No abstract

Environmental Context.Since the discovery of a diverse array of microbial life associated with hydrothermal vents on the ocean floor, where conditions are hot, reducing and acidic, scientists have been seeking insight into the mechanisms used by ‘extremophilic’ organisms (those that reside permanently under environmental extremes of temperature (hot or cold), pH (acid or alkaline), salinity, or pressure) to thrive under such seemingly inhospitable conditions. Abstract.The discovery of an abundance of microorganisms that flourish in a diverse range of environments, from the frigid waters of the Antarctic, to the superheated waters of the hydrothermal vents, at the bottom of 11-km deep ocean trenches and in salt-saturated lakes, has fuelled research aimed to understand the novel survival strategies evolved by these extreme-loving (extremophilic) organisms. Adaptations of biomolecules (proteins, nucleic acids, membranes and small molecules) evolved by extremophiles are wide ranging. Compared with a protein from a ‘regular’ organism, the extremophilic analogue might feature changes to the relative frequencies of amino acid residues that modulate the properties (e.g. conformational flexibility and stability) of the protein under conditions of the specific environmental challenge. The integrity of RNA and DNA from extremophiles may be maintained by subtle structural changes to RNA nucleobases and, in the case of (hyper)thermophiles, the expression of the enzyme reverse gyrase, which catalyses positive DNA supercoiling. The expression of small molecular weight heat-shock or related caretaker proteins also features as a common adaptive strategy for maintaining cell viability at environmental extremes. Membrane architecture in extremophiles can be modulated by the environmental temperature, with additional thermal stability in membranes from some hyperthermophiles conferred by novel (cyclised) lipid chains. In addition, a selection of osmolytes and small molecules are biosynthesised or sequestered by extremophilic organisms that have adapted to conditions of high salt and/or micronutrient deprivation.

An aerobic solution prepared from V(IV) and the cyclic dihydroxamic acid putrebactin (pbH2) in 1:1 H2O/CH3OH at pH = 2 turned from blue to orange and gave a signal in the positive ion electrospray ionization mass spectrometry (ESI-MS) at m/zobs 437.0 attributed to the monooxoV(V) species [VVO(pb)]+ ([C16H26N4O7V]+, m/zcalc 437.3). A solution prepared as above gave a signal in the 51V NMR spectrum at δV = −443.3 ppm (VOCl3, δV = 0 ppm) and was electron paramagnetic resonance silent, consistent with the presence of [VVO(pb)]+. The formation of [VVO(pb)]+ was invariant of [V(IV)]:[pbH2] and of pH values over pH = 2–7. In contrast, an aerobic solution prepared from V(IV) and the linear dihydroxamic acid suberodihydroxamic acid (sbhaH4) in 1:1 H2O/CH3OH at pH values of 2, 5, or 7 gave multiple signals in the positive and negative ion ESI-MS, which were assigned to monomeric or dimeric V(V)– or V(IV)–sbhaH4 complexes or mixed-valence V(V)/(IV)–sbhaH4 complexes. The complexity of the V-sbhaH4 system has been attributed to dimerization (2[VVO(sbhaH2)]+ ↔ [(VVO)2(sbhaH2)2]2+), deprotonation ([VVO(sbhaH2)]+ – H+ ↔ [VVO(sbhaH)]0), and oxidation ([VIVO(sbhaH2)]0 –e– ↔ [VVO(sbhaH2)]+) phenomena and could be described as the sum of two pH-dependent vectors, the first comprising the deprotonation of hydroxamate (low pH) to hydroximate (high pH) and the second comprising the oxidation of V(IV) (low pH) to V(V) (high pH). Macrocyclic pbH2 was preorganized to form [VVO(pb)]+, which would provide an entropy-based increase in its thermodynamic stability compared to V(V)–sbhaH4 complexes. The half-wave potentials from solutions of [V(IV)]:[pbH2] (1:1) or [V(IV)]:[sbhaH4] (1:2) at pH = 2 were E1/2 −335 or −352 mV, respectively, which differed from the expected trend (E1/2 [VO(pb)]+/0 < VV/IV–sbhaH4). The complex solution speciation of the V(V)/(IV)–sbhaH4 system prevented the determination of half-wave potentials for single species. The characterization of [VVO(pb)]+ expands the small family of documented V–siderophore complexes relevant to understanding V transport and assimilation in the biosphere.

To manage iron acquisition in an oxic environment, Shewanella putrefaciens produces the macrocyclic dihydroxamic acid putrebactin (PB) as its native siderophore. In this work, we have established the siderophore profile of S. putrefaciens in cultures augmented with the native PB precursor putrescine and in putrescine-depleted cultures. Compared to base medium, PB increased by two-fold in cultures of S. putrefaciens with 10 mM NaCl and 20 mM exogenous putrescine. In cultures augmented with 1,4-diaminobutan-2-one (DAB), PB decreased with only 0.02-fold PB detectable at 10 mM DAB. As an ornithine decarboxylase (ODC) inhibitor, DAB depleted levels of endogenous putrescine which attenuated downstream PB assembly. Under putrescine-depleted conditions, S. putrefaciens produced as its replacement siderophore the cadaverine-based desferrioxamine B (DFO-B), as characterised by ESI-MS of the Fe(III)-loaded form (m/z(obs) 614.13; m/z(calc) 614.27). A third siderophore, independent of DAB, was observed in low levels. LC/MS Analysis of the Fe(III)-loaded extract gave m/z(obs) 440.93, which, formulated as a 1:1 Fe(III) complex with a macrocyclic dihydroxamic acid, comprising one putrescine- and one cadaverine-based precursor (m/z(calc) 440.14). These results show that the production of native PB or non-native DFO-B by S. putrefaciens can be directed though upstream inhibition of ODC. This approach could be used to increase the molecular diversity of siderophores produced by S. putrefaciens and to map alternative diamine-dependent metabolites.