Assembly properties of bacterial actin MreB involved in Spiroplasma swimming motility.

Bacterial actin MreB forms filaments composed of antiparallel double-stranded units. The wall-less helical bacterium Spiroplasma has five MreB homologs (MreB1-5), some of which are involved in an intracellular ribbon for driving the bacterium's swimming motility. Although the interaction between MreB units is important for understanding Spiroplasma swimming, the interaction modes of each ribbon component are unclear. Here, we examined the assembly properties of Spiroplasma eriocheiris MreB5 (SpeMreB5), one of the ribbon component proteins that forms sheets. Electron microscopy revealed that sheet formation was inhibited under acidic conditions and bundle structures were formed under acidic and neutral conditions with low ionic strength. We also used solution assays and identified four properties of SpeMreB5 bundles as follows: (I) bundle formation followed sheet formation; (II) electrostatic interactions were required for bundle formation; (III) the positively charged and unstructured C-terminal region contributed to promoting lateral interactions for bundle formation; and (IV) bundle formation required Mg2+ at neutral pH but was inhibited by divalent cations under acidic pH conditions. During these studies, we also characterized two aggregation modes of SpeMreB5 with distinct responses to ATP. These properties will shed light on SpeMreB5 assembly dynamics at the molecular level.

'Candidatus Phytoplasma vignae', assigning a species description to a long-known phytoplasma occurring in northern Australia.

Gene- and genome-based approaches were used to determine whether Vigna little leaf (ViLL) phytoplasma, which occurs in northern Australia, is a distinct 'Candidatus Phytoplasma' species. The ViLL 16S rRNA gene sequences exhibited the highest known similarity to species in the 16SrXXIX-A and 16SrIX-D subgroups, namely 'Candidatus Phytoplasma omanense' (98.03-98.10%) and 'Candidatus Phytoplasma phoenicium' (96.87-97.20%), respectively. A total of 48 single-copy orthologue genes were identified to be shared among the two draft ViLL phytoplasma genomes, 30 publicly available phytoplasma genomes, and one Acholeplasma laidlawii genome as the outgroup taxon. Phylogenomic assessments using the 48 shared single-copy orthologue genes supported that ViLL and 'Ca. Phytoplasma phoenicium' were closely related yet distinct species. The 16S rRNA gene sequence analysis and phylogenomic assessment indicate that ViLL phytoplasmas are a distinct taxon. As such, a novel species, 'Candidatus Phytoplasma vignae', is proposed. Strain BAWM-336 (genome accession number JAUZLI000000000) detected in Momordica charantia (bitter melon) serves as the reference strain of this species, with infected plant material deposited in the Victorian Plant Pathology Herbarium (VPRI) as VPRI 44369.

Comparison of DNA extraction procedures for detection of Mycoplasma bovis directly from extended bovine semen straw samples using a commercial M. bovis PCR.

BACKGROUND: Mycoplasma bovis is a global pathogen of cattle but was detected for the first time in New Zealand in 2017, triggering a response under their Biosecurity Act as an "unwanted organism". Following a lengthy eradication campaign, the Ministry of Primary Industries (MPI) now requires all bovine semen destined for export to New Zealand to be screened with an M. bovis-specific real-time PCR (rtPCR) compliant with amended import health standard (IHS) test requirements aimed at preventing the accidental importation of M. bovis. The standard stipulates that semen samples cannot be centrifuged prior to DNA extraction. To comply with these strict requirements, one of the listed tests was validated together with different DNA preparation steps and compared with existing in-house procedures. DNA was extracted from semen straws using the current in-house semi-automated platform procedures for processing culture, tissue and body fluid sample submissions and was compared with the stipulated test requirements. DNA from centrifuged and unspun semen samples spiked with M. bovis was also compared. RESULTS: The rtPCR had a sensitivity and specificity of 100% (95% confidence interval = 79-100% and 74-100%, respectively) when testing DNA from other Mycoplasma species or bovine semen spiked with the latter, with a high level of repeatability for within- and between- run replicates. The consistent limit of detection was 0.001 pg/µl M. bovis DNA and between 5.3 × 102 and 7.5 × 102 CFU/ml M. bovis when artificially spiked in semen. DNA extracted using the KingFisher Flex was detected with lower Cq values than the Maxwell 16, but the comparable improvements in sensitivity were mainly associated with non-centrifuged samples (p < 0.001). None of the procedures tested impeded the detection sensitivity of M. bovis in the presence of competitor organisms Acholeplasma laidlawii, Mycoplasma bovigenitalium and Ureaplasma diversum, confirming M. bovis specificity of the polC target. CONCLUSIONS: Under the experimental conditions applied, this rtPCR test efficiently detected M. bovis in extended bovine semen straw samples from DNA extracted using both semi-automated extraction platforms, irrespective of prior centrifugation of extended semen.

