Electron donor-dependent radionuclide reduction and nanoparticle formation by Anaeromyxobacter dehalogenans strain 2CP-C

Anaeromyxobacter dehalogenans strain 2CP-C reduces U(VI) and Tc(VII) to U(IV)O_2(s) (uraninite) and Tc(IV)O_2(S) respectively. Kinetic studies with resting cells revealed that U(VI) or Tc(VII) reduction rates using H₂ as electron donor exceeded those observed in acetate-amended incubations. The reduction of U(VI) by A. dehalogenans 2CP-C resulted in extracellular accumulation of ∼5 nm uraninite nanoparticles in association with a lectin-binding extracellular polymeric substance (EPS). The electron donor did not affect UO_2(S) nanoparticle size or association with EPS, but the utilization of acetate as the source of reducing equivalents resulted in distinct UO_2(S) nanoparticle aggregates that were ∼50 nm in diameter. In contrast, reduction of Tc(VII) by A. dehalogenans 2CP-C cell suspensions produced dense clusters of TcO₂ particles, which were localized within the cell periplasm and on the outside of the outer membrane. In addition to direct reduction, A. dehalogenans 2CP-C cell suspensions reduced Tc(VII) indirectly via an Fe(II)-mediated mechanism. Fe(II) produced by strain 2CP-C from either ferrihydrite or Hanford Site sediment rapidly removed ⁹⁹Tc(VII)O₄^– from solution. These findings expand our knowledge of the radionuclide reduction processes catalysed by Anaeromyxobacter spp. that may influence the fate and transport of radionuclide contaminants in the subsurface.     

Mutations affecting the reduced nicotinamide adenine dinucleotide dehydrogenase complex of Escherichia coli

A strain carrying a point mutation affecting the NADH dehydrogenase complex of Escherichia coli has been isolated and its properties examined. The gene carrying the mutation (designated ndh) was located on the E. coli chromosome at about minute 23 and was shown to be cotransducible with the pyrC gene. Strains carrying the ndh^? allele were found to be unable to grow on mannitol and to grow very poorly on glucose unless the medium was supplemented with succinate, acetate or casamino acids.The following properties of strains carrying the ndh^? allele were established which suggest that the mutation affects the NADH dehydrogenase complex but apparently not the primary dehydrogenase. Membrane preparations possess normal to elevated levels of d-lactate oxidase and succinate oxidase activities but NADH oxidase is absent. NADH is unable to reduce ubiquinone in the aerobic steady state and reduces cytochrome b very slowly when the membranes become anaerobic. NADH dehydrogenase, measured as NADH-dichlorophenolindophenol reductase is reduced but not absent. NADH oxidase is stimulated by menadione although not by Q-3 or MK-1 and in the presence of menadione, cytochrome b is reduced normally by NADH.Further mutants affected in NADH oxidase were isolated using a screening procedure based on the growth characteristics of the original ndh^? strain. The mutations carried by these strains were all cotransducible with the pyrC gene and the biochemical properties of the additional mutants were similar to those of the original mutant.The properties of the group of ndh^? mutants established so far suggest that they are affected in the transfer of reducing equivalents from the NADH dehydrogenase complex to ubiquinone.     

Characterization and description of Anaeromyxobacter dehalogenans gen. nov., sp. nov., an aryl-halorespiring facultative anaerobic myxobacterium

Five strains were isolated which form a physiologically and phylogenetically coherent group of chlororespiring microorganisms and represent the first taxon in the Myxobacteria capable of anaerobic growth. The strains were enriched and isolated from various soils and sediments based on their ability to grow using acetate as an electron donor and 2-chlorophenol (2-CPh) as an electron acceptor. They are slender gram-negative rods with a bright red pigmentation that exhibit gliding motility and form spore-like structures. These unique chlororespiring myxobacteria also grow with 2,6-dichlorophenol, 2,5-dichlorophenol, 2-bromophenol, nitrate, fumarate, and oxygen as terminal electron acceptors, with optimal growth occurring at low concentrations (<1 mM) of electron acceptor. 2-CPh is reduced by all strains as an electron acceptor in preference to nitrate, which is reduced to ammonium. Acetate, H(2), succinate, pyruvate, formate, and lactate were used as electron donors. None of the strains grew by fermentation. The 16S ribosomal DNA (rDNA) sequences of the five strains form a coherent cluster deeply branching within the family Myxococcaceae within the class Myxobacteria and are mostly closely associated with the Myxococcus subgroup. With the exception of anaerobic growth and lack of a characteristic fruiting body, these strains closely resemble previously characterized myxobacteria and therefore should be considered part of the Myxococcus subgroup. The anaerobic growth and 9.0% difference in 16S rDNA sequence from those of other myxobacterial genera are sufficient to place these strains in a new genus and species designated Anaeromyxobacter dehalogenans. The type strain is 2CP-1 (ATCC BAA-258).     

