The pio Operon Is Essential for Phototrophic Fe(II) Oxidation in Rhodopseudomonas palustris TIE-1

Phototrophic Fe(II)-oxidizing bacteria couple the oxidation of ferrous iron [Fe(II)] to reductive CO2 fixation by using light energy, but until recently, little has been understood about the molecular basis for this process. Here we report the discovery, with Rhodopseudomonas palustris TIE-1 as a model organism, of a three-gene operon, designated the pio operon (for phototrophic iron oxidation), that is necessary for phototrophic Fe(II) oxidation. The first gene in the operon, pioA, encodes a c-type cytochrome that is upregulated under Fe(II)-grown conditions. PioA contains a signal sequence and shares homology with MtrA, a decaheme c-type cytochrome from Shewanella oneidensis MR-1. The second gene, pioB, encodes a putative outer membrane beta-barrel protein. PioB is a homologue of MtrB from S. oneidensis MR-1. The third gene, pioC, encodes a putative high potential iron sulfur protein (HiPIP) with a twin-arginine translocation (Tat) signal sequence and is similar to the putative Fe(II) oxidoreductase (Iro) from Acidithiobacillus ferrooxidans. Like PioA, PioB and PioC appear to be secreted proteins. Deletion of the pio operon results in loss of Fe(II) oxidation activity and growth on Fe(II). Complementation studies confirm that the phenotype of this mutant is due to loss of the pio genes. Deletion of pioA alone results in loss of almost all Fe(II) oxidation activity; however, deletion of either pioB or pioC alone results in only partial loss of Fe(II) oxidation activity. Together, these results suggest that proteins encoded by the pio operon are essential and specific for phototrophic Fe(II) oxidation in R. palustris TIE-1

Bacteria Are Beautiful

As a microbiologist, I'm appalled when I go to buy soap or dishwashing detergent, because these days it's very hard to find anything that doesn't say "antibacterial" on it. This is disturbing for a couple of reasons. First, there's absolutely no evidence to suggest that these antibacterial versions in any way help to keep our homes cleaner and us safer from disease-in fact, there's some evidence to suggest these products contribute to the spread of antibiotic resistance. And second, it distresses me to see the public given the perception that bacteria are bad and need to be eradicated

The fox Operon from Rhodobacter Strain SW2 Promotes Phototrophic Fe(II) Oxidation in Rhodobacter capsulatus SB1003

Anoxygenic photosynthesis based on Fe(II) is thought to be one of the most ancient forms of metabolism and is hypothesized to represent a transition step in the evolution of oxygenic photosynthesis. However, little is known about the molecular basis of this process because, until recently (Y. Jiao and D. K. Newman, J. Bacteriol. 189:1765-1773, 2007), most phototrophic Fe(II)-oxidizing bacteria have been genetically intractable. In this study, we circumvented this problem by taking a heterologous-complementation approach to identify a three-gene operon (the foxEYZ operon) from Rhodobacter sp. strain SW2 that confers enhanced light-dependent Fe(II) oxidation activity when expressed in its genetically tractable relative Rhodobacter capsulatus SB1003. The first gene in this operon, foxE, encodes a c-type cytochrome with no significant similarity to other known proteins. Expression of foxE alone confers significant light-dependent Fe(II) oxidation activity on SB1003, but maximal activity is achieved when foxE is expressed with the two downstream genes foxY and foxZ. In SW2, the foxE and foxY genes are cotranscribed in the presence of Fe(II) and/or hydrogen, with foxZ being transcribed only in the presence of Fe(II). Sequence analysis predicts that foxY encodes a protein containing the redox cofactor pyrroloquinoline quinone and that foxZ encodes a protein with a transport function. Future biochemical studies will permit the localization and function of the Fox proteins in SW2 to be determined

Characterization of the Arsenate Respiratory Reductase from Shewanella sp. Strain ANA-3

