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Unlike wastewater, pulp and paper mill effluents are generally severely deficient in bioavailable nitrogen and phosphorus. The influence of nitrogen and phosphorus limitations on steady-state or typical pulp and paper mill activated sludge floc properties and performance was studied using a bioreactor-fed synthetic raw mill effluent and seeded with mill activated sludge. Limitation of either nitrogen or phosphorus decreased growth, five-day biochemical oxygen demand, and suspended solids removal. Nitrogen limitation greatly enhanced activated sludge floc poly-β-hydroxybutyrate (PHB), but not carbohydrate or extracellular polymeric substances (EPS). In contrast, phosphorus limitation increased total floc carbohydrate and EPS, but not PHB. The flocs showed little ability to store either nitrogen or phosphorus. Nitrogen limitation, but not phosphorus limitation, produced much more negative net floc surface charge, increasing fines, while phosphorus limitation, but not nitrogen limitation, increased the floc bound water content and surface hydrophobicity and decreased fines. [ABSTRACT FROM AUTHOR]

Stachybotrys elegans is a mycoparasite of the soilborne plant pathogenic fungus Rhizoctonia solani. The mycoparasitic activity of S. elegans is correlated with the production of cell wall degrading enzymes such as chitinases. This report details the cloning by RACE-PCR and characterization of a full-length cDNA clone, sechi44, that appears to encode an extracellular endochitinase. An analysis of the sechi44 sequence indicates that this gene contains a 1269-bp ORF and encodes a 423-aa polypeptide. The SECHI44 protein has a calculated molecular weight of 44.1 kDa and pI of 5.53. Since the SECHI44 protein also appears to encode a signal peptide, an extracellular location for the corresponding protein is predicted. Comparison of SECHI44 sequence with known sequences of fungal endochitinases revealed that SECHI44 is grouped with endochitinases from other mycoparasites. Real-time quantitative RT-PCR analysis showed an elevated level of expression of sechi44 (21-fold) in chitin-rich (induced) as compared to no-carbon (non-induced) culture conditions. In dual culture, the temporal expression of sechi44 increased after 2 days of contact with R. solani, reaching a 10-fold increase after 9 days, followed by a decrease to basic expression level at 12 days. Interestingly, inhibition of sechi44 expression was observed when S. elegans hyphae were in close proximity with R. solani hyphae. [Copyright &y& Elsevier]

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.Key words: denitrification, nitrogen fixation, quantitative hybridization, activated sludge, primary clarifier.Nous avons examiné les liens existant entre l'abondance de trois gènes fonctionnels et la vitesse des réactions biochimiques correspondantes dans plusieurs communautés microbiennes de boues activées et d'effluents de papetières. Des sondes génétiques furent conçues pour des gènes clés de la dénitrification (nirS et nirK) et pour un gène de la fixation de l'azote (nifH), et furent validées à l'aide d'une panoplie de souches aux génotypes nir et nif connus. Des mesures de nombres de cellules viables se basant sur l'ATP ont été utilisées afin de procurer une estimation des tailles totales des populations. Dans certaines communautés microbiennes (cultures d'enrichissement de boues activées et échantillons multiples recueillis à partir d'un même clarificateur primaire de papetière), une forte corrélation a été observée entre l'abondance des gènes et les taux d'activités métaboliques. Toutefois, le rapport de l'abondance génétique spécifique sur les taux d'activité métabolique ont varié largement lorsque nous avons comparé divers bioréacteurs de boues non enrichies, ainsi que des communautés microbiennes de clarificateurs primaires de papetière distincts. Ces résultats nous indiquent que dans les cas où une activité biochimique n'est pas complètement induite chez une communauté bactérienne ou lorsque des communautés très différentes sont comparées, le sondage génétique quantitatif est d'avantage apte à fournir une mesure du potentiel d'une communauté à accomplir la fonction codée, que l'analyse biochimique pertinente. Cependant, la méthode de quantification des gènes employée ici a probablement sous-estimé le nombre véritable de gènes sondés présents dans la communauté microbienne, puisque les gènes nirS et nifH dans les communautés microbiennes avaient une similitude de séquence inférieure par rapport aux sondes utilisées.Mots clés : dénitrification, fixation de l'azote, hybridation quantitative, boues activées, clarificateur primaire.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]

Identifies two Sinorhizobium meliloti chromosomal loci affecting the poly-3-hydroxybutyrate degradation pathway. Isolation of complementing clones; Genetic and sequence analysis of aau-7 complementing region; Identification of aau-1; Biochemical characterization of the mutants.

