Genomic analyses of Symbiomonas scintillans show no evidence for endosymbiotic bacteria but does reveal the presence of giant viruses.

Symbiomonas scintillans Guillou et Chrétiennot-Dinet, 1999 is a tiny (1.4 µm) heterotrophic microbial eukaryote. The genus was named based on the presence of endosymbiotic bacteria in its endoplasmic reticulum, however, like most such endosymbionts neither the identity nor functional association with its host were known. We generated both amplification-free shotgun metagenomics and whole genome amplification sequencing data from S. scintillans strains RCC257 and RCC24, but were unable to detect any sequences from known lineages of endosymbiotic bacteria. The absence of endobacteria was further verified with FISH analyses. Instead, numerous contigs in assemblies from both RCC24 and RCC257 were closely related to prasinoviruses infecting the green algae Ostreococcus lucimarinus, Bathycoccus prasinos, and Micromonas pusilla (OlV, BpV, and MpV, respectively). Using the BpV genome as a reference, we assembled a near-complete 190 kbp draft genome encoding all hallmark prasinovirus genes, as well as two additional incomplete assemblies of closely related but distinct viruses from RCC257, and three similar draft viral genomes from RCC24, which we collectively call SsVs. A multi-gene tree showed the three SsV genome types branched within highly supported clades with each of BpV2, OlVs, and MpVs, respectively. Interestingly, transmission electron microscopy also revealed a 190 nm virus-like particle similar the morphology and size of the endosymbiont originally reported in S. scintillans. Overall, we conclude that S. scintillans currently does not harbour an endosymbiotic bacterium, but is associated with giant viruses.

Plasma membrane damage limits replicative lifespan in yeast and induces premature senescence in human fibroblasts.

Plasma membrane damage (PMD) occurs in all cell types due to environmental perturbation and cell-autonomous activities. However, cellular outcomes of PMD remain largely unknown except for recovery or death. In this study, using budding yeast and normal human fibroblasts, we found that cellular senescence-stable cell cycle arrest contributing to organismal aging-is the long-term outcome of PMD. Our genetic screening using budding yeast unexpectedly identified a close genetic association between PMD response and replicative lifespan regulations. Furthermore, PMD limits replicative lifespan in budding yeast; upregulation of membrane repair factors ESCRT-III (SNF7) and AAA-ATPase (VPS4) extends it. In normal human fibroblasts, PMD induces premature senescence via the Ca2+-p53 axis but not the major senescence pathway, DNA damage response pathway. Transient upregulation of ESCRT-III (CHMP4B) suppressed PMD-dependent senescence. Together with mRNA sequencing results, our study highlights an underappreciated but ubiquitous senescent cell subtype: PMD-dependent senescent cells.

Evaluation of the MilA ELISA for the diagnosis of herd infection with Mycoplasma bovis using bulk tank milk and estimation of the prevalence of M. bovis in Australia.

Infection with Mycoplasma bovis has been identified as a growing threat in dairy industries worldwide and there is an urgent need for an inexpensive and accurate herd-level screening tool to identify herds that have been exposed to M. bovis. This study aimed to evaluate the use of the MilA ELISA for testing bulk tank milk (BTM) samples for antibodies against M. bovis and estimate a suitable cut-off and diagnostic sensitivity (DSe) and specificity (DSp) for this assay. An optimal cut-off was then applied for investigating the geographical and seasonal distribution of infection with M. bovis in Australia. A total of 5554 BTM samples from 2683 dairy herds were collected during March, August and December 2017. BTM samples were tested in the MilA ELISA and a cut-off of 29 antibody units (AU) was estimated to be optimal using Bayesian latent class analysis which makes no assumption about the true disease status of herds under investigation. At this cut-off, the DSe and DSp were estimated to be 96.6% (95% highest probability density [HPD] interval: 87.0, 99.8) and 94.2% (95% HPD: 89.9, 97.4), respectively. The diagnostic specifications were found to vary markedly with stage of the production cycle, suggesting that targeted sampling was needed to maximize accuracy. We also found distinct differences in the apparent prevalence of M. bovis in different dairying regions, as well as seasonal variation. The highest apparent prevalence of M. bovis was observed in samples collected in March and an overall drop in the proportion of positive herds was seen from March to December. Overall, this study provides insights into the dynamics of BTM antibodies against M. bovis in Australian dairy herds and how the MilA ELISA can be applied for bulk tank milk testing.