Transition of the genital mollicutes from the second to the third trimester of pregnancy and its association with adverse pregnancy outcomes in GDM women: a prospective, single-center cohort study from China.

BACKGROUND: The association of genital Mollicutes infection transition with adverse pregnancy outcomes was insignificant among general pregnant women, but there remains a paucity of evidence linking this relationship in gestational diabetes mellitus (GDM) women. The aim was to investigate the association between genital Mollicutes infection and transition and adverse pregnancy outcomes in GDM women, and to explore whether this association still exist when Mollicutes load varied. METHODS: We involved pregnant women who attended antenatal care in Chongqing, China. After inclusion and exclusion criteria, we conducted a single-center cohort study of 432 GDM women with pregnancy outcomes from January 1, 2018 to December 31, 2021. The main outcome was adverse pregnancy outcomes, including premature rupture of membrane (PROM), fetal distress, macrosomia and others. The exposure was Mollicutes infection, including Ureaplasma urealyticum (Uu) and Mycoplasma hominis (Mh) collected in both the second and the third trimesters, and testing with polymerase chain reaction method. The logistic regression models were used to estimate the relationship between Mollicutes infection and adverse pregnancy outcomes. RESULTS: Among 432 GDM women, 241 (55.79%) were infected with genital Mollicutes in either the second or third trimester of pregnancy. At the end of the pregnancy follow-up, 158 (36.57%) participants had adverse pregnancy outcomes, in which PROM, fetal distress and macrosomia were the most commonly observed adverse outcomes. Compared with the uninfected group, the Mollicutes (+/-) group showed no statistical significant increase in PROM (OR = 1.05, 95% CI:0.51 ∼ 2.08) and fetal distress (OR = 1.21, 95% CI: 0.31 ∼ 3.91). Among the 77 participants who were both Uu positive in the second and third trimesters, 38 participants presented a declined Uu load and 39 presented an increased Uu load. The Uu increased group had a 2.95 odds ratio (95% CI: 1.10~8.44) for adverse pregnancy outcomes. CONCLUSION: Mollicutes infection and transition during trimesters were not statistically associated with adverse pregnancy outcomes in GDM women. However, among those consistent infections, women with increasing Uu loads showed increased risks of adverse pregnancy outcomes. For GDM women with certain Mollicutes infection and colonization status, quantitative screening for vaginal infection at different weeks of pregnancy was recommended to provide personalized fertility treatment.

Leptodelphax maculigera (Hemiptera: Delphacidae) Harbors the Corn Stunt Complex Pathogens.

The African planthopper Leptodelphax maculigera (Hemiptera: Delphacidae) has been recently reported in many places in Brazil in association with maize. Its occurrence in maize production fields in Brazil has brought concerns to the corn production chain regarding the possibility of this planthopper to be a vector for maize bushy stunt phytoplasma (MBSP), corn stunt spiroplasma (Spiroplasma kunkelii), maize rayado fino virus (MRFV), and maize striate mosaic virus (MSMV). The phytoplasma and spiroplasma, which are bacteria belonging to the class Mollicutes, and the two viruses are associated with the corn stunt disease complex. Given the presence of the African planthopper species and the corn stunt complex in Brazil, we further investigated the abundance of this planthopper species in the State of Santa Catarina, Brazil, and whether the planthopper can carry the four pathogens. We inspected 12 maize production fields in different municipalities in the state for 20 weeks, using two yellow sticky traps for each maize field. The sticky traps were replaced weekly. A total of 130 specimens of L. maculigera were captured, with a great discrepancy in quantity among locations and weeks. We detected the mollicute MBSP and the viruses MRFV and MSMV in L. maculigera, whereas S. kunkelii was absent in the assessed African planthopper samples. The molecular detection of the phytoplasma and the viruses in field-collected African planthoppers is strong evidence that this insect species has the ability to acquire those pathogens through feeding from the phloem of diseased maize plants. Nonetheless, transmission capacity needs to be experimentally proven to assert L. maculigera as a vector for the corn-stunting pathogens.