SummaryMalate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate ; malate using either NADH or NADPH as a reductant. In plants, the enzyme is important in providing malate for C4 metabolism, pH balance, stomatal and pulvinal movement, respiration, β‐oxidation of fatty acids, and legume root nodule functioning. Due to its diverse roles the enzyme occurs as numerous isozymes in various organelles. While antibodies have been produced and cDNAs characterized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and characterization of cDNAs encoding five different forms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule‐enhanced MDH (neMDH). Phylogenetic analyses show that neMDH is related to mitochondrial and glyoxysomal MDHs, but diverge from these forms early in land plant evolution. Four of the five forms could effectively complement an E. coli Mdh– mutant. RNA and protein blots show that neMDH is most highly expressed in effective root nodules. Immunoprecipitation experiments show that antibodies produced to cMDH and neMDH are immunologically distinct and that the neMDH form comprises the major form of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that can account for the extraordinarily high synthesis of malate in nodules. [ABSTRACT FROM AUTHOR]

Presents information on a study which describes the isolation of the Rhizobium meliloti NADP+ malic enzyme gene (tme) and the genetic, biochemical, and symbiotic characterizations of tme mutants. Symbiotic and growth phenotypes of tme mutants; Regulation of tme gene expression; Insertion mutagenesis of tme gene; What the presence of two malic enzyme in many bacteria suggests.

Examines the phosphoenolpyruvate carboxykinase A (pckA) gene of Rhizobium meliloti. Mapping of gene region; DNA sequence; Expression level; Carbon sources.

Discusses the role of increased pyruvate orthophosphate dikinase (PPDK) activity on the development of an alternative gluconeogenic pathway in Rhizobium meliloti. Importance of the formation of phosphoenolpyruvate; Isolation and mapping of suppressor mutations; Comparison of PPDK activity between the strains with suppressor mutations and the wild-type.

Characterizes the Rhizobium meliloti NAD+-dependent malic enzyme gene product (DME) and NADP+-dependent malic enzyme (TME). Expression of DME gene and repression of TME in free living cells and N2-fixing bacteriods; Gene mapping; Inhibition of acetylcholine on DME activity.

Many plant species, particularly legumes, protect themselves with saponins. Previously, a correlation was observed between levels of oleanolic acid-derived saponins, such as hederagenin-derived compounds, in the legume Medicago truncatula and caterpillar deterrence. Using concentrations that reflect the foliar levels of hederagenin-type saponins, the sapogenin hederagenin was not toxic to 4th instar caterpillars of the cabbage looper Trichoplusia ni nor did it act as a feeding deterrent. Female caterpillars consumed more diet than males, presumably to obtain the additional nutrients required for oogenesis, and are, thus, exposed to higher hederagenin levels. When fed the hederagenin diet, male caterpillars expressed genes encoding trypsin-like proteins (LOC113500509, LOC113501951, LOC113501953, LOC113501966, LOC113501965, LOC113499659, LOC113501950, LOC113501948, LOC113501957, LOC113501962, LOC113497819, LOC113501946, LOC113503910) as well as stress-responsive (LOC113503484, LOC113505107) proteins and cytochrome P450 6B2-like (LOC113493761) at higher levels than females. In comparison, female caterpillars expressed higher levels of cytochrome P450 6B7-like (LOC113492289). Bioinformatic tools predict that cytochrome P450s could catalyze the oxygenation of hederagenin which would increase the hydrophilicity of the compound. Expression of a Major Facilitator Subfamily (MFS) transporter (LOC113492899) showed a hederagenin dose-dependent increase in gene expression suggesting that this transporter may be involved in sapogenin efflux. These sex-related differences in feeding and detoxification should be taken into consideration in insecticide evaluations to minimize pesticide resistance. [ABSTRACT FROM AUTHOR]

