Mycoplasma agalactiae Secretion of ß-(1→6)-Glucan, a Rare Polysaccharide in Prokaryotes, Is Governed by High-Frequency Phase Variation.

UNLABELLED: Mycoplasmas are minimal, wall-less bacteria but have retained the ability to secrete complex carbohydrate polymers that constitute a glycocalyx. In members of the Mycoplasma mycoides cluster, which are important ruminant pathogens, the glycocalyx includes both cell-attached and cell-free polysaccharides. This report explores the potential secretion of polysaccharides by M. agalactiae, another ruminant pathogen that belongs to a distant phylogenetic group. Comparative genomic analyses showed that M. agalactiae possesses all the genes required for polysaccharide secretion. Notably, a putative synthase gene (gsmA) was identified, by in silico reconstruction of the biosynthetic pathway, that could be involved in both polymerization and export of the carbohydrate polymers. M. agalactiae polysaccharides were then purified in vitro and found to be mainly cell attached, with a linear ß-(1→6)-glucopyranose structure [ß-(1→6)-glucan]. Secretion of ß-(1→6)-glucan was further shown to rely on the presence of a functional gsmA gene, whose expression is subjected to high-frequency phase variation. This event is governed by the spontaneous intraclonal variation in length of a poly(G) tract located in the gsmA coding sequence and was shown to occur in most of the M. agalactiae clinical isolates tested in this study. M. agalactiae susceptibility to serum-killing activity appeared to be dictated by ON/OFF switching of ß-(1→6)-glucan secretion, suggesting a role of this phenomenon in survival of the pathogen when it invades the host bloodstream. Finally, ß-(1→6)-glucan secretion was not restricted to M. agalactiae but was detected also in M. mycoides subsp. capri PG3(T), another pathogen of small ruminants. IMPORTANCE: Many if not all bacteria are able to secrete polysaccharides, either attached to the cell surface or exported unbound into the extracellular environment. Both types of polysaccharides can play a role in bacterium-host interactions. Mycoplasmas are no exception despite their poor overall metabolic capacity. We showed here that M. agalactiae secretes a capsular ß-(1→6)-glucopyranose thanks to a specific glycosyltransferase with synthase activity. This secretion is governed by high-frequency ON/OFF phase variation that might be crucial in mycoplasma host dissemination, as cell-attached ß-(1→6)-glucopyranose increases serum-killing susceptibility. Our results provide functional genetic data about mycoplasmal glycosyltransferases with dual functions, i.e., assembly and export of the sugar polymers across the cell membrane. Furthermore, we demonstrated that nonprotein epitopes can be subjected to surface antigenic variation in mycoplasmas. Finally, the present report contributes to unravel the role of secreted polysaccharides in the virulence and pathogenicity of these peculiar bacteria.

Epidemiological surveillance of mycoplasmas belonging to the 'Mycoplasma mycoides' cluster: is DGGE fingerprinting of 16S rRNA genes suitable?

AIMS: The analysis by Denaturing Gradient Gel Electrophoresis (DGGE) of the PCR-amplified V3 region of 16S rRNA gene was previously shown to detect and differentiate a large number of human and animal mycoplasmas. In this study, we further assessed the suitability of the technique for epidemiological surveillance of mycoplasmas belonging to the 'Mycoplasma mycoides' cluster, a phylogenetic group that includes major ruminant pathogens. METHODS AND RESULTS: The V3 region of 16S rRNA genes from approx. 50 field strains was amplified and analysed by DGGE. Detection and identification results were compared with the ones obtained by antigenic testing and sequence analysis. CONCLUSIONS: The DGGE technique is robust and valuable as a first-line test, but the patterns obtained for strains belonging to the 'M. mycoides' cluster were too variable within a taxon and in contrast too conserved between taxa to allow an unequivocal identification of isolates without further analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Issues raised by the quest for a single universal test able to detect and identify any mycoplasma in one clinical sample are thoroughly documented.

A PCR for the detection of mycoplasmas belonging to the Mycoplasma mycoides cluster: application to the diagnosis of contagious agalactia.

