A Proposed Life Cycle Model of <i>Spiroplasma mirum</i> Based on Scanning Electron Microscopical Observations of Growth in Liquid Culture

Itoh, Kazuyoshi; Pan, In‐Jen; Koshimizu, Kaoru

doi:10.1111/j.1348-0421.1989.tb00968.x

Cited by 7 publications

(4 citation statements)

References 14 publications

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“…SEM imaging indicated a characteristically long and filamentous but non-helical structure. Spiroplasma cells have been shown to vary in morphology based on conditions and age of in vitro culture, with older cells or those that have been cultured in sub-optimal conditions tending to lack helical structure (Itoh, Pan & Koshimizu, 1989). Thus, the non-helical structure observed may be an artifact of our inability to cultivate Spiroplasma sp.…”

Section: Discussionmentioning

confidence: 99%

Genome-resolved insights into a novelSpiroplasmasymbiont of the Wheat Stem Sawfly (Cephus cinctus)

Yeoman

Brutscher

Esen

et al. 2019

PeerJ

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Arthropods often have obligate relationships with symbiotic microbes, and recent investigations have demonstrated that such host-microbe relationships could be exploited to suppress natural populations of vector carrying mosquitos. Strategies that target the interplay between agricultural pests and their symbionts could decrease the burden caused by agricultural pests; however, the lack of comprehensive genomic insights into naturally occurring microbial symbionts presents a significant bottleneck. Here we employed amplicon surveys, genome-resolved metagenomics, and scanning electron microscopy to investigate symbionts of the wheat stem sawfly (Cephus cinctus), a major pest that causes an estimated $350 million dollars or more in wheat yield losses in the northwestern United States annually. Through 16S rRNA gene sequencing of two major haplotypes and life stages of wheat stem sawfly, we show a novel Spiroplasma species is ever-present and predominant, with phylogenomic analyses placing it as a member of the ixodetis clade of mollicutes. Using state-of-the-art metagenomic assembly and binning strategies we were able to reconstruct a 714 Kb, 72.7%-complete Spiroplasma genome, which represents just the second draft genome from the ixodetis clade of mollicutes. Functional annotation of the Spiroplasma genome indicated carbohydrate-metabolism involved PTS-mediated import of glucose and fructose followed by glycolysis to lactate, acetate, and propionoate. The bacterium also encoded biosynthetic pathways for essential vitamins B2, B3, and B9. We identified putative Spiroplasma virulence genes: cardiolipin and chitinase. These results identify a previously undescribed symbiosis between wheat stem sawfly and a novel Spiroplasma sp., availing insight into their molecular relationship, and may yield new opportunities for microbially-mediated pest control strategies.

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Section: Discussionmentioning

confidence: 99%

Genome-resolved insights into a novelSpiroplasmasymbiont of the Wheat Stem Sawfly (Cephus cinctus)

Yeoman

Brutscher

Esen

et al. 2019

PeerJ

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“…Ten l of MH medium containing S. aureus JCM2413 (5  10 3 ) was added to the cell-culture medium, which was then incubated for 20 min in the same manner as described above for the bacteriostatic assay. Medium-treated bacterial cells in the wells were collected by centrifugation for 30 min at 3,000  g at 4C and fixed as previously described [13]. The samples were then dehydrated, infiltrated with isoamyl acetate, dried in a critical-point dryer (Hitachi, HCP-2; Hitachi, Tokyo, Japan), fixed to aluminum stubs and coated with gold about 10 Å thick in an ion coater (Hitachi E-1030).…”

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confidence: 99%

Bacteriostatic Activity of Whey Acidic Protein (WAP)

