David J. Bergmann

Structure and Sequence Conservation of hao Cluster Genes of Autotrophic Ammonia-Oxidizing Bacteria: Evidence for Their Evolutionary History

ABSTRACT Comparison of the organization and sequence of the hao (hydroxylamine oxidoreductase) gene clusters from the gammaproteobacterial autotrophic ammonia-oxidizing bacterium (aAOB) Nitrosococcus oceani and the betaproteobacterial aAOB Nitrosospira multiformis and Nitrosomonas europaea revealed a highly conserved gene cluster encoding the following proteins: hao, hydroxylamine oxidoreductase; orf2, a putative protein; cycA, cytochrome c554; and cycB, cytochrome cm552. The deduced protein sequences of HAO, c554, and cm552 were highly similar in all aAOB despite their differences in species evolution and codon usage. Phylogenetic inference revealed a broad family of multi-c-heme proteins, including HAO, the pentaheme nitrite reductase, and tetrathionate reductase. The c-hemes of this group also have a nearly identical geometry of heme orientation, which has remained conserved during divergent evolution of function. High sequence similarity is also seen within a protein family, including cytochromes cm552, NrfH/B, and NapC/NirT. It is proposed that the hydroxylamine oxidation pathway evolved from a nitrite reduction pathway involved in anaerobic respiration (denitrification) during the radiation of the Proteobacteria. Conservation of the hydroxylamine oxidation module was maintained by functional pressure, and the module expanded into two separate narrow taxa after a lateral gene transfer event between gamma- and betaproteobacterial ancestors of extant aAOB. HAO-encoding genes were also found in six non-aAOB, either singly or tandemly arranged with an orf2 gene, whereas a c554 gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c554 gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB.

Membrane-Associated Quinoprotein Formaldehyde Dehydrogenase from Methylococcus capsulatus Bath

A membrane-associated, dye-linked formaldehyde dehydrogenase (DL-FalDH) was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 micromol of Cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. Soluble NAD(P)(+)-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane monooxygenase (Tate and Dalton, Microbiology 145:159-167, 1999; Vorholt et al., J. Bacteriol. 180:5351-5356, 1998). The membrane-associated enzyme is a homotetramer with a subunit molecular mass of 49,500 Da. UV-visible absorption, electron paramagnetic resonance, and electrospray mass spectrometry suggest the redox cofactor of the DL-FalDH is pyrroloquinoline quinone (PQQ), with a PQQ-to-subunit stochiometry of approximately 1:1. The enzyme was specific for formaldehyde, oxidizing formaldehyde to formate, and utilized the cytochrome b(559/569) complex as the physiological electron acceptor.

Cytochromes P460 and c′-beta; A new family of high-spin cytochromes c

Cytochromes‐P460 of Nitrosomonas europaea and Methylococcus capsulatus (Bath), and the cytochrome c′ of M. capsulatus, believed to be involved in binding or transformation of N‐oxides, are shown to represent an evolutionarily related new family of monoheme, ∼17 kDa, cytochromes c found in the genomes of diverse Proteobacteria. All members of this family have a predicted secondary structure predominantly of beta‐sheets in contrast to the predominantly alpha‐helical cytochromes c′ found in photoheterotrophic and denitrifying Proteobacteria.

Bacterial Numbers from Landlocked Fall Chinook Salmon Eyed Eggs Subjected to Various Formalin Treatments as Determined by Scanning Electron Microscopy and Bacteriological Culture Methods

Abstract This study compared two methods of enumerating bacteria adhered to the external membrane of eggs of landlocked fall Chinook salmon Oncorhynchus tshawytscha that were subjected to different formalin treatment regimes from egg eye-up to fry hatch. Bacterial numbers were recorded by either directly counting bacteria via a scanning electron microscope (SEM) or via established bacterial culture methods that provided the number of colony-forming units. Treatment regimes consisted of a daily 15-min exposure to formalin at either 500 mg/L or 1,667 mg/ L or no formalin initially followed by exposure to a 1,667-mg/L dose starting 7 d after eye-up. A control group did not receive any formalin treatments throughout the experiment. No significant correlation was observed between the number of colony-forming units (CFUs) per square millimeter of egg membrane determined by plate culture and the number of bacteria per square millimeter counted with the SEM. Bacterial numbers determined by the SEM were 40–120 tim...

The primary structure of cytochrome P460 of Nitrosomonas europaea: presence of a c-heme binding motif.

Cytochrome P460 and hydroxyamine oxidoreductase of Nitrosomonas europaea both catalyze the oxidation of hydroxylamine and contain a 460 nm‐absorbing chromophore. The gene (cyp) encoding cytochrome P460 was cloned and sequenced. The predicted amino acid sequence contains a single c‐heme binding motif (CXXCH) near the carboxy‐terminus. Cytochrome P460 shows little sequence homology to other c‐cytochromes including hydroxyamine oxidoreductase. The presence of a signal peptide and a possible c‐heme binding site suggest that the cytochrome P460 of N. europaea is periplasmic.

