[Show abstract][Hide abstract]ABSTRACT: Oxidative stress is one of the major challenges that Shewanella encounter routinely because they thrive in redox-stratified environments prone to reactive oxygen species (ROS) formation, letting alone that ROS can be generated endogenously. As respiration is the predominant process for endogenous ROS, regulators mediating respiration have been demonstrated and/or implicated to play a role in oxidative stress response. In our efforts to unveil the involvement of global regulators for respiration in the oxidative stress response, we found that loss of the Arc system increases S. oneidensis sensitivity to H2O2 whereas neither Fnr nor Crp has a significant role. A comparison of transcriptomic profiles of the wild-type and its isogenic arcA mutant revealed that the OxyR regulon is independent of the Arc system. We then provided evidence that the enhanced H2O2 sensitivity of the arcA mutant is due to an increased H2O2 uptake rate, a result of a cell envelope defect. Although one of three proteases of the ArcA regulon when in excess is partially accountable for the envelope defect, the major contributors remain elusive. Overall, our data indicate that the Arc system influences the bacterial cell envelope biosynthesis, a physiological aspect that has not been associated with the regulator before.
[Show abstract][Hide abstract]ABSTRACT: Nearly half of flagellated microorganisms possess a multiple-flagellin system. While a functional filament can be formed from one of multiple flagellins alone in many bacteria, it is more common that one flagellin is the major constituent and others contribute. Underlying mechanisms proposed for such scenarios cover flagellin regulation of various levels, including transcription, translation, post-translational modification, secretion, and filament assembly. In Shewanella oneidensis, the flagellar filament is composed of FlaA and FlaB flagellins; the latter is the major one in terms of motility. In this study we showed that regulation of all levels except for filament assembly is indistinguishable between these two flagellins. Further analyses revealed that two amino acid residues predominately dictated functional difference with respect to motility. Given that Shewanella prefer a solid-surface associated life style, to which filaments consisting of either FlaA or FlaB are equally supportive, we envision that roles of flagella in surface adhesion and formation of bacterial communities are particularly important for their survival and proliferation in these specific niches.
No preview · Article · Apr 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract]ABSTRACT: Shewanella thrives in redox-stratified environments where accumulation of H2O2 becomes inevitable because of the chemical oxidation of reduced metals, sulfur species, or organic molecules. As a research
model, the representative species Shewanella oneidensis has been extensively studied for its response to various stresses. However, little progress has been made toward an understanding
of the physiological and genetic responses of this bacterium to oxidative stress, which is critically relevant to its application
as a dissimilatory metal-reducing bacterium. In this study, we systematically investigated the mechanism underlying the response
to H2O2 at cellular, genomic, and molecular levels. Using transcriptional profiling, we found that S. oneidensis is hypersensitive to H2O2 in comparison with Escherichia coli, and well-conserved defense genes such as ahpCF, katB, katG, and dps appear to form the first line of defense, whereas iron-sulfur-protecting proteins may not play a significant role. Subsequent
identification and characterization of an analogue of the E. coli oxyR gene revealed that S. oneidensis OxyR is the master regulator that mediates the bacterial response to H2O2-induced oxidative stress by directly repressing or activating the defense genes. The sensitivity of S. oneidensis to H2O2 is likely attributable to the lack of an inducible manganese import mechanism during stress. To cope with stress, major strategies
that S. oneidensis adopts include rapid removal of the oxidant and restriction of intracellular iron concentrations, both of which are achieved
predominantly by derepression of the katB and dps genes.
No preview · Article · Nov 2013 · Journal of bacteriology
[Show abstract][Hide abstract]ABSTRACT: Shewanella oneidensis exhibits a remarkable versatility in respiration, which largely relies on its various respiratory pathways. Most of these pathways are composed of secretory terminal reductases and multiple associated electron transport proteins that contain cofactors such as Fe-S, molybdopterin, and NiFe. The majority of these cofactors are inserted enzymatically in the cytoplasm, and thus are substrates of the twin-arginine translocation (Tat) protein export system, which transports fully folded proteins. Using genomic array footprinting, we discovered that loss of TatA or TatC caused a reduction in the growth rate of S. oneidensis under aerobic conditions. Mutational analysis of the predicted Tat substrates revealed that PetA, the Rieske Fe-S subunit of the ubiquinol-cytochrome c reductase, predominantly dictates the aerobic growth defect of tat mutants in S. oneidensis. In addition, evidence is presented that the signal sequence in PetA appears to be resistant to cleavage after the protein is inserted into the cytoplasmic membrane.
[Show abstract][Hide abstract]ABSTRACT: Comparative analysis of phenotypes of S. oneidensis tat mutants.
A. Morphology by phase-contrast microscope. B. Motility phenotype of tat mutants. The plates were incubated at 30°C for 36 h to test the motility of different strains. Verified non-motile mutant strain ΔfliD was used as the negative control. C. Pellicle formation of tat mutants. The plates were incubated at 30°C for 24 h. Verified pellicle-free mutant strain ΔaggA was used as the negative control. D. Susceptibility of tat mutants to SDS. 0.25% SDS was used to test the sensitivity of tat mutants. For B, C, and D, photos were taken in a time course manner.
[Show abstract][Hide abstract]ABSTRACT: Shewanella oneidensis is a facultative anaerobic γ-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, in this study we discovered that ampicillin has a significant impact on growth of S. oneidensis. Particularly, cell lysis occurred only with ampicillin at levels ranging from 0.49 to 6.25 µg/ml but not at 50 µg/ml. This phenotype is attributable to insufficient expression of the β-lactamase BlaA. The subsequent analysis revealed that the blaA gene is strongly induced by ampicillin at high (50 µg/ml), but not at low levels (2.5 µg/ml). In addition, we demonstrated that penicillin binding protein 5 (PBP5), the most abundant low molecular weight PBP (LMW PBP), is the only one relevant to β-lactam resistance under the tested conditions. This nonessential PBP, largely resembling its Escherichia coli counterpart in functionality, mediates expression of the blaA gene.