Iwamori

Nukumi

Itoh

et al. 2010

The Journal of Veterinary Medical Science

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ABSTRACT. We have previously reported the action of whey acidic protein (WAP) inhibiting the proliferation of mouse mammary epithelial cells in the experiments utilizing in vivo and in vitro systems. We report herein the bacteriostatic activity of WAP. Western blot analysis demonstrated successful isolation of WAP from whey fractions of rat milk by column chromatography. The WAP fraction inhibited the growth of Staphylococcus aureus JCM2413 in a dose-dependent manner, but did not inhibit the growth of Escherichia coli. The bacteriostatic activity of WAP was highest at pH 6.6 and was not affected by the presence of 150 mM NaCl. A scanning electron micrograph of bacteria treated with WAP exhibited the disruption of the bacterial cell walls. The whey acidic protein (WAP) with a four-disulfide core (4-DSC) structure (WAP motif) composed of eight cysteine residues has been identified in milk in a range of species [1,4,6,7,9,11,15]. Possible functions of WAP have been proposed based on its structural similarity to protease inhibitor [9]. However, only a few studies have attempted to determine the actual function of WAP [3,20]. We have previously reported that WAP inhibits the proliferation of mouse mammary epithelial cells in vivo and in vitro [12,[14][15][16][17] and has a function in reducing tumorigenesis and invasion of mammary cancer cells [18]. A number of antibacterial proteins with a WAP motif have been identified in a variety of tissues,-e.g., elafin [22], eppin [24], SLPI [25], SWAM1 and SWAM2 [10], although the molecular antibacterial mechanism of these proteins is not entirely clear. On the basis of homologous amino acid sequences in the WAP motif between WAP and known antibacterial proteins with WAP motifs, WAP may also have antibacterial potential. However, because the amino acid sequences of WAP motif proteins are widely divergent, with the exception of the conserved cysteine residues, it is quite difficult to predict their antibacterial activity merely by examining these sequences.In the present study, we investigated the antibacterial activity of WAP because there have been no studies investigating the effect of WAP on microbes.Milk was collected from lactating Wistar-Imamichi rats (Japan SLC, Hamamatsu, Japan) as described by Simoms et al. [21]. The viscous milk was diluted 1:3 with chilled distilled water, and fat was removed by centrifugation for 30 min at 3,100  g at 4C. The supernatant was adjusted to pH 4.6, and caseins were removed by centrifugation for 30 min at 3,100  g at 4C. The supernatant was adjusted to pH 7.0, and was then loaded on a Sephadex G-75 column (2.6  90 cm; Invitrogen, Carlsbad, CA, U.S.A.) equilibrated in 50 mM Tris-HCl (pH 7.0)/10 mM NaCl. Four milliliters of the eluate was collected at a flow rate of 0.2 ml/min at 4C and dialyzed overnight at 4C against distilled water. A part of each eluate was examined spectrophotometrically (SmartSpecTM Plus; Bio-Rad, Hercules, CA, U.S.A.) to determine the protein concentration using a protein assay kit (BioRad). The eluates wer...

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“…Spiroplasma mirum , Suckling Mouse Cataract Agent (SMCA) ( Megraud et al, 1983 ), was originally isolated from rabbit ticks ( Haemaphysalis leporis-palustis ). We purchased SMCA [CIP 103925] from ATCC and cultured and cloned it with the same method ( Itoh et al, 1989 ).…”

Section: Methodsmentioning

confidence: 99%

The genome and antigen proteome analysis of Spiroplasma mirum

Liu

Li²,

Ye³

et al. 2022

Front. Microbiol.

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Spiroplasma mirum, small motile wall-less bacteria, was originally isolated from a rabbit tick and had the ability to infect newborn mice and caused cataracts. In this study, the whole genome and antigen proteins of S. mirum were comparative analyzed and investigated. Glycolysis, pentose phosphate pathway, arginine metabolism, nucleotide biosynthesis, and citrate fermentation were found in S. mirum, while trichloroacetic acid, fatty acids metabolism, phospholipid biosynthesis, terpenoid biosynthesis, lactose-specific PTS, and cofactors synthesis were completely absent. The Sec systems of S. mirum consist of SecA, SecE, SecDF, SecG, SecY, and YidC. Signal peptidase II was identified in S. mirum, but no signal peptidase I. The relative gene order in S. mirum is largely conserved. Genome analysis of available species in Mollicutes revealed that they shared only 84 proteins. S. mirum genome has 381 pseudogenes, accounting for 31.6% of total protein-coding genes. This is the evidence that spiroplasma genome is under an ongoing genome reduction. Immunoproteomics, a new scientific technique combining proteomics and immunological analytical methods, provided the direction of our research on S. mirum. We identified 49 proteins and 11 proteins (9 proteins in common) in S. mirum by anti-S. mirum serum and negative serum, respectively. Forty proteins in S. mirum were identified in relation to the virulence. All these proteins may play key roles in the pathogeny and can be used in the future for diagnoses and prevention.

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A Proposed Life Cycle Model of Spiroplasma mirum Based on Scanning Electron Microscopical Observations of Growth in Liquid Culture

Cited by 7 publications

References 14 publications

Genome-resolved insights into a novelSpiroplasmasymbiont of the Wheat Stem Sawfly (Cephus cinctus)

Genome-resolved insights into a novelSpiroplasmasymbiont of the Wheat Stem Sawfly (Cephus cinctus)

Bacteriostatic Activity of Whey Acidic Protein (WAP)

The genome and antigen proteome analysis of Spiroplasma mirum

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