Improvement in sustainable production of switchgrass (SG,Panicum virgatumL), as a purpose-grown biomass feedstock crop, could be realized through investigation of plant–microbe interactions associated with plant growth promoting rhizobacteria (PGPR), capable of biological nitrogen fixation (BNF). The objective of this study is to increase establishment year production of SG biofuels by inoculation with a mixed PGPR inoculum. We isolated pure strains of N2-fixing, and other PGPR, from SG rhizomes. The bacteria were identified asPaenibacillus polymyxa, an N2-fixing bacterium, and other PGPR capable of solubilizing phosphate and/or producing auxins. Field trials utilizing these strains in a mixed PGPR inoculum showed that inoculated plants contained more N in tillers during anthesis but not at senescence, suggesting that more N could be cycled to belowground roots and rhizomes for winter storage. The amount of N removal in biomass and recovery of fertilizer N were also greater for inoculated than uninoculated plants. PGPR inoculation also resulted in positive N balances, suggesting improved access to N from non-fertilizer N sources, possibly through BNF and improved soil N uptake. Overall, inoculation of SG with PGPR enhanced N acquisition and could be an effective strategy to increase the establishment year production of this crop. [ABSTRACT FROM PUBLISHER]

Abstract: Nitrogen-fixing bacteria (NFB) and other plant growth promoting rhizobacteria (PGPR) have been used to increase growth and yield of corn and sugarcane, two graminaceous crops utilized as bioenergy feedstocks. Switchgrass (SG, Panicum virgatum L.), a native perennial grass of North America, is also a desirable bioenergy crop because it can be grown on marginal lands and thus does not necessarily displace food production. Yet no investigations have been conducted on the use of NFB and/or PGPR as inoculants to increase SG growth and productivity. Studies reporting high biomass yields from SG with minimal to no fertilizer nitrogen (N) input prompted us to hypothesize that SG is obtaining part of its N from an alternative source, perhaps through biological nitrogen fixation from root/rhizome associated PGPR. We isolated bacteria from SG rhizomes that had not received fertilizer N input for over ten years and which increased plant growth in the absence of the fertilizer N under growth chamber conditions. The bacteria were identified as a strain of Paenibacillus polymyxa, a N2-fixing bacterium, and several other PGPR capable of solubilizing phosphate and/or producing auxins. Our objective was to test this PGPR inoculum under field conditions with low-N inputs. Here, we present the findings regarding SG productivity in the establishment year. Switchgrass seeds inoculated with the PGPR culture had greater tiller density, and taller tillers resulting in 40% higher yield than uninoculated plants. PGPR have the potential to be effectively utilized as inoculants to enhance SG yields in a low-N input production system. [Copyright &y& Elsevier]

When nano-sized titanium dioxide (nano-TiO2) absorbs ultra-violet (UV-A) radiation, it produces reactive oxygen species that can be toxic to bacteria. We used the agronomically beneficial nitrogen-fixing bacterium Sinorhizobium meliloti strain 1021 as a model microorganism to detect nano-TiO2 toxicity. Sinorhizobium meliloti was exposed to aqueous dispersions of micrometer-sized TiO2 (micron-TiO2, 44 μm) or nanometer-sized TiO2 (nano-TiO2, 21 nm) at nominal concentrations of 0, 100, 300, 600, 900, and 1800 mg TiO2/L. There were fewer viable S. meliloti cells after exposure to nano-TiO2 under dark and UV-A light conditions. Nano-TiO2 was more toxic to S. meliloti with UV-A irradiation (100% mortality at 100 mg TiO2/L) than under dark conditions (100% mortality at 900 mg TiO2/L). Micron-TiO2 concentrations less than 300 mg TiO2/L had no effect on S. meliloti viability under dark or UV-A light conditions. Exposure to 600 mg/L or more of micron-TiO2 under UV-A light could also photo-kill S. meliloti cells (100% mortality). Further studies are needed to ascertain whether nano-TiO2 interferes with the growth of N2-fixing microorganisms in realistic agricultural environments. [ABSTRACT FROM AUTHOR]