Structure and Function of Gli123 Involved in Mycoplasma mobile Gliding.

Mycoplasma mobile is a fish pathogen that glides on solid surfaces by means of its own gliding machinery composed of internal and surface structures. In the present study, we focused on the function and structure of Gli123, a surface protein that is essential for the localization of other surface proteins. The amino acid sequence of Gli123, which is 1,128 amino acids long, contains lipoprotein-specific repeats. We isolated the native Gli123 protein from M. mobile cells and a recombinant protein, rGli123, from Escherichia coli. The isolated rGli123 complemented a nonbinding and nongliding mutant of M. mobile that lacked Gli123. Circular dichroism and rotary-shadowing electron microscopy (EM) showed that rGli123 has a structure that is not significantly different from that of the native protein. Rotary-shadowing EM suggested that Gli123 adopts two distinct globular and rod-like structures, depending on the ionic strength of the solution. Negative-staining EM coupled with single-particle analysis revealed that Gli123 forms a globular structure featuring a small protrusion with dimensions of approximately 15.7, 14.7, and 14.1 nm for the "height," major axis and minor axis, respectively. Small-angle X-ray scattering analyses indicated a rod-like structure composed of several tandem globular domains with total dimensions of approximately 34 nm in length and 6 nm in width. Both molecular structures were suggested to be dimers, based on the predicted molecular size and structure. Gli123 may have evolved by multiplication of repeating lipoprotein units and acquired a role for Gli521 and Gli349 assembly. IMPORTANCE Mycoplasmas are pathogenic bacteria that are widespread in animals. They are characterized by small cell and genome sizes but are equipped with unique abilities for infection, such as surface variation and gliding. Here, we focused on a surface-localizing protein named Gli123 that is essential for Mycoplasma mobile gliding. This study suggested that Gli123 undergoes drastic conformational changes between its rod-like and globular structures. These changes may be caused by a repetitive structure common in the surface proteins that is responsible for the modulation of the cell surface structure and related to the assembly process for the surface gliding machinery. An evolutionary process for surface proteins essential for this mycoplasma gliding was also suggested in the present study.

Comparison of Five Commercial Molecular Assays for Mycoplasma Testing of Cellular Therapy Products.

Testing of cellular therapy products for Mycoplasma is a regulatory requirement by the United States Food and Drug Administration (FDA) to ensure the sterility and safety of the product prior to release for patient infusion. The risk of Mycoplasma contamination in cell culture is high. Gold standard testing follows USP 63 which requires a 28-day agar and broth cultivation method that is impractical for short shelf-life biologics. Several commercial molecular platforms have been marketed for faster raw material and product release testing; however, little performance data are available in the literature. In this study, we performed a proof-of-principle analysis to evaluate the performance of five commercial molecular assays, including the MycoSEQ Mycoplasma detection kit (Life Technologies), the MycoTOOL Mycoplasma real-time detection kit (Roche), the VenorGEM qOneStep kit (Minerva Biolabs), the ATCC universal Mycoplasma detection kit, and the Biofire Mycoplasma assay (bioMérieux Industry) using 10 cultured Mollicutes spp., with each at four log-fold dilutions (1,000 CFU/mL to 1 CFU/mL) in biological duplicates with three replicates per condition (n = 6) to assess limit of detection (LOD) and repeatability. Additional testing was performed in the presence of tumor infiltrating lymphocytes (TILs). Based on LOD alone, the Biofire Mycoplasma assay was most sensitive followed by the MycoSEQ and MycoTOOL which were comparable. We showed that not all assays were capable of meeting the ≤10 CFU/mL LOD to replace culture-based methods according to European and Japanese pharmacopeia standards. No assay interference was observed when testing in the presence of TILs.

