Johannes Gescher

Genes Coding for a New Pathway of Aerobic Benzoate Metabolism in Azoarcus evansii

A new pathway for aerobic benzoate oxidation has been postulated for Azoarcus evansii and for a Bacillus stearothermophilus-like strain. Benzoate is first transformed into benzoyl coenzyme A (benzoyl-CoA), which subsequently is oxidized to 3-hydroxyadipyl-CoA and then to 3-ketoadipyl-CoA; all intermediates are CoA thioesters. The genes coding for this benzoate-induced pathway were investigated in the beta-proteobacterium A. evansii. They were identified on the basis of N-terminal amino acid sequences of purified benzoate metabolic enzymes and of benzoate-induced proteins identified on two-dimensional gels. Fifteen genes probably coding for the benzoate pathway were found to be clustered on the chromosome. These genes code for the following functions: a putative ATP-dependent benzoate transport system, benzoate-CoA ligase, a putative benzoyl-CoA oxygenase, a putative isomerizing enzyme, a putative ring-opening enzyme, enzymes for beta-oxidation of CoA-activated intermediates, thioesterase, and lactone hydrolase, as well as completely unknown enzymes belonging to new protein families. An unusual putative regulator protein consists of a regulator protein and a shikimate kinase I-type domain. A deletion mutant with a deletion in one gene (boxA) was unable to grow with benzoate as the sole organic substrate, but it was able to grow with 3-hydroxybenzoate and adipate. The data support the proposed pathway, which postulates operation of a new type of ring-hydroxylating dioxygenase acting on benzoyl-CoA and nonoxygenolytic ring cleavage. A beta-oxidation-like metabolism of the ring cleavage product is thought to lead to 3-ketoadipyl-CoA, which finally is cleaved into succinyl-CoA and acetyl-CoA.

Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration

ABSTRACT An extension of the respiratory chain to the cell surface is necessary to reduce extracellular electron acceptors like ferric iron or manganese oxides. In the past few years, more and more compounds were revealed to be reduced at the surface of the outer membrane of Gram-negative bacteria, and the list does not seem to have an end so far. Shewanella as well as Geobacter strains are model organisms to discover the biochemistry that enables the dissimilatory reduction of extracellular electron acceptors. In both cases, c-type cytochromes are essential electron-transferring proteins. They make the journey of respiratory electrons from the cytoplasmic membrane through periplasm and over the outer membrane possible. Outer membrane cytochromes have the ability to catalyze the last step of the respiratory chains. Still, recent discoveries provided evidence that they are accompanied by further factors that allow or at least facilitate extracellular reduction. This review gives a condensed overview of our current knowledge of extracellular respiration, highlights recent discoveries, and discusses critically the influence of different strategies for terminal electron transfer reactions.

Periplasmic Electron Transfer via the c-Type Cytochromes MtrA and FccA of Shewanella oneidensis MR-1

ABSTRACT Dissimilatory microbial reduction of insoluble Fe(III) oxides is a geochemically and ecologically important process which involves the transfer of cellular, respiratory electrons from the cytoplasmic membrane to insoluble, extracellular, mineral-phase electron acceptors. In this paper evidence is provided for the function of the periplasmic fumarate reductase FccA and the decaheme c-type cytochrome MtrA in periplasmic electron transfer reactions in the gammaproteobacterium Shewanella oneidensis. Both proteins are abundant in the periplasm of ferric citrate-reducing S. oneidensis cells. In vitro fumarate reductase FccA and c-type cytochrome MtrA were reduced by the cytoplasmic membrane-bound protein CymA. Electron transfer between CymA and MtrA was 1.4-fold faster than the CymA-catalyzed reduction of FccA. Further experiments showing a bidirectional electron transfer between FccA and MtrA provided evidence for an electron transfer network in the periplasmic space of S. oneidensis. Hence, FccA could function in both the electron transport to fumarate and via MtrA to mineral-phase Fe(III). Growth experiments with a ΔfccA deletion mutant suggest a role of FccA as a transient electron storage protein.

Bacterial translation elongation factor EF-Tu interacts and colocalizes with actin-like MreB protein

We show that translation initiation factor EF-Tu plays a second important role in cell shape maintenance in the bacterium Bacillus subtilis. EF-Tu localizes in a helical pattern underneath the cell membrane and colocalizes with MreB, an actin-like cytoskeletal element setting up rod cell shape. The localization of MreB and of EF-Tu is interdependent, but in contrast to the dynamic MreB filaments, EF-Tu structures are more static and may serve as tracks for MreB filaments. In agreement with this idea, EF-Tu and MreB interact in vivo and in vitro. Lowering of the EF-Tu levels had a minor effect on translation but a strong effect on cell shape and on the localization of MreB, and blocking of the function of EF-Tu in translation did not interfere with the localization of MreB, showing that, directly or indirectly, EF-Tu affects the cytoskeletal MreB structure and thus serves two important functions in a bacterium.

