pneumoniae cell.Mycoplasma pneumoniae causes infections of the upper and lower respiratory tracts. These bacteria are responsible for a large fraction of community-acquired pneumonias. Although usually harmless for adult patients, M. pneumoniae may cause severe disease in children or elderly people. In addition, M. pneumoniae is involved in extrapulmonary complications such as pediatric encephalitis and erythema multiforme (for reviews, see references 15, 21, and 34).M. pneumoniae and its relatives, the Mollicutes, are all characterized by the lack of a cell wall and a very close adaptation to a life within a eukaryotic host. This close adaptation is reflected by degenerative genome evolution that resulted in an extreme genome reduction. As a result, the Mollicutes are the organisms that are capable of independent life with the smallest known genome. M. pneumoniae has a genome of 816 kb and encodes only 688 proteins (18). This genome reduction is taken even further in the close relative Mycoplasma genitalium, which has only 482 protein-coding genes (18). Thus, the analysis of the Mollicutes allows us to study a minimal form of natural life. This question has recently attracted much interest and resulted in the determination of the essential gene sets of M. pneumoniae, M. genitalium, and, more recently, Mycoplasma pulmonis (6, 20). In M. genitalium, with the most reduced genomes, only 100 out of the 482 protein-coding genes are dispensable, suggesting that the remaining 382 genes form the essential gene set (7).Reductive genome evolution in M. pneumoniae is still under way: the genes for the utilization of mannitol as a carbon source seem to be present in M. pneumoniae; however, this substrate cannot be used by the bacteria. M. genitalium, which is further advanced in genome reduction, has lost the genes for mannitol transport and oxidation. It was therefore suggested that the genes for mannitol utilization in M. pneumoniae either are not expressed or encode inactive proteins (12).In M. pneumoniae as well as in other Mollicutes, pathogenicity is closely linked to carbon metabolism (13). M. pneumoniae can use glucose, fructose, and glycerol as the only carbon sources (12). Studies with Mycoplasma mycoides revealed that glycerol metabolism has a major impact on the pathogenicity of these bacteria. Oxidation of glycerol involves the glycerol 3-phosphate oxidase, which produces hydrogen peroxide rather than NADH₂, which is generated by the glycerol 3-phosphate dehydrogenase in most other bacteria (28). In addition to the induction of autoimmune responses, the formation of hydrogen peroxide is the only established mechanism by which mycoplasmas cause damage to their hosts (31, 34). Pathogenic strains of M. mycoides possess a highly active ABC transport system for glycerol in addition to the ubiquitous glycerol facilitator (33). The efficient formation of hydrogen peroxide by the membrane-bound glycerol 3-phosphate oxidase is the major virulence factor of the highly pathogenic strains of M. mycoides (28).M. pneumoniae possesses the complete set of genes for glycerol utilization, and the bacteria do indeed use this carbon source (12). The first component in glycerol metabolism is the glycerol facilitator encoded by the glpF gene. The transported glycerol is then phosphorylated by the glycerol kinase (product of glpK), and glycerol 3-phosphate is subsequently oxidized to dihydroxyacetone phosphate, a glycolytic intermediate. The relevant enzyme is annotated as glycerol 3-phosphate dehydrogenase (encoded by the gene glpD) in M. pneumoniae (17).In all organisms studied so far, glycerol metabolism is under dual control: the genes involved in glycerol utilization are expressed only if glycerol or glycerol 3-phosphate is present in the medium, and they are not expressed in the presence of glucose, the preferred carbon source (3, 4). This second mode of regulation, carbon catabolite repression, involves two distinct mechanisms in the Firmicutes, from which the Mollicutes evolved. In the presence of preferred sugars, the CcpA repressor protein binds in the promoter regions of glycerol utilization genes and prevents their expression. Moreover, the molecular inducer of the system, glycerol 3-phosphate, is formed only in the absence of glucose. This results from the low activity of the glycerol kinase. This enzyme is activated upon phosphorylation by HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). HPr can phosphorylate other proteins only in the absence of glucose, thus providing a link between glucose availability, the activity of the glycerol kinase, and the induction of the glycerol utilization genes (3). Nothing is known about the regulation of glycerol utilization in any member of the Mollicutes; however, regulatory events seem to be rare in these organisms due to the lack of regulatory proteins, among them CcpA.In this work, we studied the mechanisms of glycerol utilization in M. pneumoniae, its regulation, and its contribution to cytotoxicity. We demonstrate constitutive expression of the genes for glycerol utilization in M. pneumoniae. As observed in M. mycoides, glycerol 3-phosphate oxidation involves the formation of hydrogen peroxide and is important for damaging the host cells.     

Serological diagnosis of <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis> infection by using the mimic epitopes

Nowadays, there is lack of effective serological detection method for Mycoplasma pneumoniae (M. pneumoniae) infection in clinic. In this study, the mimic epitopes of M. pneumoniae were screened to evaluate the role in the serodiagnosis of M. pneumoniae infection. The M. pneumoniae-positive serum was used as the target for biopanning to phage display random 7-peptide library. The positive phage clones were selected and the DNA were sequenced and analyzed by BLAST. The representative phages were identified using dot immunoblotting and ELISA. The exogenous heptapeptides were synthesized and their reactions with M. pneumonia-positive serum were tested by indirect ELISA. Two heptapeptides, namely heptapeptide 1: TVNFKLY and heptapeptide 2: LPQRLRT, were screened out from the randomly selected 40 phages after the four bio-panning rounds. They had high homologies to some M. pneumoniae antigens. Besides, the representative bacteriophage containing heptapeptide 1 or 2 could react with the M. pneumonia- positive serum. The sensitivities of heptapeptide 1 and heptapeptide 2 for the diagnosis of M. pneumoniae infection were 90.1 and 80.0%, respectively, and the specificities were 94.3 and 97.1%, respectively. Therefore the two heptapeptides were the mimic epitopes of M. pneumoniae and might have potential serological diagnosis value for M. pneumoniae infection.     

Characteristics of Virulent, Attenuated, and Avirulent Mycoplasma pneumoniae Strains

Homologous pairs of virulent and attenuated or avirulent Mycoplasma pneumoniae strains were derived and compared in an effort to elucidate the mechanisms of virulence. These related strains were found to vary in growth, glycolysis, protein electrophoretic patterns, peroxide formation, morphology, and cytadsorption. Variations in the last two characteristics closely correlated with avirulence. This enables understanding of one stage in the pathogenic sequence and provides a convenient marker for avirulence. The derivation of infectious avirulent strains may make possible their use as live vaccines against M. pneumoniae disease.     

Multiple-Mutation Reaction: a Method for Simultaneous Introduction of Multiple Mutations into the glpK Gene of Mycoplasma pneumoniae

In Mycoplasma pneumoniae, the UGA opal codon specifies tryptophan rather than a translation stop site. This often makes it difficult to express Mycoplasma proteins in E. coli isolates. In this work, we developed a strategy for the one-step introduction of several mutations. This method, the multiple-mutation reaction, is used to simultaneously replace nine opal codons in the M. pneumoniae glpK gene.