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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 P 450 6B2-like (LOC113493761) at higher levels than females. In comparison, female caterpillars expressed higher levels of cytochrome P 450 6B7-like (LOC113492289). Bioinformatic tools predict that cytochrome P 450 s 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., (© 2024. The Author(s).)

When nano-sized titanium dioxide (nano-TiO 2 ) 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-TiO 2 toxicity. Sinorhizobium meliloti was exposed to aqueous dispersions of micrometer-sized TiO 2 (micron-TiO 2 , 44 μm) or nanometer-sized TiO 2 (nano-TiO 2 , 21 nm) at nominal concentrations of 0, 100, 300, 600, 900, and 1800 mg TiO 2 /L. There were fewer viable S. meliloti cells after exposure to nano-TiO 2 under dark and UV-A light conditions. Nano-TiO 2 was more toxic to S. meliloti with UV-A irradiation (100% mortality at 100 mg TiO 2 /L) than under dark conditions (100% mortality at 900 mg TiO 2 /L). Micron-TiO 2 concentrations less than 300 mg TiO 2 /L had no effect on S. meliloti viability under dark or UV-A light conditions. Exposure to 600 mg/L or more of micron-TiO 2 under UV-A light could also photo-kill S. meliloti cells (100% mortality). Further studies are needed to ascertain whether nano-TiO 2 interferes with the growth of N 2 -fixing microorganisms in realistic agricultural environments.

Plant resistance mechanisms to insect herbivory can potentially be bred into crops as an important strategy for integrated pest management. Medicago truncatula ecotypes inoculated with the rhizobium Ensifer medicae (Sinorhizobium medica) WSM419 were screened for resistance to herbivory by caterpillars of the beet armyworm, Spodoptera exigua, through leaf and whole plant choice studies; TN1.11 and F83005.5 are identified as the least and most deterrent ecotypes, respectively. In response to caterpillar herbivory, both ecotypes mount a robust burst of plant defensive jasmonate phytohormones. Restriction of caterpillars to either of these ecotypes does not adversely affect pest performance. This argues for an antixenosis (deterrence) resistance mechanism associated with the F83005.5 ecotype. Unbiased metabolomic profiling identified strong ecotype-specific differences in metabolite profile, particularly in the content of oleanolic-derived saponins that may act as antifeedants. Compared to the more susceptible ecotype, F83005.5 has higher levels of oleanolic-type zanhic acid- and medicagenic acid-derived compounds. Together, these data support saponin-mediated deterrence as a resistance mechanism of the F83005.5 ecotype and implicates these compounds as potential antifeedants that could be used in agricultural sustainable pest management strategies.

Northern peatlands are important global C reservoirs, largely because of their slow rates of microbial C mineralization. Particularly in sites that are heavily influenced by anthropogenic disturbances, there is scant information about microbial ecology and whether or not microbial community structure influences greenhouse gas production. This work characterized communities of bacteria and archaea using terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of 16S rRNA and functional genes across eight natural, mined, or restored peatlands in two locations in eastern Canada. Correlations were explored among chemical properties of peat, bacterial and archaeal community structure, and carbon dioxide (CO2) and methane (CH4) production rates under oxic and anoxic conditions. Bacteria and archaea similar to those found in other peat soil environments were detected. In contrast to other reports, methanogen diversity was low in our study, with only 2 groups of known or suspected methanogens. Although mining and restoration affected substrate availability and microbial activity, these land-uses did not consistently affect bacterial or archaeal community composition. In fact, larger differences were observed between the two locations and between oxic and anoxic peat samples than between natural, mined, and restored sites, with anoxic samples characterized by less detectable bacterial diversity and stronger dominance by members of the phylum Acidobacteria. There were also no apparent strong linkages between prokaryote community structure and CH4 or CO2 production, suggesting that different organisms exhibit functional redundancy and/or that the same taxa function at very different rates when exposed to different peat substrates. In contrast to other earlier work focusing on fungal communities across similar mined and restored peatlands, bacterial and archaeal communities appeared to be more resistant or resilient to peat substrate changes brought about by these land uses.

