Expression of a recombinant protein by an acetic acid bacterial host.

We evaluated the suitability of Komagataeibacter europaeus, a vinegar production organism adept at synthetic media growth, as a host for heterologous gene expression. Cryptic plasmids (pGE1 and pGE2 derivatives) from K. europaeus strain KGMA0119 were employed as vectors for heterologous gene expression. The focus was placed on the groES promoter as a potential inducible switch. The groES promoter was fused with the EGFP gene and introduced into a pGE1 derivative to assess its suitability. Ethanol, acetic acid, and heat stresses were examined under various conditions for induction. EGFP transcription surged 600-fold when late logarithmic phase K. europaeus cells, cultured at 30 °C, endured heat stress at 40 °C, coupled with 20% acetic acid and 30% ethanol stress after an additional 6-hour cultivation. This robust induction system was then applied to express two proteins, Tth pol from the thermophilic bacterium Thermus thermophilus strain M1 and UPV230, a restriction enzyme from the acid-tolerant microorganism Ureaplasma parvum, known to cause vaginal infections and miscarriages. Both Tth pol and UPV230 were successfully expressed in K. europaeus cells and purified. The recovery of Tth pol from K. europaeus cells (480 µg protein per liter culture) was approximately half that from E. coli (960 µg protein per liter culture). In contrast, UPV230 recovery from K. europaeus cells (640 µg protein per liter culture) was nearly 10 times higher than that from Escherichia coli (66 µg protein per liter). The data highlights the potential of acetic acid bacteria as a host for producing acidophilic proteins. The shift in recognition from a 6-base sequence to a 4-base sequence of UPV230 was observed, accompanied by a change in structure as the pH transitioned from acidic pH to near-neutral pH.

Comparison of databases useful for the analysis of vaginal microbiota in Japanese women using next-generation sequencing data (QIIME 2 software).

AIMS: Approximately 10% of children are born prematurely, and bacterial vaginosis during pregnancy is associated with preterm delivery. Highly accurate species-level vaginal microflora analysis helps control bacteria-induced preterm birth. Therefore, we aimed to conduct a bioinformatic analysis of gene sequences using 16S databases and compare their efficacy in comprehensively identifying potentially pathogenic vaginal microbiota in Japanese women. METHODS AND RESULTS: The 16 s rRNA databases, Silva, Greengenes, and the basic local alignment search tool (BLAST) were compared to determine whether the classification quality could be improved using the V3-V4 region next-generation sequencing (NGS) sequences. It was found that NGS data were aligned using the BLAST database with the QIIME 2 platform, whose classification quality was higher than that of Silva, and the combined Silva and Greengenes databases based on the mutual complementarity of the two databases. CONCLUSIONS: The reference database selected during the bioinformatic processing influenced the recognized sequence percentage, taxonomic rankings, and accuracy. This study showed that the BLAST database was the best choice for NGS data analysis of Japanese women's vaginal microbiota.

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.

Blockade of endoplasmic reticulum stress-induced cell death by Ureaplasma parvum vacuolating factor.

Previously, we found that Ureaplasma parvum internalised into HeLa cells and cytosolic accumulation of galectin-3. U. parvum induced the host cellular membrane damage and survived there. Here, we conducted vesicular trafficking inhibitory screening in yeast to identify U. parvum vacuolating factor (UpVF). U. parvum triggered endoplasmic reticulum (ER) stress and upregulated the unfolded protein response-related factors, including BiP, P-eIF2 and IRE1 in the host cells, but it blocked the induction of the downstream apoptotic factors. MicroRNA library screening of U. parvum-infected cells and UpVF-transfected cells identified miR-211 and miR-214 as the negative regulators of the apoptotic cascade under ER stress. Transient expression of UpVF induced HeLa cell death with intracellular vacuolization; however, some stable UpVF transformant survived. U. parvum-infected cervical cell lines showed resistance to actinomycin D, and UpVF stable transformant cell lines exhibited resistance to X-ray irradiation, as well as cisplatin and paclitaxel. UpVF expressing cervical cancer xenografts in nude mice also acquired resistance to cisplatin and paclitaxel. A mycoplasma expression vector based on Mycoplasma mycoides, Syn-MBA (multiple banded antigen)-UpVF, reduced HeLa cell survival compared with that of Syn-MBA after 72 hr of infection. These findings together suggest novel mechanisms for Ureaplasma infection and the possible implications for cervical cancer malignancy. TAKE AWAYS: • Ureaplasmal novel virulence factor, UpVF, was identified. • UpVF triggered ER stress but suppressed apoptotic cascade via miR-211 and -214. • UpVF conferred resistance to anticancer treatments both in vivo and in vitro. • Dual expression of MBA and UpVF in JCVI-syn3B showed host cell damage.

Development of an efficient one-step real-time reverse transcription polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection.

The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 105 copies of synthetic RNA. The one-step real-time RT-PCR method's sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2.

A case series of the dynamics of lipid mediators in patients with sepsis.