Benzoate-Coenzyme A Ligase from Thauera aromatica: an Enzyme Acting in Anaerobic and Aerobic Pathways

In the denitrifying member of the beta-Proteobacteria Thauera aromatica, the anaerobic metabolism of aromatic acids such as benzoate or 2-aminobenzoate is initiated by the formation of the coenzyme A (CoA) thioester, benzoyl-CoA and 2-aminobenzoyl-CoA, respectively. Both aromatic substrates were transformed to the acyl-CoA intermediate by a single CoA ligase (AMP forming) that preferentially acted on benzoate. This benzoate-CoA ligase was purified and characterized as a 57-kDa monomeric protein. Based on V(max)/K(m), the specificity constant for 2-aminobenzoate was 15 times lower than that for benzoate; this may be the reason for the slower growth on 2-aminobenzoate. The benzoate-CoA ligase gene was cloned and sequenced and was found not to be part of the gene cluster encoding the general benzoyl-CoA pathway of anaerobic aromatic metabolism. Rather, it was located in a cluster of genes coding for a novel aerobic benzoate oxidation pathway. In line with this finding, the same CoA ligase was induced during aerobic growth with benzoate. A deletion mutant not only was unable to grow anaerobically on benzoate or 2-aminobenzoate, but also aerobic growth on benzoate was affected. This suggests that benzoate induces a single benzoate-CoA ligase. The product of benzoate activation, benzoyl-CoA, then acts as inducer of separate anaerobic or aerobic pathways of benzoyl-CoA, depending on whether oxygen is lacking or present.

Dissimilatory iron reduction in Escherichia coli: identification of CymA of Shewanella oneidensis and NapC of E. coli as ferric reductases

Over geological time scales, microbial reduction of chelated Fe(III) or Fe(III) minerals has profoundly affected todays composition of our bio‐ and geosphere. However, the electron transfer reactions that are specific and defining for dissimilatory iron(III)‐reducing (DIR) bacteria are not well understood. Using a synthetic biology approach involving the reconstruction of the putative electron transport chain of the DIR bacterium Shewanella oneidensis MR‐1 in Escherichia coli, we showed that expression of cymA was necessary and sufficient to convert E. coli into a DIR bacterium. In intact cells, the Fe(III)‐reducing activity was limited to Fe(III) NTA as electron acceptor. In vitro biochemical analysis indicated that CymA, which is a cytoplasmic membrane‐associated tetrahaem c‐type cytochrome, carries reductase activity towards Fe(III) NTA, Fe(III) citrate, as well as to AQDS, a humic acid analogue. The in vitro specific activities of Fe(III) citrate reductase and AQDS reductase of E. coli spheroplasts were 10× and 30× higher, respectively, relative to the specific rates observed in intact cells, suggesting that access of chelated and insoluble forms of Fe(III) and AQDS is restricted in whole cells. Interestingly, the E. coli CymA orthologue NapC also carried ferric reductase activity. Our data support the argument that the biochemical mechanism of Fe(III) reduction per se was not the key innovation leading to environmental relevant DIR bacteria. Rather, the evolution of an extension of the electron transfer pathway from the Fe(III) reductase CymA to the cell surface via a system of periplasmic and outer membrane cytochrome proteins enabled access to diffusion‐impaired electron acceptors.

A mineralogical, geochemical, and microbiogical assessment of the antimony- and arsenic-rich neutral mine drainage tailings near Pezinok, Slovakia

Abstract The mineralogical composition of mining wastes deposited in voluminous tailing impoundments around the world is the key factor that controls retention and release of pollutants. Here we report a detailed mineralogical, geochemical, and microbiological investigation of two tailing impoundments near the town of Pezinok, Slovakia. The primary objective of this study was the mineralogy that formed in the impoundment after the deposition of the tailings (so-called tertiary minerals). Tertiary minerals include oxyhydroxides of Fe, Sb, As, Ca and are present as grains and as rims on primary ore minerals. X-ray microdiffraction data show that the iron oxyhydroxides with abundant As are X-ray amorphous. The limiting (lowest) Fe/As (wt/wt%) ratio in this material is 1.5; beyond this ratio, the hydrous ferric oxide does not retain arsenic. The grains with less As and little to moderate amounts of Sb are goethite; the grains where Sb dominates over Fe are poorly crystalline tripuhyite (FeSbO4). Even the most heavily contaminated samples (up to 29 wt% As2O5) are populated with diverse communities of microorganisms including typical arsenic-resistant heterotrophic species as well as iron reducers and sulfur oxidizers. Several recovered clones cluster within phylogenetic groups that are solely based on environmental sequences and do not contain a single cultivated species, thus calling for more work on such extreme environments.

