Recombinant Mycoplasma mobile Probable GTP-binding protein EngB (engB)

What is Mycoplasma mobile and why is it significant for research?

Mycoplasma mobile is a flask-shaped mycoplasma bacterium (approximately 1 μm × 0.3 μm) originally isolated from a fish species called tench (Tinca tinca). It belongs to the Mycoplasma hominis group and is believed to be pathogenic. Unlike most other mycoplasmas which are non-motile, M. mobile exhibits robust gliding motility, making it a unique model organism for studying bacterial movement mechanisms . M. mobile grows optimally at around 20°C, lower than the 37°C preference of most well-studied mycoplasmas, yet maintains a doubling time of approximately 10 hours, comparable to mycoplasmas with mammalian hosts . Its complete genome sequence has revealed numerous features that contribute to its distinct characteristics, including its motility apparatus.

What are GTP-binding proteins and what role do they play in bacterial systems?

GTP-binding proteins (G proteins) function as molecular switches in various cellular processes. While the search results don't specifically address the role of EngB in M. mobile, we can infer from related research that bacterial GTP-binding proteins typically regulate essential cellular functions including protein synthesis, cell division, and signal transduction. In mycoplasmas, which have highly reduced genomes, these proteins likely play critical roles in fundamental cellular processes. The probable GTP-binding protein EngB is part of the sophisticated molecular machinery that allows M. mobile to function despite having one of the smallest genomes among self-replicating organisms.

What is the genomic context of the engB gene in Mycoplasma mobile?

The complete genome of Mycoplasma mobile consists of 777,079 base pairs with an extremely low GC content of 24.9% . While the search results don't specifically identify the genomic location of engB in M. mobile, we know that M. mobile's genome encodes approximately 635 proteins, including 109 proteins that appear unique to this species . Based on comparative genomics, proteins in M. mobile can be categorized as follows:

How does the structure of M. mobile EngB compare to similar proteins in other bacterial species?

Based on information about the related protein in Mycoplasma penetrans, the EngB protein contains characteristic sequence motifs typical of GTP-binding proteins, including the GTP-binding domain . The amino acid sequence of M. penetrans EngB includes a GTP-binding domain indicated by the consensus sequence GXXXXGK(S/T), which is typical of this protein family . While we don't have the exact sequence for M. mobile EngB in the search results, it would likely share significant homology with the M. penetrans version due to conserved functional domains.

What is the relationship between EngB and the unique gliding motility of Mycoplasma mobile?

Recent research has clarified the molecular structure of M. mobile's motility apparatus. The motility mechanism is powered by ATPases that use rotational catalytic mechanisms . While the search results don't explicitly connect EngB to gliding motility, GTP-binding proteins often play regulatory roles in complex cellular processes. The gliding speed of M. mobile increases linearly with temperature, reaching approximately 2.3 μm/s at 22.5°C and 3.3 μm/s at 27.5°C . The maximum force generated by M. mobile during gliding has been measured at approximately 26-28 pN across different temperatures (17.5 to 27.5°C) .
Researchers have proposed that M. mobile's gliding mechanism consists of at least two steps: one that generates force and another that allows displacement . GTP-binding proteins like EngB might be involved in regulatory aspects of this complex machinery, potentially in signaling pathways that coordinate the multiple components of the gliding apparatus.

How does ATP hydrolysis contribute to the function of Mycoplasma mobile's motility apparatus, and might EngB play a regulatory role?

The gliding machinery of M. mobile is powered by twin motor structures that utilize ATP hydrolysis. Recent cryo-electron microscopy studies have revealed at near-atomic resolution the ATPases that power this machinery . The molecular structures of the two units that make up the twin motor are similar to known ATP synthases, but they combine to form an unprecedented complex structure . While the search results do not explicitly detail EngB's role, GTP-binding proteins often function as regulatory elements in molecular machines. EngB might coordinate ATP utilization or regulate conformational changes in the motility apparatus in response to environmental signals.

What are the optimal conditions for expressing recombinant M. mobile EngB in laboratory settings?

Based on related recombinant protein work, successful expression of M. mobile proteins typically requires consideration of the organism's unique genetic characteristics, particularly its extremely low GC content (24.9%) . For recombinant expression, mammalian cell systems have been successfully used for expressing mycoplasma proteins, as seen with the M. penetrans EngB . When designing expression constructs, researchers should account for M. mobile's unusual codon usage patterns resulting from its AT-rich genome.
The expression and purification protocol would likely involve:

• Codon optimization for the expression system

• Addition of appropriate affinity tags (His-tag, etc.)

• Temperature optimization during expression (considering M. mobile's preference for lower temperatures around 20°C)

• Purification under conditions that maintain protein stability and activity

What methodologies can be employed to study the GTPase activity of recombinant EngB from M. mobile?

For analyzing GTPase activity of recombinant EngB, researchers could employ several approaches:

• Colorimetric assays measuring inorganic phosphate release during GTP hydrolysis

• Coupled-enzyme assays that link GTP hydrolysis to measurable spectrophotometric changes

• Radioactive assays using [γ-32P]GTP to quantify hydrolysis rates

• HPLC-based methods to analyze nucleotide conversion
  When designing these experiments, researchers should consider control conditions that include:

• Testing activity across a range of temperatures (especially 20-30°C, given M. mobile's optimal growth temperature)

• Examining dependence on divalent cations (typically Mg2+)

• Investigating potential regulatory factors that might enhance or inhibit GTPase activity

How does recent cryo-electron microscopy research advance our understanding of M. mobile's molecular machinery?

Recent advances using cryo-electron microscopy have revealed the detailed structure of M. mobile's gliding machinery at near-atomic resolution . A research team led by Professor Makoto Miyata at Osaka Metropolitan University has been investigating M. mobile's motility mechanisms since 1997 and recently uncovered the structure of the ATPases that power the gliding apparatus . This research revealed that while the molecular structures of the two units composing the twin motor are similar to known ATP synthases, they combine to form a novel complex structure previously undocumented .
This structural insight facilitates understanding of how ATP hydrolysis energy is converted into mechanical movement, providing a foundation for future molecular-level investigations of bacterial motility systems . Professor Miyata notes that these findings have potential applications in nanobot actuator development and in creating treatments for mycoplasma infections .

What potential interactions might exist between EngB and the recently characterized motility apparatus of M. mobile?

While the search results don't directly address interactions between EngB and the motility apparatus, the recently characterized twin motor structure of M. mobile's gliding machinery presents intriguing possibilities for regulatory interactions. GTP-binding proteins often function as molecular switches, and EngB could potentially:

• Regulate the assembly of motility complex components

• Coordinate the energy utilization between the twin motor units

• Modulate the activity of the motility apparatus in response to environmental signals

• Participate in the directional control of movement Research investigating these potential interactions would represent a significant advance in understanding the complete regulatory network governing M. mobile's unique motility.