水谷 雅希

ABSTRACT Mycoplasmas have been widely investigated for their pathogenicity, as well as for genomics and synthetic biology. Conventionally, transformation of mycoplasmas was not highly efficient, and due to the low transformation efficiency, large amounts of DNA and recipient cells were required for that purpose. Here, we report a robust and highly efficient transformation method for the minimal cell JCVI-syn3B, which was created through streamlining the genome of Mycoplasma mycoides . When the growth states of JCVI-syn3B were examined in detail by focusing on such factors as pH, color, absorbance, colony forming unit, and transformation efficiency, it was found that the growth phase after the lag phase can be divided into three distinct phases, of which the highest transformation efficiency was observed during the early exponential growth phase. Notably, the transformation efficiency of up to 4.4 × 10 ⁻² transformants per cell per microgram of plasmid DNA was obtained. A method to obtain several hundred to several thousand transformants with less than 0.2 mL of culture with approximately 1 × 10 ⁷ –10 ⁸ cells and 10 ng of plasmid DNA was developed. Moreover, a transformation method using a frozen stock of transformation-ready cells was established. These procedures and information could simplify and enhance the transformation process of minimal cells, facilitating advanced genetic engineering and biological research using minimal cells. IMPORTANCE Mycoplasmas are parasitic and pathogenic bacteria for many animals. They are also useful bacteria to understand the cellular process of life and for bioengineering because of their simple metabolism, small genomes, and cultivability. Genetic manipulation is crucial for these purposes, but transformation efficiency in mycoplasmas is typically quite low. Here, we report a highly efficient transformation method for the minimal genome mycoplasma JCVI-syn3B. Using this method, transformants can be obtained with only 10 ng of plasmid DNA, which is around one-thousandth of the amount required for traditional mycoplasma transformations. Moreover, a convenient method using frozen stocks of transformation-ready cells was established. These improved methods play a crucial role in further studies using minimal cells.

Cloning and transfer of long-stranded DNA in the size of a bacterial whole genome has become possible by recent advancements in synthetic biology. For the whole genome cloning and whole genome transplantation, bacteria with small genomes have been mainly used, such as mycoplasmas and related species. The key benefits of whole genome cloning include the effective maintenance and preservation of an organism's complete genome within a yeast host, the capability to modify these genome sequences through yeast-based genetic engineering systems, and the subsequent use of these cloned genomes for further experiments. This approach provides a versatile platform for in-depth genomic studies and applications in synthetic biology. Here, we cloned an entire genome of an insect-associated bacterium, Spiroplasma chrysopicola, in yeast. The 1.12 Mbp whole genome was successfully cloned in yeast, and sequences of several clones were confirmed by Illumina sequencing. The cloning efficiency was high, and the clones contained only a few mutations, averaging 1.2 nucleotides per clone with a mutation rate of 4 × 10⁻⁶. The cloned genomes could be distributed and used for further research. This study serves as an initial step in the synthetic biology approach to Spiroplasma.

<jats:p><jats:italic>Mycoplasma pneumoniae</jats:italic>, a human pathogenic bacterium, binds to sialylated oligosaccharides and glides on host cell surfaces <jats:italic>via</jats:italic> a unique mechanism. Gliding motility is essential for initiating the infectious process. In the present study, we measured the stall force of an <jats:italic>M. pneumoniae</jats:italic> cell carrying a bead that was manipulated using optical tweezers on two strains. The stall forces of M129 and FH strains were averaged to be 23.7 and 19.7 pN, respectively, much weaker than those of other bacterial surface motilities. The binding activity and gliding speed of the M129 strain on sialylated oligosaccharides were eight and two times higher than those of the FH strain, respectively, showing that binding activity is not linked to gliding force. Gliding speed decreased when cell binding was reduced by addition of free sialylated oligosaccharides, indicating the existence of a drag force during gliding. We detected stepwise movements, likely caused by a single leg under 0.2-0.3 mM free sialylated oligosaccharides. A step size of 14-19 nm showed that 25-35 propulsion steps per second are required to achieve the usual gliding speed. The step size was reduced to less than half with the load applied using optical tweezers, showing that a 2.5 pN force from a cell is exerted on a leg. The work performed in this step was 16-30% of the free energy of the hydrolysis of ATP molecules, suggesting that this step is linked to the elementary process of <jats:italic>M. pneumoniae</jats:italic> gliding. We discuss a model to explain the gliding mechanism, based on the information currently available.</jats:p>

<jats:p><jats:italic>Mycoplasma gallisepticum</jats:italic>, an avian-pathogenic bacterium, glides on host tissue surfaces by using a common motility system with <jats:italic>Mycoplasma pneumoniae</jats:italic>. In the present study, we observed and analyzed the gliding behaviors of <jats:italic>M. gallisepticum</jats:italic> in detail by using optical microscopes. <jats:italic>M. gallisepticum</jats:italic> glided at a speed of 0.27 ± 0.09 μm/s with directional changes relative to the cell axis of 0.6 ± 44.6 degrees/5 s without the rolling of the cell body. To examine the effects of viscosity on gliding, we analyzed the gliding behaviors under viscous environments. The gliding speed was constant in various concentrations of methylcellulose but was affected by Ficoll. To investigate the relationship between binding and gliding, we analyzed the inhibitory effects of sialyllactose on binding and gliding. The binding and gliding speed sigmoidally decreased with sialyllactose concentration, indicating the cooperative binding of the cell. To determine the direct energy source of gliding, we used a membrane-permeabilized ghost model. We permeabilized <jats:italic>M. gallisepticum</jats:italic> cells with Triton X-100 or Triton X-100 containing ATP and analyzed the gliding of permeabilized cells. The cells permeabilized with Triton X-100 did not show gliding; in contrast, the cells permeabilized with Triton X-100 containing ATP showed gliding at a speed of 0.014 ± 0.007 μm/s. These results indicate that the direct energy source for the gliding motility of <jats:italic>M. gallisepticum</jats:italic> is ATP.</jats:p> <jats:p>IMPORTANCE <jats:italic>Mycoplasmas</jats:italic>, the smallest bacteria, are parasitic and occasionally commensal. <jats:italic>Mycoplasma gallisepticum</jats:italic> is related to human pathogenic <jats:italic>Mycoplasmas</jats:italic>—<jats:italic>Mycoplasma pneumoniae</jats:italic> and <jats:italic>Mycoplasma genitalium</jats:italic>—which cause so-called ‘walking pneumonia’ and non-gonococcal urethritis, respectively. These <jats:italic>Mycoplasmas</jats:italic> trap sialylated oligosaccharides, which are common targets among influenza viruses, on host trachea or urinary tract surfaces and glide to enlarge the infected areas. Interestingly, this gliding motility is not related to other bacterial motilities or eukaryotic motilities. Here, we quantitatively analyze cell behaviors in gliding and clarify the direct energy source. The results provide clues for elucidating this unique motility mechanism.</jats:p>