Quantifying functional gene populations: comparing gene abundance and corresponding enzymatic activity using denitrification and nitrogen fixation in pulp and paper mill effluent treatment systems.

The relationship between the abundance of three functional genes and their corresponding biochemical reaction rates was investigated in several activated sludge and mill effluent microbial communities. Gene probes were prepared for two key denitrification genes (nirS and nirK) and for one nitrogen-fixation gene (nifH) and were validated using a variety of strains of known nir and nif genotype. ATP-based measures of viable cell numbers were used to provide total population sizes. In certain microbial communities (activated sludge enrichment cultures and multiple samples taken from the same mill primary clarifier), a strong correlation was observed between gene abundance and biochemical activity rates. However, when comparing several different nonenriched activated sludge bioreactors and separate primary clarifier microbial communities, the ratio of specific gene abundance to biochemical activity rates varied widely. These results suggest that in cases where a microbial community is not fully induced for a given biochemical activity or when very different communities are compared, quantitative gene probing can give a better measure of a community's potential to carry out the encoded function than can the relevant biochemical assay. However, the gene quantitation method employed here probably underestimated the true number of probed genes present in the microbial communities due to nirS and nifH genes in the communities having reduced DNA sequence similarity with the probes used.

Coliform bacteria and nitrogen fixation in pulp and paper mill effluent treatment systems.

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N(2) fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase (nifH) gene probing, and bacterial isolations. In situ N(2) fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH, of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH. In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N(2)-fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N(2) fixation (0.87 to 4.90 mg of N liter(-1) day(-1)) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.

Cloning and analysis of Pycnoporus cinnabarinus cellobiose dehydrogenase.

We have cloned and sequenced a gene encoding cellobiose dehydrogenase (CDH) from Pycnoporus cinnabarinus (Pci). PCR primers that may recognize a homologous cdh were designed using regions of complete conservation of amino acid sequence within the known sequences of Trametes versicolor (Tv) and Phanerochaete chrysosporium (Pc) CDH. Upstream primers hybridized to regions encoding the heme domain, whereas downstream primers recognized highly conserved regions within the flavin domain. Eight different primer pairs yielded three PCR products close in size to the control amplification, which used cloned Tv cdh as template. The PCR products that were close to the control size were cloned, and one of these, a 1.8-kb product, was completely sequenced. The PCR product was highly homologous to both Tv and Pch cdh, and contained eight putative introns. The cloned product was used to isolate a full-length clone encoding CDH from a Pci genomic library. Pci cdh encoded a protein with 83% identity with Tv CDH and 74% identity with Pch CDH. Northern blot analysis revealed that Pci cdh was transcribed as a single mRNA species and was expressed in the presence of cellulose as the carbon source.

Cloning and sequencing of a gene encoding cellobiose dehydrogenase from Trametes versicolor.

Cellobiose dehydrogenase (CDH) is an enzyme produced under lignocellulose-degrading conditions by Trametes versicolor strain 52J (Tv) and several other wood-degrading fungi, including Phanerochaete chrysosporium (Pc). In order to understand better the nature and properties of this enzyme, we isolated a genomic clone of Tv cdh using heterologous probes derived from the sequence of Pc cdh. DNA sequence analysis revealed that Tv cdh consists of 3091 bp of coding sequence interrupted by 14 introns. Southern blotting showed that the gene was present in a single copy in the strain of Tv analyzed. Tv cdh was shown by Northern blot analysis to be expressed as a single transcript under cellulolytic conditions. RT-PCR of poly(A)+ RNA isolated under cellulolytic conditions was used to generate a near full-length cDNA copy of the cdh mRNA. The deduced protein encoded by Tv cdh consists of 768 amino acids (aa), including a predicted 19 aa signal peptide. The protein had 73% identity to the corresponding protein from Pc, which is the only other CDH-encoding gene that has been cloned. Based upon its deduced primary structure and alignment to similar sequences, Tv CDH shares a general structural organization with Pc CDH and other hemoflavoenzymes. Amino acid residues H-109 and M-61 in the N-terminal heme domain are hypothesized to function in heme binding; the C-terminal flavin domain contained a consensus sequence for flavin binding between residues 217-222. Although the protein is known to bind to cellulose, no obvious homology to bacterial or fungal cellulose binding domains was observed. However, a strong homology was observed to a region of Pc CDH that is hypothesized to be involved in cellulose binding.

Purification and Characterization of Cellobiose Dehydrogenases from the White Rot Fungus Trametes versicolor.