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., (2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Though there is overlap, plant responses to caterpillar herbivory show distinct variations from mechanical wounding. In particular, effectors in caterpillar oral secretions modify wound-associated plant responses. Previous studies have focused on transcriptional and protein abundance differences in response to caterpillar herbivory. This study investigated Spodoptera exigua caterpillar-specific post-translational modification of Arabidopsis thaliana soluble leaf proteins by liquid chromatography/electrospray ionization/mass spectroscopy/mass spectroscopy (LC/ESI/MS/MS). Given that caterpillar labial saliva contains oxidoreductases, such as glucose oxidase, particular attention was paid to redox-associated modifications, such as the oxidation of protein cysteine residues. Caterpillar- and saliva-specific protein modifications were observed. Differential phosphorylation of the jasmonic acid biosynthetic enzyme, lipoxygenase 2, and a chaperonin protein is seen in plants fed upon by caterpillars with intact salivary secretions compared to herbivory by larvae with impaired labial salivary secretions. Often a systemic suppression of photosynthesis is associated with caterpillar herbivory. Of the five proteins modified in a caterpillar-specific manner (a transcription repressor, a DNA-repair enzyme, PS I P700, Rubisco and Rubisco activase), three are associated with photosynthesis. Oxidative modifications are observed, such as caterpillar-specific denitrosylation of Rubisco activase and chaperonin, cysteine oxidation of Rubisco, DNA-repair enzyme, and chaperonin and caterpillar-specific 4-oxo-2-nonenal modification of the DNA-repair enzyme.

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-beta3-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.

Specific primers were developed to amplify a 227 bp segment of the arbuscular mycorrhizal fungus Glomus intraradices gene encoding glucose-6-phosphate dehydrogenase (G6PDH), an enzyme involved in the pentose phosphate pathway. G6PDH gene expression was measured by real-time quantitative reverse transcriptase-polymerase chain reaction in response to phosphorus (P) concentrations in the growth medium of colonized transformed carrot roots. We investigated the effects of different P concentration treatments on carbon (C) metabolism within the intraradical mycelia of G. intraradices. The results showed a significant (P=0.017) down-regulation of G6PDH expression in the intraradical mycelia of G. intraradices cultures grown in high P than low P conditions but no significant difference in regulation in excessive P concentrations when compared with the low P or high P concentrations. These results indicate that a reduction in the C flow from the host could be occurring as a result of elevated P and that a decrease in fungal G6PDH gene expression occurs, but not in the short term (less than 2 h). Reduced C flow from the host could lead to reduced fungal growth and root colonization, as was observed under high soil P conditions.

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 (NH(4)Cl), phosphorus (KH(2)PO(4)), 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.

Aims: The aim of this study was to identify and characterize a compound produced by the plant growth promoting bacterium, Bacillus thuringiensis non-Bradyrhizobium Endophytic Bacterium 17., Methods and Results: The bacterial peptide was analysed and purified via HPLC. Using the disk diffusion assay this peptide inhibited the growth of 16/19 B. thuringiensis strains, 4/4 Bacillus cereus strains, among others, as well as a Gram-negative strain Escherichia coli MM294 (pBS42). Both bactericidal and bacteristatic effects were observed on B. cereus ATCC 14579 and bactericidal effects were observed on B. thuringiensis ssp. thuringiensis Bt1267. The molecular weight of the peptide was estimated via SDS-PAGE and confirmed with Matrix Assisted Laser Desorption Ionization Quadrapole Time of Flight mass spectrometry; its weight is 3162 Da. The peptide is biologically active after exposure to 100 degrees C for 15 min, and within the pH range 1.00-9.25. Its activity disappeared when treated with proteinase K and protease, but not with alpha-amylase or catalase., Conclusions: We conclude that this is the first report of a bacteriocin produced by a plant growth promoting rhizobacteria (B. thuringiensis) species and have named the bacteriocin thuricin 17., Significance and Impact of the Study: Our work has characterized a bacteriocin produced by a plant growth promoting bacterium. This strain is previously reported to increase soya bean nodulation.

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.

