Studies On Molecular Mechanism Of Ampicillin Resistance In Shewanella Oneidensis

Shewanella oneidensis is a facultative anaerobic y-proteobacterium possessing remarkably diverse respiratory capacities for reducing various organic and inorganic substrates. As a veteran research model for investigating redox transformations of environmental contaminants the bacterium is well known to be a naturally ampicillin-resistant microorganism. However, we discovered an unexpected response of S. oneidensis to ampicillin:the antibiotic at relatively low levels (0.49-6.25μg/ml) rather than high levels have a more significantly inhibitory effect for growth. To decipher the scientific explaination of the special phenomenon, this study focused on the molecular mechanism of ampicillin resistance in S. oneidensis. The results were as follows:Expression of blaA underlies unexpected ampicillin induced cell lysis of Shewanella oneidensis.The response of S. oneidensis to β-lactams was studied by the pellicle (air-liquid interface biofilm) formation assay and antibiotic susceptibility assay. Pellicle formed by S. oneidensis was delayed by ampicillin only when the agent was served at levels ranging from0.49to6.25μg/ml. After determining growth of S. oneidensis in shaking cultures in the presence or absence of ampicillin, we found that the postponed pellicle formation was due to cell lysis induced by ampicillin of low levels. Further analysis revealed that the resistance of S. oneidensis to ampicillin was mainly caused, if not exclusively, by the production of BlaA, one of seven β-lactamases encoded in the genome. Insufficient production of BlaA, which was strongly induced by ampicillin at high levels, was largely accountable for cell lysis. Additionally, we demonstrated that penicillin binding protein5(PBP5), the most abundant low molecular weight PBP (LMW PBP), played an important role in the bacterial (3-lactam resistance. In addition to function as a "trap" for (3-lactams, PBP5appeared to mediate expression of BlaA via an unknown signal relay.Effects of metal ions on ampicillin susceptibility of S. oneidensis.The addition of EDTA exacerbated cell lysis induced by ampicillin of low levels, while cell lysis was alleviated by most of metal ions. Using antibiotic susceptibility assay, we found that metal ions improved the ampicillin resistance, especially zinc. The enhancement of ampicillin resistance induced by zinc was dependent on the production of BlaA. However, the effect of zinc was not resulted from the activation of metallo-β-lactamases or the expression of blaA. It was more probable that outer membrane permeability was decreased in the presence of metal ions, leading to the restriction of antibiotics diffusion, though loss of porins SO0312or SO3060did not affect the enhancing effect of zinc. In addition, the susceptibility of S. oneidensis to other kinds of antibiotics was also affected by metal ions.The regulation of blaA expression in S. oneidensis.After determining the activities of blaA promoter and ampicillin susceptibility, we found that the regulation of BlaA was markedly different from the case of AmpC in Citrobacter freundii, but both were closely related to peptidoglycan recycling. In contrast to AmpC, the expression of BlaA was significantly increased by the inactivation of AmpG in S. oneidensis, causing a hyper-resistance to ampicillin. A PblaA and lacZ fusion strain WT/PblaA-lacZ was also constructed to study the expression of BlaA via colonies colors. Using WT/PblaA-lacZ as a parent strain, we performed the transposon mutagenesis for screening insertional mutants that improved the expression of BlaA. Further analysis revealed that the insertional site of transposon mutants was converged on mrcA and lppC, which encode high molecular weight PBP1a and lipoprotein LppC respectively. LppC shared a high similarity with LpoA of Escherichia coli. Thus, it was likely that LppC and PBP1a could form a complex responsible for peptidoglycan synthesis in S. oneidensis. Given that the two complexes PBP1a-LpoA and PBP1b-LpoB cannot simultaneously lose in bacteria cells, genes encoding PBP1b-LpoB in S. oneidensis were also determined by construction of synthetically lethal mutants. In this bacterium, the functions of these two distinct multienzyme complexes differed from each other greatly. The loss of PBP1a-LpoA complex resulted in growth defect and constitutive expression of BlaA, which caused a hype-resistance to ampicillin. However, neither growth nor ampicillin resistance was affected by PBPlb-LpoB complex. Moreover, the expression of BlaA induced by mrcA deletion was independent on AmpG. The result suggested that the signal molecules for blaA induction were probably located in periplasm.