The white rot fungus Trametes versicolor degrades lignocellulosic material at least in part by oxidizing the lignin via a number of secreted oxidative and peroxidative enzymes. An extracellular reductive enzyme, cellobiose dehydrogenase (CDH), oxidizes cellobiose and reduces insoluble Mn(IV)O(inf2), commonly found as dark deposits in decaying wood, to form Mn(III), a powerful lignin-oxidizing agent. CDH also reduces ortho-quinones and produces sugar acids which can promote manganese peroxidase and therefore ligninolytic activity. To better understand the role of CDH in lignin degradation, proteins exhibiting cellobiose-dependent quinone-reducing activity were isolated and purified from cultures of T. versicolor. Two distinct proteins were isolated; the proteins had apparent molecular weights of 97,000 and 81,000 and isoelectric points of 4.2 and 6.4, respectively. The larger CDH (CDH 4.2) contained both flavin and heme cofactors, whereas the smaller contained only a flavin (CDH 6.4). These CDH enzymes were rapidly reduced by cellobiose and lactose and somewhat more slowly by cellulose and certain cello-oligosaccharides. Both glycoproteins were able to reduce a very wide range of quinones and organic radical species but differed in their ability to reduce metal ion complexes. Temperature and pH optima for CDH 4.2 were affected by the reduced substrate. Although CDH 4.2 showed rather high substrate specificity among the ortho-quinones, it could also rapidly reduce a structurally very diverse collection of other species, from negatively charged triiodide ions to positively charged hexaquo ferric ions. CDH 6.4 showed a higher K(infm) and a lower V(infmax) and turnover number than did CDH 4.2 for all substrates tested. Furthermore, CDH 6.4 did not reduce the transition metals Fe(III), Cu(II), and Mn(III) at concentrations likely to be physiologically relevant, while CDH 4.2 was able to rapidly reduce even very low concentrations of these ions. The reduction of Fe(III) and Cu(II) by CDH 4.2 may be important in sustaining a Fenton's-type reaction, which produces hydroxyl radicals that can cleave both lignin and cellulose. Unlike the CDH proteins from Phanerochaete chrysosporium, CDH 4.2 and CDH 6.4 are unable to produce hydrogen peroxide.

Production and Characterization of Trametes versicolor Mutants Unable To Bleach Hardwood Kraft Pulp.

Protoplasts of the monokaryotic strain 52J of Trametes versicolor were treated with UV light and screened for the inability to produce a colored precipitate on guaiacol-containing agar plates. Mutants unable to oxidize guaiacol had absent or very low secretion of laccase and manganese peroxidase (MnP) proteins. All isolates unable to secrete MnP were also unable to bleach or delignify kraft pulp. One mutant strain, M49, which grew normally but did not oxidize guaiacol, was tested further with a number of other substrates whose degradation has been associated with delignification by white rot fungi. Compared with the parent, 52J, mutant M49, secreting no MnP and low laccase, could not brighten or delignify kraft pulp, produced less ethylene from 2-keto methiolbutyric acid, released much less (sup14)CO(inf2) from [(sup14)C]DHP (a synthetic lignin-like polymerizate), and produced much less methanol from pulp. This mutant also displayed decreased abilities to oxidize the dyes poly B-411, poly R-478, and phenol red compared with the wild-type strain and was also unable to decolorize kraft bleachery effluent or mineralize its organochlorine. Addition of purified MnP in conjunction with H(inf2)O(inf2), MnSO(inf4), and an Mn(III) chelator to M49 cultures partially restored methanol production, pulp delignification, and biobleaching in some cases.

Comparative study of iron acquisition by biotype 1 and biotype 2 strains of Actinobacillus pleuropneumoniae.

Four strains of the swine pathogen, Actinobacillus pleuropneumoniae, namely, the type strain (ATCC 27088; biotype 1), the 'reference' strain of biotype 2 (Bertschinger 2008/76), and two additional biotype 1 strains, strain BC181, which is less virulent than the type strain, and strain K17, which was isolated from a lamb, were investigated with respect to iron acquisition. All strains produced iron-repressible outer membrane proteins. However, only the type and biotype 2 strains could acquire iron from porcine transferrin and no organism could utilize human, bovine or ovine transferrin, or ovine or porcine lactoferrin; haemoglobin supported good growth of all strains except strain K17. In all cases, iron acquisition from transferrin and haemoglobin required direct contact between the organisms and the proteins indicating the existence of specific receptors. An affinity isolation technique, using biotinylated porcine transferrin plus streptavidin-agarose, allowed the isolation of the following polypeptides from total membranes of organisms grown under iron-restricted conditions: 99 kDa and 64 kDa from ATCC 27088; 93 kDa from Bertschinger 2008/76; 95 kDa (trace amounts) and 60 kDa from BC181; none from K17. These results indicate that the 93-99 kDa polypeptides are involved in the acquisition of iron from porcine transferrin and that the inability of strain K17 to use transferrin as an iron source is due, probably, to the lack of, or a defect in, an analogous component.

