Dianne K. Newman

Genetic approaches to study of biofilms.

Interest in the study of microbial biofilms has increased greatly in recent years due in large part to the profound impact biofilms have in clinical, industrial, and natural settings. Traditionally, the study of biofilms has been approached from an ecological or engineering perspective, using a combination of classical microbiology and advanced microscopy. We and others have begun to use genetic approaches to understand the development of these complex communities. To begin we must answer the question: What is a biofilm? This definition, by necessity, may be quite broad because it is clear that many organisms can attach to a variety of surfaces under diverse environmental conditions. Therefore, in the context of this article we will operationally define a biofilm as bacteria that are attached to a surface in sufficient numbers to be detected macroscopically.

A role for excreted quinones in extracellular electron transfer

Respiratory processes in bacteria are remarkable because of their ability to use a variety of compounds, including insoluble minerals, as terminal electron acceptors. Although much is known about microbial electron transport to soluble electron acceptors, little is understood about electron transport to insoluble compounds such as ferric oxides. In anaerobic environments, humic substances can serve as electron acceptors and also as electron shuttles to ferric oxides. To explore this process, we identified mutants in Shewanella putrefaciens that are unable to respire on humic substances. Here we show that these mutants contain disruptions in a gene that is involved in the biosynthesis of menaquinone. During growth, the wild type releases a menaquinone-related redox-active small molecule into the medium that complements the mutants. This finding raises the possibility that electron transfer to a variety of oxidants, including poorly soluble minerals, may be mediated by microbially excreted quinones that have yet to be identified.

The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa

Certain members of the fluorescent pseudomonads produce and secrete phenazines. These heterocyclic, redox‐active compounds are toxic to competing organisms, and the cause of these antibiotic effects has been the focus of intense research efforts. It is largely unknown, however, how pseudomonads themselves respond to – and survive in the presence of – these compounds. Using Pseudomonas aeruginosa DNA microarrays and quantitative RT‐PCR, we demonstrate that the phenazine pyocyanin elicits the upregulation of genes/operons that function in transport [such as the resistance‐nodulation‐cell division (RND) efflux pump MexGHI‐OpmD] and possibly in redox control (such as PA2274, a putative flavin‐dependant monooxygenase), and downregulates genes involved in ferric iron acquisition. Strikingly, mexGHI‐opmD and PA2274 were previously shown to be regulated by the PA14 quorum sensing network that controls the production of virulence factors (including phenazines). Through mutational analysis, we show that pyocyanin is the physiological signal for the upregulation of these quorum sensing‐controlled genes during stationary phase and that the response is mediated by the transcription factor SoxR. Our results implicate phenazines as signalling molecules in both P. aeruginosa PA14 and PAO1.

Extracellular electron transfer

Abstract. Results from several laboratories indicate that extracellular electron transfer may be a general mechanism whereby microoorganisms generate energy for cell growth and/or maintenance. Specifically, bacteria can use redox-active organic small molecules, generated outside or inside the cells, to shuttle electrons between reduced and oxidized compounds. Electron shuttling has now been reported for several different bacterial species, and exchanges of shuttling compounds may even syntrophically link diverse organisms in nature. Biofilm systems in both geological and clinical settings are likely to be important environments for metabolisms that employ extracellular electron transfer. Both structural and functional analyses suggest that electron shuttles and some virulence factors may be related to one another.

Deposition of banded iron formations by anoxygenic phototrophic Fe(II)-oxidizing bacteria

The mechanism of banded iron formation (BIF) deposition is controversial, but classically has been interpreted to reflect ferrous iron [Fe(II)] oxidation by molecular oxygen after cyanobacteria evolved on Earth. Anoxygenic photoautotrophic bacteria can also catalyze Fe(II) oxidation under anoxic conditions. Calculations based on experimentally determined Fe(II) oxidation rates by these organisms under light regimes representative of ocean water at depths of a few hundred meters suggest that, even in the presence of cyanobacteria, anoxygenic phototrophs living beneath a wind-mixed surface layer provide the most likely explanation for BIF deposition in a stratified ancient ocean and the absence of Fe in Precambrian surface waters.

Redox-active antibiotics control gene expression and community behavior in divergent bacteria.

It is thought that bacteria excrete redox-active pigments as antibiotics to inhibit competitors. In Pseudomonas aeruginosa, the endogenous antibiotic pyocyanin activates SoxR, a transcription factor conserved in Proteo- and Actinobacteria. In Escherichia coli, SoxR regulates the superoxide stress response. Bioinformatic analysis coupled with gene expression studies in P. aeruginosa and Streptomyces coelicolor revealed that the majority of SoxR regulons in bacteria lack the genes required for stress responses, despite the fact that many of these organisms still produce redox-active small molecules, which indicates that redox-active pigments play a role independent of oxidative stress. These compounds had profound effects on the structural organization of colony biofilms in both P. aeruginosa and S. coelicolor, which shows that “secondary metabolites” play important conserved roles in gene expression and development.

