Mechanisms underlying the potent antimicrobial effects of plasma-activated seawater (PASW) on fish spoilage bacterium Shewanella putrefaciens.

Plasma-activated seawater (PASW) presents a promising approach for marine fish preservation, yet its antimicrobial efficacy and mechanisms remain unclear. This study found that PASW exhibits superior bactericidal properties against the fish spoilage bacterium Shewanella putrefaciens compared to plasma-activated water (PAW), and increased effectiveness in preserving fish fillets. To clarify the mechanisms, a detailed investigation was conducted, including the generation of reactive oxygen/nitrogen species (ROS/RNS) and active halogen species in PASW, and their antimicrobial efficacy. Findings showed greater nitrite and hydrogen peroxide production in PASW relative to PAW, as well as the conversion of chloride/bromide ions into active species, which collectively enhanced PASW's antimicrobial activity. The synergistic action of ROS/RNS and active chlorine/bromine species in PASW promoted the generation of intracellular ROS, causing increased membrane damage, redox imbalance, and consequently higher bacterial mortality. This study enhances our understanding of PASW's antimicrobial effects and highlights its potential applications in the seafood industry.

Coculture of Acinetobacter johnsonii and Shewanella putrefaciens Contributes to the ABC Transporter that Impacts Cold Adaption in the Aquatic Food Storage Environment.

Acinetobacter johnsonii and Shewanella putrefaciens were identified as specific spoilage organisms in aquatic food. The interactions among specific spoilage organisms under cold stress have a significant impact on the assembly of microbial communities, which play crucial roles in the spoilage and cold adaptation processes. The limited understanding of A. johnsonii and S. putrefaciens interactions in the cold adaptation mechanism hinders the elucidation of their roles in protein and metabolism levels. 4D quantitative proteomic analysis showed that the coculture of A. johnsonii and S. putrefaciens responds to low temperatures through ABC transporter proteins, resulting in phospholipid transport and inner membrane components. SapA and FtsX proteins were significantly upregulated, while LolC, LolD, LolE, PotD, PotA, PotB, and PotC proteins were significantly downregulated. Metabolome assays revealed that metabolites of glutathione and spermidine/putrescin were significantly upregulated, while metabolites of arginine/lysine/ornithine were significantly downregulated and involved in the ABC transporter metabolism. The results of ultramicroscopic analyses showed that the coculture of A. johnsonii and S. putrefaciens surface combined with the presence of the leakage of intracellular contents, suggesting that the bacteria were severely damaged and wrinkled to absorb metabolic nutrients and adapt to cold temperatures.

Effects of exogenous riboflavin or cytochrome addition on the cathodic reduction of Cr(VI) in microbial fuel cell with Shewanella putrefaciens.

Bioreduction of Cr(VI) is recognized as a cost-effective and environmentally friendly method, attracting widespread interest. However, the slow rate of Cr(VI) bioreduction remains a practical challenge. Additionally, the direct removal efficiency of microbes for high concentrations of Cr(VI) is not ideal due to the toxicity. Therefore, this study investigated the effects of exogenous riboflavin or cytochrome on the cathodic reduction of Cr(VI) in microbial fuel cells. The results demonstrated that the exogenous riboflavin or cytochrome effectively improved the voltage output of the cells, with riboflavin increasing the voltage by 52.08%. Within the first 24 h, the Cr(VI) removal ratio in the normal, cytochrome, and riboflavin groups was 14.3%, 29.3%, and 53.8%, respectively. And the final removal ratio was 55.1%, 69.1%, and 98.0%, respectively. These results showed different enhancement effects of riboflavin and cytochrome on Cr(VI) removal. The analysis of riboflavin and cytochrome contents revealed that the additions did not have a significant impact on the autocrine riboflavin of S. putrefaciens, but affected the autocrine cytochrome. SEM, XPS, and FTIR results confirmed the presence of reduced Cr(III) on the cathode, which formed precipitate and adhered to the cathode surface. The EDS analysis showed that the amount of Cr on the cathode in normal, cytochrome, and riboflavin groups was 4.71%, 6.37%, 7.56%, respectively, which was consistent with the voltage and Cr(VI) removal data. These findings demonstrated the significant enhancement of exogenous riboflavin or cytochrome on Cr(VI) reduction, thereby providing data reference for the future bio-assisted remediation of Cr(VI) pollution.

