Study On Regulation Of Hormone Expression In Shewanella Oneidensis

Shewanella, a group of facultative anaerobic γ-proteobacteria, are widely distributed in environments. They have drawn a lot of attention because of their diverse respiratory capacities in reducing various organic and inorganic substrates, which is mainly based on researches on the model species, S. oneidensis. Although outer-membrane porins have been implicated in transport of many organic and inorganic substrates into the periplasm, little is known about the distribution and function of these proteins, or how they respond to environmental cues in S. oneidensis. In this study, we examined the profile of porin proteins, and determined the major ones with respect to expression levels.SO3896 and SO3545 are two major porins of S. oneidensisAmong the seven non-specific porins annontated in S. oneidensis genome, two with molecular masses of～40 kDa SO3896 and SO3545 had been identified to be the most abundant, by using SDS-PAGE and MS/MS analyses. Subsequent mutational analysis confirmed their identification.Functional characterization of porins in S. oneidensisSome studies found that zinc enhanced bacteria antibiotic resistance through reducing certain porin expression. Using antibiotic susceptibility assay, we found that zinc enhanced ampicillin resistance in both S. oneidensis wild-type and porin mutant strains to the same extent. This indicates that the enhancement of ampicillin resistance induced by zinc is independent of porins. However, the resistance to another β-lactam imipenem was not affected by zinc, suggesting that the role of zinc on ampicillin resistance was a sort of specific.Regulation of expression of two major porins in S. oneidensisTo investigate the molecular mechanism by which the roles of these two abundant porins play and how they are regulated, we carried out experiments at both transcriptional and translational levels. Sequencing, promoter fusion, and SDS-PAGE analyses revealed a binding site of Fur on SO3896 promoter region, which is located in the 220-240 AT-rich region before the start codon. Fur positively regulated SO3896 expression and iron was not required for the regulation. In addition, expression of SO3896 decreased about 10-fold when crp or cyaA was deleted. This deficiency cannot be complemented by expression of SO3896 driven by its native promoter in multiple copies, suggesting that both Crp and its cognite binding site are indispensable for SO3896 expression. Moreover, it can be inferred from promoter determination and SDS-PAGE assays that post-transcriptional regulation exists in SO3896 expression.During further investigation of the significance of Crp regulation on SO3896 expression, we learned that both ASO3896 and Acrp exhibited deficiency in fumarate, iron, and nitrate utilization, suggesting that a critical role of SO3896 in anaerobic respiration, presumably by adjusting transport of these substrates into the periplasm.Unexpectedly, the expression of SO3896 and SO3545 were not affected by osmotic stress and the EnvZ/OmpR two-component system. Additonally, expression of SO3545 was independent of Fur and Crp in S. oneidensis.