[Show abstract][Hide abstract]ABSTRACT: Ampicillin susceptibility assay for various strains, in which one of predicted β-lactamases was deleted. Three-microliter cultures of the late-exponential phase (∼0.6 of OD600) were dropped on LB agar plates supplemented with ampicillin varying in concentrations. Plates were incubated at 30°C and results were photographed at18 h.
[Show abstract][Hide abstract]ABSTRACT: Expression of napA, narfA, narP in S. oneidensis strains. WT/napA represents expression of napA in WT strain. A.
lacZ-based reporter analysis of the nap and nrfA promoters in ΔnarP. Expression of nap and nrfA in the wild type and ΔnarP cells cultured aerobically in the presence of nitrate and nitrite, respectively, were shown. B. qRT-PCR analysis of napA, nrfA, and narP in the wild-type, Δcrp, and Δfnr cells grown with 5 mM nitrite aerobically. Abundance is given relative to 16 S rRNA.
[Show abstract][Hide abstract]ABSTRACT: Sequence comparison of the S. oneidensis and E. coli NarP proteins. The predicted phosphorylation residues, Asp57 in S. oneidensis NarP and Asp59 in E. coli NarP, were marked.
[Show abstract][Hide abstract]ABSTRACT: We have previously illustrated the nitrate/nitrite respiratory pathway of Shewanella oneidensis, which is renowned for its remarkable versatility in respiration. Here we investigated the systems regulating the pathway with a reliable approach which enables characterization of mutants impaired in nitrate/nitrite respiration by guaranteeing biomass. The S. oneidensis genome encodes an Escherichia coli NarQ/NarX homolog SO3981 and two E. coli NarP/NarL homologs SO1860 and SO3982. Results of physiological characterization and mutational analyses demonstrated that S. oneidensis possesses a single two-component system (TCS) for regulation of nitrate/nitrite respiration, consisting of the sensor kinase SO3981(NarQ) and the response regulator SO3982(NarP). The TCS directly controls the transcription of nap and nrfA (genes encoding nitrate and nitrite reductases, respectively) but regulates the former less tightly than the latter. Additionally, phosphorylation at residue 57 of SO3982 is essential for its DNA-binding capacity. At the global control level, Crp is found to regulate expression of narQP as well as nap and nrfA. In contrast to NarP-NarQ, Crp is more essential for nap rather than nrfA.
[Show abstract][Hide abstract]ABSTRACT: Physiological characterization of mutant strains. The experiments were performed at least three times. In all panels, error bars (Standard deviation <5%) were omitted for clarification. A. Aerobic growth of S. oneidensis strains in the presence of 5 mM NaNO3 (solid line) or NaNO2 (dash line). Mutants used here have been previously confirmed by genetic complementation . B. Nitrite reduction of S. oneidensis ΔcymA. 5 mM nitrite was initially added. ΔcymAc represents the ΔcymA strain containing a copy of cymA with its own promoter on pHG101. The ΔnapA and ΔnapAc strains were included as the control. C. Growth of S. oneidensis ΔnarP and ΔSO1860 in the presence of 5 mM nitrate under anaerobic conditions. ΔnarPc represents the ΔnarP strain containing a copy of narP under the control of ParcA within pHG102 .
[Show abstract][Hide abstract]ABSTRACT: Although solid surface-associated biofilm development of S. oneidensis has been extensively studied in recent years, pellicles formed at the air-liquid interface are largely overlooked. The goal of this work was to understand basic requirements and mechanism of pellicle formation in S. oneidensis.
We demonstrated that pellicle formation can be completed when oxygen and certain cations were present. Ca(II), Mn(II), Cu(II), and Zn(II) were essential for the process evidenced by fully rescuing pellicle formation of S. oneidensis from the EDTA treatment while Mg (II), Fe(II), and Fe(III) were much less effective. Proteins rather than DNA were crucial in pellicle formation and the major exopolysaccharides may be rich in mannose. Mutational analysis revealed that flagella were not required for pellicle formation but flagellum-less mutants delayed pellicle development substantially, likely due to reduced growth in static media. The analysis also demonstrated that AggA type I secretion system was essential in formation of pellicles but not of solid surface-associated biofilms in S. oneidensis.
This systematic characterization of pellicle formation shed lights on our understanding of biofilm formation in S. oneidensis and indicated that the pellicle may serve as a good research model for studying bacterial communities.
[Show abstract][Hide abstract]ABSTRACT: Shewanella are renowned for their ability to utilize a wide range of electron acceptors (EA) for respiration, which has been partially accredited to the presence of a large number of the c-type cytochromes. To investigate the involvement of c-type cytochrome proteins in aerobic and anaerobic respiration of Shewanella oneidensis Mr -1, 36 in-frame deletion mutants, among possible 41 predicted, c-type cytochrome genes were obtained. The potential involvement of each individual c-type cytochrome in the reduction of a variety of EAs was assessed individually as well as in competition experiments. While results on the well-studied c-type cytochromes CymA(SO4591) and MtrC(SO1778) were consistent with previous findings, collective observations were very interesting: the responses of S. oneidensis Mr -1 to low and highly toxic metals appeared to be significantly different; CcoO, CcoP and PetC, proteins involved in aerobic respiration in various organisms, played critical roles in both aerobic and anaerobic respiration with highly toxic metals as EA. In addition, these studies also suggested that an uncharacterized c-type cytochrome (SO4047) may be important to both aerobiosis and anaerobiosis.