The luxS deletion reduces the spoilage ability of Shewanella putrefaciens: An analysis focusing on quorum sensing and activated methyl cycle.

The luxS mutant strains of Shewanella putrefaciens (SHP) were constructed to investigate the regulations of gene luxS in spoilage ability. The potential regulations of AI-2 quorum sensing (QS) system and activated methyl cycle (AMC) were studied by analyzing the supplementation roles of key circulating substances mediated via luxS, including S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), homocysteine (Hcy) and 4,5-dihydroxy-2,3-pentanedione (DPD). Growth experiments revealed that the luxS deletion led to certain growth limitations of SHP, which were associated with culture medium and exogenous additives. Meanwhile, the decreased biofilm formation and diminished hydrogen sulfide (H2S) production capacity of SHP were observed after luxS deletion. The relatively lower total volatile base nitrogen (TVB-N) contents and higher sensory scores of fish homogenate with luxS mutant strain inoculation also indicated the weaker spoilage-inducing effects after luxS deletion. However, these deficiencies could be offset with the exogenous supply of circulating substances mentioned above. Our findings suggested that the luxS deletion would reduce the spoilage ability of SHP, which was potentially attributed to the disorder of AMC and AI-2 QS system.

Chelating Effect of Siderophore Desferrioxamine-B on Uranyl Biomineralization Mediated by Shewanella putrefaciens.

In contaminated water and soil, little is known about the role and mechanism of the biometabolic molecule siderophore desferrioxamine-B (DFO) in the biogeochemical cycle of uranium due to complicated coordination and reaction networks. Here, a joint experimental and quantum chemical investigation is carried out to probe the biomineralization of uranyl (UO22+, referred to as U(VI) hereafter) induced by Shewanella putrefaciens (abbreviated as S. putrefaciens) in the presence of DFO and Fe3+ ion. The results show that the production of mineralized solids {hydrogen-uranium mica [H2(UO2)2(PO4)2·8H2O]} via S. putrefaciens binding with UO22+ is inhibited by DFO, which can both chelate preferentially UO22+ to form a U(VI)-DFO complex in solution and seize it from U(VI)-biominerals upon solvation. However, with Fe3+ ion introduced, the strong specificity of DFO binding with Fe3+ causes re-emergence of biomineralization of UO22+ {bassetite [Fe(UO2)2(PO4)2·8(H2O)]} by S. putrefaciens, owing to competitive complexation between Fe3+ and UO22+ for DFO. As DFO possesses three hydroxamic functional groups, it forms hexadentate coordination with Fe3+ and UO22+ ions via these functional groups. The stability of the Fe3+-DFO complex is much higher than that of U(VI)-DFO, resulting in some DFO-released UO22+ to be remobilized by S. putrefaciens. Our finding not only adds to the understanding of the fate of toxic U(VI)-containing substances in the environment and biogeochemical cycles in the future but also suggests the promising potential of utilizing functionalized DFO ligands for uranium processing.

Genome analysis of Shewanella putrefaciens 4H revealing the potential mechanisms for the chromium remediation.

Microbial remediation of heavy metal polluted environment is ecofriendly and cost effective. Therefore, in the present study, Shewanella putrefaciens stain 4H was previously isolated by our group from the activated sludge of secondary sedimentation tank in a dyeing wastewater treatment plant. The bacterium was able to reduce chromate effectively. The strains showed significant ability to reduce Cr(VI) in the pH range of 8.0 to 10.0 (optimum pH 9.0) and 25-42 ℃ (optimum 30 ℃) and were able to reduce 300 mg/L of Cr(VI) in 72 h under parthenogenetic anaerobic conditions. In this paper, the complete genome sequence was obtained by Nanopore sequencing technology and analyzed chromium metabolism-related genes by comparative genomics The genomic sequence of S. putrefaciens 4H has a length of 4,631,110 bp with a G + C content of 44.66% and contains 4015 protein-coding genes and 3223,  2414, 2343 genes were correspondingly annotated into the COG, KEGG, and GO databases. The qRT-PCR analysis showed that the expression of chrA, mtrC, and undA genes was up-regulated under Cr(VI) stress. This study explores the Chromium Metabolism-Related Genes of S. putrefaciens 4H and will help to deepen our understanding of the mechanisms of Cr(VI) tolerance and reduction in this strain, thus contributing to the better application of S. putrefaciens 4H in the field of remediation of chromium-contaminated environments.