A cytochrome bb'-type quinol oxidase in Bacillus subtilis strain 168.

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa(3), which is a cytochrome c oxidase, and cytochrome aa(3) and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa(3) and caa(3) terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b(558)b(595)b'.     

Aerobic and anaerobic metabolism in a facultative anaerobe Ep01 lacking cytochrome, quinone and catalase

Abstract A facultative anaerobe, strain Ep01 produced a mixture of pyruvate, formate, acetate and ethanol from glucose anaerobically, and acetate and pyruvate aerobically. Cell extract of anaerobic-grown cells contained active pyruvate formatelyase, aldehyde dehydrogenase and alcohol dehydrogenase, while cell extract of aerobic grown cells contained an active pyruvate dehydrogenase system, NaDH oxidase and NADH peroxidase. Levels of acetate kinase and phosphate acetyltransferase activities were not significantly different in cells grown under either condition. Based on the metabolic products and the emzyme activities, we propose the presence of two metabolic pathways in strain Ep01, namely, a pathway to form formate, acetate and ethanol under anaerobic conditions, and a pathway to form under aerobic conditions. This explains why strain Ep01 can grow well both under strictly anaerobic conditions and well-aerated conditions.     

Comprehensive set of integrative plasmid vectors for copper-inducible gene expression in Myxococcus xanthus

Myxococcus xanthus is widely used as a model system for studying gliding motility, multicellular development, and cellular differentiation. Moreover, M. xanthus is a rich source of novel secondary metabolites. The analysis of these processes has been hampered by the limited set of tools for inducible gene expression. Here we report the construction of a set of plasmid vectors to allow copper-inducible gene expression in M. xanthus. Analysis of the effect of copper on strain DK1622 revealed that copper concentrations of up to 500 μM during growth and 60 μM during development do not affect physiological processes such as cell viability, motility, or aggregation into fruiting bodies. Of the copper-responsive promoters in M. xanthus reported so far, the multicopper oxidase cuoA promoter was used to construct expression vectors, because no basal expression is observed in the absence of copper and induction linearly depends on the copper concentration in the culture medium. Four different plasmid vectors have been constructed, with different marker selection genes and sites of integration in the M. xanthus chromosome. The vectors have been tested and gene expression quantified using the lacZ gene. Moreover, we demonstrate the functional complementation of the motility defect caused by lack of PilB by the copper-induced expression of the pilB gene. These versatile vectors are likely to deepen our understanding of the biology of M. xanthus and may also have biotechnological applications.     

Uranium(VI) reduction by Anaeromyxobacter dehalogenans strain 2CP-C

Previous studies demonstrated growth of Anaeromyxobacter dehalogenans strain 2CP-C with acetate or hydrogen as the electron donor and Fe(III), nitrate, nitrite, fumarate, oxygen, or ortho-substituted halophenols as electron acceptors. In this study, we explored and characterized U(VI) reduction by strain 2CP-C. Cell suspensions of fumarate-grown 2CP-C cells reduced U(VI) to U(IV). More-detailed growth studies demonstrated that hydrogen was the required electron donor for U(VI) reduction and could not be replaced by acetate. The addition of nitrate to U(VI)-reducing cultures resulted in a transitory increase in U(VI) concentration, apparently caused by the reoxidation of reduced U(IV), but U(VI) reduction resumed following the consumption of N-oxyanions. Inhibition of U(VI) reduction occurred in cultures amended with Fe(III) citrate, or citrate. In the presence of amorphous Fe(III) oxide, U(VI) reduction proceeded to completion but the U(VI) reduction rates decreased threefold compared to control cultures. Fumarate and 2-chlorophenol had no inhibitory effects on U(VI) reduction, and both electron acceptors were consumed concomitantly with U(VI). Since cocontaminants (e.g., nitrate, halogenated compounds) and bioavailable ferric iron are often encountered at uranium-impacted sites, the metabolic versatility makes Anaeromyxobacter dehalogenans a promising model organism for studying the complex interaction of multiple electron acceptors in U(VI) reduction and immobilization.     