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, supplemented with carbon (glucose), nitrogen (NH4Cl), phosphorus (KH2PO4), or combined nutrients (CNP), with or without hexadecane, a model compound representing aliphatic hydrocarbons used to simulate a pollutant. In fall 1999 and fall 2001, comparable denitrification activities and catabolic potentials were observed in the biofilms, implying that denitrifying populations showed similar activity patterns and catabolic potentials during the fall from year to year in this river ecosystem, when environmental conditions were similar. Both nirS and nirK denitrification genes were detected by PCR amplification, suggesting that both denitrifying bacterial subpopulations can potentially contribute to total denitrification. Between 91.7 and 99.8% of the consumed N was emitted in the form of N2, suggesting that emission of N2O, a major potent greenhouse gas, by South Saskatchewan River biofilms is low. Denitrification was markedly stimulated by the addition of CNP, and nirS and nirK genes were predominant only in the presence of CNP. In contrast, individual nutrients had no impact on denitrification and on the occurrence of nirS and nirK genes detected by PCR amplification. Similarly, only CNP resulted in significant increases in algal and bacterial biomass relative to control biofilms. Biomass measurements indicated a linkage between autotrophic and heterotrophic populations in the fall 1999 biofilms. Correlation analyses demonstrated a significant relationship (P < or = 0.05) between the denitrification rate and the biomass of algae and heterotrophic bacteria but not cyanobacteria. At the concentration assessed (1 ppb), hexadecane partially inhibited denitrification in both years, slightly more in the fall of 2001. This study suggested that the response of the anaerobic heterotrophic biofilm community may be cyclic and predictable from year to year and that there are interactive effects between nutrients and the contaminant hexadecane.

To isolate Sinorhizobium meliloti mutants deficient in malate dehydrogenase (MDH) activity, random transposon Tn5tac1 insertion mutants were screened for conditional lethal phenotypes on complex medium. Tn5tac1 has an outward-oriented isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter (Ptac). The insertion in strain Rm30049 was mapped to the mdh gene, which was found to lie directly upstream of the genes encoding succinyl-CoA synthetase (sucCD) and 2-oxoglutarate dehydrogenase (sucAB and lpdA). Rm30049 required IPTG for wild-type growth in complex media, and had a complex growth phenotype in minimal media with different carbon sources. The mdh:: Tn5tacl insertion eliminated MDH activity under all growth conditions, and activities of succinyl-CoA synthetase, 2-oxoglutarate dehydrogenase, and succinate dehydrogenase were affected by the addition of IPTG. Reverse-transcriptase polymerase chain reaction (RT-PCR) studies confirmed that expression from Ptac was induced by IPTG and leaky in its absence. Alfalfa plants inoculated with Rm30049 were chlorotic and stunted, with small white root nodules, and had shoot dry weight and percent-N content values similar to those of uninoculated plants. Cosmid clone pDS15 restored MDH activity to Rm30049, complemented both the mutant growth and symbiotic phenotypes, and was found to carry six complete (sdhB, mdh, sucCDAB) and two partial (IpdA, sdhA) tricarboxylic acid cycle genes.

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.

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 44.1kDa 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.

The effect of contamination with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) on an indigenous soil bacterial community was examined in two uncontaminated loam soil columns possessing native grasses. One column was spiked twice with RDX crystals for a total RDX load of 1000 mg (kg soil)(-1). The reduced metabolite of RDX degradation, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine, was observed in the column leachate, suggesting anaerobic degradation of RDX. Denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA from both contaminated and uncontaminated columns produced identical banding patterns which were stable over the course of the experimental period. The bacterial diversity remained high in the contaminated column, as determined by restriction fragment length polymorphism and rarefaction analyses of random 16S rDNA clones. These combined results suggested that long-term exposure to 1000 mg RDX (kg soil)(-1) did not produce an observable effect on bacterial diversity or the numerically dominant members of the indigenous soil bacterial community.

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 K3Fe(CN)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.

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.