Examines the nitrogen fixation in pulp and paper mill effluent treatment systems in Eastern Canada. Components of pulp mill-primary qualifier microbial communities; Use of acetylene reduction assays, quantitative nitrogenase, gene probing and bacterial isolation; Prevalence of klebsiellas in pulp and paper mill water system.

We previously reported the construction of metagenomic libraries in the IncP cosmid vector pRK7813, enabling heterologous expression of these broad-host-range libraries in multiple bacterial hosts. Expressing these libraries in Sinorhizobium meliloti, we have successfully complemented associated phenotypes of polyhydroxyalkanoate synthesis mutants. DNA sequence analysis of three clones indicates that the complementing genes are homologous to, but substantially different from, known polyhydroxyalkanaote synthase-encoding genes. Thus we have demonstrated the ability to isolate diverse genes for polyhydroxyalkanaote synthesis by functional complementation of defined mutants. Such genes might be of use in the engineering of more efficient systems for the industrial production of bioplastics. The use of functional complementation will also provide a vehicle to probe the genetics of polyhydroxyalkanaote metabolism and its relation to carbon availability in complex microbial assemblages. [ABSTRACT FROM AUTHOR]

Biofilms were cultivated on polycarbonate strips in rotating annular reactors using South Saskatchewan River water during the fall of 1999 and the fall of 2001. The reactors were supplemented with carbon (glucose), nitrogen (NH4Cl), phosphorus (KH2PO4), or combined nutrients (CNP), with or without hexadecane. The impact of these treatments on nitrification and on the exopolysaccharide composition of river biofilms was determined. The results showed that the biofilms had higher NH4+ oxidation, NO3– production, and N2O production activities in fall 1999 than fall 2001 when grown with CNP but had higher activities in fall 2001 than fall 1999 when grown with individual nutrients. The exopolysaccharide amounts and proportions were generally higher in fall 1999 than fall 2001, as a consequence of the higher nutrient levels in the river water in the first year of this study. The addition of P and especially CNP stimulated NH4+ oxidation by the biofilms, showing a P limitation in this river ecosystem. The presence of hexadecane negatively affected these activities and lowered the amounts of exopolysaccharides in CNP and P biofilms in fall 1999 but increased the biofilm activities and exopolysaccharide amounts in CNP biofilm in fall 2001. Antagonistic, synergistic, and independent effects between nutrients and hexadecane were also observed. This study demonstrated that the biofilm autotrophic nitrification activity in the South Saskatchewan River was limited by P, that this activity and the exopolysaccharide amounts and proportions were dependent on the nutrient concentrations in the river water, and suggested that exopolysaccharides may play a protective role for biofilm microorganisms against toxic pollutants. [ABSTRACT FROM AUTHOR]

The goal of this study was to initiate investigation of the genetics of bacterial poly-3-hydroxybutyrate (PHB) metabolism at the community level. We constructed metagenome libraries from activated sludge and soil microbial communities in the broad-host-range IncP cosmid pRK7813. Several unique clones were isolated from these libraries by functional heterologous complementation of a Sinorhizobium meliloti bdhA mutant, which is unable to grow on the PHB cycle intermediate D-3-hydroxybutyrate due to absence of the enzyme D-3-hydroxybutyrate dehydrogenase activity. Clones that conferred D-3-hydroxybutyrate utilization on Escherichia coli were also isolated. Although many of the S. meliloti bdhA mutant complementing clones restored D-3-hydroxybutyrate dehydrogenase activity to the mutant host, for some of the clones this activity was not detectable. This was also the case for almost all of the clones isolated in the E. coli selection. Further analysis was carried out on clones isolated in the S. meliloti complementation. Transposon mutagenesis to locate the complementing genes, followed by DNA sequence analysis of three of the genes, revealed coding sequences that were broadly divergent but lay within the diversity of known short-chain dehydrogenase/reductase encoding genes. In some cases, the amino acid sequence identity between pairs of deduced BdhA proteins was <35%, a level at which detection by nucleic acid hybridization based methods would probably not be successful. [ABSTRACT FROM AUTHOR]