Function of Ubiquinone in Escherichia coli: a Mutant Strain Forming a Low Level of Ubiquinone

A ubiquinone-deficient mutant of Escherichia coli K-12 forming 20% of the normal amount of ubiquinone was compared with a normal strain. This lowered concentration of ubiquinone is still four times the concentration of cytochrome b(1). The mutant strain grew more slowly than the normal strain on a minimal medium with glucose as sole source of carbon and gave a lower aerobic growth yield than the normal strain. The reduced nicotinamide adenine dinucleotide (NADH) oxidase rate in membranes from the mutant strain was 40% of the oxidase rate in membranes from the normal strain, and the percentage reduction of cytochrome b(1) in the aerobic steady state, with NADH as substrate, was increased in membranes from the mutant strain. It is concluded that ubiquinone is required for maximum oxidase activity at the relatively high concentration (27 times that of cytochrome b(1)) found in normal cells. The results are discussed in relation to a scheme previously advanced for ubiquinone function in E. coli.     

Isolation, oxygen sensitivity, and virulence of NADH oxidase mutants of the anaerobic spirochete Brachyspira (Serpulina) hyodysenteriae, etiologic agent of swine dysentery.

Brachyspira (Serpulina) hyodysenteriae, the etiologic agent of swine dysentery, uses the enzyme NADH oxidase to consume oxygen. To investigate possible roles for NADH oxidase in the growth and virulence of this anaerobic spirochete, mutant strains deficient in oxidase activity were isolated and characterized. The cloned NADH oxidase gene (nox; GenBank accession no. U19610) on plasmid pER218 was inactivated by replacing 321 bp of coding sequence with either a gene for chloramphenicol resistance (cat) or a gene for kanamycin resistance (kan). The resulting plasmids, respectively, pCmDeltaNOX and pKmDeltaNOX, were used to transform wild-type B. hyodysenteriae B204 cells and generate the antibiotic-resistant strains Nox-Cm and Nox-Km. PCR and Southern hybridization analyses indicated that the chromosomal wild-type nox genes in these strains had been replaced, through allelic exchange, by the inactivated nox gene containing cat or kan. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis revealed that both nox mutant cell lysates were missing the 48-kDa Nox protein. Soluble NADH oxidase activity levels in cell lysates of Nox-Cm and Nox-Km were reduced 92 to 96% compared to the activity level in parent strain B204. In an aerotolerance test, cells of both nox mutants were at least 100-fold more sensitive to oxygen exposure than were cells of the wild-type parent strain B204. In swine experimental infections, both nox mutants were less virulent than strain B204 in that fewer animals were colonized by the mutant cells and infected animals displayed mild, transient signs of disease, with no deaths. These results provide evidence that NADH oxidase serves to protect B. hyodysenteriae cells against oxygen toxicity and that the enzyme, in that role, contributes to the pathogenic ability of the spirochete.     

The Myxococcus xanthus wbgB gene encodes a glycosyltransferase homologue required for lipopolysaccharide O-antigen biosynthesis

Myxococcus xanthus is a gram-negative soil bacterium that initiates a complex developmental program in response to starvation. A transposon insertion (Tn5-lac omega109) mutant with developmental deficiencies was isolated and characterized in this study. A strain containing this insertion mutation in an otherwise wild-type background showed delayed developmental aggregation for about 12 h and sporulated at 1-2% of the wild-type level. Tn5-lac omega109 was found to have disrupted the M. xanthus wbgB gene, which is located 2.1 kb downstream of the M. xanthus lipopolysacharide (LPS) O-antigen biosynthesis genes wzm wzt wbgA. The deduced polypeptide sequence of WbgB shares significant similarity with bacterial glycosyltransferases including M. xanthus WbgA. The wbgB::Tn5-lac omega109 mutant was found to be defective in LPS O-antigen synthesis by immunochemical analysis. Further mutational analysis indicated that the defects of the wbgB::Tn5-lac omega109 mutant were not the result of polar effects on downstream genes. Various motility assays demonstrated that the Tn5-lac omega109 mutation affected both social (S) and adventurous (A) gliding motility of M. xanthus cells. The pleiotrophic effects of wbgB mutations indicate the importance of LPS O-antigen biosynthesis for various cellular functions in M. xanthus.     

Hydrogenase mutants of Alcaligenes eutrophus H16 show alterations in the electron transport system

Mutations in the genes coding for the soluble and the membrane-bound hydrogenase of Alcaligenes eutrophus strain H16 significantly affected the expression of respiratory chain components. In lithoautotrophically grown wild type cells electron flow mainly proceeded via the cytochrome c oxidases. Mutants defective in the membrane-bound hydrogenase contained a 2- to 3-fold higher cytochrome a content than the wild type and cytochrome c oxidase of the aa3-type was preferentially used by these cells for substrate oxidation. Mutants impaired in the soluble hydrogenase revealed slow growth on hydrogen, presumably due to inefficient reverse electron flow mechanisms which provide the cells with NADH for autotrophic CO2-fixation. In this class of mutants the two quinol oxidases of the o- and d-type in addition to the co-type oxidase were the predominant electron-transport branches.     