A Mycoplasma gallisepticum Glycerol ABC Transporter Involved in Pathogenicity.

MalF has been shown to be required for virulence in the important avian pathogen Mycoplasma gallisepticum To characterize the function of MalF, predicted to be part of a putative ABC transporter, we compared metabolite profiles of a mutant with a transposon inserted in malF (MalF-deficient ST mutant 04-1; ΔmalF) with those of wild-type bacteria using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Of the substrates likely to be transported by an ABC transport system, glycerol was detected at significantly lower abundance in the ΔmalF mutant, compared to the wild type. Stable isotope labeling using [U-13C]glycerol and reverse transcription-quantitative PCR analysis indicated that MalF was responsible for the import of glycerol into M. gallisepticum and that, in the absence of MalF, the transcription of gtsA, which encodes a second transporter, GtsA, was upregulated, potentially to increase the import of glycerol-3-phosphate into the cell to compensate for the loss of MalF. The loss of MalF appeared to have a global effect on glycerol metabolism, suggesting that it may also play a regulatory role, and cellular morphology was also affected, indicating that the change to glycerol metabolism may have a broader effect on cellular organization. Overall, this study suggests that the reduced virulence of the ΔmalF mutant is due to perturbed glycerol uptake and metabolism and that the operon including malF should be reannotated as golABC to reflect its function in glycerol transport.IMPORTANCE Many mycoplasmas are pathogenic and cause disease in humans and animals. M. gallisepticum causes chronic respiratory disease in chickens and infectious sinusitis in turkeys, resulting in economic losses in poultry industries throughout the world. Expanding our knowledge about the pathogenesis of mycoplasma infections requires better understanding of the specific gene functions of these bacteria. In this study, we have characterized the metabolic function of a protein involved in the pathogenicity of M. gallisepticum, as well as its effect on expression of selected genes, cell phenotype, and H2O2 production. This study is a key step forward in elucidating why this protein plays a key role in virulence in chickens. This study also emphasizes the importance of functional characterization of mycoplasma proteins, using tools such as metabolomics, since prediction of function based on homology to other bacterial proteins is not always accurate.

A combined metabolomic and bioinformatic approach to investigate the function of transport proteins of the important pathogen Mycoplasma bovis.

Mycoplasma bovis is an economically important pathogen of the cattle industry worldwide, and there is an urgent need for a more effective vaccine to control the diseases caused by this organism. Although the M. bovis genome sequence is available, very few gene functions of M. bovis have been experimentally determined, and a better understanding of the genes involved in pathogenesis are required for vaccine development. In this study, we compared the metabolite profiles of wild type M. bovis to a number of strains that each contained a transposon insertion into a putative transporter gene. Transport systems are thought to play an important role in survival of mycoplasmas, as they rely on the host for many nutrients. We also performed 13C-stable isotope labelling on strains with transposon insertions into putative glycerol transporters. Integration of metabolomic and bioinformatic analyses revealed unexpected results (when compared to genome annotation) for two mutants, with a putative amino acid transporter (MBOVPG45_0533) appearing more likely to transport nucleotide sugars, and a second mutant, a putative dicarboxylate/amino acid:cation (Na+ or H+) symporter (DAACS), more likely to function as a biopterin/folate transporter. This study also highlighted the apparent redundancy in some transport and metabolic pathways, such as the glycerol transport systems, even in an organism with a reduced genome. Overall, this study highlights the value of metabolomics for revealing the likely function of a number of transporters of M. bovis.

