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Sulfhydryl groups of pyocin R1. Morphology and activity modified with sulfhydryl reagents
Abstract:
Electron microscopic observation of pyocin R1 with the negative staining technique demonstrated that pyocin R1 retained its phage tail-like shape of an extended sheath even when it was inactivated by treatment with p-chloromercuribenzoic acid (PCMB) or 4-(p-sulfophenylazo)-2-mercuriphenol (SAMP). Thus it was shown that the contraction and extension of the sheath does not occur reversibly on the modification of sulfhydryl groups accompanying the change of activity. The activity lost under these conditions was restored to the original level by treatment with 2-mercaptoethanol (2-ME). Numbers of sulfhydryl groups in the pyocin R1 particle were determined to be 208 mol and 152 mol per mol (11.8 x 10(6) daltons) by spectrophotometric titration with SAMP and by membrane-filter assay with radioactive PCMB, respectively. Most of these cysteine residues appeared to be localized in the substructure other than the sheath and core. It was also shown that all of these sulfhydryl groups were not necessary for expression of its activity but a part of them were essential for adsorption to the sensitive cells.
Insights
Pyocin R1
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Pyocins are bacteriocins produced by Pseudomonas aeruginosa.
- Pyocin R1 possesses a contractile sheath similar to bacteriophage tails.
- The role of sulfhydryl groups in pyocin R1 activity and structure is not fully understood.
Purpose of the Study:
- To investigate the structural and functional role of sulfhydryl groups in pyocin R1.
- To determine the number and localization of sulfhydryl groups in pyocin R1.
- To elucidate the mechanism of pyocin R1 inactivation and reactivation.
Main Methods:
- Negative staining electron microscopy to observe pyocin R1 structure.
- Chemical modification of sulfhydryl groups using p-chloromercuribenzoic acid (PCMB) and 4-(p-sulfophenylazo)-2-mercuriphenol (SAMP).
- Spectrophotometric titration and radioactive labeling assays to quantify sulfhydryl groups.
- Reactivation of pyocin R1 activity using 2-mercaptoethanol (2-ME).
Main Results:
- Pyocin R1 maintained its phage tail-like shape after inactivation with PCMB or SAMP.
- Inactivation of sulfhydryl groups did not cause reversible sheath contraction or extension.
- Lost pyocin R1 activity was restored by treatment with 2-ME.
- Quantification revealed 208-152 sulfhydryl groups per pyocin R1 particle.
- Most sulfhydryl groups were located outside the sheath and core.
- Only a subset of sulfhydryl groups is essential for pyocin R1 activity, particularly for cell adsorption.
Conclusions:
- The sheath contraction/extension mechanism is not directly linked to reversible sulfhydryl group modification.
- Sulfhydryl groups are crucial for pyocin R1 adsorption to sensitive cells.
- Specific sulfhydryl groups are essential for pyocin R1's biological activity.