Creation of metal-complexing agents, reduction of manganese dioxide, and promotion of manganese peroxidase-mediated Mn(III) production by cellobiose:quinone oxidoreductase from Trametes versicolor.

Culture supernatants of the white-rot fungus Trametes versicolor were found to contain Mn(III)-complexing agents able to effectively promote manganese-dependent peroxidase (MnP)-mediated oxidation of phenol red. The high molecular weight fractions of these supernatants contained carbohydrate polymers that functioned as effective Mn(III)-complexing agents. Gluconic and glucuronic acids were also found to be effective Mn(III)-complexing ligands capable of supporting MnP-mediated phenol red oxidation, as was the cellobionic acid formed from cellobiose by cellobiose:quinone oxidoreductase (CBQase) (EC 1.1.5.1). The formation of cellulose-derived sugar acid manganese-complexing agents by CBQase increased in the presence of wood pulp and cellulolytic enzymes. CBQase, while oxidizing cellobiose, reduced insoluble Mn(IV)O2 to Mn(II) or Mn(III) by a reaction whose rate is determined by the nature of the manganese-complexing agents present. A model is proposed to explain how oxidation of carbohydrate and reduction of MnO2 and quinones by CBQase may complement oxidation by MnP, thus promoting lignin biodegradation.

Manganese Peroxidase, Produced by Trametes versicolor during Pulp Bleaching, Demethylates and Delignifies Kraft Pulp.

Previous work has shown that Trametes (Coriolus) versicolor bleaches kraft pulp brownstock with the concomitant release of methanol. In this work, the fungus is shown to produce both laccase and manganese peroxidase (MnP) but not lignin peroxidase during pulp bleaching. MnP production was enhanced by the presence of pulp and/or Mn(II) ions. The maximum level of secreted MnP was coincident with the maximum rate of fungal bleaching. Culture filtrates isolated from bleaching cultures produced Mn(II)- and hydrogen peroxide-dependent pulp demethylation and delignification. Laccase and MnP were separated by ion-exchange chromatography. Purified MnP alone produced most of the demethylation and delignification exhibited by the culture filtrates. On the basis of the methanol released and the total and phenolic methoxyl contents of the pulp, it appears that MnP shows a preference for the oxidation of phenolic lignin substructures. The extensive increase in brightness observed in the fungus-treated pulp was not found with MnP alone. Therefore, either the MnP effect must be optimized or other enzymes or compounds from the fungus are also required for brightening.

A new assay for lignin-type peroxidases employing the dye azure B.

The discovery in 1983 of fungal "ligninases" capable of catalyzing the peroxidation of nonphenolic aromatic lignin components has been seen as a major advance in understanding how certain basidiomycete fungi can completely degrade lignin. The ability of these lignin-type peroxidases to covert millimolar concentrations of veratryl alcohol to veratraldehyde, indicated by a change in the A310 of veratraldehyde, has become the standard assay for routine quantitation of LP activity. A new assay based on the oxidation of micromolar concentrations of the dye Azure B is presented. Although it is as simple and rapid as the veratryl alcohol assay, it appears to overcome some of the shortcomings of that assay. In particular, interference from UV- and short-wavelength visible-light-absorbing materials is greatly reduced and assay specificity is improved.

Lignin Peroxidase Activity Is Not Important in Biological Bleaching and Delignification of Unbleached Kraft Pulp by Trametes versicolor.

The discovery in 1983 of fungal lignin peroxidases able to catalyze the oxidation of nonphenolic aromatic lignin model compounds and release some CO(2) from lignin has been seen as a major advance in understanding how fungi degrade lignin. Recently, the fungus Trametes versicolor was shown to be capable of substantial decolorization and delignification of unbleached industrial kraft pulps over 2 to 5 days. The role, if any, of lignin peroxidase in this biobleaching was therefore examined. Several different assays indicated that T. versicolor can produce and secrete peroxidase proteins, but only under certain culture conditions. However, work employing a new lignin peroxidase inhibitor (metavanadate ions) and a new lignin peroxidase assay using the dye azure B indicated that secreted lignin peroxidases do not play a role in the T. versicolor pulp-bleaching system. Oxidative activity capable of degrading 2-keto-4-methiolbutyric acid (KMB) appeared unique to ligninolytic fungi and always accompanied pulp biobleaching.