Biofilm communities cultivated in rotating annular bioreactors using water from the South Saskatchewan River were assessed for the effects of seasonal variations and nutrient (C, N, and P) additions. Confocal laser microscopy revealed that while control biofilms were consistently dominated by bacterial biomass, the addition of nutrients shifted biofilms of summer and fall water samples to phototrophic-dominated communities. In nutrient-amended biofilms, similar patterns of nitrification, denitrification, and hexadecane mineralization rates were observed for winter and spring biofilms; fall biofilms had the highest rates of nitrification and hexadecane mineralization, and summer biofilms had the highest rates of denitrification. Very low rates of all measured activities were detected in control biofilms (without nutrient addition) regardless of season. Nutrient addition caused large increases in hexadecane mineralization and denitrification rates but only modest increases, if any, in nitrification rates, depending upon the season. Generally, both alkB and nirK were more readily PCR amplified from nutrient-amended biofilms. Both genes were amplified from all samples except for nirK from the fall control biofilm. It appears that bacterial production in the South Saskatchewan River water is limited by the availability of nutrients and that biofilm activities and composition vary with nutrient availability and time of year. [ABSTRACT FROM AUTHOR]

Examines the effect of cool soil temperature on soybean plant growth and yield in Quebec. Increase in nitrogen fixation and yield due to the addition of genistein to soybean inocula; Details on the law temperature limitation of soybean nodulation; Improvement of growth and yields of soybean grown in cool spring soils.

After isolation from a pulp mill wastewater treatment facility, two yeast strains, designated SPT1 and SPT2, were characterized and used in the development of mediated biochemical oxygen demand (BOD) biosensors for wastewater. 18S rRNA gene sequence analysis revealed a one nucleotide difference between the sequence of SPT1 and those of Candida sojae and Candida viswanthii. While SPT2 had the highest overall homology to Pichia norvegensis, at only 73.5%, it is clearly an ascomycete, based on BLAST comparisons and phylogenetic analyses. Neighbor-joining dendrograms indicated that SPT1 clustered with several Candida spp., and that SPT2 clustered with Starmera spp., albeit as a very deep branch. Physiological tests, microscopic observations, and fatty acid analysis confirmed that SPT1 and SPT2 are novel yeast strains. Physiological tests also indicated that both strains had potential for use in mediated biosensors for estimation of BOD in wastewater. The lower detection limits of SPT1- and SPT2-based K[sub 3] Fe(CN)[sub 6] -mediated biosensors for a pulp-mill effluent were 2 and 1 mg BOD/L, respectively. Biosensor-response times for effluents from eight different pulp mills were in the range of 5 min. Reliability and sensitivity of the SPT1- and SPT2-based biosensors were good, but varied with the wastewater.Key words: yeast characterization, 18S rRNA gene sequence, pulp-mill wastewater, BOD[sub 5] , mediated BOD biosensor.Deux nouvelles souches de levure, désignées SPT1 et SPT2, ont été isolées dans un service de traitement des eaux résiduaires de moulin de pâte à papier, caractérisées et utilisées dans le développement de biosenseurs de demande biochimique en oxygène (DBO) pour l'eau usagée. L'analyse de la sequence de gène 18S rARN a indiqué des différences d'un nucléotide entre l'ordre de SPT1 et ceux de Candida sojae et Candida viswanthii. SPT2 s'alignait le plus a Pichia norvegensis, avec seulement une homologie de 73,5%, elle est évidemment une ascomycete, par comparisons BLAST et analyses phylogénetiques. Les dendrogrammes « neighbour-joining » ont indiqué que SPT1 groupait avec plusieurs espèces de Candida, tandis que SPT2 groupait avec des espèces Starmera, quoique comme branche plus profound. Les tests physiologiques, les observations microscopiques et l'analyse d'acide gras ont confirmé que SPT1 et SPT2 ont des nouvelles souches de levure. Les tests physiologiques ont également indiqué que les deux souches ont offert des possibilités intéressantes pour l'usage dans les biosenseurs d'évaluation de la DBO d'eau usagée. Les limites de détection inférieures des biosenseurs K[sub 3] Fe(CN)[sub 6] à base de SPT1 et SPT2 pour un effluent de moulin de pâte à papier étaient 2 et 1 mg DBO/L, respectivement. Les temps de réponse de biosenseur pour des effluents de huit moulins de pâte à papier différents étaient dans l'intervalle de 5 min. La fiabilité et la sensibilité des biosenseurs à base de SPT1 et de SPT2 étaient bonnes, mais variable avec l'eau usagée.Mots clés : caractérisation de levure, sequence de gène 18S rARN, eaux résiduaires de moulin de pâte à papier, DBO[sub 5] , biosenseur de DBO. [ABSTRACT FROM AUTHOR]