Characterization of the Positive Transcription Regulator PfaR for Improving Eicosapentaenoic Acid Production in Shewanella putrefaciens W3-18-1.

The biosynthetic pathway of eicosapentaenoic acid (EPA) has previously been reported in marine bacteria, while the regulatory mechanism remains poorly understood. In this study, a putative transcriptional regulator PfaR encoded adjacent to the PFA biosynthesis gene cluster (pfaEABCD) was computationally and experimentally characterized. Comparative analyses on the wild type (WT) strain, in-frame deletion, and overexpression mutants revealed that PfaR positively regulated EPA synthesis at low temperature. RNA-Seq and real-time quantitative PCR analyses demonstrated that PfaR stimulated the transcription of pfaABCD. The transcription start site of pfaR was mapped by using primer extension and highly conserved promoter motifs bound by the housekeeping Sigma 70 factor that were identified in the upstream of pfaR. Moreover, overexpression of PfaR in WT strain W3-18-1 at low temperature could improve EPA productivity from 0.07% to 0.13% (percentage of EPA to dry weight, mg/mg) of dry weight. Taken together, these findings could provide important implications into the transcriptional control and metabolic engineering in terms of EPA productivity for industrial strains. IMPORTANCE We have experimentally confirmed that PfaR is a positive transcription regulator that promotes EPA synthesis at low temperature in Shewanella putrefaciens W3-18-1. Overexpression of PfaR in WT strain W3-18-1 could lead to a 1.8-fold increase in EPA productivity at low temperature. It is further shown that PfaR may be regulated by housekeeping Sigma 70 factor at low temperature.

Study on spoilage potential and its molecular basis of Shewanella putrefaciens in response to cold conditions by Label-free quantitative proteomic analysis.

To elucidate how Shewanella putrefaciens survives and produces spoilage products in response to cold conditions, the metabolic and protease activity of S. putrefaciens DSM6067 cultured at three different temperatures (30 °C, 10 °C, and 4 °C) was studied by determining the bacterial growth, total volatile basic nitrogen (TVB-N), biogenic amines, extracellular protease activity, as well as the differential expressed proteins via Label-free quantitative proteomics analysis. The lag phase of the strain cultured at 10 °C and 4 °C was about 20 h and 120 h longer than at 30 °C, respectively. The TVB-N increased to 89.23 mg N/100 g within 28 h at 30 °C, and it needed at least 72 h and 224 h at 10 °C and 4 °C, respectively. Cold temperatures (10 °C and 4 °C) also inhibited the yield factors and the extracellular protease activity per cell at the lag phase. However, the protease activity per cell and the yield factors of the sample cultivated at 10 °C and 4 °C well recovered, especially at the mid and latter stages of the log phase. The further quantitative proteomic analysis displayed a complex biological network to tackle cold stress: cold stress responses, nutrient uptake, and energy conservation strategy. It was observed that the protease and peptidase were upregulated, so as to the degradation pathways of serine, arginine, and aspartate, which might lead to the accumulation of spoilage products. This study highlighted the spoilage potential of S. putrefaciens still should be concerned even at low temperatures.

Single Strain-Triggered Biogeochemical Cycle of Arsenic.

