Host subversion of bacterial metallophore usage drives copper intoxication.

IMPORTANCE: During infection, bacteria must overcome the dual threats of metal starvation and intoxication. This work reveals that the zinc-withholding response of the host sensitizes S. aureus to copper intoxication. In response to zinc starvation, S. aureus utilizes the metallophore staphylopine. The current work revealed that the host can leverage the promiscuity of staphylopine to intoxicate S. aureus during infection. Significantly, staphylopine-like metallophores are produced by a wide range of pathogens, suggesting that this is a conserved weakness that the host can leverage to toxify invaders with copper. Moreover, it challenges the assumption that the broad-spectrum metal binding of metallophores is inherently beneficial to bacteria.

Host subversion of bacterial metallophore usage drives copper intoxication.

Microorganisms can acquire metal ions in metal-limited environments using small molecules called metallophores. While metals and their importers are essential, metals can also be toxic, and metallophores have limited ability to discriminate metals. The impact of the metallophore-mediated non-cognate metal uptake on bacterial metal homeostasis and pathogenesis remains to be defined. The globally significant pathogen Staphylococcus aureus uses the Cnt system to secrete the metallophore staphylopine in zinc-limited host niches. Here, we show that staphylopine and the Cnt system facilitate bacterial copper uptake, potentiating the need for copper detoxification. During in vivo infection, staphylopine usage increased S. aureus susceptibility to host-mediated copper stress, indicating that the innate immune response can harness the antimicrobial potential of altered elemental abundances in host niches. Collectively, these observations show that while the broad-spectrum metal-chelating properties of metallophores can be advantageous, the host can exploit these properties to drive metal intoxication and mediate antibacterial control. IMPORTANCE: During infection bacteria must overcome the dual threats of metal starvation and intoxication. This work reveals that the zinc-withholding response of the host sensitizes Staphylococcus aureus to copper intoxication. In response to zinc starvation S. aureus utilizes the metallophore staphylopine. The current work revealed that the host can leverage the promiscuity of staphylopine to intoxicate S. aureus during infection. Significantly, staphylopine-like metallophores are produced by a wide range of pathogens, suggesting that this is a conserved weakness that the host can leverage to toxify invaders with copper. Moreover, it challenges the assumption that the broad-spectrum metal binding of metallophores is inherently beneficial to bacteria.

Impact of the pentose phosphate pathway on metabolism and pathogenesis of Staphylococcus aureus.

Staphylococcus aureus is an important pathogen that leads to significant disease through multiple routes of infection. We recently published a transposon sequencing (Tn-seq) screen in a mouse acute pneumonia model and identified a hypothetical gene (SAUSA300_1902, pgl) with similarity to a lactonase of Escherichia coli involved in the pentose phosphate pathway (PPP) that was conditionally essential. Limited studies have investigated the role of the PPP in physiology and pathogenesis of S. aureus. We show here that mutation of pgl significantly impacts ATP levels and respiration. RNA-seq analysis of the pgl mutant and parent strains identified compensatory changes in gene expression for glucose and gluconate as well as reductions in the pyrimidine biosynthesis locus. These differences were also evident through unbiased metabolomics studies and 13C labeling experiments that showed mutation of pgl led to reductions in pyrimidine metabolism including decreases in ribose-5P, UMP and GMP. These nucleotide reductions impacted the amount of extracellular DNA in biofilms and reduced biofilm formation. Mutation also limited the capacity of the strain to resist oxidant damage induced by hydrogen peroxide and paraquat and subsequent intracellular survival inside macrophages. Changes in wall teichoic acid impacted susceptibility to hydrogen peroxide. We demonstrated the importance of these changes on virulence in three different models of infection, covering respiratory, skin and septicemia, demonstrating the need for proper PPP function in all models. This work demonstrates the multifaceted role metabolism can play in multiple aspects of S. aureus pathogenesis.

Copper ions inhibit pentose phosphate pathway function in Staphylococcus aureus.

