Utilization of multiple microbial tools to evaluate efficacy of restoration strategies to improve recreational water quality at a Lake Michigan Beach (Racine, WI).

Hydro-meteorological conditions facilitate transport of fecal indicator bacteria (FIB) to the nearshore environment, affecting recreational water quality. North Beach (Racine, Wisconsin, United States), is an exemplar public beach site along Lake Michigan, where precipitation-mediated surface runoff, wave encroachment, stormwater and tributary outflow were demonstrated to contribute to beach advisories. Multiple restoration actions, including installation of a stormwater retention wetland, were successfully deployed to improve recreational water quality. Implementation of molecular methods (e.g. human microbial source tracking markers and Escherichia coli (E. coli) qPCR) assisted in identifying potential pollution sources and improving public health response time. However, periodic water quality failures still occur. As local beach managers reassess restoration measures in response to climatic changes, use of expanded microbial methods (including bacterial community profiling) may contribute to a better understanding of these dynamic environments. In this 2-year study (2015 and 2019), nearshore/offshore Lake Michigan, stormwater, and tributary samples were collected to determine if, 1) the constructed wetland (~50 m from the shoreline) continued to provide stormwater separation/retention and 2) mixing between onshore sources, Root River and Lake Michigan, was increasing due to rising precipitation/lake levels. Monthly rainfall totals were 1.5× higher in 2019 than 2015, coinciding with a 0.63 m lake-level rise. The prevalence of more intense, onshore winds also increased, facilitating interaction between potential reservoirs of FIB with nearshore water through wind driven waves and lake intrusion, e.g. beach sands and the adjacent Root River. While a strong relationship existed between wet weather wetland and North Beach nearshore E. coli concentrations (all sites), bacterial communities were strikingly different. Conversely, bacterial community overlap existed between the Root River mouth and nearshore/offshore sites. These results suggest the constructed wetland can accommodate the climate-related changes observed in this study. Future restoration activities could be directed towards upstream tributary sources in order to minimize microbial contaminants entering Lake Michigan.

Effect of soluble corn fiber supplementation for 1 year on bone metabolism in children, the MetA-bone trial: Rationale and design.

Calcium intake is critical for adequate bone mineralization in adolescence, but it is usually inadequate in US adolescents. A strategy to maximize bone mineralization is to increase calcium absorption, which could be achieved by soluble corn fiber (SCF). There are no studies determining the long-term effects of SCF on bone mass in children. OBJECTIVES: To determine the effect of one-year SCF supplementation compared to placebo on bone mass and bone biomarkers in children with low habitual calcium intake. We hypothesize that SCF supplementation will result in a higher bone mineral content and higher levels of bone formation and lower bone resorption biomarkers. METHODS: 240 healthy children (10-13 years), with usual low calcium intake, will be randomized to four experimental groups for 1 year: (1) SCF (12 g/d); (2) SCF (12 g/d) + 600 mg/d of calcium; (3) Placebo (maltodextrin); and (4) Placebo +600 mg/d of calcium. The supplements have been pre-mixed with a flavored powder beverage and participants will only need to dilute it in water and drink this twice per day. Bone will be measured using dual energy x-ray absorptiometry (DXA) at baseline, 6 and 12 months. Serum bone biomarkers will be measured at baseline and at 12 months. CONCLUSIONS: If supplementing diets with SCF lead to higher bone mass during adolescence, this could help achieve the genetic potential for PBM and to start adult life with stronger bones. If successful, SCF can be incorporated into diets for promoting bone health in adolescents.

Investigation of carryover effect of prior fibre consumption on growth, serum and tissue metabolic markers in Ossabaw pigs fed a high-fat diet.

Carryover effect of prior fibre consumption on metabolic markers was investigated. Treatments were arranged in 2 × 2 factorial with 2 fibre sources, 4% inulin or cellulose (Solka-Floc®) and fat levels (5 or 15%) for the low-fat diet (LFD) and high-fat diet (HFD) respectively. Pigs were fed the two fibre diets for the first 56d (nursery phase), and thereafter fed either the LFD or HFD containing no added fibre source from d56 to 140 (growing phase). Pigs on the HFD were heavier (p = .05) than those on LF (64.61 vs. 68.38 kg), regardless of prior fibre type consumed. Pigs that were fed cellulose during the nursery and later fed the HFD had the highest ADG (p < .05). Feeding the HFD resulted in higher back fat (BF) (13.41 and 18.18 ± 0.12 mm for LFD and HFD, respectively; p < .01). The HFD resulted in higher (p < .01) insulin (0.014 and 0.016 ± 0.001 mg/L for LF and HF respectively) and glucose (100.89 and 125.03 ± 4.39 mg/dl for LF and HF respectively) concentrations in the serum. Inulin increased (p ≤ .02) jejunal expression of SREBP-1c and CL-4, but reduced (p < .05) TNFɑ and IL-6 expression in the ileum. Alpha-diversity was significantly different (p < .05) between the inulin and cellulose fed pigs at the end of the nursery and finishing phases. Therefore, inulin feeding before a HFD may lead to reduction in ADG and inflammatory markers in the small intestine of pigs, and thus prevent future metabolic disorders.

