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Research Article | Spotlight

The Variable Internal Structure of the Mycoplasma penetrans Attachment Organelle Revealed by Biochemical and Microscopic Analyses: Implications for Attachment Organelle Mechanism and Evolution

Steven L. Distelhorst, Dominika A. Jurkovic, Jian Shi, Grant J. Jensen, Mitchell F. Balish
Piet A. J. de Boer, Editor
Steven L. Distelhorst
aDepartment of Microbiology, Miami University, Oxford, Ohio, USA
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Dominika A. Jurkovic
aDepartment of Microbiology, Miami University, Oxford, Ohio, USA
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Jian Shi
bDivision of Biology and Bioengineering, California Institute of Technology, Pasadena, California, USA
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Grant J. Jensen
bDivision of Biology and Bioengineering, California Institute of Technology, Pasadena, California, USA
cHoward Hughes Medical Institute, California Institute of Technology, Pasadena, California, USA
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Mitchell F. Balish
aDepartment of Microbiology, Miami University, Oxford, Ohio, USA
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Piet A. J. de Boer
Case Western Reserve University School of Medicine
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DOI: 10.1128/JB.00069-17
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  • FIG 1
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    FIG 1

    SEM images of M. penetrans whole cells and detergent-insoluble structures. (A) Field of M. penetrans cells. Arrows, connecting filaments. Scale bar, 1 μm. (B) Individual M. penetrans cell. Arrow, AO. Scale bar, 200 nm. (C and D) Detergent-insoluble structures from M. penetrans cells extracted with Triton X-100 (C) or Tween 20 (D). Asterisks, wide ends of structures. Scale bar, 200 nm.

  • FIG 2
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    FIG 2

    AO-associated objects and their lengths. (A) Distribution of the lengths of TXI objects observed by SEM. (B) Distribution of the lengths of nucleoid-free zones observed by light and fluorescence microscopy. (C) Distribution of the lengths of TWI objects observed by SEM. (D) Correspondence of the lengths of nucleoid-free zones to the fractions of the cell length occupied by the nucleoid-free zones in 52 cells with a single nucleoid-free zone. (E and F) Correspondence of the lengths of nucleoid-free zones to the fractions of the cell length occupied by the nucleoid-free zones in 16 cells with two nucleoid-free zones, a shorter one (E) and a longer one (F). (G) Example of nucleoid-free zones. Black, cell outline viewed by phase-contrast microscopy; white, nucleoid stained with DAPI. Arrows, nucleoid-free zones, measured along the long axis of the cell for panel B. Scale bar, 500 nm.

  • FIG 3
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    FIG 3

    Internal organization of M. penetrans observed by ECT. (A to C) Sections of different whole cells. (D to F) Corresponding schematics. In panels A to C, ∼20-nm granules are ribosomes (reviewed in reference 70). The insertion site of a connecting filament was observed at one cell pole (C and F). Black lines, cell membranes and boundaries of ribosome-free zones; circles, ribosomes (positions are schematic and do not represent actual ribosomes); arrow in panel F, location of connecting filament. Scale bars, 100 nm.

  • FIG 4
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    FIG 4

    SDS-PAGE of the M. penetrans whole-cell lysate and TWI and TXI proteins. A total of 22 μg each of protein from the whole-cell lysate (lane 2), the TWI fraction (lane 3), and the TXI fraction (lane 4) was separated by SDS-PAGE for comparison and for excision of candidate bands chosen for identification. Lane 1 contains protein standards, with sizes indicated to the left. The 66-kDa band in lane 4 was present only in some preparations. Numbers in the gel indicate specific bands chosen for MALDI-TOF mass spectrometry and correspond to protein identifications in Table 1.

  • FIG 5
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    FIG 5

    RT-PCR analysis of the putative cytoskeletal operon of M. penetrans. (A) Agarose gel containing PCR products from RT-generated transcripts that span the gene junctions numbered in panel B. Top, products amplified from reverse-transcribed RNA using a primer complementary to a region at the 3′ end of mype1570; bottom, products amplified from genomic DNA used as a positive control. (B) Schematic of genes encoding MYPE1520 to MYPE1570, showing the genomic orientation and relative sizes, including the 5′ and 3′ flanking genes. Numbers 1 to 7 refer to the positions of the primer pairs used to span gene junctions for RT-PCR.