BACKGROUND: Bioactive lipid mediators play a crucial role during infection. Previously, we showed the expression level of FAAH mRNA in septic patients was lower than in healthy controls. CASE PRESENTATION: Four patients with a Sequential Organ Failure Assessment (SOFA) score of <7 recovered from sepsis. One patient with SOFA score of 12 on day 7 died on day 21. In the fatal case, eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid, and linoleic acid-derived lipid mediators, including 9-hydroxyoctadecadienoic acid (9-HODE), 13-HODE, 9,10-dihydroxy-12-octadecenoic acid (9,10-DiHOME), and 12,13-DiHOME, were elevated on day 1. Increase in anti-inflammatory prostaglandin E1 ethanolamide together with persistently lower transcription level of FAAH mRNA was detected on day 7 in the fatal case. CONCLUSION: Lipidomic analysis on day 1 revealed elevated linoleic acid metabolites, whereas on day 7, elevated prostaglandin E1 ethanolamide and low level of FAAH mRNA transcription were observed in the fatal case of sepsis.

Type II restriction modification system in Ureaplasma parvum OMC-P162 strain.

Ureaplasma parvum serovar 3 strain, OMC-P162, was isolated from the human placenta of a preterm delivery at 26 weeks' gestation. In this study, we sequenced the complete genome of OMC-P162 and compared it with other serovar 3 strains isolated from patients with different clinical conditions. Ten unique genes in OMC-P162, five of which encoded for hypothetical proteins, were identified. Of these, genes UPV_229 and UPV_230 formed an operon whose open reading frames were predicted to code for a DNA methyltransferase and a hypothetical protein, respectively. DNA modification analysis of the OMC-P162 genome identified N4-methylcytosine (m4C) and N6-methyladenine (m6A), but not 5-methylocytosine (m5C). UPV230 recombinant protein displayed endonuclease activity and recognized the CATG sequence, resulting in a blunt cut between A and T. This restriction enzyme activity was identical to that of the cultivated OMC-P162 strain, suggesting that this restriction enzyme was naturally expressed in OMC-P162. We designated this enzyme as UpaP162. Treatment of pT7Blue plasmid with recombinant protein UPV229 completely blocked UpaP162 restriction enzyme activity. These results suggest that the UPV_229 and UPV_230 genes act as a type II restriction-modification system in Ureaplasma OMC-P162.

Complete Genome Sequence of Ureaplasma parvum Serovar 3 Strain SV3F4, Isolated in Japan.

Here, we present the complete genome sequence of Ureaplasma parvum serovar 3, clinical strain SV3F4, isolated from a Japanese patient with a history of an infectious abortion.

Whole surface image of Mycoplasma mobile, suggested by protein identification and immunofluorescence microscopy.

Mycoplasma mobile, a freshwater fish pathogen featured with robust gliding motility, binds to the surface of the gill, where it then colonizes. Here, to obtain a whole image of its cell surface, we identified the proteins exposed on the surface using the following methods. (i) The cell surface was labeled with sulfosuccinimidyl-6-(biotinamido) hexanoate and recovered by an avidin column. (ii) The cells were subjected to phase partitioning using Triton X-114, and the hydrophobic proteins were recovered. (iii) The membrane fraction was analyzed by two-dimensional gel electrophoresis. These recovered proteins were subjected to peptide mass fingerprinting, and a final list of 36 expressed surface proteins was established. The ratio of identified proteins to whole surface proteins was estimated through two-dimensional gel electrophoresis of the membrane fraction. The localization of three newly found proteins, Mvsps C, E, and F, has been clarified by immunofluorescence microscopy. Integrating all information, a whole image of the cell surface showed that the proteins for gliding that were localized at the base of the protrusion of flask-shaped M. mobile account for more than 12% of all surface proteins and that Mvsps, surface variants that were localized at both parts other than the neck, account for 49% of all surface proteins.

"Mycoplasmal antigen modulation," a novel surface variation suggested for a lipoprotein specifically localized on Mycoplasma mobile.

Mycoplasma mobile, a pathogen of freshwater fish, glides easily across surfaces, colonizes on the fish gill, and causes necrosis. The cell surface is differentiated into three parts: the head, neck, and body. Mobile variable surface proteins (Mvsps) localizing at each of these parts may be involved in surface variation including phase variation and antigenic variation, although no proof exists. In this study, we examined this possibility by focusing on MvspI, the largest Mvsp. Immunofluorescence microscopy showed that MvspI is expressed on the surfaces of all cells. When anti-MvspI antibody was added at concentrations over 0.8 nM, MvspI was observed to decrease over time. After 72 h of cultivation with the antibody, the fluorescence intensity and amount of MvspI decreased up to 13 and 39%, respectively, compared to those of cells grown without antibody. These changes were reversed by the removal of the antibody. Such effects were not observed when another antibody targeting other Mvsps was used, suggesting that the decrease is specific to the relationship between MvspI and the antibody. Cell growth was also inhibited by the antibody, but the decrease in MvspI could not be explained by the selective growth of MvspI-negative variants or by the inhibition of growth with other conditions. The decrease in MvspI caused by the antibody binding may suggest a novel type of surface variation, designated here as "mycoplasmal antigen modulation."