Latest Advances in Laboratory Detection of Mycoplasma genitalium.

Mycoplasma genitalium is an important sexually transmitted pathogen affecting both men and women. Its extremely slow growth in vitro and very demanding culture requirements necessitate the use of molecular-based diagnostic tests for its detection in clinical specimens. The recent availability of U.S. Food and Drug Administration (FDA)-cleared commercial molecular-based assays has enabled diagnostic testing to become more widely available in the United States and no longer limited to specialized reference laboratories. Advances in the knowledge of the epidemiology and clinical significance of M. genitalium as a human pathogen made possible by the availability of molecular-based testing have led to updated guidelines for diagnostic testing and treatment that have been published in various countries. This review summarizes the importance of M. genitalium as an agent of human disease, explains the necessity of obtaining a microbiological diagnosis, describes currently available diagnostic methods, and discusses how the emergence of antimicrobial resistance has complicated treatment alternatives and influenced the development of diagnostic tests for resistance detection, with an emphasis on developments over the past few years.

The observation of taxonomic boundaries for the 16SrII and 16SrXXV phytoplasmas using genome-based delimitation.

Within the 16SrII phytoplasma group, subgroups A-X have been classified based on restriction fragment length polymorphism of their 16S rRNA gene, and two species have been described, namely 'Candidatus Phytoplasma aurantifolia' and 'Ca. Phytoplasma australasia'. Strains of 16SrII phytoplasmas are detected across a broad geographic range within Africa, Asia, Australia, Europe and North and South America. Historically, all members of the 16SrII group share ≥97.5 % nucleotide sequence identity of their 16S rRNA gene. In this study, we used whole genome sequences to identify the species boundaries within the 16SrII group. Whole genome analyses were done using 42 phytoplasma strains classified into seven 16SrII subgroups, five 16SrII taxa without official 16Sr subgroup classifications, and one 16SrXXV-A phytoplasma strain used as an outgroup taxon. Based on phylogenomic analyses as well as whole genome average nucleotide and average amino acid identity (ANI and AAI), eight distinct 16SrII taxa equivalent to species were identified, six of which are novel descriptions. Strains within the same species had ANI and AAI values of >97 %, and shared ≥80 % of their genomic segments based on the ANI analysis. Species also had distinct biological and/or ecological features. A 16SrII subgroup often represented a distinct species, e.g., the 16SrII-B subgroup members. Members classified within the 16SrII-A, 16SrII-D, and 16SrII-V subgroups as well as strains classified as sweet potato little leaf phytoplasmas fulfilled criteria to be included as members of a single species, but with subspecies-level relationships with each other. The 16SrXXV-A taxon was also described as a novel phytoplasma species and, based on criteria used for other bacterial families, provided evidence that it could be classified as a distinct genus from the 16SrII phytoplasmas. As more phytoplasma genome sequences become available, the classification system of these bacteria can be further refined at the genus, species, and subspecies taxonomic ranks.

Prevalence of Mollicutes among men who have sex with men and transgender women aged 15 to 19 years in Salvador, North-eastern Brazil.