Membrane Tetraheme Cytochrome cm552 of the Ammonia-Oxidizing Nitrosomonas europaea: A Ubiquinone Reductase†

Cytochrome c(m552) (cyt c(m552)) from the ammonia-oxidizing Nitrosomonas europaea is encoded by the cycB gene, which is preceded in a gene cluster by three genes encoding proteins involved in the oxidation of hydroxylamine: hao, hydroxylamine oxidoreductase; orf2, a putative membrane protein; cycA, cyt c(554). By amino acid sequence alignment of the core tetraheme domain, cyt c(m552) belongs to the NapC/TorC family of tetra- or pentaheme cytochrome c species involved in electron transport from membrane quinols to a variety of periplasmic electron shuttles leading to terminal reductases. However, cyt c(m552) is thought to reduce quinone with electrons originating from HAO. In this work, the tetrahemic 27 kDa cyt c(m552) from N. europaea was purified after extraction from membranes using Triton X-100 with subsequent exchange into n-dodecyl beta-d-maltoside. The cytochrome had a propensity to form strong SDS-resistant dimers likely mediated by a conserved GXXXG motif present in the putative transmembrane segment. Optical spectra of the ferric protein contained a broad ligand-metal charge transfer band at approximately 625 nm indicative of a high-spin heme. Mossbauer spectroscopy of the reduced (57)Fe-enriched protein revealed the presence of high-spin and low-spin hemes in a 1:3 ratio. Multimode EPR spectroscopy of the native state showed signals from an electronically interacting high-spin/low-spin pair of hemes. Upon partial reduction, a typical high-spin heme EPR signal was observed. No EPR signals were observed from the other two low-spin hemes, indicating an electronic interaction between these hemes as well. UV-vis absorption data indicate that CO (ferrous enzyme) or CN(-) (ferric or ferrous enzyme) bound to more than one and possibly all hemes. Other anionic ligands did not bind. The four ferrous hemes of the cytochrome were rapidly oxidized in the presence of oxygen. Comparative modeling, based on the crystal structure and conserved residues of the homologous NrfH protein from Desulfovibrio of cyt c(m552), predicted some structural elements, including a Met-ligated high-spin heme in a quinone-binding pocket, and likely axial ligands to all four hemes.

Effect of Flavobacterium columnare inoculation, antibiotic treatments and resident bacteria on rainbow trout Oncorhynchus mykiss eyed egg survival and external membrane structure.

This study was conducted to evaluate the potential pathogenicity of the bacterium Flavobacterium columnare on rainbow trout Oncorhynchus mykiss eyed eggs. Survival to hatching was unaffected by the inclusion in the incubation water of either 300 colony-forming units (CFU) ml(-1) or 3000 CFU ml(-1) of F. columnare at either 10 or 12 degrees C in either McConaughy or Shasta strain eyed eggs. Bacterial numbers, obtained via scanning electron microscopy or culture, and external membrane morphology were also not significantly different among eggs receiving different concentrations of F. columnare. Initial F. columnare burdens were significantly and positively correlated to the presence of biofilm on the egg external membrane, and biofilm was in turn significantly correlated with increased membrane degradation. The use of either streptomycin or tetracyclin antibiotics significantly reduced bacterial numbers on McConaughy strain eggs, and more eggs survived to hatch in those dishes treated with antibiotics.

Grass rhizosheaths: associated bacterial communities and potential for nitrogen fixation.

ABSTRACT. Rhizosheaths are structures composed of mucilage secreted from plants and adherent soil particles that form a cylinder around the root. Using scanning electron microscopy (SEM), we observed bacteria associated with rhizosheaths of the grasses Achnatherum hymenoides, Calamovilfa longifolia, Hesperostipa comata, and Pascopyrum smithii from a sand dune area in Harding County in northwestern South Dakota. The greatest numbers of bacteria, observed with SEM (529 mm-2), and the greatest number of culturable bacteria (9.9 × 107 CFU · g-1 or 5178 CFU · mm-2) were on rhizosheaths of C. longifolia. Rhizosheaths of all the grasses examined contained a higher density of bacteria than the surrounding soil. Nitrogen fixation, as assayed by reduction of acetylene to ethylene, was present in some rhizosheaths. Bacterial nifH gene sequences amplified from bacteria associated with rhizosheaths were most similar to those from Alcaligenes latus and Mesorhizobium loti.

Mechanism of N-Oxidation and Electron Transfer in the Ammonia Oxidizing Autotrophs

In Nitrosomonas electrons flow from hydroxylamine oxidoreductase to ammonia monoxygenase, to NAD or to cytochrome oxidase or a nitrite-reducing system. The pathway includes the extrinsic membrane tetraheme cytochrome c-554 and, possibly, a membrane-anchored tetraheme c-cytochrome (CycB). Cyc554 and cycB share an operon in the HAO gene cluster. CycB has high homology with nirT, napC and torC suggesting a role in anaerobic metabolism. A small fraction of active HAO is found in the membrane. In the AMO operon, the gene-derived amino acid sequence suggests, for subunits-A and -B, 6 or 2 membrane-spanning helices, respectively. AmoA could provide a proton wire or pore.

A Survey of Bacteria in the Ovarian Fluid of Landlocked Fall Chinook Salmon and their Relationship with Egg Survival

Abstract A survey of the bacteria in the ovarian fluid of landlocked fall Chinook salmon Oncorhynchus tshawytscha from Lake Oahe, South Dakota, was conducted to investigate possible effects on egg mortality. Bacteria were isolated from the ovarian fluid of 15 out of 19 spawning females. A number of bacteria, including known and potentially pathogenic species, were identified: Acinetobacter spp., Aeromonas hydrophila, Aeromonas veronii, Arthrobacter spp., Pseudomonas spp. (including P. putida), Moraxella spp., and Microbacterium spp. With the exception of Aeromonas hydrophila, none of these bacteria had previously been isolated from Chinook salmon ovarian fluid. There was no observable association between the aeromonads or pseudomonads and egg survival. However, there was a weak but significant positive correlation between bacterial density (colony-forming units/mL of ovarian fluid) and egg survival; the significance of this correlation is uncertain.