Phenazines and Other Redox-Active Antibiotics Promote Microbial Mineral Reduction

ABSTRACT Natural products with important therapeutic properties are known to be produced by a variety of soil bacteria, yet the ecological function of these compounds is not well understood. Here we show that phenazines and other redox-active antibiotics can promote microbial mineral reduction. Pseudomonas chlororaphis PCL1391, a root isolate that produces phenazine-1-carboxamide (PCN), is able to reductively dissolve poorly crystalline iron and manganese oxides, whereas a strain carrying a mutation in one of the phenazine-biosynthetic genes (phzB) is not; the addition of purified PCN restores this ability to the mutant strain. The small amount of PCN produced relative to the large amount of ferric iron reduced in cultures of P. chlororaphis implies that PCN is recycled multiple times; moreover, poorly crystalline iron (hydr)oxide can be reduced abiotically by reduced PCN. This ability suggests that PCN functions as an electron shuttle rather than an iron chelator, a finding that is consistent with the observation that dissolved ferric iron is undetectable in culture fluids. Multiple phenazines and the glycopeptidic antibiotic bleomycin can also stimulate mineral reduction by the dissimilatory iron-reducing bacterium Shewanella oneidensis MR1. Because diverse bacterial strains that cannot grow on iron can reduce phenazines, and because thermodynamic calculations suggest that phenazines have lower redox potentials than those of poorly crystalline iron (hydr)oxides in a range of relevant environmental pH (5 to 9), we suggest that natural products like phenazines may promote microbial mineral reduction in the environment.

Dissimilatory arsenate and sulfate reduction in Desulfotomaculum auripigmentum sp. nov.

Abstract A newly discovered arsenate-reducing bacterium, strain OREX-4, differed significantly from strains MIT-13 and SES-3, the previously described arsenate-reducing isolates, which grew on nitrate but not on sulfate. In contrast, strain OREX-4 did not respire nitrate but grew on lactate, with either arsenate or sulfate serving as the electron acceptor, and even preferred arsenate. Both arsenate and sulfate reduction were inhibited by molybdate. Strain OREX-4, a gram-positive bacterium with a hexagonal S-layer on its cell wall, metabolized compounds commonly used by sulfate reducers. Scorodite (FeAsO42· H2O) an arsenate-containing mineral, provided micromolar concentrations of arsenate that supported cell growth. Physiologically and phylogenetically, strain OREX-4 was far-removed from strains MIT-13 and SES-3: strain OREX-4 grew on different electron donors and electron acceptors, and fell within the gram-positive group of the Bacteria, whereas MIT-13 and SES-3 fell together in the ɛ-subdivision of the Proteobacteria. Together, these results suggest that organisms spread among diverse bacterial phyla can use arsenate as a terminal electron acceptor, and that dissimilatory arsenate reduction might occur in the sulfidogenic zone at arsenate concentrations of environmental interest. 16S rRNA sequence analysis indicated that strain OREX-4 is a new species of the genus Desulfotomaculum, and accordingly, the name Desulfotomaculum auripigmentum is proposed.

Shewanella oneidensis MR-1 Uses Overlapping Pathways for Iron Reduction at a Distance and by Direct Contact under Conditions Relevant for Biofilms

ABSTRACT We developed a new method to measure iron reduction at a distance based on depositing Fe(III) (hydr)oxide within nanoporous glass beads. In this “Fe-bead” system, Shewanella oneidensis reduces at least 86.5% of the iron in the absence of direct contact. Biofilm formation accompanies Fe-bead reduction and is observable both macro- and microscopically. Fe-bead reduction is catalyzed by live cells adapted to anaerobic conditions, and maximal reduction rates require sustained protein synthesis. The amount of reactive ferric iron in the Fe-bead system is available in excess such that the rate of Fe-bead reduction is directly proportional to cell density; i.e., it is diffusion limited. Addition of either lysates prepared from anaerobic cells or exogenous electron shuttles stimulates Fe-bead reduction by S. oneidensis, but iron chelators or additional Fe(II) do not. Neither dissolved Fe(III) nor electron shuttling activity was detected in culture supernatants, implying that the mediator is retained within the biofilm matrix. Strains with mutations in omcB or mtrB show about 50% of the wild-type levels of reduction, while a cymA mutant shows less than 20% of the wild-type levels of reduction and a menF mutant shows insignificant reduction. The Fe-bead reduction defect of the menF mutant can be restored by addition of menaquinone, but menaquinone itself cannot stimulate Fe-bead reduction. Because the menF gene encodes the first committed step of menaquinone biosynthesis, no intermediates of the menaquinone biosynthetic pathway are used as diffusible mediators by this organism to promote iron reduction at a distance. CymA and menaquinone are required for both direct and indirect mineral reduction, whereas MtrB and OmcB contribute to but are not absolutely required for iron reduction at a distance.

Biosynthesis of 2-methylbacteriohopanepolyols by an anoxygenic phototroph

Sedimentary 2-methyhopanes have been used as biomarker proxies for cyanobacteria, the only known bacterial clade capable of oxygenic photosynthesis and the only group of organisms found thus far to produce abundant 2-methylbacteriohopanepolyols (2-MeBHPs). Here, we report the identification of significant quantities of 2-MeBHP in two strains of the anoxygenic phototroph Rhodopseudomonas palustris. Biosynthesis of 2-MeBHP can occur in the absence of O2, deriving the C-2 methyl group from methionine. The relative abundance of 2-MeBHP varies considerably with culture conditions, ranging from 13.3% of total bacteriohopanepolyol (BHP) to trace levels of methylation. Analysis of intact BHPs reveals the presence of methylated bacteriohopane-32,33,34,35-tetrol but no detectable methylation of 35-aminobacteriohopane-32,33,34-triol. Our results demonstrate that an anoxygenic photoautotroph is capable of generating 2-MeBHPs and show that the potential origins of sedimentary 2-methylhopanoids are more diverse than previously thought.