BACKGROUND: Some species of Mollicutes have been associated with different pathologies of the urogenital tract in humans, with a high prevalence among adult men who have sex with men (MSM) and transgender women (TGW). However, few studies have been performed to investigate its prevalence among adolescents. In this study, we estimated the initial prevalence of Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), and Ureaplasma parvum (UP); the rate of misdiagnosis at different anatomical sites; and the associated factors with positive tests for Mollicutes among MSM and TGW aged 15 to 19 years enrolled in the PrEP1519 study. METHODS: PrEP-1519 is the first study to investigate the effectiveness of pre-exposure prophylaxis for human immunodeficiency virus among adolescent MSM and TGW aged 15 to 19 in Latin America. Oral, anal, and urethral swabs were taken from 246 adolescents upon enrolment in the study to detect MG, MH, UU, and UP by quantitative polymerase chain reaction (qPCR). Bivariate and multivariate analyses were conducted by Poisson regression and 95% confidence intervals (95% CI) were estimated. RESULTS: The prevalence of Mollicutes was 32.1%. UU was the most prevalent species (20.7%), followed by MH (13.4%), MG (5.7%), and UP (3.2%); 67.3% of the positive samples would have been missed if only urethral samples had been taken. Receptive anal sex (prevalence ratio [PR] = 1.79; 95% CI = 1.07-3.01) and clinical suspicion of sexually transmitted infection (PR = 1.62; 95% CI = 1.01-2.61) were factors associated with the detection of Mollicutes in general. Group sex (PR = 1.98; 95% CI = 1.12-3.50) and receptive anal sex (PR = 2.36; 95% CI = 0.95-5.86) were associated with the detection of Mycoplasma spp. No sociodemographic, clinical, or behavioural variable was significantly associated with the detection of Ureaplasma spp. CONCLUSIONS: A high prevalence of Mollicutes was observed among adolescent MSM and TGW, especially at extragenital sites. Further research is required to understand the epidemiological profile of high-risk adolescents in different regions and contexts, and to investigate the pathogenesis of Mollicutes in the oral and anal mucosa before routine screening can be recommended in clinical practice.

Amendment of a thermophile-fermented compost to humus improves the growth of female larvae of the Hercules beetle Dynastes hercules (Coleoptera: scarabaeidae).

AIMS: Hercules beetle is a popular pet and large adult individuals are considered valuable. Incorporating compost prepared from marine animals and fermented by thermophilic bacteria into the humus benefits the gut microflora of several livestock. Here, we evaluated whether this compost improves the growth of the Hercules beetle (Dynastes hercules hercules) larvae. METHODS AND RESULTS: We mixed the compost grains with the humus at a final concentration of 1% (w/w) and transferred ∼90 days old Hercules beetle larvae to fresh humus with or without the compost. After 72 days rearing period, only the female larvae reared in the humus with compost exhibited superior growth, compared with those grown in compost-free humus. The gut bacterial composition was determined at 0 and 46 day after transferring the larvae to humus with or without compost. Improved growth of the female larvae was associated with increased abundance of Mollicutes and decreased abundance of Gammaproteobacteria. CONCLUSION: The thermophile-fermented compost has a probiotic effect on the female Hercules beetle larvae that is mediated by altered gut microflora.

Composition, Antibiofilm, and Antibacterial Potential of Volatile Oils from Geopropolis of Different Stingless Bees' Species.

We aimed to characterize and investigate the antibacterial potential of the native stingless bees geopropolis volatile oils (VO) for the search of potentially new bioactive compounds. Geopropolis samples from Melipona bicolor schencki, M. compressipes manaosensis, M. fasciculata, M. quadrifasciata, M. marginata and M. seminigra merrillae were collected from hives in South Brazil. VO were obtained by hydrodistillation and characterised by gas chromatography coupled to mass spectrometry (GC/MS). Antimicrobial activity was assessed by microplate dilution method. The lowest MIC against cell walled bacteria was 219±0 µg mL-1 from M. quadrifasciata geopropolis VO with Staphylococcus aureus. The M. b. schencki geopropolis VO minimal inhibition concentration (MIC) was 424±0 µg mL-1 against all the mycoplasma strains evaluated. Fractionation resulted in the reduction of 50 % of the MIC value from the original oil. However, its compounds' synergism seems to be essential to this activity. Antibiofilm assays demonstrated 15.25 % eradication activity and 13.20 % inhibition of biofilm formation after 24 h for one subfraction at 2× its MIC as the best results found. This may be one of the essential mechanisms by which geopropolis VOs perform their antimicrobial activity.

Corrigendum: Hyperammonemia After Lung Transplantation: Systematic Review and a Mini Case Series.

[This corrects the article DOI: 10.3389/ti.2022.10433.].

Hyperammonemia After Lung Transplantation: Systematic Review and a Mini Case Series.