Metabolite profiling of Mycoplasma gallisepticum mutants, combined with bioinformatic analysis, can reveal the likely functions of virulence-associated genes.

Mycoplasma gallisepticum is an economically important pathogen of commercial poultry. An improved understanding of M. gallisepticum pathogenesis is required to develop better control methods. We recently identified a number of M. gallisepticum mutants with defects in colonization and persistence in chickens using signature-tagged transposon mutagenesis. Loss of virulence was associated with mutations in a putative oligopeptide/dipeptide (opp/dpp) ATP-binding cassette (ABC) transporter (where the transposon was inserted into the MGA_0220 (oppD1) gene and two hypothetical proteins (encoded by MGA_1102 and MGA_0588), one of which (MGA_1102) contains a putative peptidase motif. To further characterise the function of these proteins, we compared the metabolome of each transposon mutant with that of wild type bacteria. Two independent LC/MS analyses revealed consistent significant decreases in the abundances of several amino acids and the dipeptide alanyl-glycine (Ala-Gly) in the MGA_0220 mutant, consistent with this protein being a peptide transporter. Similarly, lysine and Ala-Gly were significantly decreased in the MGA_1102 mutant, consistent with our bioinformatic analysis suggesting that MGA_1102 encodes a membrane-located peptidase. Few differences were observed in metabolite levels in the MGA_0588 mutant, suggesting that the disrupted protein has a non-metabolic role. Overall, this study indicates that metabolomics is a useful tool in the functional analysis of mutants.

Analysis of the Mycoplasma bovis lactate dehydrogenase reveals typical enzymatic activity despite the presence of an atypical catalytic site motif.

The lactate dehydrogenase (LDH) of Mycoplasma genitalium has been predicted to also act as a malate dehydrogenase (MDH), but there has been no experimental validation of this hypothesized dual function for any mollicute. Our analysis of the metabolite profile of Mycoplasma bovis using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) detected malate, suggesting that there may be MDH activity in M. bovis. To investigate whether the putative l-LDH enzyme of M. bovis has a dual function (MDH and LDH), we performed bioinformatic and functional biochemical analyses. Although the amino acid sequence and predicted structural analysis of M. bovisl-LDH revealed unusual residues within the catalytic site, suggesting that it may have the flexibility to possess a dual function, our biochemical studies using recombinant M. bovis -LDH did not detect any MDH activity. However, we did show that the enzyme has typical LDH activity that could be inhibited by both MDH substrates oxaloacetate (OAA) and malate, suggesting that these substrates may be able to bind to M. bovis LDH. Inhibition of the conversion of pyruvate to lactate by OAA may be one method the mycoplasma cell uses to reduce the potential for accumulation of intracellular lactate.

The Mycoplasma gallisepticum virulence factor lipoprotein MslA is a novel polynucleotide binding protein.

Although lipoproteins of mycoplasmas are thought to play a crucial role in interactions with their hosts, very few have had their biochemical function defined. The gene encoding the lipoprotein MslA in Mycoplasma gallisepticum has recently been shown to be required for virulence, but the biochemical function of this gene is not known. Although this gene has no significant sequence similarity to any gene of known function, it is located within an operon in M. gallisepticum that contains a homolog of a gene previously shown to be a nonspecific exonuclease. We mutagenized both genes to facilitate expression in Escherichia coli and then examined the functions of the recombinant proteins. The capacity of MslA to bind polynucleotides was examined, and we found that the protein bound single- and double-stranded DNA, as well as single-stranded RNA, with a predicted binding site of greater than 1 nucleotide but less than or equal to 5 nucleotides in length. Recombinant MslA cleaved into two fragments in vitro, both of which were able to bind oligonucleotides. These findings suggest that the role of MslA may be to act in concert with the lipoprotein nuclease to generate nucleotides for transport into the mycoplasma cell, as the remaining genes in the operon are predicted to encode an ABC transporter.