The microbial metabolism of arsenic plays a prominent role in governing the biogeochemical cycle of arsenic. Although diverse microbes are known to be involved in the redox transformation of inorganic arsenic, the underlying mechanisms about the arsenic redox cycle mediated by a single microbial strain remain unclear yet. Herein, we discover that Shewanella putrefaciens CN32, a well-known arsenate-respiring and dissimilatory metal-reducing bacterium, could mediate the reversible arsenic redox transformation under aerobic conditions. Genetic analysis shows that S. putrefaciens CN32 contains both ars and arr operon but lacks an As(III) oxidase encoding gene. Arsenic(V) reduction tests demonstrate that the ars operon is advantageous but not essential for As(V) respiration in S. putrefaciens CN32. The Arr complex encoded by the arr operon not only plays a crucial role in arsenate respiration under anaerobic conditions but also participates in the sequential process of As(V) reduction and As(III) oxidation under aerobic conditions. The Arr enzyme also contributes to the microbial As(III) resistance. The expression and catalysis directionality of Arr in S. putrefaciens CN32 are regulated by the carbon source types. Our results highlight the complexity of arsenic redox biotransformation in environments and provide new insights into the important contribution of Arr to the As biogeochemical cycle in nature.

Effect of humic acid on bioreduction of facet-dependent hematite by Shewanella putrefaciens CN-32.

Interfacial reactions between iron (Fe) (hydr)oxide surfaces and the activity of bacteria during dissimilatory Fe reduction affect extracellular electron transfer. The presence of organic matter (OM) and exposed facets of Fe (hydr)oxides influence this process. However, the underlying interfacial mechanism of facet-dependent hematite and its toxicity toward microbes during bioreduction in the presence of OM remains unknown. Herein, humic acid (HA), as typical OM, was selected to investigate its effect on the bioreduction of hematite {100} and {001}. When HA concentration was increased from 0 to 500 mg L-1, the bioreduction rates increased from 0.02 h-1 to 0.04 h-1 for hematite {100} and from 0.026 h-1 to 0.05 h-1 for hematite {001}. Since hematite {001} owned lower resistance than hematite {100} irrespective of the HA concentration, and hematite {100} was less favorable for reduction. Microscopy-based analysis showed that more hematite {001} nanoparticles adhered to the cell surface and were bound more closely to the bacteria. Moreover, less cell damage was observed in the HA-hematite {001} treatments. As the reaction progressed, some bacterial cells died or were inactivated; confocal laser scanning microscopy showed that bacterial survival was higher in the HA-hematite {001} treatments than in the HA-hematite {100} treatments after bioreduction. Spectroscopic analysis revealed that facet-dependent binding was primarily realized by surface complexation of carboxyl functional groups with structural Fe atoms, and that the binding order of HA functional groups and hematite was affected by the exposed facets. The exposed facets of hematite could influence the electrochemical properties and activity of bacteria, as well as the binding of bacteria and Fe oxides in the presence of OM, thereby governing the extracellular electron transfer and concomitant bioreduction of Fe (hydr)oxides. These results provide new insights into the interfacial reactions between OM and facet-dependent Fe oxides in anoxic, OM-rich soil and sediment environments.

Comparative Transcriptome Analysis of Shewanella putrefaciens WS13 Biofilms Under Cold Stress.

Shewanella putrefaciens is a Gram-negative bacterium that can cause seafood spoilage under low-temperature conditions. The bacterium easily forms biofilms to enhance its survival in challenging environments. Our previous research revealed that the biofilm formed by S. putrefaciens WS13 under the low temperature (4 °C) has larger biomass and tighter structure than at an optimum growth temperature (30 °C). In this study, comparative transcriptome analysis was further performed to get insights into the global-level of gene expression in the biofilm formed by S. putrefaciens WS13 under the refrigerating and optimal temperatures using Illumina RNA-Sequencing technique. The results revealed that a total of 761 genes were differentially expressed, of which 497 were significantly up-regulated and 264 were significantly down-regulated (p<0.05). The qRT-PCR results of randomly selected differentially expressed genes (DEGs) confirmed the RNA sequencing results. Comparison of transcriptome data revealed 28 significantly changed metabolic pathways under the cold stress, including the down-regulated chemotaxis, and motility, and up-regulated tryptophan metabolism, histidine biosynthesis, and quorum sensing, which benefited the biofilm formation of S. putrefaciens WS13 under the adverse circumstance. This study provided useful data for better understanding of the biofilm formation of S. putrefaciens, and also laid a theoretical foundation for novel vaccine and drug targets against the severe spoilage bacterium under the cold stress.

Surface biomineralization of uranium onto Shewanella putrefaciens with or without extracellular polymeric substances.

