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. 2019 Feb;11(1):67-74.

Mutagenesis of the rpoS gene involved in alteration of outer membrane composition

Sayyed Shahryar Rahpeyma  1 , Jamshid Raheb  1
Affiliations

Mutagenesis of the rpoS gene involved in alteration of outer membrane composition

Sayyed Shahryar Rahpeyma et al. Iran J Microbiol. 2019 Feb.

Abstract

Background and objectives: rpoS is a bacterial sigma factor of RNA polymerase which is involved in the expression of the genes which control regulons and play a critical role in survival against stresses. Few suitable vectors are available which could be maintained successfully in Flexibacter chinesis cells and could in particular be used as a suicide vector to make mutation in the rpoS gene. The aim of this study was to investigate if rpoS mutagenesis has impact on bacterial morphology in addition to cell division.

Materials and methods: A 0.603 kb BamHI-PstI fragment subclone of pICRPOS38Ω was cloned into linearized pLYLO3. The final construct, pLRPOS38 suicide vector, was introduced into Flexibacter chinesis. Then the cytoplasm of mutant strain and wild-type were investigated by transmission electron microscopy.

Results: After successful subcloning of suicide vector into F. chinesis, based on TEM study, it was demonstrated that mutation in rpoS gene leads to decomposition of outer membrane of F. chinesis.

Conclusion: A suitable vector to make suicide mutation in rpoS was constructed for F. chinesi.

Keywords: Flexibacter chinesis; Suicide vector; Transmission electron microscopy; Transposon; rpoS.

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Figures

Fig. 1.
Fig. 1.
a) Miniprep of pICRPOS38 was digested with a number of restriction enzymes. Lane 1 is λ ladder of 1 kb DNA as a molecular size. Lane 2 is pICRPOS38 digested with EcoRI and BamHI b) Restriction digestion of pICRPOS38 at a unique PmlI site. Lane 1 is λ ladder of 1 kb DNA as a molecular size. Lane 2 is pICRPOS38 digested with PmlI. Lane 3 is pICRPOS38 digested with PmlI and EcoRI. Lane 4 is pICRPOS38 digested with PmlI and BamHI. Lane 5 is pICRPOS38 digested with PmlI, EcoRI and BamHI.
Fig. 2.
Fig. 2.
Restriction digestion of pICRPOS38Ω. Lane 1 is λ ladder of 1 kb DNA as a molecular size. Lane 2 is pICRPOS38Ω digested with PstI. Lane 3 is undigested pICRPOS38Ω as a control. Lane 4 is pICRPOS38Ω digested with HindII and Lane 5 is pICRPOS38Ω digested with BamHI.
Fig. 3.
Fig. 3.
Restriction digestion of pKRPOS38Ω. Lane 1 is λ ladder of 1 kb DNA as a molecular size. Lane 2 is pKRPOS38Ω digested with SalI and BamHI.
Fig. 4.
Fig. 4.
Colony blot hybridization for Tn4351 detection and a none-spreading mutant of F. chinesis. a. DNA from erythromycin-resistance colonies was transferred to nylon membranes in duplicate and probed with radiolabeled pVOHI to detect Tn4351. The positive colons are arrowed. b. DNA from erythromycin-resistance colonies was transferred to nylon membranes in duplicate and probed with radiolabeled R751 to detect the delivery vector. The positive controls are arrowed. c. A none-spreading colony is arrowed.
Fig. 5.
Fig. 5.
Restriction digestion of construct pLRPOS38. A miniprep of pICRPOS38 was digested. Lane 1 is λ ladder of 1 kb DNA as a molecular size. Lane 2, 3 and 4 show pLRPOS38 digested with BamHI and PstI sites resulting in a0.6 kb partial fragment of rpoS gene and 6 kb linearized pLYLO3 vector.
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