Endophytic bacteria reside within plant tissues and have often been found to promote plant growth. Fourteen strains of putative endophytic bacteria, not including endosymbiotic Bradyrhizobium strains, were isolated from surface-sterilized soybean (Glycine max. (L.) Merr.) root nodules. These isolates were designated as non-Bradyrhizobium endophytic bacteria (NEB). Three isolates (NEB4, NEB5, and NEB17) were found to increase soybean weight when plants were co-inoculated with one of the isolates and Bradyrhizobium japonicum under nitrogen-free conditions, compared with plants inoculated with B. japonicum alone. In the absence of B. japonicum, these isolates neither nodulated soybean, nor did they affect soybean growth. All three isolates were Gram-positive spore-forming rods. While Biolog tests indicated that the three isolates belonged to the genus Bacillus, it was not possible to determine the species. Phylogenetic analysis of 16S rRNA gene hypervariant region sequences demonstrated that both NEB4 and NEB5 are Bacillus subtilis strains, and that NEB17 is a Bacillus thuringiensis strain.Key words: root nodule, endophytic bacteria, plant-growth-promoting bacteria, Bacillus subtilis, Bacillus thuringiensis.Les bactéries endophytes sont des bactéries qui résident à l'intérior des tissus végétaux. Plusieurs études ont démontré la capacité de ces bactéries à favoriser la croissance des plantes. Quatorze souches de bactéries endophytes ont été isolées à partir de nodules de soya (Glycinemax (L.) Merr.) stérilisés en surface. Ces isolats ont été désignées comme étant des bactéries endophytes n'appartenant pas au genre Bradyrhizobium (NEB). Lorsque co-inoculés avec Bradyrhizobium japonicum, trois isolats (NEB4, NEB5 et NEB17) se sont avérées capable d'augmenter le poids de plantes soya. En absence de la souche de B. japonicum, ces même isolats ont été incapable d'affecter la croissance des plants ou de noduler. NEB4, NEB5 et NEB17 sont des bactéries Gram positives formant des spores. Des essais Biolog n'ont pas permis l'obtention d'une identification au niveau de l'espèce, mais ont quand même indiqué que les trois souches appartiennent au genre Bacillus. L'analyse phylogénétique des séquences d'une région très variable du gène 16S rARN a démontré que NEB4 et NEB5 sont des souches appartenant à l'espèce Bacillus subtilis, et que NEB17 est une souche appartnenant à l'espèce Bacillus thuringiensis.Mots clés : nodosités racinaires, bactérie endophyte, bactérie favorisant la croissance des plantes, Bacillus subtilis, Bacillus thuringiensis.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]