Mechanisms of Bacterial Extracellular Electron Exchange.

The biochemical mechanisms by which microbes interact with extracellular soluble metal ions and insoluble redox-active minerals have been the focus of intense research over the last three decades. The process presents two challenges to the microorganism. Firstly, electrons have to be transported at the cell surface, which in Gram-negative bacteria presents an additional problem of electron transfer across the ~6nm of the outer membrane. Secondly, the electrons must be transferred to or from the terminal electron acceptors or donors. This review covers the known mechanisms that bacteria use to transport electrons across the cell envelope to external electron donors/acceptors. In Gram-negative bacteria, electron transfer across the outer membrane involves the use of an outer membrane ß-barrel and cytochrome. These can be in the form of a porin-cytochrome protein, such as Cyc2 of Acidithiobacillus ferrooxidans, or a multiprotein porin-cytochrome complex like MtrCAB of Shewanella oneidensis MR-1. For mineral-respiring organisms, there is the additional challenge of transferring the electrons from the cell to mineral surface. For the strict anaerobe Geobacter sulfurreducens this requires electron transfer through conductive pili to associated cytochrome OmcS that directly reduces Fe(III)oxides, while the facultative anaerobe S. oneidensis MR-1 accomplishes mineral reduction through direct membrane contact, contact through filamentous extensions and soluble flavin shuttles, all of which require the outer membrane cytochromes MtrC and OmcA in addition to secreted flavin.

Analysis of nitroxide spin label motion in a protein-protein complex using multiple frequency EPR spectroscopy.

X- and W-band EPR spectra, at room and low temperatures, are reported for nitroxide spin labels attached to cysteine residues selectively introduced into two proteins, the DNase domain of colicin-E9 and its immunity protein, Im9. The dynamics of each site of attachment on the individual proteins and in the tight DNase-Im9 complex have been analysed by computer simulations of the spectra using a model of Brownian dynamics trajectories for the spin label and protein. Ordering potentials have been introduced to describe mobility of labels restricted by the protein domain. Label mobility varies with position from completely immobilised, to motionally restricted and to freely rotating. Bi-modal dynamics of the spin label have been observed for several sites. We show that W-band spectra are particularly useful for detection of anisotropy of spin label motion. On complex formation significant changes are observed in the dynamics of labels at the binding interface region. This work reveals multi-frequency EPR as a sensitive and valuable tool for detecting conformational changes in protein structure and dynamics especially in protein-protein complexes.

Osmotically induced shape changes of large unilamellar vesicles measured by dynamic light scattering.

Static and dynamic light scattering measurements have been used to characterize the size, size distribution, and shape of extruded vesicles under isotonic conditions. Dynamic light scattering was then used to characterize osmotically induced shape changes by monitoring changes in the hydrodynamic radius (R(h)) of large unilamellar vesicles (LUVs). These changes are compared to those predicted for several shapes that appear in trajectories through the phase diagram of the area difference elasticity (ADE) model (. Phys. Rev. E. 52:6623-6634). Measurements were performed on dioleoylphosphatidylcholine (DOPC) vesicles using two membrane-impermeant osmolytes (NaCl and sucrose) and a membrane-permeant osmolyte (urea). For all conditions, we were able to produce low-polydispersity, nearly spherical vesicles, which are essential for resolving well-defined volume changes and consequent shape changes. Hyper-osmotic dilutions of DOPC vesicles in urea produced no change in R(h), whereas similar dilutions in NaCl or sucrose caused reductions in vesicle volume resulting in observable changes to R(h). Under conditions similar to those of this study, the ADE model predicts an evolution from spherical to prolate then oblate shapes on increasing volume reduction of LUVs. However, we found that DOPC vesicles became oblate at all applied volume reductions.

Complete denitration of nitroglycerin by bacteria isolated from a washwater soakaway.