To gain a better insight of how Copper (Cu) ions toxify cells, metabolomic analyses were performed in S. aureus strains that lacks the described Cu ion detoxification systems (ΔcopBL ΔcopAZ; cop-). Exposure of the cop- strain to Cu(II) resulted in an increase in the concentrations of metabolites utilized to synthesize phosphoribosyl diphosphate (PRPP). PRPP is created using the enzyme phosphoribosylpyrophosphate synthetase (Prs) which catalyzes the interconversion of ATP and ribose 5-phosphate to PRPP and AMP. Supplementing growth medium with metabolites requiring PRPP for synthesis improved growth in the presence of Cu(II). A suppressor screen revealed that a strain with a lesion in the gene coding adenine phosphoribosyltransferase (apt) was more resistant to Cu. Apt catalyzes the conversion of adenine with PRPP to AMP. The apt mutant had an increased pool of adenine suggesting that the PRPP pool was being redirected. Over-production of apt, or alternate enzymes that utilize PRPP, increased sensitivity to Cu(II). Increasing or decreasing expression of prs resulted in decreased and increased sensitivity to growth in the presence of Cu(II), respectively. We demonstrate that Prs is inhibited by Cu ions in vivo and in vitro and that treatment of cells with Cu(II) results in decreased PRPP levels. Lastly, we establish that S. aureus that lacks the ability to remove Cu ions from the cytosol is defective in colonizing the airway in a murine model of acute pneumonia, as well as the skin. The data presented are consistent with a model wherein Cu ions inhibits pentose phosphate pathway function and are used by the immune system to prevent S. aureus infections.

Repurposing Anthelmintics: Rafoxanide- and Copper-Functionalized SBA-15 Carriers against Methicillin-Resistant Staphylococcus aureus.

The development of materials that can more efficiently administer antimicrobial agents in a controlled manner is urgently needed due to the rise in microbial resistance to traditional antibiotics. While new classes of antibiotics are developed and put into widespread usage, existing, inexpensive compounds can be repurposed to fight bacterial infections. Here, we present the synthesis of amine-functionalized SBA-15 mesoporous silica nanomaterials with physisorbed rafoxanide (RFX), a commonly used salicylanilide anthelmintic, and anchored Cu(II) ions that exhibit enhanced antimicrobial efficacy against the pathogenic bacterium Staphylococcus aureus. The synthesized nanomaterials are structurally characterized by a combination of physicochemical, thermal, and optical methods. Additionally, release studies are carried out in vitro to determine the effects of pH and the synthetic sequence used to produce the materials on Cu(II) ion release. Our results indicate that SBA-15 mesoporous silica nanocarriers loaded with Cu(II) and RFX exhibit 10 times as much bactericidal action against wild-type S. aureus as the nanocarrier loaded with only RFX. Furthermore, the synthetic sequence used to produce the nanomaterials could significantly affect (enhance) their bactericidal efficacy.

Bayesian Modeling and Intrabacterial Drug Metabolism Applied to Drug-Resistant Staphylococcus aureus.

We present the application of Bayesian modeling to identify chemical tools and/or drug discovery entities pertinent to drug-resistant Staphylococcus aureus infections. The quinoline JSF-3151 was predicted by modeling and then empirically demonstrated to be active against in vitro cultured clinical methicillin- and vancomycin-resistant strains while also exhibiting efficacy in a mouse peritonitis model of methicillin-resistant S. aureus infection. We highlight the utility of an intrabacterial drug metabolism (IBDM) approach to probe the mechanism by which JSF-3151 is transformed within the bacteria. We also identify and then validate two mechanisms of resistance in S. aureus: one mechanism involves increased expression of a lipocalin protein, and the other arises from the loss of function of an azoreductase. The computational and experimental approaches, discovery of an antibacterial agent, and elucidated resistance mechanisms collectively hold promise to advance our understanding of therapeutic regimens for drug-resistant S. aureus.