Background: Hyperammonemia after lung transplantation (HALT) is a rare but serious complication with high mortality. This systematic review delineates possible etiologies of HALT and highlights successful strategies used to manage this fatal complication. Methods: Seven biomedical databases and grey literature sources were searched using keywords relevant to hyperammonemia and lung transplantation for publications between 1995 and 2020. Additionally, we retrospectively analyzed HALT cases managed at our institution between January 2016 and August 2018. Results: The systematic review resulted in 18 studies with 40 individual cases. The mean peak ammonia level was 769 µmol/L at a mean of 14.1 days post-transplant. The mortality due to HALT was 57.5%. In our cohort of 120 lung transplants performed, four cases of HALT were identified. The mean peak ammonia level was 180.5 µmol/L at a mean of 11 days after transplantation. HALT in all four patients was successfully treated using a multimodal approach with an overall mortality of 25%. Conclusion: The incidence of HALT (3.3%) in our institution is comparable to prior reports. Nonetheless, ammonia levels in our cohort were not as high as previously reported and peaked earlier. We attributed these significant differences to early recognition and prompt institution of multimodal treatment approach.

Tree of motility - A proposed history of motility systems in the tree of life.

Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility.

Integrative characterization of the near-minimal bacterium Mesoplasma florum.

The near-minimal bacterium Mesoplasma florum is an interesting model for synthetic genomics and systems biology due to its small genome (~ 800 kb), fast growth rate, and lack of pathogenic potential. However, fundamental aspects of its biology remain largely unexplored. Here, we report a broad yet remarkably detailed characterization of M. florum by combining a wide variety of experimental approaches. We investigated several physical and physiological parameters of this bacterium, including cell size, growth kinetics, and biomass composition of the cell. We also performed the first genome-wide analysis of its transcriptome and proteome, notably revealing a conserved promoter motif, the organization of transcription units, and the transcription and protein expression levels of all protein-coding sequences. We converted gene transcription and expression levels into absolute molecular abundances using biomass quantification results, generating an unprecedented view of the M. florum cellular composition and functions. These characterization efforts provide a strong experimental foundation for the development of a genome-scale model for M. florum and will guide future genome engineering endeavors in this simple organism.

Mariniplasma anaerobium gen. nov., sp. nov., a novel anaerobic marine mollicute, and proposal of three novel genera to reclassify members of Acholeplasma clusters II-IV.

A novel strictly anaerobic chemoorganotrophic bacterium, designated Mahy22T, was isolated from sulfidic bottom water of a shallow brackish meromictic lake in Japan. Cells of the strain were Gram-stain-negative, non-motile and coccoid in shape with diameters of about 600-800 nm. The temperature range for growth was 15-37 °C, with optimum growth at 30-32 °C. The pH range for growth was pH 6.2-8.9, with optimum growth at pH 7.2-7.4. The strain grew with NaCl concentrations of 5% or below (optimum, 2-3%). Growth of the strain was enhanced by the addition of thiosulfate. The major cellular fatty acids were C16:0 and anteiso-C15:0. Respiratory quinones were not detected. The complete genome sequence of strain Mahy22T possessed a 1 885 846 bp circular chromosome and a 12 782 bp circular genetic element. The G+C content of the genome sequence was 30.1 mol%. Phylogenetic analysis based on the 16S rRNA gene revealed that the novel strain belonged to the family Acholeplasmataceae, class Mollicutes. The closest relative of strain Mahy22T with a validly published name was Acholeplasma palmae J233T with a 16S rRNA gene sequence similarity of 90.5%. Based on the results of polyphasic analysis, the name Mariniplasma anaerobium gen. nov., sp. nov. is proposed to accommodate strain Mahy22T, along with reclassification of some Acholeplasma species into Alteracholeplasma gen. nov., Haploplasma gen. nov. and Paracholeplasma gen. nov.

'Candidatus Phytoplasma stylosanthis', a novel taxon with a diverse host range in Australia, characterised using multilocus sequence analysis of 16S rRNA, secA, tuf, and rp genes.