Microbial arsenate respiration contributes to the mobilization of arsenic from the solid to the soluble phase in various locales worldwide. To begin to predict the extent to which As(V) respiration impacts arsenic geochemical cycling, we characterized the expression and activity of the Shewanella sp. strain ANA-3 arsenate respiratory reductase (ARR), the key enzyme involved in this metabolism. ARR is expressed at the beginning of the exponential phase and persists throughout the stationary phase, at which point it is released from the cell. In intact cells, the enzyme localizes to the periplasm. To purify ARR, a heterologous expression system was developed in Escherichia coli. ARR requires anaerobic conditions and molybdenum for activity. ARR is a heterodimer of ~131 kDa, composed of one ArrA subunit (~95 kDa) and one ArrB subunit (~27 kDa). For ARR to be functional, the two subunits must be expressed together. Elemental analysis of pure protein indicates that one Mo atom, four S atoms associated with a bis-molybdopterin guanine dinucleotide cofactor, and four to five [4Fe-4S] are present per ARR. ARR has an apparent melting temperature of 41°C, a Km of 5 µM, and a Vmax of 11,111 µmol of As(V) reduced min–1 mg of protein–1 and shows no activity in the presence of alternative electron acceptors such as antimonite, nitrate, selenate, and sulfate. The development of a heterologous overexpression system for ARR will facilitate future structural and/or functional studies of this protein family

Phototrophic Fe(II) Oxidation Promotes Organic Carbon Acquisition by Rhodobacter capsulatus SB1003

Anoxygenic phototrophic Fe(II) oxidation is usually considered to be a lithoautotrophic metabolism that contributes to primary production in Fe-based ecosystems. In this study, we employed Rhodobacter capsulatus SB1003 as a model organism to test the hypothesis that phototrophic Fe(II) oxidation can be coupled to organic carbon acquisition. R. capsulatus SB1003 oxidized Fe(II) under anoxic conditions in a light-dependent manner, but failed to grow lithoautotrophically on soluble Fe(II). When provided with Fe(II)-citrate, however, growth was observed that was dependent upon microbially catalyzed Fe(II) oxidation, resulting in the formation of Fe(III)-citrate. Subsequent photochemical breakdown of Fe(III)-citrate yielded acetoacetic acid, that supported growth in the light but not the dark. Deletion of genes (RR00247-RR00248) that encode homologs of atoA and atoD, required for acetoacetic acid utilization, severely impaired the ability of R. capsulatus SB1003 to grow on Fe(II)-citrate. The growth yield achieved by R. capsulatus SB1003 in the presence of citrate cannot be explained by lithoautotrophic growth on Fe(II) enabled by indirect effects of the ligand (such as altering the thermodynamics of Fe(II) oxidation or preventing cell encrustation). Together, these results demonstrate that R. capsulatus SB1003 grows photoheterotrophically on Fe(II)-citrate. Nitrilotriacetic acid (NTA) also supported light-dependent growth on Fe(II), suggesting that Fe(II) oxidation may be a general mechanism whereby some Fe(II)-oxidizing bacteria mine otherwise inaccessible organic carbon sources

Redox Reactions of Phenazine Antibiotics with Ferric (Hydr)oxides and Molecular Oxygen

Phenazines are small redox-active molecules produced by a variety of bacteria. Beyond merely serving as antibiotics, recent studies suggest that phenazines play important physiological roles, including one in iron acquisition. Here we characterize the ability of four electrochemically reduced natural phenazines—pyocyanin (PYO), phenazine-1-carboxylate (PCA), phenazine-1-carboxamide, and 1-hydroxyphenazine (1-OHPHZ)—to reductively dissolve ferrihydrite and hematite in the pH range 5–8. Generally, the reaction rate is higher for a phenazine with a lower reduction potential, with the reaction between PYO and ferrihydrite at pH 5 being an exception; the rate decreases as the pH increases; the rate is higher for poorly crystalline ferrihydrite than for highly crystalline hematite. Ferric (hydr)oxide reduction by reduced phenazines can potentially be inhibited by oxygen, where O_2 competes with Fe(III) as the final oxidant. The reactivity of reduced phenazines with O_2 decreases in the order: PYO > 1-OHPHZ > PCA. Strikingly, reduced PYO, which is the least reactive phenazine with ferrihydrite and hematite at pH 7, is the most reactive phenazine with O_2. These results imply that different phenazines may perform different functions in environments with gradients of iron and O_2