Tools, Strains, and Strategies To Effectively Conduct Anaerobic and Aerobic Transcriptional Reporter Screens and Assays in Staphylococcus aureus.

Transcriptional reporters are reliable and time-tested tools to study gene regulation. In Staphylococcus aureus, ß-galactosidase (lacZ)-based genetic screens are not widely used because of the necessity of selectable markers for strain construction and the production of staphyloxanthin pigment, which obfuscates results. We describe a series of vectors that allow for markerless insertion of codon-optimized lacZ-based transcriptional reporters. The vectors code for different ribosomal binding sites, allowing for tailored lacZ expression. A ΔcrtM::kanR deletion insertion mutant was constructed that prevents the synthesis of staphyloxanthin, thereby permitting blue-white screening without the interference of carotenoid production. We demonstrate the utility of these vectors to monitor aerobic and anaerobic transcriptional activities. For the latter, we describe the use of a ferrocyanide-ferricyanide redox system [Fe(CN)63-/4-] permitting blue-white screening in the absence of oxygen. We also describe additional reporter systems and methods for monitoring transcriptional activity during anaerobic culture, including an FAD-binding fluorescent protein (EcFbFP), alpha-hemolysin (hla), or lipase (geh). The systems and methods described are compatible with vectors utilized to create and screen high-density transposon mutant libraries. IMPORTANCE Staphylococcus aureus is a human pathogen and a leading cause of infectious disease-related illness and death worldwide. For S. aureus to successfully colonize and invade host tissues, it must tightly control the expression of genes encoding virulence factors. Oxygen tension varies greatly at infection sites, and many abscesses are devoid of oxygen. In this study, we have developed novel tools and methods to study how and when S. aureus alters transcription of genes. A key advantage of these methods and tools is that they can be utilized in the presence and absence of oxygen. A better understanding of anaerobic gene expression in S. aureus will provide important insights into the regulation of genes in low-oxygen environments.

Growth and Stress Tolerance Comprise Independent Metabolic Strategies Critical for Staphylococcus aureus Infection.

Staphylococcus aureus is an important pathogen that leads to high morbidity and mortality. Although S. aureus produces many factors important for pathogenesis, few have been validated as playing a role in the pathogenesis of S. aureus pneumonia. To gain a better understanding of the genetic elements required for S. aureus pathogenesis in the airway, we performed an unbiased genome-wide transposon sequencing (Tn-seq) screen in a model of acute murine pneumonia. We identified 136 genes important for bacterial survival during infection, with a high proportion involved in metabolic processes. Phenotyping 80 individual deletion mutants through diverse in vitro and in vivo assays demonstrated that metabolism is linked to several processes, which include biofilm formation, growth, and resistance to host stressors. We further validated the importance of 23 mutations in pneumonia. Multivariate and principal-component analyses identified two key metabolic mechanisms enabling infection in the airway, growth (e.g., the ability to replicate and form biofilms) and resistance to host stresses. As deep validation of these hypotheses, we investigated the role of pyruvate carboxylase, which was important across multiple infection models and confirmed a connection between growth and resistance to host cell killing. Pathogenesis is conventionally understood in terms of the host-pathogen interactions that enable a pathogen to neutralize a host's immune response. We demonstrate with the important bacterial pathogen S. aureus that microbial metabolism influences key traits important for in vivo infection, independent from host immunomodulation. IMPORTANCE Staphylococcus aureus is an important bacterial pathogen that causes significant morbidity and mortality, infecting numerous bodily sites, including the respiratory tract. To identify the bacterial requirements for lung infection, we conducted a genome-wide screen in a mouse model of acute pneumonia. We discovered that metabolic genes were overrepresented in those required for lung infection. In contrast to the conventional view of pathogenesis focusing on immunomodulation, we demonstrate through phenotyping of deletion mutants in several functional assays that replicative ability and tolerance against host defenses form two key metabolic dimensions of bacterial infection. These dimensions are independent for most pathways but are coupled in central carbon metabolism and highlight the critical role of bacterial metabolism in survival against host defenses during infection.

