Acrylate Reductase of an Anaerobic Electron Transport Chain of the Marine Bacterium Shewanella woodyi.

Many microorganisms are capable of anaerobic respiration in the absence of oxygen, by using different organic compounds as terminal acceptors in electron transport chain. We identify here an anaerobic respiratory chain protein responsible for acrylate reduction in the marine bacterium Shewanella woodyi. When the periplasmic proteins of S. woodyi were separated by ion exchange chromatography, acrylate reductase activity copurified with an ArdA protein (Swoo_0275). Heterologous expression of S. woodyi ardA gene (swoo_0275) in Shewanella oneidensis MR-1 cells did not result in the appearance in them of periplasmic acrylate reductase activity, but such activity was detected when the ardA gene was co-expressed with an ardB gene (swoo_0276). Together, these genes encode flavocytochrome c ArdAB, which is thus responsible for acrylate reduction in S. woodyi cells. ArdAB was highly specific for acrylate as substrate and reduced only methacrylate (at a 22-fold lower rate) among a series of other tested 2-enoates. In line with these findings, acrylate and methacrylate induced ardA gene expression in S. woodyi under anaerobic conditions, which was accompanied by the appearance of periplasmic acrylate reductase activity. ArdAB-linked acrylate reduction supports dimethylsulfoniopropionate-dependent anaerobic respiration in S. woodyi and, possibly, other marine bacteria.

Expression of filaments of the Geobacter extracellular cytochrome OmcS in Shewanella oneidensis.

The physiological role of Geobacter sulfurreducens extracellular cytochrome filaments is a matter of debate and the development of proposed electronic device applications of cytochrome filaments awaits methods for large-scale cytochrome nanowire production. Functional studies in G. sulfurreducens are stymied by the broad diversity of redox-active proteins on the outer cell surface and the redundancy and plasticity of extracellular electron transport routes. G. sulfurreducens is a poor chassis for producing cytochrome nanowires for electronics because of its slow, low-yield, anaerobic growth. Here we report that filaments of the G. sulfurreducens cytochrome OmcS can be heterologously expressed in Shewanella oneidensis. Multiple lines of evidence demonstrated that a strain of S. oneidensis, expressing the G. sulfurreducens OmcS gene on a plasmid, localized OmcS on the outer cell surface. Atomic force microscopy revealed filaments with the unique morphology of OmcS filaments emanating from cells. Electron transfer to OmcS appeared to require a functional outer-membrane porin-cytochrome conduit. The results suggest that S. oneidensis, which grows rapidly to high culture densities under aerobic conditions, may be suitable for the development of a chassis for producing cytochrome nanowires for electronics applications and may also be a good model microbe for elucidating cytochrome filament function in anaerobic extracellular electron transfer.

Rapid reaction studies on the chemistry of flavin oxidation in urocanate reductase.

Urocanate reductase (UrdA) is a bacterial flavin-dependent enzyme that reduces urocanate to imidazole propionate, enabling bacteria to use urocanate as an alternative respiratory electron acceptor. Elevated serum levels of imidazole propionate are associated with the development of type 2 diabetes, and, since UrdA is only present in humans in gut bacteria, this enzyme has emerged as a significant factor linking the health of the gut microbiome and insulin resistance. Here, we investigated the chemistry of flavin oxidation by urocanate in the isolated FAD domain of UrdA (UrdA') using anaerobic stopped-flow experiments. This analysis unveiled the presence of a charge-transfer complex between reduced FAD and urocanate that forms within the dead time of the stopped-flow instrument (∼1 ms), with flavin oxidation subsequently occurring with a rate constant of ∼60 s-1. The pH dependence of the reaction and analysis of an Arg411Ala mutant of UrdA' are consistent with Arg411 playing a crucial role in catalysis by serving as the active site acid that protonates urocanate during hydride transfer from reduced FAD. Mutational analysis of urocanate-binding residues suggests that the twisted conformation of urocanate imposed by the active site of UrdA' facilitates urocanate reduction. Overall, this study provides valuable insight into the mechanism of urocanate reduction by UrdA.

Characterization of a Novel Shewanella algae Arginine Decarboxylase Expressed in Escherichia coli.