Bradyrhizobium japonicum mutants with altered nod gene induction characteristics were isolated by screening mutants for genistein-independent nod gene expression. Plasmid pZB32, carrying a nodY::lacZ transcriptional gene fusion, was introduced into B. japonicum cells that had been subjected to UV mutagenesis. Ten independent transformants producing a blue color on plates containing 5bromo-4chloro-3indolyl-beta-D-galactopyranoside but lacking genistein, indicative of constitutive expression of the nodY::lacZ reporter gene, were isolated. Beta-galactosidase activity assays revealed that while all of the 10 strains were sensitive to low concentrations of genistein, none exhibited truly constitutive nodY::lacZ expression in liquid culture. Soybean plants inoculated with three of the mutants were chlorotic and stunted, with shoot dry weights close to those of the uninoculated plants, indicating the absence of nitrogen fixation. Differences in the kinetics of nodY::lacZ expression and lipochitin oligosaccharide Nod signal production suggested that the strains carried different mutations. Some of these strains may be useful in mitigating the low root zone temperature-associated delay in soybean nodulation at the northern extent of soybean cultivation.

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.

Two new yeast strains (SPT1 and SPT2) were isolated and immobilized on glassy carbon electrodes to form microbial biosensors for estimation of biochemical oxygen demand (BOD). Ferricyanide was proven to be the most efficient mediator to shuttle electrons from the redox center of reduced microbial enzymes to the electrode in the presence of excess glucose/glutamic acid (GGA). With a 3-fold greater metabolic assimilation capability and greater responses to various effluent samples, SPT1 was selected for sensor-BOD measurements. BOD estimations for the GGA standard resulted in an extended linear range: 2-100 mg/l. Response reproducibility was +/-10% for a GGA standard containing 10 mg BOD/l. For analysis of pulp mill effluents, the BOD detection limit was 2 mg/l with a response time of 5 min.

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.

We have identified two Sinorhizobium meliloti chromosomal loci affecting the poly-3-hydroxybutyrate degradation pathway. One locus was identified as the gene acsA, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase. Analysis of the acsA nucleotide sequence revealed that this gene encodes a putative protein with a molecular weight of 72,000 that shows similarity to acetyl-CoA synthetase in other organisms. Acetyl-CoA synthetase activity was not affected in cell extracts of glucose-grown acsA::Tn5 mutants; instead, acetoacetyl-CoA synthetase activity was drastically reduced. These findings suggest that acetoacetyl-CoA synthetase, rather than CoA transferase, activates acetoacetate to acetoacetyl-CoA in the S. meliloti poly-3-hydroxybutyrate cycle. The second locus was identified as phbC, encoding poly-3-hydroxybutyrate synthase, and was found to be required for synthesis of poly-3-hydroxybutyrate deposits.

Malate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate reversible 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, beta-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.

The formation of phosphoenolpyruvate (PEP) is a major step in the gluconeogenic pathway in which tricarboxylic acid (TCA) cycle intermediates are converted to hexose sugars. In Rhizobium (now Sinorhizobium) meliloti this step is catalysed by the enzyme PEP carboxykinase (PCK) which converts oxaloacetate to PEP. R. meliloti Pck- mutants grow very poorly with TCA cycle intermediates as the sole source of carbon. Here, the isolation and mapping of suppressor mutations which allow Pck- mutants to grow on succinate and other TCA cycle intermediates is reported. Tn5 insertions which abolished the suppressor phenotype and mapped to the suppressor locus were located within the pod gene encoding pyruvate orthophosphate dikinase (PPDK). Strains carrying suppressor mutations had increased PPDK activity compared to the wild-type. The suppressor phenotype was dependent on the combined activities of malic enzyme and PPDK, which thus represent an alternative route for the formation of PEP in R. meliloti. PPDK activity was not required for symbiotic N2 fixation.