The influence of extracellular polymeric substances (EPS) on the interaction between uranium [U(VI)] and Shewanella putrefaciens (S. putrefaciens), especially the U(VI) biomineralization process occurring on whole cells and cell components of S. putrefaciens was investigated in this study. The removal efficiency of U(VI) by S. putrefaciens was decreased by 22% after extraction of EPS. Proteins were identified as the main components of EPS by EEM analysis and were determined to play a major role in the biosorption of uranium. SEM-EDS results showed that U(VI) was distributed around the whole cell as 500-nanometer schistose structures, which consisted primarily of U and P. However, similar uranium lamellar crystal were wrapped only on the surface of EPS-free S. putrefaciens cells. FTIR and XPS analysis indicated that phosphorus- and nitrogen-containing groups played important roles in complexing U (VI). XRD and U LIII-edge EXAFS analyses demonstrated that the schistose structure consisted of hydrogen uranyl phosphate [H2(UO2)2(PO4)2•8H2O]. Our study provides new insight into the mechanisms of induced uranium crystallization by EPS and cell wall membranes of living bacterial cells under aerobic conditions.

Plant growth-promoting rhizobacteria Shewanella putrefaciens and Cronobacter dublinensis enhance drought tolerance of pearl millet by modulating hormones and stress-responsive genes.

Drought is a major abiotic stress that affects crop productivity. Endophytic bacteria have been found to alleviate the adverse effects of drought on plants. In the present study, we evaluated the effects of two endophytic bacteria Shewanella putrefaciens strain MCL-1 and Cronobacter dublinensis strain MKS-1 on pearl millet (Pennisetum glaucum (L.) R. Br.) under drought stress conditions. Pearl millet plants were grown under three water levels: field capacity (FC), mild drought stress (MD), and severe drought stress (SD). The effects of inoculation on plant growth, physiological attributes, phytohormone content, and drought stress-responsive genes were assessed. The inoculation of pearl millet seeds with endophytes significantly improved shoot and root dry weight and root architecture of plants grown under FC and drought stress conditions. There was a significant increase in relative water content and proline accumulation in the inoculated plants. Among the phytohormones analyzed, the content of ABA and IAA was significantly higher in endophyte-treated plants under all moisture regimes than in uninoculated plants. C. dublinensis-inoculated plants had higher GA content than uninoculated plants under all moisture regimes. The expression level of genes involved in phytohormone biosynthesis (SbNCED, SbGA20oX, and SbYUC) and coding drought-responsive transcription factors (SbAP2, SbSNAC1 and PgDREB2A) was significantly higher under SD in endophyte-inoculated plants than in uninoculated plants. Thus, these endophytic bacteria presumably enhanced the tolerance of pearl millet to drought stress by modulating root growth, plant hormones, physiology and the expression of genes involved in drought tolerance.

Release of Pb adsorbed on graphene oxide surfaces under conditions of Shewanella putrefaciens metabolism.

In this study, Pb(II) was used as a target heavy metal pollutant, and the metabolism of Shewanella putrefaciens (S. putrefaciens) was applied to achieve reducing conditions to study the effect of microbial reduction on lead that was preadsorbed on graphene oxide (GO) surfaces. The results showed that GO was transformed to its reduced form (r-GO) by bacteria, and this process induced the release of Pb(II) adsorbed on the GO surfaces. After 72 hr of exposure in an S. putrefaciens system, 5.76% of the total adsorbed Pb(II) was stably dispersed in solution in the form of a Pb(II)-extracellular polymer substance (EPS) complex, while another portion of Pb(II) released from GO-Pb(II) was observed as lead phosphate hydroxide (Pb10(PO4)6(OH)2) precipitates or adsorbed species on the surface of the cell. Additionally, increasing pH induced the stripping of oxidative debris (OD) and elevated the content of dispersible Pb(II) in aqueous solution under the conditions of S. putrefaciens metabolism. These research results provide valuable information regarding the migration of heavy metals adsorbed on GO under reducing conditions due to microbial metabolism.

Antibacterial Activity and Mechanism of Linalool against Shewanella putrefaciens.