Four axenic bacterial species capable of biodegrading nitroglycerin (glycerol trinitrate [GTN]) were isolated from soil samples taken from a washwater soakaway at a disused GTN manufacturing plant. The isolates were identified by 16S rRNA gene sequence homology as Pseudomonas putida, an Arthrobacter species, a Klebsiella species, and a Rhodococcus species. Each of the isolates utilized GTN as its sole nitrogen source and removed nitro groups sequentially from GTN to produce glycerol dinitrates and mononitrates (GMN), with the exception of the Arthrobacter strain, which achieved removal of only the first nitro group within the time course of the experiment. The Klebsiella strain exhibited a distinct preference for removal of the central nitro group from GTN, while the other five strains exhibited no such regioselectivity. All strains which removed a second nitro group from glycerol 1,2-dinitrate showed regiospecific removal of the end nitro group, thereby producing glycerol 2-mononitrate. Most significant was the finding that the Rhodococcus species was capable of removing the final nitro group from GMN and thus achieved complete biodegradation of GTN. Such complete denitration of GTN has previously been shown only in mixed bacterial populations and in cultures of Penicillium corylophilum Dierckx supplemented with an additional carbon and nitrogen source. Hence, to the best of our knowledge, this is the first report of a microorganism that can achieve complete denitration of GTN.

Physical properties of liposomes and proteoliposomes prepared from Escherichia coli polar lipids.

Reconstituted proteoliposomes serve as experimental systems for the study of membrane enzymes. Osmotic shifts and other changes in the solution environment may influence the structures and membrane properties of phospholipid vesicles (including liposomes, proteoliposomes and biological membrane vesicles) and hence the activities of membrane-associated proteins. Polar lipid extracts from Escherichia coli are commonly used in membrane protein reconstitution. The solution environment influenced the phase transition temperature and the diameter of liposomes and proteoliposomes prepared from E. coli polar lipid by extrusion. Liposomes prepared from E. coli polar lipids differed from dioleoylphosphatidylglycerol liposomes in Young's elastic modulus, yield point for solute leakage and structural response to osmotic shifts, the latter indicated by static light scattering spectroscopy. At high concentrations, NaCl caused aggregation of E. coli lipid liposomes that precluded detailed interpretation of light scattering data. Proteoliposomes and liposomes prepared from E. coli polar lipids were similar in size, yield point for solute leakage and structural response to osmotic shifts imposed with sucrose as osmolyte. These results will facilitate studies of bacterial enzymes implicated in osmosensing and of other enzymes that are reconstituted in E. coli lipid vesicles.

Myelin basic protein component C1 in increasing concentrations can elicit fusion, aggregation, and fragmentation of myelin-like membranes.

Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid.

Microbial degradation of the plant sulpholipid.

Five bacterial strains capable of using sulphoquinovose (6-deoxy-6-sulpho-D-glucopyranose) as a sole source of carbon have been isolated and partially characterized.

Mechanism for biotransformation of nonylphenol polyethoxylates to Xenoestrogens in Pseudomonas putida.

A strain of Pseudomonas putida isolated from activated sewage grew aerobically on the xenoestrogen precursor, nonylphenol polyethoxylate (NPEOx, where x is the number of ethoxylate units) as sole carbon source. Comparative growth yields on NPEOav6, NPEOav9, and NPEOav20 (mixtures with average ethoxylate numbers as indicated) were consistent with utilization of all but two ethoxylate units, and the final accumulating metabolite was identified by gas chromatography-mass spectroscopy as nonylphenol diethoxylate (NPEO2). There was no growth on nonylphenol or polyethylene glycols, and there was no evidence for production of carboxylic acid analogs of NPEOx. Biodegradation kinetics measured by high-pressure liquid chromatography (HPLC) for each component in NPEOx mixtures showed that biodegradation proceeded via successive exoscission of the ethoxylate chain and not by direct scission between the second and third ethoxylate residues. The NPEOx-degrading activity was inducible by substrate, and cell extracts of NPEOav9-induced cells were also active on the pure alcohol ethoxylate, dodecyl octaethoxylate (AEO8), producing sequentially, under either aerobic or anaerobic conditions, AEO7, AEO6, AEO5, etc., thus demonstrating that the pathway involved removal of single ethoxylate units. HPLC analysis of 2,4-dinitrophenylhydrazone derivatives revealed acetaldehyde (ethanal) as the sole aldehydic product from either NPEOav9 or AEO8 under either aerobic or anaerobic conditions. We propose a mechanism for biotransformation which involves an oxygen-independent hydroxyl shift from the terminal to the penultimate carbon of the terminal ethoxylate unit of NPEOx and dissociation of the resulting hemiacetal to release acetaldehyde and the next-lower homolog, NPEOx-1, which then undergoes further cycles of the same reaction until x = 2.

Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter.

Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the alpha/beta barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively.

A 13C-NMR study of the mechanism of bacterial metabolism of monomethyl sulfate.

Two different mechanisms have been proposed previously for initiating the biodegradation of monomethyl sulfate (MeSO4) in bacteria. For a Hyphomicrobium species, a sulfatase enzyme has been proposed to hydrolyse MeSO4 to methanol and inorganic sulfate. For an Agrobacterium sp., an alternative proposal involves monooxygenation of MeSO4 (hydroxylation) to produce methanediol monosulfate, which decomposes spontaneously to formaldehyde and inorganic sulfate. In the present study, 13C-NMR was used to monitor metabolic intermediates of [13C]MeSO4 in real time in each species in order to resolve the issue of mechanism of biodegradation. Agrobacterium sp. M3C grew on MeSO4 but not on methanol. MeSO4-grown cells catabolised [13C]MeSO4 but not [13C]methanol, and [13C]methanol did not accumulate from MeSO4 in the presence of a known inhibitor of methanol dehydrogenase (cyclopropanol). Hyphomicrobium MS223 grew on MeSO4 and, in contrast with the Agrobacterium sp., also on methanol. The normally rapid metabolism of [13C]methanol by methanol-grown cells was arrested by cyclopropanol, but metabolism of [13C]MeSO4 by MeSO4-grown cells was unaffected. Moreover there was no accumulation of [13C]methanol from [13C]MeSO4 under conditions in which methanol dehydrogenase was shown to be inactive. The results provided strong evidence against the intermediacy of methanol in the biodegradation of MeSO4 in either species, and thereby render untenable mechanisms involving sulfatase-mediated hydrolysis of MeSO4. The data are consistent with the hydroxylation of MeSO4 via a monooxygenation mechanism and subsequent spontaneous hydrolysis of the methanediol monosulfate intermediate.

Biodegradation of Glycerol Trinitrate and Pentaerythritol Tetranitrate by Agrobacterium radiobacter.

Bacteria capable of metabolizing highly explosive and vasodilatory glycerol trinitrate (GTN) were isolated under aerobic and nitrogen-limiting conditions from soil, river water, and activated sewage sludge. One of these strains (from sewage sludge) chosen for further study was identified as Agrobacterium radiobacter subgroup B. A combination of high-pressure liquid chromatography and nuclear magnetic resonance analyses of the culture medium during the growth of A. radiobacter on basal salts-glycerol-GTN medium showed the sequential conversion of GTN to glycerol dinitrates and glycerol mononitrates. Isomeric glycerol 1,2-dinitrate and glycerol 1,3-dinitrate were produced simultaneously and concomitantly with the disappearance of GTN, with significant regioselectivity for the production of the 1,3-dinitrate. Dinitrates were further degraded to glycerol 1- and 2-mononitrates, but mononitrates were not biodegraded. Cells were also capable of metabolizing pentaerythritol tetranitrate, probably to its trinitrate and dinitrate analogs. Extracts of broth-grown cells contained an enzyme which in the presence of added NADH converted GTN stoichiometrically to nitrite and the mixture of glycerol dinitrates. The specific activity of this enzyme was increased 160-fold by growth on GTN as the sole source of nitrogen.

Rapid screening for bacterial phenotypes capable of biodegrading anionic surfactants: development and validation of a microtitre plate method.