Contagious agalactia is a mycoplasmal infection caused by Mycoplasma agalactiae, Mycoplasma mycoides subsp. mycoides LC, M. mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens. Identification of the causative organisms is usually performed by isolation and classical biochemical and serological tests, though this is a lengthy and cumbersome process for mycoplasmas. Specific PCR assays have been developed for the identification of Mycoplasma agalactiae and M. putrefaciens. For members of the M. mycoides cluster existing PCR tests are based on the amplification of highly conserved genes coding for ribosomal proteins, hence a possibility of cross-reactions. The gene glk, coding for a glucokinase, that is found in this cluster is very distantly related to any other bacterial glucokinase described so far. It was therefore chosen as target to design a new PCR test. The validation was performed independently in three laboratories in France and India using over 100 mycoplasma strains of various geographical origins. All strains belonging to the M. mycoides cluster were detected by amplification of the expected PCR product (428 bp) while no amplification was obtained from M. agalactiae strains. Our results demonstrate the universality of this PCR in spite of the great heterogeneity found within this cluster. This new tool may be of great help for the implementation of control measures directed towards contagious agalactia.

Mycoplasmas, plants, insect vectors: a matrimonial triangle.

Plant pathogenic mycoplasmas were discovered by electron microscopy, in 1967, long after the discovery and culture in 1898 of the first pathogenic mycoplasma of animal origin, Mycoplasma mycoides. Mycoplasmas are Eubacteria of the class Mollicutes, a group of organisms phylogenetically related to Gram-positive bacteria. Their more characteristic features reside in the small size of their genomes, the low guanine (G) plus cytosine (C) content of their genomic DNA and the lack of a cell wall. Plant pathogenic mycoplasmas are responsible for several hundred diseases and belong to two groups: the phytoplasmas and the spiroplasmas. The phytoplasmas (previously called MLOs, for mycoplasma like organisms) were discovered first; they are pleiomorphic, and have so far resisted in vitro cultivation. Phytoplasmas represent the largest group of plant pathogenic Mollicutes. Only three plant pathogenic spiroplasmas are known today. Spiroplasma citri, the agent of citrus stubborn was discovered and cultured in 1970 and shown to be helical and motile. S. kunkelii is the causal agent of corn stunt. S. phoeniceum, responsible for periwinkle yellows, was discovered in Syria. There are many other spiroplasmas associated with insects and ticks. Plant pathogenic mycoplasmas are restricted to the phloem sieve tubes in which circulates the photosynthetically-enriched sap, the food for many phloem-feeding insects (aphids, leafhoppers, psyllids, etc.). Interestingly, phytopathogenic mycoplasmas are very specifically transmitted by leafhoppers or psyllid species. In this paper, the most recent knowledge on phytopathogenic mycoplasmas in relation with their insect and plant habitats is presented as well as the experiments carried out to control plant mycoplasma diseases, by expression of mycoplasma-directed-antibodies in plants (plantibodies).

Characterization of FruR as a putative activator of the fructose operon of Spiroplasma citri.

The role of fruR, the first gene of the Spiroplasma citri fructose operon, was investigated. In vivo transcription of the fructose operon is greatly enhanced by the presence of fructose in the growth medium while glucose has no effect. When fruR is not expressed, transcription of the fructose operon is not stimulated by fructose, and fructose fermentation is decreased, indicating that FruR is an activator of the fructose operon. The promoter of the fructose operon was localized by primer extension, and a direct T-rich repeat was found to overlap the -35 box. This repeat could be the binding site of FruR. The presence of fructose in the culture medium also decreases the toxicity of methyl alpha-glucoside, however FruR is not involved in this regulation. This is the first description of transcription regulation of a mollicute operon.

Fructose utilization and phytopathogenicity of Spiroplasma citri.