Redox stress response and UV tolerance in the acidophilic iron-oxidizing bacteria Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans.

The oxidative stress response represents a sum of antioxidative mechanisms that are essential for determining the adaptation and abundance of microorganisms in the environment. Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans are chemolithotrophic bacteria that obtain their energy from the oxidation of ferrous ion. Both microorganisms are important for bioleaching of sulfidic ores and both are tolerant to high levels of heavy metals and other factors that can induce oxidative stress. In this work, we compared the tolerance and response of L. ferriphilum and At. ferrooxidans to Fe3+, H2O2, K2CrO4, and UV-C radiation. We evaluated growth, generation of reactive oxygen species (ROS), oxidative damage to lipid membranes and DNA, and the activity of antioxidative proteins in cells exposed to these stressors. L. ferriphilum had higher cell density, lower ROS content and less lipid and DNA damage than At. ferrooxidans. Consistent with this, the activity levels of thioredoxin and superoxide dismutase in L. ferriphilum were upregulated and higher than in At. ferrooxidans. This indicated that L. ferriphilum has a higher capacity to respond to oxidative stress and to manage redox homeostasis. This capacity could largely contribute to the high abundance of this species in natural and anthropogenic sites.

Cetylpyridinium Trichlorostannate: Synthesis, Antimicrobial Properties, and Controlled-Release Properties via Electrical Resistance Tomography.

Cetylpyridinium trichlorostannate (CPC-Sn), comprising cetylpyridinium chloride (CPC) and stannous chloride, was synthesized and characterized via single-crystal X-ray diffraction measurements indicating stoichiometry of C21H38NSnCl3 where the molecules are arranged in a 1:1 ratio with a cetylpyridinium cation and a [SnCl3]- anion. CPC-Sn has shown potential for application as a broad-spectrum antimicrobial agent, to reduce bacteria-generated volatile sulfur compounds and to produce advanced functional materials. In order to investigate its controlled-release properties, electrical resistance tomography was implemented. The results demonstrate that CPC-Sn exhibits extended-release properties in an aqueous environment as opposed to the CPC counterpart.

Staphylococcus aureus lacking a functional MntABC manganese import system has increased resistance to copper.

S. aureus USA300 isolates utilize the copBL and copAZ gene products to prevent Cu intoxication. We created and examined a ΔcopAZ ΔcopBL mutant strain (cop-). The cop- strain was sensitive to Cu and accumulated intracellular Cu. We screened a transposon (Tn) mutant library in the cop- background and isolated strains with Tn insertions in the mntABC operon that permitted growth in the presence of Cu. The mutations were in mntA and they were recessive. Under the growth conditions utilized, MntABC functioned in manganese (Mn) import. When cultured with Cu, strains containing a mntA::Tn accumulated less Cu than the parent strain. Mn(II) supplementation improved growth when cop- was cultured with Cu and this phenotype was dependent upon the presence of MntR, which is a repressor of mntABC transcription. A ΔmntR strain had an increased Cu load and decreased growth in the presence of Cu, which was abrogated by the introduction of mntA::Tn. Over-expression of mntABC increased cellular Cu load and sensitivity to Cu. The presence of a mntA::Tn mutation protected iron-sulfur (FeS) enzymes from inactivation by Cu. The data presented are consistent with a model wherein defective MntABC results in decreased cellular Cu accumulation and protection to FeS enzymes from Cu poisoning.

Effect of Sodium Chloride on Pyrite Bioleaching and Initial Attachment by Sulfobacillus thermosulfidooxidans.