Effects of dietary resistant starch content on metabolic status, milk composition, and microbial profiling in lactating sows and on offspring performance.

In the present study, the effects of dietary resistant starch (RS) content on serum metabolite and hormone concentrations, milk composition, and faecal microbial profiling in lactating sows, as well as on offspring performance was investigated. Sixteen sows were randomly allotted at breeding to two treatments containing low- and high-RS contents from normal and high-amylose corn varieties, respectively, and each treatment had eight replicates (sows). Individual piglet body weight (BW) and litter size were recorded at birth and weaning. Milk samples were obtained on day 10 after farrowing for composition analysis. On day 2 before weaning, blood and faecal samples were collected to determine serum metabolite and hormone concentrations and faecal microbial populations, respectively. Litter size at birth and weaning were not influenced (p > 0.05) by the sow dietary treatments. Although feeding the RS-rich diet to sows reduced (p = 0.004) offspring birth BW, there was no difference in piglet BW at weaning (p > 0.05). High-RS diet increased (p < 0.05) serum triacylglycerol and nonesterified fatty acid concentrations and milk total solid content, and tended (p = 0.09) to increase milk fat content in lactating sows. Feeding the RS-rich diet to sows increased (p < 0.01) faecal bacterial population diversity. These results indicate that high-RS diets induce fatty acid mobilization and a greater intestinal bacterial richness in lactating sows, as well as a greater nutrient density in maternal milk, without affecting offspring performance at weaning.

Molecular analysis of bacterial community based on 16S rDNA and functional genes in activated sludge enriched with 2,4-dichlorophenoxyacetic acid (2,4-D) under different cultural conditions.

Differential emergence and diversity of bacterial communities from activated sludge in response to varied cultural conditions using 2,4-dichlorophenoxyacetic acid (2,4-D) were investigated by coupling molecular analyses based on 16S rDNA with functional genes. We employed three different cultural conditions: (1) a culture sequentially fed a high concentration (300 mg/L) of 2,4-D (HS); (2) a culture continuously fed a low concentration (10 mg/L) of 2,4-D (LC); and (3) a serial batch culture in which 1% (v/v) of culture was transferred to a fresh medium containing a high concentration (300 mg/L) of 2,4-D (HB). The HS and LC bioreactors were operated for 3 months and HB was repeatedly transferred for 1 month. The 2,4-D was stably degraded under all the cultural conditions tested. PCR amplification and cloning-based analysis of functional genes using community DNAs from the cultures revealed five different oxygenase genes that may be involved in the initial step of 2,4-D degradation. All five gene-types were present in HS, while one of the five genes, type V (tftA) was not detected in LC. Quantitative PCR analysis showed that in HS, Ralstonia eutropha JMP 134 type-tfdA4 (type I) was the most abundant in copy number (2.0 +/- 0.1 x 10(7) copies/microg DNA) followed by RASC type-tfdA (type II) (1.8 +/- 1.0 x 10(6) copies/microg DNA), putative cadA-like gene (type IV) (2.6 +/- 0.8 x 10(5) copies/microg DNA), cadA gene (type III) (1.3 +/- 1.0 x 10(4) copies/microg DNA), and tftA gene (type V) (3.5 +/- 1.1 x 10(3) copies/microg DNA). Similar results were obtained in LC. In contrast, HB contained only type I and type III genes, and the type I gene was five orders of magnitude greater in copy number than the type III gene. Denaturing gel gradient electrophoresis (DGGE) analysis of PCR, amplified 16S rDNA fragments of bacterial communities in the three different cultures showed low similarity coefficient values (< or =0.35) when compared to the original activated sludge, suggesting that 2,4-D amendment caused a drastic change in the bacterial community. Particularly, HB showed only six bands (16-18 bands in the other cultures) and very low similarity coefficient values when compared to the other communities (0.10 to HS, 0.17 to LC, and 0.0 to original sludge). These results indicated that serial batch culturing (HB) resulted in a phylogenetically limited number of 2,4-D degrading bacteria carrying limited catabolic genes whereas more diverse 2,4-D degraders and catabolic genes were present in HS and LC. Therefore, the approach used for monitoring should be taken into account when one evaluates the population dynamics of contaminant-degrading bacteria at bioremediation sites.

Analysis of environmental Escherichia coli isolates for virulence genes using the TaqMan PCR system.