Arginine decarboxylase (ADC) catalyzes the decarboxylation of arginine to form agmatine, an important physiological and pharmacological amine, and attracts attention to the enzymatic production of agmatine. In this study, we for the first time overexpressed and characterized the marine Shewanella algae ADC (SaADC) in Escherichia coli. The recombinant SaADC showed the maximum activity at pH 7.5 and 40 °C. The SaADC displayed previously unreported substrate inhibition when the substrate concentration was higher than 50 mM, which was the upper limit of testing condition in other reports. In the range of 1-80 mM L-arginine, the SaADC showed the Km, kcat, Ki, and kcat/Km values of 72.99 ± 6.45 mM, 42.88 ± 2.63 s-1, 20.56 ± 2.18 mM, and 0.59 s/mM, respectively, which were much higher than the Km (14.55 ± 1.45 mM) and kcat (12.62 ± 0.68 s-1) value obtained by assaying at 1-50 mM L-arginine without considering substrate inhibition. Both the kcat values of SaADC with and without substrate inhibition are the highest ones to the best of our knowledge. This provides a reference for the study of substrate inhibition of ADCs.

Decolorization of the benzidine-based azo dye Congo red by the new strain Shewanella xiamenensis G5-03 / Descoloração do corante azo derivado da benzidina vermelho Congo por uma nova linhagem de Shewanella xiamenensis G5-03

Shewanella xiamenensis G5-03 was observed to decolorize the azo dye Congo red in synthetic wastewater. The influence of some factors on the dye decolorization efficiency was evaluated. The optimal decolorization conditions were temperature 30-35 °C, pH 10.0, incubation time 10 h, and static condition. The kinetic of Congo red decolorization fitted to the Michaelis–Menten model (Vmax = 111.11 mg L-¹ h-¹ and Km = 448.3 mg L-¹). The bacterium was also able to degrade benzidine, a product of azo bond breakage of the Congo red, which contributed to reduce the phytotoxicity. The ability of S. xiamenensis G5-03 for simultaneous decolorization and degradation of Congo red shows its potential application for the biological treatment of wastewaters containing azo dyes.

Shewanella xiamenensis G5-03 foi capaz de descolorir o corante azo vermelho Congo em água residuária sintética. A influência de alguns fatores na eficiência da descoloração do corante foi avaliada. As condições ótimas de descoloração foram temperatura de 30-35 °C, pH 10,0 e condições estáticas. A cinética de descoloração do vermelho Congo se ajustou ao modelo de Michaelis–Menten (Vmax = 111,11 mg L-¹ h-¹ and Km = 448,3 mg L-¹). A bactéria também foi capaz de degradar a benzidina, um produto da quebra da ligação azo do vermelho Congo, o que contribuiu para a redução da fitotoxicidade. A habilidade da S. xiamenensis G5-03 em simultaneamente descolorir e degradar o vermelho Congo demostra seu potencial de aplicação no tratamento de águas residuárias contendo corantes azo.

Conformational rearrangements enable iterative backbone N-methylation in RiPP biosynthesis.

Peptide backbone α-N-methylations change the physicochemical properties of amide bonds to provide structural constraints and other favorable characteristics including biological membrane permeability to peptides. Borosin natural product pathways are the only known ribosomally encoded and posttranslationally modified peptides (RiPPs) pathways to incorporate backbone α-N-methylations on translated peptides. Here we report the discovery of type IV borosin natural product pathways (termed 'split borosins'), featuring an iteratively acting α-N-methyltransferase and separate precursor peptide substrate from the metal-respiring bacterium Shewanella oneidensis. A series of enzyme-precursor complexes reveal multiple conformational states for both α-N-methyltransferase and substrate. Along with mutational and kinetic analyses, our results give rare context into potential strategies for iterative maturation of RiPPs.

A Kinetic Investigation of the Early Steps in Cytochrome c Nitrite Reductase (ccNiR)-Catalyzed Reduction of Nitrite.