The Biolog microtitre plate assay, which is based on tetrazolium dye reduction as an indicator of sole-carbon-source utilization, has been evaluated as a rapid method to investigate the biodegradation of five classes of anionic surfactant by pure and mixed cultures of bacteria. The assay gave reproducible results over a fourfold range of inoculum optical density, and the surfactant concentration was selected to provide a compromise between the length of the lag period prior to colour production and the maximum colour produced. A kinetic model was developed and used to analyse the appearance of colour in the assay and was found to give rise to three biologically significant parameters describing the processes underlying the assay. No false-positives were obtained with environmental isolates. The small number of false-negatives obtained (< 8% of the total) could be explained by the methodology used to prepare the bacterial inoculum. All isolates which were positive in the Biology assay were shown to be both primary and ultimate degraders of the test surfactant. These results show that the method provides a useful means of studying the biodegradation of anionic surfactants by both pure and mixed cultures of bacteria and will find use in the rapid analysis of biodegradation kinetics and specificities of larger numbers of individual isolates than hitherto possible. In addition, an important benefit of the methodology is that it can be used for direct analysis of the biodegradation potential of whole bacterial communities without having to make an artificial selection during laboratory growth.

Purification and characterization of the short-chain alkylsulphatase of coryneform B1a.

Using a combination of streptomycin sulphate precipitation, and DEAE-cellulose and butyl-agarose chromatography, an alkylsulphatase active towards short-chain alkyl sulphates has been purified approx. 70-fold from extracts of coryneform B1a grown on butyl-1-sulphate. The enzyme protein is dimeric with a subunit molecular mass of 77.6 kDa, has an isoelectric point of pI 7.2, and converts butyl-1-sulphate stoichiometrically into butan-1-ol and inorganic sulphate. Stoichiometric incorporation of 18O from H2(18)O into sulphate during the reaction showed that enzymic hydrolysis occurred at the O-S bond of the C-O-S ester linkage. The enzyme was active on C3-C7 linear primary alkyl sulphates but not on higher (C8,9) or lower (C1,2) homologues, although the latter pair were competitive inhibitors. The specificity constant (kcat./Km) was highest for pentyl sulphate (Km 1.89 +/- 0.38 mM; kcat. 6.86 +/- 0.52 s-1) and decreased for higher and lower homologues. No activity was detected towards C3-C9 racemic alkyl-2-sulphates, D- or L-enantiomers of butyl-2-sulphate, the symmetrical secondary alkyl sulphates pentyl-3-sulphate, heptyl-4-sulphate, nonyl-5-sulphate, C1-C8 alkane sulphonates, choline sulphate, or butyric acid-4-sulphate; none of these compounds (except the symmetrical esters and butyric acid-4-sulphate, which were not tested) was demonstrably inhibitory. The enzyme was compared with other alkylsulphatases in terms of substrate specificity and mode of action.

Socioeconomic studies of high-level nuclear waste disposal.

The socioeconomic investigations of possible impacts of the proposed repository for high-level nuclear waste at Yucca Mountain, Nevada, have been unprecedented in several respects. They bear on the public decision that sooner or later will be made as to where and how to dispose permanently of the waste presently at military weapons installations and that continues to accumulate at nuclear power stations. No final decision has yet been made. There is no clear precedent from other countries. The organization of state and federal studies is unique. The state studies involve more disciplines than any previous efforts. They have been carried out in parallel to federal studies and have pioneered in defining some problems and appropriate research methods. A recent annotated bibliography provides interested scientists with a compact guide to the 178 published reports, as well as to relevant journal articles and related documents.

Biodegradation of sulphosuccinate: direct desulphonation of a secondary sulphonate.