In Australia, Stylosanthes little leaf (StLL) phytoplasma has been detected in Stylosanthes scabra Vogel, Arachis pintoi Krapov, Saccharum officinarum L., Carica papaya L., Medicago sativa L., and Solanum tuberosum L. The 16S rRNA gene sequence of StLL phytoplasma strains from S. scabra, C. papaya, S. officinarum and S. tuberosum were compared and share 99.93-100 % nucleotide sequence identity. Phylogenetic comparisons between the 16S rRNA genes of StLL phytoplasma and other 'Candidatus Phytoplasma' species indicate that StLL represents a distinct phytoplasma lineage. It shares its most recent known ancestry with 'Ca. Phytoplasma luffae' (16SrVIII-A), with which it has 97.17-97.25 % nucleotide identity. In silico RFLP analysis of the 16S rRNA amplicon using iPhyClassifier indicate that StLL phytoplasmas have a unique pattern (similarity coefficient below 0.85) that is most similar to that of 'Ca. Phytoplasma luffae'. The unique in silico RFLP patterns were confirmed in vitro. Nucleotide sequences of genes that are more variable than the 16S rRNA gene, namely tuf (tu-elongation factor), secA (partial translocation gene), and the partial ribosomal protein (rp) gene operon (rps19-rpl22-rps3), produced phylogenetic trees with similar branching patterns to the 16S rRNA gene tree. Sequence comparisons between the StLL 16S rRNA spacer region confirmed previous reports of rrn interoperon sequence heterogeneity for StLL, where the spacer region of rrnB encodes a complete tRNA-Isoleucine gene and the rrnA spacer region does not. Together these results suggest that the Australian phytoplasma, StLL, is unique according to the International Organization for Mycoplasmology (IRPCM) recommendations. The novel taxon 'Ca. Phytoplasma stylosanthis' is proposed, with the most recent strain from a potato crop in Victoria, Australia, serving as the reference strain (deposited in the Victorian Plant Pathology Herbarium as VPRI 43683).

North American Fireflies Host Low Bacterial Diversity.

Although there are numerous studies of firefly mating flashes, lantern bioluminescence, and anti-predation lucibufagin metabolites, almost nothing is known about their microbiome. We therefore used 16S rRNA community amplicon sequencing to characterize the gut and body microbiomes of four North American firefly taxa: Ellychnia corrusca, the Photuris versicolor species complex, Pyractomena borealis, and Pyropyga decipiens. These firefly microbiomes all have very low species diversity, often dominated by a single species, and each firefly type has a characteristic microbiome. Although the microbiomes of male and female fireflies did not differ from each other, Ph. versicolor gut and body microbiomes did, with their gut microbiomes being enriched in Pseudomonas and Acinetobacter. Ellychnia corrusca egg and adult microbiomes were unique except for a single egg microbiome that shared a community type with E. corrusca adults, which could suggest microbial transmission from mother to offspring. Mollicutes that had been previously isolated from fireflies were common in our firefly microbiomes. These results set the stage for further research concerning the function and transmission of these bacterial symbionts.

Dihydrouridine synthesis in tRNAs is under reductive evolution in Mollicutes.

Dihydrouridine (D) is a tRNA-modified base conserved throughout all kingdoms of life and assuming an important structural role. The conserved dihydrouridine synthases (Dus) carries out D-synthesis. DusA, DusB and DusC are bacterial members, and their substrate specificity has been determined in Escherichia coli. DusA synthesizes D20/D20a while DusB and DusC are responsible for the synthesis of D17 and D16, respectively. Here, we characterize the function of the unique dus gene encoding a DusB detected in Mollicutes, which are bacteria that evolved from a common Firmicute ancestor via massive genome reduction. Using in vitro activity tests as well as in vivo E. coli complementation assays with the enzyme from Mycoplasma capricolum (DusBMCap), a model organism for the study of these parasitic bacteria, we show that, as expected for a DusB homolog, DusBMCap modifies U17 to D17 but also synthetizes D20/D20a combining therefore both E. coli DusA and DusB activities. Hence, this is the first case of a Dus enzyme able to modify up to three different sites as well as the first example of a tRNA-modifying enzyme that can modify bases present on the two opposite sides of an RNA-loop structure. Comparative analysis of the distribution of DusB homologs in Firmicutes revealed the existence of three DusB subgroups namely DusB1, DusB2 and DusB3. The first two subgroups were likely present in the Firmicute ancestor, and Mollicutes have retained DusB1 and lost DusB2. Altogether, our results suggest that the multisite specificity of the M. capricolum DusB enzyme could be an ancestral property.