The decaheme enzyme cytochrome c nitrite reductase (ccNiR) catalyzes reduction of nitrite to ammonium in a six-electron, eight-proton process. With a strong reductant as the electron source, ammonium is the sole product. However, intermediates accumulate when weaker reductants are employed, facilitating study of the ccNiR mechanism. Herein, the early stages of Shewanella oneidensis ccNiR-catalyzed nitrite reduction were investigated by using the weak reductants N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and ferrocyanide. In stopped-flow experiments, reduction of nitrite-loaded ccNiR by TMPD generated a transient intermediate, identified as FeH1II(NO2-), where FeH1 represents the ccNiR active site. FeH1II(NO2-) accumulated rapidly and was then more slowly converted to the two-electron-reduced moiety {FeH1NO}7; ccNiR was not reduced beyond the {FeH1NO}7 state. The midpoint potentials for sequential reduction of FeH1III(NO2-) to FeH1II(NO2-) and then to {FeH1NO}7 were estimated to be 130 and 370 mV versus the standard hydrogen electrode, respectively. FeH1II(NO2-) does not accumulate at equilibrium because its reduction to {FeH1NO}7 is so much easier than the reduction of FeH1III(NO2-) to FeH1II(NO2-). With weak reductants, free NO• was released from nitrite-loaded ccNiR. The release of NO• from {FeH1NO}7 is exceedingly slow (k ∼ 0.001 s-1), but it is somewhat faster (k ∼ 0.050 s-1) while FeH1III(NO2-) is being reduced to {FeH1NO}7; then, the release of NO• from the undetectable transient {FeH1NO}6 can compete with reduction of {FeH1NO}6 to {FeH1NO}7. CcNiR appears to be optimized to capture nitrite and minimize the release of free NO•. Nitrite capture is achieved by reducing bound nitrite with even weak electron donors, while NO• release is minimized by stabilizing the substitutionally inert {FeH1NO}7 over the more labile {FeH1NO}6.

Thermal destabilization mechanism of cytochrome c' from psychrophilic Shewanella violacea.

Cytochrome c' is a nitric oxide (NO)-binding heme protein found in Gram negative bacteria. The thermal stability of psychrophilic Shewanella violacea cytochrome c' (SVCP) is lower than those of its homologues from other 2 psychrophilic Shewanella species, indicating that thermal destabilization mechanism for low-temperature adaptation accumulates in SVCP. In order to understand this mechanism at the amino acid level, here the stability and function of SVCP variants, modeled using the 2 homologues, were examined. The variants exhibited increased stability, and they bound NO similar to the wild type. The vulnerability as to the SVCP stability could be attributed to less hydrogen bond at the subunit interface, more flexible loop structure, and less salt bridge on the protein surface, which appear to be its destabilization mechanism. This study provides an example for controlling stability without spoiling function in psychrophilic proteins.

Structural characterization of the microbial enzyme urocanate reductase mediating imidazole propionate production.

The human microbiome can produce metabolites that modulate insulin signaling. Type 2 diabetes patients have increased circulating concentrations of the microbially produced histidine metabolite, imidazole propionate (ImP) and administration of ImP in mice resulted in impaired glucose tolerance. Interestingly, the fecal microbiota of the patients had increased capacity to produce ImP, which is mediated by the bacterial enzyme urocanate reductase (UrdA). Here, we describe the X-ray structures of the ligand-binding domains of UrdA in four different states, representing the structural transitions along the catalytic reaction pathway of this unexplored enzyme linked to disease in humans. The structures in combination with functional data provide key insights into the mechanism of action of UrdA that open new possibilities for drug development strategies targeting type 2 diabetes.

The degradation activities for three seaweed polysaccharides of Shewanella sp. WPAGA9 isolated from deep-sea sediments.

Seaweed oligosaccharides possess great bioactivities. However, different microbial strains are required to degrade multiple polysaccharides due to their limited biodegradability, thereby increasing the cost and complexity of production. Shewanella sp. WPAGA9 was isolated from deep-sea sediments in this study. According to the genomic and biochemical analyses, the extracellular fermentation broth of WPAGA9 had versatile degradation abilities for three typical seaweed polysaccharides including agar, carrageenan, and alginate. The maximum enzyme activities of the extracellular fermentation broth of WPAGA9 were 71.63, 76.4, and 735.13 U/ml for the degradation of agar, alginate, and carrageenan, respectively. Moreover, multiple seaweed oligosaccharides can be produced by the extracellular fermentation broth of WPAGA9 under similar optimum conditions. Therefore, WPAGA9 can simultaneously degrade three types of seaweed polysaccharides under similar conditions, thereby greatly reducing the production cost of seaweed oligosaccharides. This finding indicates that Shewanella sp. WPAGA9 is an ideal biochemical tool for producing multiple active seaweed oligosaccharides at low costs and is also an important participant in the carbon cycle process of the deep-sea environment.

Psychrophile-based simple biocatalysts for effective coproduction of 3-hydroxypropionic acid and 1,3-propanediol.