Spiroplasma citri is a plant-pathogenic mollicute. Recently, the so-called nonphytopathogenic S. citri mutant GMT 553 was obtained by insertion of transposon Tn4001 into the first gene of the fructose operon. Additional fructose operon mutants were produced either by gene disruption or selection of spontaneous xylitol-resistant strains. The behavior of these spiroplasma mutants in the periwinkle plants has been studied. Plants infected via leafhoppers with the wild-type strain GII-3 began to show symptoms during the first week following the insect-transmission period, and the symptoms rapidly became severe. With the fructose operon mutants, symptoms appeared only during the fourth week and remained mild, except when reversion to a fructose+ phenotype occurred. In this case, the fructose+ revertants quickly overtook the fructose- mutants and the symptoms soon became severe. When mutant GMT 553 was complemented with the fructose operon genes that restore fructose utilization, severe pathogenicity, similar to that of the wild-type strain, was also restored. Finally, plants infected with the wild-type strain and grown at 23 degrees C instead of 30 degrees C showed late symptoms, but these rapidly became severe. These results are discussed in light of the role of fructose in plants. Fructose utilization by the spiroplasmas could impair sucrose loading into the sieve tubes by the companion cells and result in accumulation of carbohydrates in source leaves and depletion of carbon sources in sink tissues.

Fructose utilization and pathogenicity of Spiroplasma citri: characterization of the fructose operon.

Transposon Tn4001 mutagenesis of Spiroplasma citri wild-type (wt) strain GII-3 led to the isolation and characterization of non-phytopathogenic mutant GMT 553. In this mutant, transposon Tn4001 is inserted within the first gene of the fructose operon. This operon comprises three genes. The first gene (fruR) codes for a putative transcriptional regulator protein belonging to the deoxyribonucleoside repressor (DeoR) family. Sequence similarities and functional complementation of mutant GMT 553 with different combinations of the wt genes of the fructose operon showed that the second gene (fruA) codes for the permease of the phosphoenolpyruvate:fructose phosphotransferase system (fructose PTS), and the third, fruK, for the 1-phosphofructokinase (1-PFK). Transcription of the fructose operon in wt strain GII-3 resulted in two messenger RNAs, one of 2.8kb and one of 3.8kb. Insertion of Tn4001 in the genome of mutant GMT 553 abolished transcription of the fructose operon, and resulted in the inability of this mutant to use fructose. Functional complementation experiments demonstrated that fructose utilization was restored with fruR-fruA-fruK, fruA-fruK or fruA only, but not with fruR or fruR-fruA. This is the first time that an operon for sugar utilization has been functionally characterized in the mollicutes.

The unique organization of the rpoB region of Spiroplasma citri: a restriction and modification system gene is adjacent to rpoB.

A 6.5-kb DNA fragment containing the gene (rpoB) encoding the RNA polymerase (RNAP) beta subunit, from the mollicute Spiroplasma citri (Sc), was cloned and sequenced. The classical eubacterial organization, with the genes (rplK, A, J and L) encoding ribosomal proteins L11, L1, L10 and L12 located immediately upstream from rpoB, was not found in the Sc DNA. Instead, an open reading frame (hsdS) potentially encoding a component of a type I restriction and modification system was identified upstream from rpoB, and sequences showing similarities with insertion elements were found between hsdS and rpoB.

Insusceptibility of members of the class Mollicutes to rifampin: studies of the Spiroplasma citri RNA polymerase beta-subunit gene.

In order to study the mechanism of insusceptibility of Spiroplasma citri to rifampin, we have cloned and sequenced its rpoB gene, which encodes the beta subunit of RNA polymerase. By comparison of the deduced amino acid sequence with sequences of beta subunits from susceptible and resistant bacteria, it was possible to identify several differences in the so-called Rif region (encompassing rpoB codons 500 to 575 in the Escherichia coli sequence). We constructed a chimeric rpoB gene made of the E. coli rpoB gene in which the Rif region was replaced by the equivalent region from S. citri. E. coli cells harboring this chimeric gene were resistant to rifampin. Subsequent experiments involving site-directed mutagenesis demonstrated that a single amino acid substitution (asparagine at position 526) was able to provide high-level rifampin resistance in E. coli.