The transcription factors ActR and SoxR differentially affect the phenazine tolerance of Agrobacterium tumefaciens

Bacteria in soils encounter redox‐active compounds, such as phenazines, that can generate oxidative stress, but the mechanisms by which different species tolerate these compounds are not fully understood. Here, we identify two transcription factors, ActR and SoxR, that play contrasting yet complementary roles in the tolerance of the soil bacterium Agrobacterium tumefaciens to phenazines. We show that ActR promotes phenazine tolerance by proactively driving expression of a more energy‐efficient terminal oxidase at the expense of a less efficient alternative, which may affect the rate at which phenazines abstract electrons from the electron transport chain (ETC) and thereby generate reactive oxygen species. SoxR, on the other hand, responds to phenazines by inducing expression of several efflux pumps and redox‐related genes, including one of three copies of superoxide dismutase and five novel members of its regulon that could not be computationally predicted. Notably, loss of ActR is far more detrimental than loss of SoxR at low concentrations of phenazines, and also increases dependence on the otherwise functionally redundant SoxR‐regulated superoxide dismutase. Our results thus raise the intriguing possibility that the composition of an organism's ETC may be the driving factor in determining sensitivity or tolerance to redox‐active compounds

Magnetosome vesicles are present before magnetite formation, and MamA is required for their activation

Bacterial magnetosomes are intracellular compartments that house highly ordered magnetite crystals. By using Magnetospirillum sp. AMB-1 as a model system, we show that magnetosome vesicles exist in the absence of magnetite, biomineralization of magnetite proceeds simultaneously in multiple vesicles, and biomineralization proceeds from the same location in each vesicle. The magnetosome-associated protein, MamA, is required for the formation of functional magnetosome vesicles and displays a dynamic subcellular localization throughout the growth cycle of magnetotactic bacteria. Together, these results suggest that the magnetosome precisely coordinates magnetite biomineralization and can serve as a model system for the study of organelle biogenesis in noneukaryotic cells

A Putative ABC Transporter, HatABCDE, Is among Molecular Determinants of Pyomelanin Production in Pseudomonas aeruginosa

Pyomelanin overproduction is a common phenotype among Pseudomonas aeruginosa isolates recovered from cystic fibrosis and urinary tract infections. Its prevalence suggests that it contributes to the persistence of the producing microbial community, yet little is known about the mechanisms of its production. Using transposon mutagenesis, we identified factors that contribute to melanogenesis in a clinical isolate of P. aeruginosa. In addition to two enzymes already known to be involved in its biosynthesis (homogentisate dioxygenase and hydroxyphenylpyruvate dioxygenase), we identified 26 genes that encode regulatory, metabolic, transport, and hypothetical proteins that contribute to the production of homogentisic acid (HGA), the monomeric precursor of pyomelanin. One of these, PA14_57880, was independently identified four times and is predicted to encode the ATP-binding cassette of an ABC transporter homologous to proteins in Pseudomonas putida responsible for the extrusion of organic solvents from the cytosol. Quantification of HGA production by P. aeruginosa PA14 strains missing the predicted subcomponents of this transporter confirmed its role in HGA production: mutants unable to produce the ATP-binding cassette (PA14_57880) or the permease (PA14_57870) produced substantially less extracellular HGA after growth for 20 h than the parental strain. In these mutants, concurrent accumulation of intracellular HGA was observed. In addition, quantitative real-time PCR revealed that intracellular accumulation of HGA elicits upregulation of these transport genes. Based on their involvement in homogentisic acid transport, we rename the genes of this operon hatABCDE

What Genetics Offers Geobiology

For over 50 years, the Parker Brothers’ board game “Clue” has maintained its position as the classic family detective game. A murder has been committed in the mansion, but we don’t know where, by whom, or how. Was it Professor Plum in the study with a knife, or Miss Scarlett in the ballroom with a candlestick? Through rolls of the dice, fragments of information patiently accumulated piece-by-piece, and the application of logic, players construct a case to figure out “whodunit”. Because there are several potential solutions to the problem, the key challenge is to figure out what happened by understanding how it happened