3-Hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO) have tremendous potential markets in many industries. This study evaluated the simultaneous biosynthesis of the 2 compounds using the new psychrophile-based simple biocatalyst (PSCat) reaction system. The PSCat method is based on the expression of glycerol dehydratase, 1,3-propanediol dehydrogenase, and aldehyde dehydrogenase from Klebsiella pneumoniae in Shewanella livingstonensis Ac10 and Shewanella frigidimarina DSM 12253, individually. Heat treatment at 45 °C for 15 min deactivated the intracellular metabolic flux, and the production process was started after adding substrate, cofactor, and coenzyme. In the solo production process after 1 h, the maximum production of 3-HP was 62.0 m m. For 1,3-PDO, the maximum production was 25.0 m m. In the simultaneous production process, productivity was boosted, and the production of 3-HP and 1,3-PDO increased by 13.5 and 4.9 m m, respectively. Hence, the feasibility of the individual production and the simultaneous biosynthesis system were verified in the new PSCat approach.

Optimizing Eicosapentaenoic Acid Production by Grafting a Heterologous Polyketide Synthase Pathway in the Thraustochytrid Aurantiochytrium.

Eicosapentaenoic acid (EPA) is an essential nutritional supplement for human health. The most prominent dietary source of EPA is fish oil, which is unsustainable because of the decline in fishery resources and serious environmental pollution. Alternatively, a heterologous polyketide synthase pathway for EPA biosynthesis was assembled in Thraustochytrid Aurantiochytrium. A 2A peptide-based facile assembly platform that can achieve multigene expression as a polycistron was first established. The platform was then applied to express the EPA biosynthetic gene cluster from Shewanella japonica in Aurantiochytrium. In the shake flask fermentation, the lipid and PUFA yields of the mutant were increased by 26.9 and 36.0%, respectively, and led to about 5-fold increase of the EPA yield. The final EPA titer reached 2.7 g/L in fed-batch fermentation. This study provides a novel metabolic engineering strategy to regulate the EPA ratio in microalgal oil for human nutritional supplementation.

blaOXA -731 , a new chromosome-encoded blaOXA -48 -like variant in Shewanella sp. from the aquatic environment in Myanmar.

Shewanella sp., the progenitors of blaOXA-48 -like genes are increasingly reported with the possession of different blaOXA-48 -like variants. This study aims to characterize blaOXA-731 , a new variant of a blaOXA-48 -like gene identified in Shewanella sp. isolated from the aquatic environment in Myanmar. Phylogenetic analysis of the blaOXA-731 sequence with other blaOXA-48 -like variants showed that it has the highest nucleotide identity of 86.09% with blaOXA-48 . However, the active site motifs in OXA-731 were 100% identical to that in OXA-48. Whole-genome sequencing analysis showed that blaOXA-731 is not surrounded by any mobile genetic elements. The genetic context of blaOXA-731 was found as similar to other blaOXA-48 -like genes previously identified in Shewanella sp. S1 nuclease pulsed-field gel electrophoresis followed by Southern blotting confirmed the location of blaOXA-731 in the chromosome of the Shewanella genome. Cloning and expression studies showed that OXA-731 has ß-lactamase activity similar to OXA-48 and OXA-181, but it has no significant carbapenemase activity. Our results showed the significance of blaOXA-48 -like-carrying Shewanella sp. in the spreading of blaOXA-48 -like genes in the community.

Structural and enzymatic analysis of a dimeric cholylglycine hydrolase like acylase active on N-acyl homoserine lactones.

The prevalence of substrate cross-reactivity between AHL acylases and ß-lactam acylases provides a glimpse of probable links between quorum sensing and antibiotic resistance in bacteria. Both these enzyme classes belong to the N-terminal nucleophile (Ntn)-hydrolase superfamily. Penicillin V acylases alongside bile salt hydrolases constitute the cholylglycine hydrolase (CGH) group of the Ntn-hydrolase superfamily. Here we report the ability of two acylases, Slac1 and Slac2, from the marine bacterium Shewanella loihica-PV4 to hydrolyze AHLs. Three-dimensional structure of Slac1reveals the conservation of the Ntn hydrolase fold and CGH active site, making it a unique CGH exclusively active on AHLs. Slac1homologs phylogenetically cluster separate from reported CGHs and AHL acylases, thereby representing a functionally distinct sub-class of CGH that might have evolved as an adaptation to the marine environment. We hypothesize that Slac1 could provide the structural framework for understanding this subclass, and further our understanding of the evolutionary link between AHL acylases and ß-lactam acylases.

HD-[HD-GYP] Phosphodiesterases: Activities and Evolutionary Diversification within the HD-GYP Family.