The bacterial biodegradation of a secondary sulphonate, sulphosuccinate, has been shown to occur by direct desulphonation. A bacterium, designated Pseudomonas sp. BS1, was isolated from activated sewage sludge, for its capacity to grow on sulphosuccinate as the sole source of carbon and energy. Cultures grown on sulphosuccinate were able to convert this substrate to sulphite which was subsequently oxidized rapidly to sulphate. The sequence of desulphonation and carbon-chain catabolism of sulphosuccinate was determined from measurements of the kinetics of sulphite and 14CO2 release from specifically radiolabelled sulpho[1,4-14C]succinate and sulpho[2,3-14C]succinate, which were synthesized from the corresponding maleic anhydrides. When each radiolabelled compound was incubated separately with washed-cell suspensions of Pseudomonas BS1, sulphite was released before 14CO2, as shown by chemical assay and radiorespirometry, respectively. Differences in the kinetics and extent of 14CO2 release from the 1,4- and 2,3-labelled substrates were consistent with entry of the intact C4 chain into the citric acid cycle. When carrier oxaloacetate was added to incubation mixtures containing resting-cell suspensions and radiolabelled sulphosuccinate, a radiolabelled metabolite with the same HPLC retention time as oxaloacetate accumulated. No radioactive metabolites accumulated when carrier oxaloacetate was replaced with succinate, fumarate or malate. Collectively, the data indicated co-production of sulphite and oxaloacetate from sulphosuccinate, which is interpreted in terms of an oxidative desulphonation mechanism.

SDS-degrading bacteria attach to riverine sediment in response to the surfactant or its primary biodegradation product dodecan-1-ol.

A laboratory-scale river microcosm was used to investigate the effect of the anionic surfactant sodium dodecyl sulphate (SDS) on the attachment of five Pseudomonas strains to natural river-sediment surfaces. Three of the Pseudomonas strains were chosen for their known ability to express alkylsulphatase enzymes capable of hydrolysing SDS, and the other two for their lack of such enzymes. One strain from each category was isolated from the indigenous bacterial population present in the river sediment used; other isolates were from soil or sewage. The alkylsulphatase phenotypes were confirmed by gel zymography of cell extracts. Addition of SDS to mixed suspensions of river sediment with any one of the biodegradation-competent strains stimulated the attachment of bacteria to the sediment particles. In contrast, the attachment of biodegradation-incompetent strains was weak and, moreover, was unaffected by SDS. The SDS-stimulated attachment for competent organisms coincided with rapid biodegradation of the surfactant. The primary intermediate of SDS biodegradation, dodecan-1-ol, accumulated transiently, and the numbers of attached bacteria correlated closely with the amount of dodecan-1-ol present. Direct addition of dodecan-1-ol also stimulated attachment but the effect was more immediate compared with SDS, when there was a lag period of approximately 2 h. To account for these observations, a model is proposed in which SDS stimulates the attachment of biodegradation-competent bacteria through its conversion to dodecan-1-ol, and it is hypothesized that the observed reversibility of the attachment is due to the subsequent removal of dodecan-1-ol by further bacterial metabolism.

Germline and somatic abnormalities of chromosome 7 in Wilms' tumor.

Although a gene (WT1) located at chromosome 11p13 is implicated in the development of Wilms' tumor (WT), there is evidence that genes on other chromosomes are also involved. A WT patient presented with a constitutional balanced translocation between chromosomes 1 and 7, t(1;7)(q42;p15), the breakpoints of which could represent a WT predisposition gene in this patient. Cytogenetic analysis of the tumor from this patient revealed an acquired abnormality of the other chromosome 7, resulting in an isochromosome of the long arm and a 46,XY,t(1;7)(q42;p15)c,i(7)(q10) karyotype. The regions of the translocation breakpoints were investigated in a series of 24 WTs using Southern blot analysis. This confirmed the monosomy of 7p and trisomy of 7q in the tumor of the translocation patient, and in addition a loss of chromosome 7p alleles was identified in a WT of a bilaterally affected patient. In addition, two WTs were shown to have an extra copy of chromosome 7 alleles. Multiple copies of chromosome 1q alleles, probably resulting from secondary changes, were observed in two WTs, one of which was also associated with a trisomy of chromosome 7. These results indicate that 7p may contain a tumor suppressor gene involved in WT development, and that duplications of 7q also may play a role in WT development.