Isolation and identification of a putative porcine transferrin receptor from Actinobacillus pleuropneumoniae biotype 1.

Each of two affinity isolation methods, the first based on biotinylated porcine transferrin plus streptavidin-agarose, and the second on Sepharose-coupled porcine transferrin, followed by SDS-PAGE, allowed the isolation and identification of two potential porcine-transferrin-binding polypeptides (approximately 64 kDa and 99 kDa) from total membranes of Actinobacillus pleuropneumoniae grown under iron-restricted conditions. Both polypeptides were iron-repressible and were identified as potential receptor candidates as they were not isolated when biotinylated human transferrin was used instead of biotinylated porcine transferrin. The 64 kDa polypeptide was the more easily removed from Sepharose-coupled porcine transferrin and only the 99 kDa polypeptide appeared to be an outer-membrane protein. While these results suggest that the 99 kDa polypeptide represents the porcine transferrin receptor of A. pleuropneumoniae, and that the 64 kDa polypeptide represents an associated protein serving an accessory role, other interpretations are also possible.

Responses of Haemophilus pleuropneumoniae to iron restriction: changes in the outer membrane protein profile and the removal of iron from porcine transferrin.

Outer membranes from Haemophilus pleuropneumoniae grown under iron-replete and iron-restricted conditions in vitro were analysed by means of SDS-PAGE and immunoblotting. Iron restriction resulted in the appearance of two or more novel polypeptides in the molecular size range of 96-102 kD and an increased amount of a 79 kD polypeptide. These polypeptides were recognized by porcine immune sera indicating their production by H. pleuropneumoniae during growth in vivo. Although soluble siderophore production could not be detected, growth of the organisms on an iron-restricted medium was enhanced by the presence of porcine transferrin but not by bovine or human transferrin. The results suggest that H. pleuropneumoniae possesses a specific transferrin receptor, perhaps in the form of an iron-regulated outer membrane protein.

Manganese complexes and the generation and scavenging of hydroxyl free radicals.

In a wide variety of biological systems non-enzyme complexes of the metals copper (Cu) and iron (Fe) have been shown to enhance oxygen radical damage by increasing the production of an oxidative species generally believed to be the hydroxyl free radical (.OH) via "Fenton" and possibly "Haber-Weiss" type reactions. However, the behavior of the chemically and biologically similar transition metal manganese (Mn) with .OH is unknown. Unlike Fe and Cu, inorganic complexes of Mn are known to exist in high concentrations in certain cells. Three different oxygen free radical generating systems and four .OH detection methods were used to investigate the activity of biologically relevant inorganic Mn complexes. These complexes were compared to compounds reported to scavenge and generate .OH. The direct and indirect effects of Mn on the .OH flux were compared by attempting to distinguish the effects of hydrogen peroxide (H2O2), superoxide (O2-), and .OH through the use of selective scavengers and generators. Mn-EDTA and biologically relevant Mn-pyrophosphates and polyphosphates, in contrast to Fe-EDTA, do not generate .OH in these systems. The results suggest that Mn in various forms does, indeed, inhibit oxy-radical damage mediated by .OH, but only if the .OH production is dependent on the presence of O2- or H2O2. Thus, with .OH, as with O2- and H2O2, Mn complexes appear to behave in a fundamentally different fashion from Cu and Fe.

Manganese poisoning and the attack of trivalent manganese upon catecholamines.

Human manganese poisoning or manganism results in damage to the substantia nigra of the brain stem, a drop in the level of the inhibitory neurotransmitter dopamine, and symptoms resembling those of Parkinson's disease. Manganic (Mn3+) manganese ions were shown to be readily produced by O-2 in vitro and spontaneously under conditions obtainable in the human brain. Mn3+ as its pyrophosphate complex was shown to rapidly and efficiently carry out four-electron oxidations of dopamine, its precursor dopa (3,4-dihydroxyphenylalanine), and its biosynthetic products epinephrine and norepinephrine. Mn3+-pyrophosphate was shown to specifically attack dihydroxybenzene derivatives, but only those with adjacent hydroxyl groups. Further, the addition of Mn2+-pyrophosphate to a system containing a flux of O2- and dopamine greatly accelerated the oxidation of dopamine. The oxidation of dopamine by Mn3+ neither produced nor required O2, and Mn3+ was far more efficient than Mn2+, Mn4+ (MnO2), O2-, or H2O2 in oxidizing the catecholamines. A higher oxidation state, Mn(OH)3, formed spontaneously in an aqueous Mn(OH)2 precipitate and slowly darkened, presumably being oxidized to MnO2. Like reagent MnO2, it weakly catalyzed dopamine oxidation. However, both MnO2 preparations showed dramatically increased abilities to oxidize dopamine in the presence of pyrophosphate due to enhancement of the spontaneous formation of the Mn3+ complex. These results strongly suggest that the pathology of manganese neurotoxicity is dependent on the ease with which simple Mn3+ complexes are formed under physiological conditions and the efficiency with which they destroy catecholamines.