Cyclic dinucleotides are signaling molecules that modulate many processes, including immune response and virulence factor production. Their cellular levels in bacteria are fine-tuned by metal-dependent phosphodiesterases, namely, the EAL and HD-GYP proteins, with HD-GYPs belonging to the larger HD domain superfamily. In this study, we first focus on the catalytic properties and the range of metal ions and substrates of the HD-[HD-GYP] subfamily, consisting of two HD domains. We identified SO3491 as a homologue of VCA0681 and the second example of an HD-[HD-GYP]. Both proteins hydrolyze c-di-GMP and 3'3'c-GAMP and coordinate various metal ions, but only Fe and to a lesser extent Co support hydrolysis. The proteins are active only in the diferrous form and not in the one-electron more oxidized FeIIFeIII state. Although the C-terminal HD-GYP domain is essential for activity, the role of the N-terminal HD domain remains unknown. We show that the N-terminal site is important for protein stability, influences the individual apparent kcat and KM (but not kcat/KM), and cannot bind c-di-GMP, thus precluding its involvement in cyclic dinucleotide sensing. We proceeded to perform phylogenetic analyses to examine the distribution and functional relationships of the HD-[HD-GYP]s to the rest of the HD-GYPs. The phylogeny provides a correlation map that draws a link between the evolutionary and functional diversification of HD-GYPs, serving as a template for predicting the chemical nature of the metallocofactor, level of activity, and reaction outcome.

Plasticity of the peroxidase AhpC links multiple substrates to diverse disulfide-reducing pathways in Shewanella oneidensis.

AhpC is a bacterial representative of 2-Cys peroxiredoxins (Prxs) with broad substrate specificity and functional plasticity. However, details underpinning these two important attributes of AhpC remain unclear. Here, we studied the functions and mechanisms of regulation of AhpC in the facultative Gram-negative anaerobic bacterium Shewanella oneidensis, in which AhpC's physiological roles can be conveniently assessed through its suppression of a plating defect due to the genetic loss of a major catalase. We show that successful suppression can be achieved only when AhpC is produced in a dose- and time-dependent manner through a complex mechanism involving activation of the transcriptional regulator OxyR, transcription attenuation, and translation reduction. By analyzing AhpC truncation variants, we demonstrate that reactivity with organic peroxides (OPs) rather than H2O2 is resilient to mutagenesis, implying that OP reduction is the core catalytic function of AhpC. Intact AhpC could be recycled only by its cognate reductase AhpF, and AhpC variants lacking the Prx domain or the extreme C-terminal five residues became promiscuous electron acceptors from the thioredoxin reductase TrxR and the GSH reductase Gor in addition to AhpF, implicating an additional dimension to functional plasticity of AhpC. Finally, we show that the activity of S. oneidensis AhpC is less affected by mutations than that of its Escherichia coli counterpart. These findings suggest that the physiological roles of bacterial AhpCs are adapted to different oxidative challenges, depending on the organism, and that its functional plasticity is even more extensive than previously reported.

Nitrite modulates aminoglycoside tolerance by inhibiting cytochrome heme-copper oxidase in bacteria.

As a bacteriostatic agent, nitrite has been used in food preservation for centuries. When used in combination with antibiotics, nitrite is reported to work either cooperatively or antagonistically. However, the mechanism underlying these effects remains largely unknown. Here we show that nitrite mediates tolerance to aminoglycosides in both Gram-negative and Gram-positive bacteria, but has little interaction with other types of antibiotics. Nitrite directly and mainly inhibits cytochrome heme-copper oxidases (HCOs), and by doing so, the membrane potential is compromised, blocking uptake of aminoglycosides. In contrast, reduced respiration (oxygen consumption rate) resulting from nitrite inhibition is not critical for aminoglycoside tolerance. While our data indicate that nitrite is a promising antimicrobial agent targeting HCOs, cautions should be taken when used with other antibiotics, aminoglycosides in particular.