The wild-type NAD(+)-dependent malic enzyme (dme) gene of Rhizobium (now Sinorhizobium) meliloti was cloned and localized to a 3.1 kb region isolated on the cosmid pTH69. This cosmid complemented the symbiotic nitrogen fixation (Fix-) phenotype of R. meliloti dme mutants. The dme gene was mapped by conjugation to between the cys-11 and leu-53 markers on the R. meliloti chromosome. beta-Galactosidase activities measured in bacterial strains carrying either dme-lacZ or tme-lacZ gene fusions (the tme gene encodes NADP(+)-dependent malic enzyme) indicated that the dme gene was expressed constitutively in free-living cells and in N2-fixing bacteroids whereas expression of the tme gene was repressed in bacteroids. The R. meliloti dme gene product (DME) was overexpressed in and partially purified from Escherichia coli. The properties of this enzyme, together with those of the NADP(+)-dependent malic enzyme (TME) partially purified from R. meliloti dme mutants, were determined. Acetyl-CoA inhibited DME but not TME activity. This result supports the hypothesis that DME, together with pyruvate dehydrogenase, forms a pathway in which malate is converted to acetyl-CoA.

The bacterium Rhizobium meliloti, which forms N2-fixing root nodules on alfalfa, has two distinct malic enzymes; one is NADP+ dependent, while a second has maximal activity when NAD+ is the coenzyme. The diphosphopyridine nucleotide (NAD+)-dependent malic enzyme (DME) is required for symbiotic N2 fixation, likely as part of a pathway for the conversion of C4-dicarboxylic acids to acetyl coenzyme A in N2-fixing bacteroids. Here, we report the cloning and localization of the tme gene (encoding the triphosphopyridine nucleotide [NADP+]-dependent malic enzyme) to a 3.7-kb region. We constructed strains carrying insertions within the tme gene region and showed that the NADP+ -dependent malic enzyme activity peak was absent when extracts from these strains were eluted from a DEAE-cellulose chromatography column. We found that NADP+ -dependent malic enzyme activity was not required for N2 fixation, as tme mutants induced N2-fixing root nodules on alfalfa. Moreover, the apparent NADP+ -dependent malic enzyme activity detected in wild-type (N2-fixing) bacteroids was only 20% of the level detected in free-living cells. Much of that residual bacteroid activity appeared to be due to utilization of NADP+ by DME. The functions of DME and the NADP+ -dependent malic enzyme are discussed in light of the above results and the growth phenotypes of various tme and dme mutants.

The pckA gene of Rhizobium meliloti, encoding phosphoenolpyruvate carboxykinase, was isolated from a genomic cosmid library by complementation of the succinate growth phenotype of a Pck- mutant. The gene region was mapped by subcloning and Tn5 insertion mutagenesis. The DNA sequence for a 2-kb region containing the structural gene and its promoter was determined. The pckA gene encodes as 536-amino-acid protein that shows homology with other ATP-dependent Pck enzymes. The promoter was identified following primer extension analysis and is similar to sigma 70-like promoters. Expression analysis with a pckA::lacZ gene fusion indicated that the pckA gene was strongly induced at the onset of stationary phase in complex medium. When defined carbon sources were tested, the expression level of the pckA gene was found to be high when cells were grown in minimal media with succinate or arabinose as the sole carbon source but almost absent when glucose, sucrose, or glycerol was the sole carbon source. Glucose and sucrose were not found to strongly repress pckA induction by succinate.

DEAE-cellulose chromatography of extracts of free-living Rhizobium meliloti cells revealed separate NAD(+)-dependent and NADP(+)-dependent malic enzyme activities. The NAD+ malic enzyme exhibited more activity with NAD+ as cofactor, but also showed some activity with NADP+. The NADP+ malic enzyme only showed activity when NADP+ was supplied as cofactor. Three independent transposon-induced mutants of R. meliloti which lacked NAD+ malic enzyme activity (dme-) but retained NADP+ malic enzyme activity were isolated. In an otherwise wild-type background, the dme mutations did not alter the carbon utilization phenotype; however, nodules induced by these mutants failed to fix N2. Structurally, these nodules appeared to develop like wild-type nodules up to the stage where N2-fixation would normally begin. These results support the proposal that NAD+ malic enzyme, together with pyruvate dehydrogenase, functions in the generation of acetyl-CoA required for TCA cycle function in N2-fixing bacteroids which metabolize C4-dicarboxylic acids supplied by the plant.