Biomining applies microorganisms to extract valuable metals from usually sulfidic ores. However, acidophilic iron (Fe)-oxidizing bacteria tend to be sensitive to chloride ions which may be present in biomining operations. This study investigates the bioleaching of pyrite (FeS2), as well as the attachment to FeS2 by Sulfobacillus thermosulfidooxidans DSM 9293T in the presence of elevated sodium chloride (NaCl) concentrations. The bacteria were still able to oxidize iron in the presence of up to 0.6M NaCl (35 g/L), and the addition of NaCl in concentrations up to 0.2M (~12 g/L) did not inhibit iron oxidation and growth of S. thermosulfidooxidans in leaching cultures within the first 7 days. However, after approximately 7 days of incubation, ferrous iron (Fe2+) concentrations were gradually increased in leaching assays with NaCl, indicating that iron oxidation activity over time was reduced in those assays. Although the inhibition by 0.1M NaCl (~6 g/L) of bacterial growth and iron oxidation activity was not evident at the beginning of the experiment, over extended leaching duration NaCl was likely to have an inhibitory effect. Thus, after 36 days of the experiment, bioleaching of FeS2 with 0.1M NaCl was reduced significantly in comparison to control assays without NaCl. Pyrite dissolution decreased with the increase of NaCl. Nevertheless, pyrite bioleaching by S. thermosulfidooxidans was still possible at NaCl concentrations as high as 0.4M (~23 g/L NaCl). Besides, cell attachment in the presence of different concentrations of NaCl was investigated. Cells of S. thermosulfidooxidans attached heterogeneously on pyrite surfaces regardless of NaCl concentration. Noticeably, bacteria were able to adhere to pyrite surfaces in the presence of NaCl as high as 0.4M. Although NaCl addition inhibited iron oxidation activity and bioleaching of FeS2, the presence of 0.2M seemed to enhance bacterial attachment of S. thermosulfidooxidans on pyrite surfaces in comparison to attachment without NaCl.

Deciphering the Role of Multiple Thioredoxin Fold Proteins of Leptospirillum sp. in Oxidative Stress Tolerance.

Thioredoxin fold proteins (TFPs) form a family of diverse proteins involved in thiol/disulfide exchange in cells from all domains of life. Leptospirillum spp. are bioleaching bacteria naturally exposed to extreme conditions like acidic pH and high concentrations of metals that can contribute to the generation of reactive oxygen species (ROS) and consequently the induction of thiol oxidative damage. Bioinformatic studies have predicted 13 genes that encode for TFP proteins in Leptospirillum spp. We analyzed the participation of individual tfp genes from Leptospirillum sp. CF-1 in the response to oxidative conditions. Genomic context analysis predicted the involvement of these genes in the general thiol-reducing system, cofactor biosynthesis, carbon fixation, cytochrome c biogenesis, signal transduction, and pilus and fimbria assembly. All tfp genes identified were transcriptionally active, although they responded differentially to ferric sulfate and diamide stress. Some of these genes confer oxidative protection to a thioredoxin-deficient Escherichia coli strain by restoring the wild-type phenotype under oxidative stress conditions. These findings contribute to our understanding of the diversity and complexity of thiol/disulfide systems, and of adaptations that emerge in acidophilic microorganisms that allow them to thrive in highly oxidative environments. These findings also give new insights into the physiology of these microorganisms during industrial bioleaching operations.

Expression and regulation of the mer operon in Thermus thermophilus.

Mercury (Hg) is a highly toxic and widely distributed heavy metal, which some Bacteria and Archaea detoxify by the reduction of ionic Hg (Hg[II]) to the elemental volatile form, Hg(0). This activity is specified by the mer operon. The mer operon of the deeply branching thermophile Thermus thermophilus HB27 encodes for, an O-acetyl-l-homoacetylserine sulfhydrylase (Oah2), a transcriptional regulator (MerR), a hypothetical protein (hp) and a mercuric reductase (MerA). Here, we show that this operon has two convergently expressed and differentially regulated promoters. An upstream promoter, P oah , controls the constitutive transcription of the entire operon and a second promoter (P mer ), located within merR, is responsive to Hg(II). In the absence of Hg(II), the transcription of merA is basal and when Hg(II) is present, merA transcription is induced. This response to Hg(II) is controlled by MerR and genetic evidence suggests that MerR acts as a repressor and activator of P mer . When the whole merR, including P mer , is removed, merA is transcribed from P oah independently of Hg(II). These results suggest that the transcriptional regulation of mer in T. thermophilus is both similar to, and different from, the well-documented regulation of proteobacterial mer systems, possibly representing an early step in the evolution of mer-operon regulation.