Hydrogen production driven by formate oxidation in Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a potent hydrogen producer in the deficiency of exogenous electron acceptors. The electron transfer pathway for hydrogen production remains unclear, although enzymes for hydrogen production have been identified in S. oneidensis MR-1. In this study, we investigated the electron transfer pathway from formate to hydrogen, given that formate is commonly a key chemical for bacterial hydrogen production. We revealed that two formate dehydrogenases FdhA1B1C1 and FdhA2B2C2, rather than FdnGHI, played a dominant role in formate-driven hydrogen production. Menaquinone was indispensable for the electron transfer from formate to hydrogen, which excluded the presence of formate hydrogen-lyase in S. oneidensis MR-1. A previously proposed formate dehydrogenase subunit HydC was identified as a menaquinone-binding subunit of [FeFe] hydrogenase HydAB, and the hydABC operon is conserved in bacteria living in diverse environments. A formate exporter FocA and transcriptional regulator FhlA were identified for their effect on formate metabolism and hydrogen production. FhlA positively affected the metabolism of formate and hydrogen by regulating the expression of fdhA2B2C2, fdnGHI, focA, and dld-II. Overall, the electron transfer pathway deciphered in this work will facilitate the improvement of biohydrogen production by S. oneidensis MR-1.Key Points• The electron transfer pathway from formate to hydrogen in MR-1 is deciphered.• Menaquinone is indispensable for hydrogen production.• A cytochrome b subunit transfers electrons from menaquinone to [FeFe] hydrogenase.

PBP1a glycosyltransferase and transpeptidase activities are both required for maintaining cell morphology and envelope integrity in Shewanella oneidensis.

In rod-shaped Gram-negative bacteria, penicillin binding protein 1a (PBP1a) and 1b (PBP1b) form peptidoglycan-synthesizing complexes with the outer membrane lipoprotein LpoA and LpoB, respectively. Escherichia coli mutants lacking PBP1b/LpoB are sicker than those lacking PBP1a/LpoA. However, we previously found that mutants lacking PBP1a/LpoA but not PBP1b/LpoB are deleterious in Shewanella oneidensis. Here, we show that S. oneidensis PBP1a (SoPBP1a) contains conserved signature motifs with its E. coli counterpart, EcPBP1a. Although EcPBP1a play a less prominent role in E. coli, it is capable of substituting for the SoPBP1a in a manner dependent on SoLpoA. In S. oneidensis, expression of PBP1b is lower than PBP1a, and therefore the additional expression of SoPBP1b at low levels can functionally compensate for the absence of SoPBP1a. Importantly, S. oneidensis PBP1a variants lacking either glycosyltransferase (GTase) or transpeptidase (TPase) activity fail to maintain normal morphology and cell envelope integrity. Similarly, SoPBP1b variants also fail to compensate for the loss of SoPBP1a. Furthermore, overproduction of variants of SoPBP1a, but not SoPBP1b, has detrimental effects on cell morphology in S. oneidensis wild type cells. Overall, our results indicate that the combined enzymatic activities of SoPBP1a are essential for cell wall homeostasis.

Promiscuous Enzymes Cause Biosynthesis of Diverse Siderophores in Shewanella oneidensis.

The siderophore synthetic system in Shewanella species is able to synthesize dozens of macrocyclic siderophores in vitro with synthetic precursors. In vivo, however, although three siderophores are produced naturally in Shewanella algae B516, which carries a lysine decarboxylase (AvbA) specific for siderophore synthesis, only one siderophore can be detected from many other Shewanella species. In this study, we examined a siderophore-overproducing mutant of Shewanella oneidensis which lacks an AvbA counterpart, and we found that it can also produce these three siderophores. We identified both SpeC and SpeF as promiscuous decarboxylases for both lysine and ornithine to synthesize the siderophore precursors cadaverine and putrescine, respectively. Intriguingly, putrescine is mainly synthesized from arginine through an arginine decarboxylation pathway in a constitutive manner, not liable to the concentrations of iron and siderophores. Our results provide further evidence that the substrate availability plays a determining role in siderophore production. Furthermore, we provide evidence to suggest that under iron starvation conditions, cells allocate more putrescine for siderophore biosynthesis by downregulating the expression of the enzyme that transforms putrescine into spermidine. Overall, this study provides another example of the great flexibility of bacterial metabolism that is honed by evolution to better fit living environments of these bacteria.IMPORTANCE The simultaneous production of multiple siderophores is considered a general strategy for microorganisms to rapidly adapt to their ever-changing environments. In this study, we show that some Shewanella spp. may downscale their capability for siderophore synthesis to facilitate adaptation. Although S. oneidensis lacks an enzyme specifically synthesizing cadaverine, it can produce it by using promiscuous ornithine decarboxylases. Despite this ability, this bacterium predominately produces the primary siderophore while restraining the production of secondary siderophores by regulating substrate availability. In addition to using the arginine decarboxylase (ADC) pathway for putrescine synthesis, cells optimize the putrescine pool for siderophore production. Our work provides an insight into the coordinated synthesis of multiple siderophores by harnessing promiscuous enzymes in bacteria and underscores the importance of substrate pools for the biosynthesis of natural products.