| As the main peptidoglycan synthase,high molecular weight penicillin binding proteins(PBPs)are targets for β-lactam antibiotics.In Escherichia coli,PBP1 a and PBP1b form peptidoglycan synthase complexes with the outer membrane lipoproteins LpoA and LpoB,respectively.The two complexes participate in the synthesis of peptidoglycan and their function is redundancy,while inactivation of both leads to cell death.Shewanella oneidensis is a Gram-negative bacterium.Previous studies showed that S.oneidensis also contains PBP1a/LpoA and PBP1b/LpoB,but with different physiological functions.The growth of PBP1a/LpoA-deficient strains was slowed down in Lysogeny Broth(LB)medium,and the resistance to β-lactam was enhanced,while the deletion of PBP1b/LpoB has no such effect on cells.Based on these,we systematically compared the phenotypic differences between these two complexes,and investigated the correlated mechanism.The results are as follows.Deletion of PBP1a/LpoA(ΔmrcAΔlpoA)causes numerous phenotypic defects that are not observed in PBP1b/LpoB-deficient strains,including aberrant cell morphology,growth in NaCl-less medium(LB medium without NaCl),and enhanced sensitivity to sodium dodecyl sulfate(SDS).Further studies showed that deletion of PBP1a/LpoA enhanced the uptake of vancomycin and N-phenyl-1-naphthylamine(NPN),indicating that out membrane permeability increased and cell wall integrity was destroyed.Based on the above physiological phenotypic differences,it was found that the phenotype of theΔmrcAΔlpoA could be restored when the concentration of inducer was low by inducing PBP1b expression inΔmrcAΔlpoA strains.However,the phenotype of theΔmrcAΔlpoA could not be restored when the concentration of inducer was high.In addition,PBP1b is composed of three domains,and the deletion of any domain could not restore the phenotype of theΔmrcAΔlpoA.Therefore,the enhanced expression of PBP1b can compensate for PBP1a/LpoA in function.Subsequently,a transposon mutant library was constructed to screen for suppressors of the PBP1a/LpoA mutation.A total of 13 suppressors were obtained.The inserted sites in suppressors were identified by the arbitrarily primed PCR and the BLASTn analysis.The results showed that hrpB,sspA and vanZ were inserted by the transposon at high frequency.According to gene annotation,hrpB encodes an ATP-dependent helicase that locates upstream of mrcB(encoding PBP1b).The sspA encodes stringent starvation protein A and vanZ encodes VanZ family protein.And the molecular mechanism underlying phenotype recovery of hrpB suppressors was studied.Unlike the suppressors,the in-frame deletion of hrpB does not restore the phenotype ofΔmrcAΔlpoA strain,indicating that the phenotype recovery is not due to the inactivation of hrpB.Considering the fact that the transposon used in this study contains a hidden promoter,it is speculated that the expression of mrcB in hrpB suppressors is enhanced,thus functionally compensating PBP1 a.Results of qRT-PCR showed that the expression of mrcB in hrpB suppressors was significantly increased compared withΔmrcAΔlpoA strains.So the phenotype recovery of hrpB suppressors was due to the enhanced expression of PBP1b.Consistent with the sspA suppressors phenotype,the in-frame deletion of sspA restores the phenotype ofΔmrcAΔlpoA and can be complemented genetically.The qRT-PCR analysis showed that the expression of PBP1b enhanced because of inactivation of sspA,indicating that sspA negatively regulates PBP1b at the transcriptional level.Further study showed that SspA could regulate PBP1b indirectly,perhaps via affecting nucleoprotein H-NS. |