The ClpCP Complex Modulates Respiratory Metabolism in Staphylococcus aureus and Is Regulated in a SrrAB-Dependent Manner.

The staphylococcal respiratory regulator (SrrAB) modulates energy metabolism in Staphylococcus aureus Studies have suggested that regulated protein catabolism facilitates energy homeostasis. Regulated proteolysis in S. aureus is achieved through protein complexes composed of a peptidase (ClpQ or ClpP) in association with an AAA+ family ATPase (typically, ClpC or ClpX). In the present report, we tested the hypothesis that SrrAB regulates a Clp complex to facilitate energy homeostasis in S. aureus Strains deficient in one or more Clp complexes were attenuated for growth in the presence of puromycin, which causes enrichment of misfolded proteins. A ΔsrrAB strain had increased sensitivity to puromycin. Epistasis experiments suggested that the puromycin sensitivity phenotype of the ΔsrrAB strain was a result of decreased ClpC activity. Consistent with this, transcriptional activity of clpC was decreased in the ΔsrrAB mutant, and overexpression of clpC suppressed the puromycin sensitivity of the ΔsrrAB strain. We also found that ClpC positively influenced respiration and that it did so upon association with ClpP. In contrast, ClpC limited fermentative growth, while ClpP was required for optimal fermentative growth. Metabolomics studies demonstrated that intracellular metabolic profiles of the ΔclpC and ΔsrrAB mutants were distinct from those of the wild-type strain, supporting the notion that both ClpC and SrrAB affect central metabolism. We propose a model wherein SrrAB regulates energy homeostasis, in part, via modulation of regulated proteolysis.IMPORTANCE Oxygen is used as a substrate to derive energy by the bacterial pathogen Staphylococcus aureus during infection; however, S. aureus can also grow fermentatively in the absence of oxygen. To successfully cause infection, S. aureus must tailor its metabolism to take advantage of respiratory activity. Different proteins are required for growth in the presence or absence of oxygen; therefore, when cells transition between these conditions, several proteins would be expected to become unnecessary. In this report, we show that regulated proteolysis is used to modulate energy metabolism in S. aureus We report that the ClpCP protein complex is involved in specifically modulating aerobic respiratory growth but is dispensable for fermentative growth.

Superoxide Dismutase and Pseudocatalase Increase Tolerance to Hg(II) in Thermus thermophilus HB27 by Maintaining the Reduced Bacillithiol Pool.

