Habitat preferences of ground beetle (Coleoptera: Carabidae) species in the northern Black Hills of South Dakota.

Ground beetles (Coleoptera: Carabidae) are a major component of terrestrial invertebrate communities and have been used as bioindicators of habitat change and disturbance. The Black Hills of South Dakota is a small area with a high biodiversity, but the ground beetles of this region are little studied. The habitat preferences of ground beetles in the Black Hills are unknown, and baseline data must be collected if these beetles are to be used in the future as bioindicators. Ground beetles (Coleoptera: Carabidae) were collected from pitfall traps at two sites in each of five kinds of habitats (grassland, bur oak-ironwood forests, ponderosa pine-common juniper forests, aspen-pine forests, and a spruce forest) from which habitat structure characteristics and plant abundance data also were collected. In total, 27 species of ground beetles were identified. Although some species, such as Dicaelus sculptilis Say were found in most habitats, other species showed distinct habitat preferences: Poecilus lucublandus (Say) preferred oak forests, Pasimachus elongatus LeConte preferred grasslands, and Calathus ingratus Dejean preferred high-elevation aspen-pine forests. Pterostichus adstrictus Escholtz was found only in woodlands, and Carabus taedatus Say strictly in higher elevation (over 1,500 m) aspen or coniferous woods, and may represent relict populations of boreal species. Elevation, exposure to sunlight, and cover of woody plants strongly influence the structure of carabid communities in the Black Hills.

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, approximately 17kDa, 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.

Structure and sequence conservation of hao cluster genes of autotrophic ammonia-oxidizing bacteria: evidence for their evolutionary history.

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 c(554); and cycB, cytochrome c(m)(552). The deduced protein sequences of HAO, c(554), and c(m)(552) 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 c(m)(552), 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 c(554) gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c(554) gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB.

Insight into covalent flavinylation and catalysis from redox, spectral, and kinetic analyses of the R474K mutant of the flavoprotein subunit of p-cresol methylhydroxylase.

Each flavoprotein subunit (PchF) of p-cresol methylhydroxylase (PCMH) has flavin adenine dinucleotide (FAD) covalently tethered to Tyr384. The PCMH structure suggests that Arg474 in PchF is required for self-catalytic covalent flavinylation and for substrate oxidation. The replacement of Arg474 with Lys was carried out to probe the subtleties of the role of Arg474 in these processes. In nearly all of the aspects examined, the mutant protein showed compromised properties relative to the wild-type protein, including the tenacity of noncovalent FAD binding to the apo-protein, the rate of covalent flavinylation, the affinity of the covalent flavoprotein for PchC (the cytochrome subunit), the k(cat) for substrate oxidation, and the affinity for substrate analogues in the formation of FAD-charge-transfer complexes (CT complexes). Nevertheless, because the mutant retains these attributes, the comparison allows for an examination of the role of this residue in the various properties of the enzyme. A correlation is proposed to exist between nu(m), the frequency for the absorbance maximum of the CT complex with a substrate analogue, and k(cat), the steady-state rate constant for oxidation of p-cresol by various forms of PCMH and PchF; both nu(m) and k(cat) can be expressed as functions of the ionization potential of the donor (I(D)) and the electron affinity of the acceptor (E(A)). This correlation is a better predictor of the rate constant for substrate oxidation than is the magnitude of the redox potential, E(m,7), of the bound FAD, which was determined for the various mutant enzyme species and compared with those of the wild type.

Cytochrome P460 of Nitrosomonas europaea. Formation of the heme-lysine cross-link in a heterologous host and mutagenic conversion to a non-cross-linked cytochrome c'.

The heme of cytochrome P460 of Nitrosomonas europaea, which is covalently crosslinked to two cysteines of the polypeptide as with all c-type cytochromes, has an additional novel covalent crosslink to lysine 70 of the polypeptide [Arciero, D.M. & Hooper, A.B. (1997) FEBS Lett.410, 457-460]. The protein can catalyze the oxidation of hydroxylamine. The gene for this protein, cyp, was expressed in Pseudomonas aeruginosa strain PAO lacI, resulting in formation of a holo-cytochrome P460 which closely resembled native cytochrome P460 purified from N. europaea in its UV-visible spectroscopic, ligand binding and catalytic properties. Mutant versions of cytochrome P460 of N. europaea in which Lys70 70 was replaced by Arg, Ala, or Tyr, retained ligand-binding ability but lost catalytic ability and differed in optical spectra which, instead, closely resembled those of cytochromes c'. Tryptic fragments containing the c-heme joined only by two thioether linkages were observed by MALDI-TOF for the mutant cytochromes P460 K70R and K70A but not in wild-type cytochrome P460, consistent with the structural modification of the c-heme only in the wild-type cytochrome. The present observations support the hypothesized evolutionary relationship between cytochromes P460 and cytochromes c' in N. europaea and M. capsulatus[Bergmann, D.J., Zahn, J.A., & DiSpirito, A.A. (2000) Arch. Microbiol. 173, 29-34], confirm the importance of a heme-crosslink to the spectroscopic properties and catalysis and suggest that the crosslink might form auto-catalytically.