New enzymes involved in aerobic benzoate metabolism in Azoarcus evansii

A new principle of aerobic aromatic metabolism has been postulated, which is in contrast to the known pathways. In various bacteria the aromatic substrate benzoate is first converted to its coenzyme A (CoA) thioester, benzoyl‐CoA, which is subsequently attacked by an oxygenase, followed by a non‐oxygenolytic fission of the ring. We provide evidence for this hypothesis and show that benzoyl‐CoA conversion in the bacterium Azoarcus evansii requires NADPH, O2 and two protein components, BoxA and BoxB. BoxA is a homodimeric 46 kDa iron‐sulphur‐flavoprotein, which acts as reductase. In the absence of BoxB, BoxA catalyses the benzoyl‐CoA stimulated artificial transfer of electrons from NADPH to O2 via free FADH2 to produce H2O2. Physiologically, BoxA uses NADPH to reduce BoxB, a monomeric 55 kDa iron‐protein that acts as benzoyl‐CoA oxygenase. The product of benzoyl‐CoA oxidation was identified by NMR spectroscopy as its dihydrodiol derivative, 2,3‐dihydro‐2,3‐dihydroxybenzoyl‐CoA. This suggests that BoxBA act as a benzoyl‐CoA dioxygenase/reductase. Unexpectedly, benzoyl‐CoA transformation by BoxBA was greatly stimulated when another enoyl‐CoA hydratase/isomerase‐like protein, BoxC, was added that catalysed the further transformation of the dihydrodiol product formed from benzoyl‐CoA. The benzoyl‐CoA oxygenase system has very low similarity to known (di)oxygenase systems and is the first member of a new enzyme family.

Involvement and specificity of Shewanella oneidensis outer membrane cytochromes in the reduction of soluble and solid‐phase terminal electron acceptors

The formation of outer membrane (OM) cytochromes seems to be a key step in the evolution of dissimilatory iron-reducing bacteria. They are believed to be the endpoints of an extended respiratory chain to the surface of the cell that establishes the connection to insoluble electron acceptors such as iron or manganese oxides. The gammaproteobacterium Shewanella oneidensis MR-1 contains the genetic information for five putative OM cytochromes. In this study, the role and specificity of these proteins were investigated. All experiments were conducted using a markerless deletion mutant in all five OM cytochromes that was complemented via the expression of single, plasmid-encoded genes. MtrC and MtrF were shown to be potent reductases of chelated ferric iron, birnessite, and a carbon anode in a microbial fuel cell. OmcA-producing cells were unable to catalyze iron and electrode reduction, although the protein was correctly produced and oriented. However, OmcA production resulted in a higher birnessite reduction rate compared with the mutant. The presence of the decaheme cytochrome SO_2931 as well as the diheme cytochrome SO_1659 did not rescue the phenotype of the deletion mutant.

Proof of principle for an engineered microbial biosensor based on Shewanella oneidensis outer membrane protein complexes.

Shewanella oneidensis is known for its ability to respire on extracellular electron acceptors. The spectrum of these acceptors also includes anode surfaces. Based on this activity, a versatile S. oneidensis based biosensor strain was constructed in which electricity production can be modulated. Construction started with the identification of a usable rate-limiting step of electron transfer to an anode. Thereafter, the sensor strain was genetically engineered to produce a protein complex consisting of the three proteins MtrA, MtrB and MtrF. This complex is associated to the outer membrane and most probably enables membrane spanning electron transfer. MtrF is an outer membrane cytochrome that catalyzes electron transfer reactions on the cell surface. Under anoxic conditions, wild type cells do not express MtrF but rather MtrC as electron transferring outer membrane cytochrome. Still, our analysis revealed that MtrF compared to MtrC overexpression is less toxic to the cell which gives MtrF a superior position for biosensor based applications. Transcription of mtrA, mtrB and mtrF was linked up to an inducible promoter system, which positively reacts to rising l-arabinose concentrations. Anode reduction mediated by this strain was linearly dependent on the arabinose content of the medium. This linear dependency was detectable over a wide range of arabinose concentrations. The l-arabinose biosensor presented in this study proves the principle of an outer membrane complex based sensing method which could be easily modified to different specificities by a simple change of the regulatory elements.