Tables

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  • TABLE 1

    TXI and TWI proteins identified by MALDI-TOF

    ProteinBand no. in Fig. 4Functional annotationSize (kDa)
    MYPE15303NAa 122
    MYPE15501NA386
    MYPE15604NA99
    MYPE15704NA93
    MYPE40002NA99
    MYPE51006Pyruvate dehydrogenase subunit E2, PdhC51
    MYPE66308P42 lipoprotein (P35 family)42
    MYPE68109P35 lipoprotein (P35 family)38
    MYPE69603Lipoprotein138
    MYPE87505Oligopeptide ABC transporter ATP-binding protein, OppF92
    MYPE96409Lactate dehydrogenase, Ldh34
    MYPE100907Ribosomal subunit S3, RpsC48
    • ↵a NA, not applicable.

  • TABLE 2

    Comparison of AO protein features

    Species and proteinCoiled coilMolecular mass of >70 kDaExtreme pI (<5 or >9)Total no. of features
    M. pneumoniae
        HMW1 (MPN447)XXX3
        HMW2 (MPN310)XXX3
        HMW3 (MPN453) XX2
        P24 (MPN312) 0
        P41 (MPN311)X X2
        P65 (MPN309)X X2
        P200 (MPN567) XX2
        TopJ (MPN119) XX2
    M. mobile
        MMOB0150 0
        MMOB1620 0
        MMOB1630X X2
        MMOB1640 0
        MMOB1650 X 1
        MMOB1660 0
        MMOB1670 0
        MMOB4530 X1
        MMOB4860 X1
        MMOB5430 X1
    M. penetrans
        MYPE1530XXX3
        MYPE1550XXX3
        MYPE1560XXX3
        MYPE1570XXX3
        MYPE4000XXX3
        MYPE5100 0
        MYPE6630 0
        MYPE6810 0
        MYPE6960 XX2
        MYPE8750XXX3
        MYPE9640 0
        MYPE10090 0
  • TABLE 3

    Primers used for RT-PCR

    Primer designationSequence (5′ to 3′)TargetProduct size (bp)
    1510end(up)AATGAGCAGTAAGTGCTAGCMYPE1510-MYPE1520603
    1520begin(down)GTCTTAGGAATTTGAACAGGTG
    1520end(up)GGAAAAAGAAGCTGTCACAGMYPE1520-MYPE1530566
    1530begin(down)GGTTGATTGTCAGCAGAACC
    1530end(up)TTCCAGCTCCTGCATATGACMYPE1530-MYPE1540527
    1540begin(down)GTCAGTTCCCTGAAATTGTTC
    1540end(up)CTAAATTGTTCCAATATTGAATAATCGCMYPE1540-MYPE1550454
    1550begin(down)ATAGATGAGATTCTTTATCAAAGTTCTC
    1550end(up)ATCAAACCCAAGCGATTCTGMYPE1550-MYPE1560541
    1560begin(down)TCAACATCGTCTTGTCTTGG
    1560end(up)TGCTCCTAGTAGTGAGTTGCMYPE1560-MYPE1570696
    1570begin(down)CTTCTCTAGCTTGAACTTGG
    1570end(up)TTTGGAAGACCAGCAGGAAGMYPE1570-MYPE1580380
    1580begin(down)GTGTTGCCAGGTCTAAGATC
    1580RT(down)CATAGCCGTTTGAACAGTGTNAa NA
    • ↵a NA, not applicable.

Additional Files

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    • Supplemental file 1 -

      Data Set S1, RNA-Seq data

      XLS, 655K

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The Variable Internal Structure of the Mycoplasma penetrans Attachment Organelle Revealed by Biochemical and Microscopic Analyses: Implications for Attachment Organelle Mechanism and Evolution
Steven L. Distelhorst, Dominika A. Jurkovic, Jian Shi, Grant J. Jensen, Mitchell F. Balish
Journal of Bacteriology May 2017, 199 (12) e00069-17; DOI: 10.1128/JB.00069-17

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The Variable Internal Structure of the Mycoplasma penetrans Attachment Organelle Revealed by Biochemical and Microscopic Analyses: Implications for Attachment Organelle Mechanism and Evolution
Steven L. Distelhorst, Dominika A. Jurkovic, Jian Shi, Grant J. Jensen, Mitchell F. Balish
Journal of Bacteriology May 2017, 199 (12) e00069-17; DOI: 10.1128/JB.00069-17
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    • ABSTRACT
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KEYWORDS

Bacterial Structures
Mycoplasma penetrans
Organelles
Mycoplasma
cytoskeleton
electron microscopy
evolution
fractionation
mass spectrometry
transcriptomics

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