Different effects of 2-n-heptyl-4-hydroxyquinoline-N-oxide on oxygen and nitrate respiration in Klebsiella aerogenes

The inhibition of the oxidase and respiratory nitrate reductase activity in membrane preparations from Klebsiella aerogenes by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) has been investigated. Addition of HQNO only slightly affected the aerobic steady-state reduction of cytochrome b₅₅₉ with NADH, but caused a significantly lower nitrate reducing steady-state of this cytochrome. The changes in the redox states of the cytochromes during a slow transition from anaerobic to aerobic conditions in the presence and absence of HQNO showed that the inhibition site of HQNO is located before cytochrome d. Inhibition patterns obtained upon titration of the NADH oxidase and NADH nitrate reductase activity with HQNO indicated one site of inhibitor interaction in the NADH nitrate reductase pathway and suggested a multilocated inhibition of the NADH oxidase pathway. Difference spectra with ascorbate-dichlorophenolindophenol as electron donor indicated the presence of a cytochrome b₅₆₃ component which was not oxidized by nitrate, but was rapidly oxidized by oxygen. The latter oxidation was prevented by HQNO. A scheme for the electron transport to oxygen and nitrate is presented. In the pathway to oxygen, HQNO inhibits both at the electron-accepting side of cytochrome b₅₅₉ and at the electron-donating side of cytochrome b₅₆₃, whereas in the pathway to nitrate, inhibition occurs only at the electron-accepting side of cytochrome b₅₅₉.     

Environmental detection of octahaem cytochrome c hydroxylamine/hydrazine oxidoreductase genes of aerobic and anaerobic ammonium-oxidizing bacteria

Bacterial aerobic ammonium oxidation and anaerobic ammonium oxidation (anammox) are important processes in the global nitrogen cycle. Key enzymes in both processes are the octahaem cytochrome c (OCC) proteins, hydroxylamine oxidoreductase (HAO) of aerobic ammonium-oxidizing bacteria (AOB), which catalyses the oxidation of hydroxylamine to nitrite, and hydrazine oxidoreductase (HZO) of anammox bacteria, which converts hydrazine to N(2). While the genomes of AOB encode up to three nearly identical copies of hao operons, genome analysis of Candidatus'Kuenenia stuttgartiensis' showed eight highly divergent octahaem protein coding regions as possible candidates for the HZO. Based on their phylogenetic relationship and biochemical characteristics, the sequences of these eight gene products grouped in three clusters. Degenerate primers were designed on the basis of available gene sequences with the aim to detect hao and hzo genes in various ecosystems. The hao primer pairs amplified gene fragments from 738 to 1172 bp and the hzo primer pairs amplified gene fragments from 289 to 876 bp in length, when tested on genomic DNA isolated from a variety of AOB and anammox bacteria. A selection of these primer pairs was also used successfully to amplify and analyse the hao and hzo genes in community DNA isolated from different ecosystems harbouring both AOB and anammox bacteria. We propose that OCC protein-encoding genes are suitable targets for molecular ecological studies on both aerobic and anaerobic ammonium-oxidizing bacteria.     

Random transposition by Tn916 in Desulfitobacterium dehalogenans allows for isolation and characterization of halorespiration-deficient mutants

To allow for the molecular analysis of halorespiration by the strictly anaerobic gram-positive bacterium Desulfitobacterium dehalogenans, halorespiration-deficient mutants were selected and characterized following insertional mutagenesis by the conjugative transposon Tn916. To facilitate rapid screening of transconjugants, a highly efficient method for the growth of single colonies on solidified medium has been developed. A streptomycin-resistant mutant of D. dehalogenans was isolated and mated with Enterococcus faecalis JH2-2 carrying Tn916. Insertion of one or two copies of Tn916 into the chromosome of D. dehalogenans was observed. From a total of 2,500 transconjugants, 24 halorespiration-deficient mutants were selected based upon their inability to use 3-chloro-4-hydroxyphenylacetic acid as an electron acceptor. Physiological characterization led to the definition of three phenotypic classes of mutants that differed in their ability to use the additional terminal electron acceptors nitrate and fumarate. The activities of hydrogenase and formate dehydrogenase were determined, and the transposon insertion sites in selected mutants representing the different classes were analyzed on the sequence level following amplification by inverse PCR. The results of the molecular characterization as well as the pleiotropic phenotypes of most mutants indicate that genes coding for common elements shared by the different respiratory chains present in the versatile D. dehalogenans have been disrupted.