Mercury (Hg) is a widely distributed, toxic heavy metal with no known cellular role. Mercury toxicity has been linked to the production of reactive oxygen species (ROS), but Hg does not directly perform redox chemistry with oxygen. How exposure to the ionic form, Hg(II), generates ROS is unknown. Exposure of Thermus thermophilus to Hg(II) triggered ROS accumulation and increased transcription and activity of superoxide dismutase (Sod) and pseudocatalase (Pcat); however, Hg(II) inactivated Sod and Pcat. Strains lacking Sod or Pcat had increased oxidized bacillithiol (BSH) levels and were more sensitive to Hg(II) than the wild type. The ΔbshA Δsod and ΔbshA Δpcat double mutant strains were as sensitive to Hg(II) as the ΔbshA strain that lacks bacillithiol, suggesting that the increased sensitivity to Hg(II) in the Δsod and Δpcat mutant strains is due to a decrease of reduced BSH. Treatment of T. thermophilus with Hg(II) decreased aconitase activity and increased the intracellular concentration of free Fe, and these phenotypes were exacerbated in Δsod and Δpcat mutant strains. Treatment with Hg(II) also increased DNA damage. We conclude that sequestration of the redox buffering thiol BSH by Hg(II), in conjunction with direct inactivation of ROS-scavenging enzymes, impairs the ability of T. thermophilus to effectively metabolize ROS generated as a normal consequence of growth in aerobic environments.IMPORTANCEThermus thermophilus is a deep-branching thermophilic aerobe. It is a member of the Deinococcus-Thermus phylum that, together with the Aquificae, constitute the earliest branching aerobic bacterial lineages; therefore, this organism serves as a model for early diverged bacteria (R. K. Hartmann, J. Wolters, B. Kröger, S. Schultze, et al., Syst Appl Microbiol 11:243-249, 1989, https://doi.org/10.1016/S0723-2020(89)80020-7) whose natural heated habitat may contain mercury of geological origins (G. G. Geesey, T. Barkay, and S. King, Sci Total Environ 569-570:321-331, 2016, https://doi.org/10.1016/j.scitotenv.2016.06.080). T. thermophilus likely arose shortly after the oxidation of the biosphere 2.4 billion years ago. Studying T. thermophilus physiology provides clues about the origin and evolution of mechanisms for mercury and oxidative stress responses, the latter being critical for the survival and function of all extant aerobes.

Physiological Studies of Chlorobiaceae Suggest that Bacillithiol Derivatives Are the Most Widespread Thiols in Bacteria.

Low-molecular-weight (LMW) thiols mediate redox homeostasis and the detoxification of chemical stressors. Despite their essential functions, the distribution of LMW thiols across cellular life has not yet been defined. LMW thiols are also thought to play a central role in sulfur oxidation pathways in phototrophic bacteria, including the Chlorobiaceae Here we show that Chlorobaculum tepidum synthesizes a novel LMW thiol with a mass of 412 ± 1 Da corresponding to a molecular formula of C14H24N2O10S, which suggests that the new LMW thiol is closely related to bacillithiol (BSH), the major LMW thiol of low-G+C Gram-positive bacteria. The Cba. tepidum LMW thiol structure was N-methyl-bacillithiol (N-Me-BSH), methylated on the cysteine nitrogen, the fourth instance of this modification in metabolism. Orthologs of bacillithiol biosynthetic genes in the Cba. tepidum genome and the CT1040 gene product, N-Me-BSH synthase, were required for N-Me-BSH synthesis. N-Me-BSH was found in all Chlorobiaceae examined as well as Polaribacter sp. strain MED152, a member of the Bacteroidetes A comparative genomic analysis indicated that BSH/N-Me-BSH is synthesized not only by members of the Chlorobiaceae, Bacteroidetes, Deinococcus-Thermus, and Firmicutes but also by Acidobacteria, Chlamydiae, Gemmatimonadetes, and Proteobacteria. Thus, BSH and derivatives appear to be the most broadly distributed LMW thiols in biology.IMPORTANCE Low-molecular-weight thiols are key metabolites that participate in many basic cellular processes: central metabolism, detoxification, and oxidative stress resistance. Here we describe a new thiol, N-methyl-bacillithiol, found in an anaerobic phototrophic bacterium and identify a gene that is responsible for its synthesis from bacillithiol, the main thiol metabolite in many Gram-positive bacteria. We show that the presence or absence of this gene in a sequenced genome accurately predicts thiol content in distantly related bacteria. On the basis of these results, we analyzed genome data and predict that bacillithiol and its derivatives are the most widely distributed thiol metabolites in biology.

Cobalamin Protection against Oxidative Stress in the Acidophilic Iron-oxidizing Bacterium Leptospirillum Group II CF-1.