The demand for reduced chemical preservative usage is currently growing, and natural preservatives are being developed to protect seafood. With its excellent antibacterial properties, linalool has been utilized widely in industries. However, its antibacterial mechanisms remain poorly studied. Here, untargeted metabolomics was applied to explore the mechanism of Shewanella putrefaciens cells treated with linalool. Results showed that linalool exhibited remarkable antibacterial activity against S. putrefaciens, with 1.5 µL/mL minimum inhibitory concentration (MIC). The growth of S. putrefaciens was suppressed completely at 1/2 MIC and 1 MIC levels. Linalool treatment reduced the membrane potential (MP); caused the leakage of alkaline phosphatase (AKP); and released the DNA, RNA, and proteins of S. putrefaciens, thus destroying the cell structure and expelling the cytoplasmic content. A total of 170 differential metabolites (DMs) were screened using metabolomics analysis, among which 81 species were upregulated and 89 species were downregulated after linalool treatment. These DMs are closely related to the tricarboxylic acid (TCA) cycle, glycolysis, amino acid metabolism, pantothenate and CoA biosynthesis, aminoacyl-tRNA biosynthesis, and glycerophospholipid metabolism. In addition, linalool substantially affected the activity of key enzymes, such as succinate dehydrogenase (SDH), pyruvate kinase (PK), ATPase, and respiratory chain dehydrogenase. The results provided some insights into the antibacterial mechanism of linalool against S. putrefaciens and are important for the development and application of linalool in seafood preservation.

An ATP-dependent partner switch links flagellar C-ring assembly with gene expression.

Bacterial flagella differ in their number and spatial arrangement. In many species, the MinD-type ATPase FlhG (also YlxH/FleN) is central to the numerical control of bacterial flagella, and its deletion in polarly flagellated bacteria typically leads to hyperflagellation. The molecular mechanism underlying this numerical control, however, remains enigmatic. Using the model species Shewanella putrefaciens, we show that FlhG links assembly of the flagellar C ring with the action of the master transcriptional regulator FlrA (named FleQ in other species). While FlrA and the flagellar C-ring protein FliM have an overlapping binding site on FlhG, their binding depends on the ATP-dependent dimerization state of FlhG. FliM interacts with FlhG independent of nucleotide binding, while FlrA exclusively interacts with the ATP-dependent FlhG dimer and stimulates FlhG ATPase activity. Our in vivo analysis of FlhG partner switching between FliM and FlrA reveals its mechanism in the numerical restriction of flagella, in which the transcriptional activity of FlrA is down-regulated through a negative feedback loop. Our study demonstrates another level of regulatory complexity underlying the spationumerical regulation of flagellar biogenesis and implies that flagellar assembly transcriptionally regulates the production of more initial building blocks.

Highly efficient removal of victoria blue R and bioelectricity generation from textile wastewater using a novel combined dual microbial fuel cell system.

A novel combined dual microbial fuel cell (MFC) system was developed for the continuous removal of Victoria Blue R (VBR) and electricity generation. Anaerobic and aerobic VBR-degrading bacteria, Shewanella putrefaciens and Acinetobacter calcoaceticus, respectively, were applied simultaneously. The effects of various factors on the performance of the novel system in the continuous mode were investigated, and optimal operating parameters for the system were determined. The optimal liquid retention time for continuous treatment was 36 h. The optimal external resistances of connected MFCs were 390 Ω and 1300 Ω. When artificial wastewater containing 1000 mg/l of VBR was fed continuously into the system, the VBR removal efficiency achieved was 98.7%. In addition, the acute toxicity of the effluent was decreased by a factor of 21.1-22.3, indicating that the system could detoxify VBR intermediates. VBR degradation involved a stepwise demethylation process, which occurred mainly in the first MFC, whereas aromatic ring opening, sequential deamination reaction, and carbon oxidation occurred mainly in the second MFC. When actual VBR-containing wastewater (75-262 mg/l) was introduced, the removal efficiencies of VBR, chemical oxygen demand, colority, NH3, and bioelectricity generation were >99.8%, >96.6%, >88.0%, 100%, and >194.8 mW/m2, respectively and the original inoculated strains remained dominant. Therefore, the combined dual MFC system could be applied to the treatment of actual VBR-containing wastewater.