Superoxide dismutase and oxygen toxicity defenses in the genus Neisseria.

Among aerotolerant cells, Neisseria gonorrhoeae is very unusual because despite its obligately aerobic lifestyle and frequent isolation from purulent exudates containing polymorphonuclear leukocytes vigorously evolving O2- and H2O2, it contains no superoxide dismutase (SOD). Strains (14) of N. gonorrhoeae were compared with each other and with strains of Neisseria meningitidis, Neisseria mucosa, and Neisseria subflava under identical growth conditions for their contents of the oxy-protective enzymes catalase, peroxidase, and SOD, as well as respiratory chain proteins and activity. The absence of SOD from N. gonorrhoeae strains was demonstrated under a variety of oxygen-stress conditions. The neisserial species showed very different SOD, catalase, and peroxidase profiles. These profiles correlated well with the tolerance of the species to various intra- and extracellular oxygen insults. The high tolerance of N. gonorrhoeae for extracellular O2- and H2O2 appeared to be due to very high constitutive levels of peroxidase and catalase activity combined with a cell envelope impervious to O2-. Nevertheless, N. gonorrhoeae 19424 was much more sensitive to an intracellular flux of O2- than were the other (SOD-containing) neisserial species. The responses of N. gonorrhoeae and N. meningitidis respiratory and oxy-protective enzymes to growth under high and low oxygen tensions were followed, and a novel response, the apparent repression of the respiratory chain intermediates, respiration, and SOD, peroxidase, and catalase activity, was observed. The gonococcal catalase was partially purified and characterized. The results suggest that the very active terminal oxidase, low pO2 natural habitat, O2-stable catalase, and possibly the high glutathione content of the organism explain its aerobic survival in the absence of SOD.

Sulphur acquisition by Neisseria meningitidis.

Group B Neisseria meningitidis (SD1C) was grown on defined medium supplemented with each of a variety of sulphur compounds as the sole source of sulphur. The organism grew on sulphate, sulphite, bisulphite, thiosulphate, dithionite, hydrosulphide, thiocyanate, L-cysteine, L-cystine, reduced glutathione, methionine, mercaptosuccinate, and lanthionine, but not on dithionate unless previously sulphur starved. Good growth was seen on concentrations of sulphate or thiosulphate as low as 10 microM. When pregrown on and subsequently starved for sulphate, the meningococcus showed enhanced transport capacity for this ion. Optimal conditions for assessing sulphur transport by active sulphur-limited cells were determined. The maximal sulphate uptake velocity was 9.3 nmol sulphate X mg protein-1 X min-1, and the apparent Km was 1.4 microM, far below human nasopharyngeal or serum sulphate levels.

Manganese acquisition by Lactobacillus plantarum.

Lactobacillus plantarum has an unusually high Mn(II) requirement for growth and accumulated over 30 mM intracellular Mn(II). The acquisition of Mn(II) by L. plantarum occurred via a specific active transport system powered by the transmembrane proton gradient. The Mn(II) uptake system has a Km of 0.2 microM and a Vmax of 24 nmol mg-1 of protein min-1. Above a medium Mn(II) concentration of 200 microM, the intracellular Mn(II) level was independent of the medium Mn(II) and unresponsive to oxygen stresses but was reduced by phosphate limitation. At a pH of 5.5, citrate, isocitrate, and cis-aconitate effectively promoted MN(II) uptake, although measurable levels of 1,5-[14C]citrate were not accumulated. When cells were presented with equimolar Mn(II) and Cd(II), Cd(II) was preferentially taken up by the Mn(II) transport system. Both Mn(II) and Cd(II) uptake were greatly increased by Mn(II) starvation. Mn(II) uptake by Mn(II)-starved cells was subject to a negative feedback regulatory mechanism functioning less than 1 min after exposure of the cells to Mn(II) and independent of protein synthesis. When presented with a relatively large amount of exogenous Mn(II), Mn(II)-starved cells exhibited a measurable efflux of their internal Mn(II), but the rate was only a small fraction of the maximal Mn(II) uptake rate.