Members of the genus Leptospirillum are aerobic iron-oxidizing bacteria belonging to the phylum Nitrospira. They are important members of microbial communities that catalyze the biomining of sulfidic ores, thereby solubilizing metal ions. These microorganisms live under extremely acidic and metal-loaded environments and thus must tolerate high concentrations of reactive oxygen species (ROS). Cobalamin (vitamin B12) is a cobalt-containing tetrapyrrole cofactor involved in intramolecular rearrangement reactions and has recently been suggested to be an intracellular antioxidant. In this work, we investigated the effect of the exogenous addition of cobalamin on oxidative stress parameters in Leptospirillum group II strain CF-1. Our results revealed that the external supplementation of cobalamin reduces the levels of intracellular ROSs and the damage to biomolecules, and also stimulates the growth and survival of cells exposed to oxidative stress exerted by ferric ion, hydrogen peroxide, chromate and diamide. Furthermore, exposure of strain CF-1 to oxidative stress elicitors resulted in the transcriptional activation of the cbiA gene encoding CbiA of the cobalamin biosynthetic pathway. Altogether, these data suggest that cobalamin plays an important role in redox protection of Leptospirillum strain CF-1, supporting survival of this microorganism under extremely oxidative environmental conditions. Understanding the mechanisms underlying the protective effect of cobalamin against oxidative stress may help to develop strategies to make biomining processes more effective.

Diversity of cultivable fungi associated with Antarctic marine sponges and screening for their antimicrobial, antitumoral and antioxidant potential.

The diversity of sponge-associated fungi has been poorly investigated in remote geographical areas like Antarctica. In this study, 101 phenotypically different fungal isolates were obtained from 11 sponge samples collected in King George Island, Antarctica. The analysis of ITS sequences revealed that they belong to the phylum Ascomycota. Sixty-five isolates belong to the genera Geomyces, Penicillium, Epicoccum, Pseudeurotium, Thelebolus, Cladosporium, Aspergillus, Aureobasidium, Phoma, and Trichocladium but 36 isolates could not be identified at genus level. In order to estimate the potential of these isolates as producers of interesting bioactivities, antimicrobial, antitumoral and antioxidant activities of fungal culture extracts were assayed. Around 51% of the extracts, mainly from the genus Geomyces and non identified relatives, showed antimicrobial activity against some of the bacteria tested. On the other hand, around 42% of the extracts showed potent antitumoral activity, Geomyces sp. having the best performance. Finally, the potential of the isolated fungi as producers of antioxidant activity seems to be moderate. Our results suggest that fungi associated with Antarctic sponges, particularly Geomyces, would be valuable sources of antimicrobial and antitumoral compounds. To our knowledge, this is the first report describing the biodiversity and the metabolic potential of fungi associated with Antarctic marine sponges.

Thiol/Disulfide system plays a crucial role in redox protection in the acidophilic iron-oxidizing bacterium Leptospirillum ferriphilum.

Thiol/disulfide systems are involved in the maintenance of the redox status of proteins and other molecules that contain thiol/disulfide groups. Leptospirillum ferriphilum DSM14647, an acidophilic bacterium that uses Fe(2+) as electron donor, and withstands very high concentrations of iron and other redox active metals, is a good model to study how acidophiles preserve the thiol/disulfide balance. We studied the composition of thiol/disulfide systems and their role in the oxidative stress response in this extremophile bacterium. Bioinformatic analysis using genomic data and enzymatic assays using protein extracts from cells grown under oxidative stress revealed that the major thiol/disulfide system from L. ferriphilum are a cytoplasmic thioredoxin system (composed by thioredoxins Trx and thioredoxin reductase TR), periplasmic thiol oxidation system (DsbA/DsbB) and a c-type cytochrome maturation system (DsbD/DsbE). Upon exposure of L. ferriphilum to reactive oxygen species (ROS)-generating compounds, transcriptional activation of the genes encoding Trxs and the TR enzyme, which results in an increase of the corresponding activity, was observed. Altogether these data suggest that the thioredoxin-based thiol/disulfide system plays an important role in redox protection of L. ferriphilum favoring the survival of this microorganism under extreme environmental oxidative conditions.