A transcriptome analysis of the antibacterial mechanism of flavonoids from Sedum aizoon L. against Shewanella putrefaciens.

Flavonoids from Sedum aizoon L. (FSAL) possess prominent antibacterial activity against Shewanella putrefaciens isolated from sea food. In the current study, the involved molecular mechanisms were investigated using transcriptome analyses combined with bioinformatics analysis in vitro for the first time. Results showed that treatment of FSAL (1.0 MIC) damaged the permeability and integrity of cell membrane and induced 721 differentially expressed genes (DEGs) in tested bacteria at transcriptional levels, including 107 DEGs were up-regulated and 614 DEGs were down-regulated. In addition, the RNA-Seq analysis revealed that the majority of DEGs mainly involved in pathways of lipopolysaccharide biosynthesis, glycerophospholipid metabolism, biosynthesis of amino acids, purine metabolism, ABC transporters and response to stimulus. In summary, the integrated results indicated that the intervention of FSAL induced destruction of cell wall and membrane, disorder of the metabolic process and redox balance, and damage of nucleic acids in S. putrefaciens, at last resulted in the death of cells. This study provided new insights into the anti- S. putrefaciens molecular mechanism underlying the treatment of FSAL, which may be served as the basis guide for identifying potential antimicrobial targets and application of FSAL in food safety.

γ-proteobacteria eject their polar flagella under nutrient depletion, retaining flagellar motor relic structures.

Bacteria switch only intermittently to motile planktonic lifestyles under favorable conditions. Under chronic nutrient deprivation, however, bacteria orchestrate a switch to stationary phase, conserving energy by altering metabolism and stopping motility. About two-thirds of bacteria use flagella to swim, but how bacteria deactivate this large molecular machine remains unclear. Here, we describe the previously unreported ejection of polar motors by γ-proteobacteria. We show that these bacteria eject their flagella at the base of the flagellar hook when nutrients are depleted, leaving a relic of a former flagellar motor in the outer membrane. Subtomogram averages of the full motor and relic reveal that this is an active process, as a plug protein appears in the relic, likely to prevent leakage across their outer membrane; furthermore, we show that ejection is triggered only under nutritional depletion and is independent of the filament as a possible mechanosensor. We show that filament ejection is a widespread phenomenon demonstrated by the appearance of relic structures in diverse γ-proteobacteria including Plesiomonas shigelloides, Vibrio cholerae, Vibrio fischeri, Shewanella putrefaciens, and Pseudomonas aeruginosa. While the molecular details remain to be determined, our results demonstrate a novel mechanism for bacteria to halt costly motility when nutrients become scarce.

Chaperone-assisted expression and purification of putrescine monooxygenase from Shewanella putrefaciens-95.

A putrescine monooxygenase from Shewanella putrefaciens 95 (SpPMO) is the initial enzyme catalyzing the hydroxylation of putrescine to N-hydroxyl putrescine, the precursor for the synthesis of a siderophore putrebactin was identified. This PMO clustered together with known characterized NMOs from Shewanella baltica, Bordetella pertussis, Erwinia amylovora, Streptomyces sp. Gordonia rubripertincta, Pseudomonas aeruginosa and outgrouped from Escherichia coli, Nocardia farcinica, and Rhodococcus erythropolis. The deduced SpPMO protein showed 53% and 36% sequence identity with other characterized bacterial NMOs from Erwinia amylovora and Gordonia rubripertincta respectively. In this investigation, we have cloned the complete 1518bp coding sequence of pubA from Shewanella putrefaciens 95 encoding the corresponding protein SpPMO. It comprises 505 amino acid residues in length and has approximately a molecular weight of 54 kDa. Chaperone-assisted heterologous expression of SpPMO in pET151Topo expression vector under the control of bacteriophage T7 promoter permitted a stringent IPTG dependent expression. It has been successfully cloned, overexpressed and purified as a soluble His6 -tagged enzyme using E. coli as a cloning and expression host. The expression of recombinant SpPMO was confirmed by Western blotting using anti-His6 antibody. The purified protein showed FAD and NADPH dependent N-hydroxylation activity. This study has paved a way to understand the hydroxylation step of putrebactin synthesis which can be further investigated by studying its kinetic mechanism and physiological role.