AIMS: To assess the presence of virulence genes in environmental and foodborne Escherichia coli isolates using the TaqMan PCR system. METHODS AND RESULTS: Three TaqMan pathogen detection kits called O157:H7, StxI and StxII were used to investigate the presence of virulence genes in Escherichia coli isolates. All 54 foodborne E. coli O157:H7 isolates showed expected results using these kits. Ninety (15%) of 604 environmental isolates gave positive amplification with an O157:H7-specific kit. TaqMan PCR amplification products from these 90 isolates were analysed by agarose gel electrophoresis, and 90% (81 of 90) of the environmental samples contained the expected PCR product. Sixty-six of these 90 were chosen for serotyping tests and only 35% (23 of 66) showed agglutination with both anti-O157 and anti-H7 antibodies. Further ribotyping of 16 sero-positive isolates in an automated Riboprinter did not identify these to be O157:H7. Multiplex PCR with primers for eaeA, stxI and stxII genes was used to confirm the TaqMan results in 10 selected environmental isolates. CONCLUSIONS: All three TaqMan pathogen detection kits were useful for virulence gene analysis of prescreened foodborne O157:H7 isolates, while the O157:H7-specific kit may not be suitable for virulence gene analysis of environmental E. coli isolates, because of high false positive identification. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to rapidly identify the presence of pathogenic E. coli in food or environmental samples is essential to avert outbreaks. These results are of importance to microbiologists seeking to use TaqMan PCR to rapidly identify pathogenic E. coli in environmental samples. Furthermore, serotyping may not be a reliable method for identification of O157:H7 strains.

Functional and structural adaptations of bacterial communities growing on particulate substrates under stringent nutrient limitation.

Biomass recycle reactors (BRRs) were used as a model system to study the functional and structural adaptations of mixed bacterial communities in response to the imposition of increasingly severe nutrient limitation. BRRs were fed synthetic media containing either spinach homogenate or autoclaved yeast cells to simulate the complex mixtures of particulate carbon sources that are often present in nature. In the BRRs fed spinach homogenate, the biomass (measured as particulate protein) exhibited a physiological response similar to previous studies as detected by 40-80% reductions in respiratory potential and by relatively stable catabolic ectoenzyme activities. Concomitant adaptations in bacterial community structure were detected by PCR-DGGE and RT-PCR-DGGE of 16S rDNA and 16S rRNA fragments, respectively. The microbial community structure was dynamic even after the biomass had reached a quasi-steady state with respect to physiological measurements. In the BRRs fed yeast cells, respiratory potentials increased 2- to 5-fold during the initial portion of the BRR run and alpha-glucosidase and beta-glucosidase activities increased 2- to 4-fold. Substantial bacterial community shifts were also detected in both the rDNA and rRNA profiles, indicating that this community was also structurally dynamic. These experiments suggest that phylogenetically different bacteria sustained the functional activities in these ecosystems in response to increasingly stringent nutrient limitation.

Effect of starvation length upon microbial activity in a biomass recycle reactor.

The kinetics of substrate degradation and bacterial growth was determined in a microbial community from a biomass recycle reactor that had been deprived of substrate feed for 0-32 days. Starvation caused changes in bacterial numbers, community composition, and physiological state. Substrate starvation for less than 1 day resulted in modest (less than threefold) changes in endogenous respiration rate, ATP content, and biomass level. During a starvation period of 32 days, there were substantial changes in microbial community composition, as assessed by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR amplicons of a portion of the 16S rDNA or by phospholipid fatty acid (PLFA) analysis. When the starved communities were stimulated with organic nutrients, the growth kinetics was a function of the length of the starvation period. For starvation periods of 2-8 days prior to nutrient addition, there was a phase of suboptimal exponential growth (S-phase) in which the exponential growth rate was about 30% of the ultimate unrestricted growth rate. S-phase lasted for 2-8 h and then unrestricted growth occurred at rates of 0.3-0.4 h(-1). At starvation times of 12 and 20 days, a lag phase preceded S-phase and the unrestricted growth phase.

Variable selection in high-dimensional multivariate binary data with application to the analysis of microbial community DNA fingerprints.

In order to understand the relevance of microbial communities on crop productivity, the identification and characterization of the rhizosphere soil microbial community is necessary. Characteristic profiles of the microbial communities are obtained by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16S rDNA from soil extracted DNA. These characteristic profiles, commonly called community DNA fingerprints, can be represented in the form of high-dimensional binary vectors. We address the problem of modeling and variable selection in high-dimensional multivariate binary data and present an application of our methodology in the context of a controlled agricultural experiment.

Fate of the biological control agent Pseudomonas aureofaciens TX-1 after application to turfgrass.

The fate and impact of Pseudomonas aureofaciens TX-1 following application as a biocontrol agent for fungi in turfgrass were studied. The organism was applied with a modified irrigation system by using a preparation containing 1 x 10(6) P. aureofaciens TX-1 CFU ml(-1) about 100 times between May and August. We examined the impact of this repeated introduction of P. aureofaciens TX-1 (which is known to produce the antimicrobial compound phenazine-1-carboxylic acid) on the indigenous microbial community of the turfgrass system and on establishment of introduced bacteria in the soil system. A PCR primer-DNA hybridization probe combination was developed to accurately monitor the fate of P. aureofaciens TX-1 following application in irrigation water. To assess the impact of frequent P. aureofaciens TX-1 applications on the indigenous bacterial community, turfgrass canopy, thatch, and rhizosphere samples were obtained during the growing season from control and treated plots and subjected to DNA extraction procedures and denaturing gradient gel electrophoresis (DGGE). PCR amplification and hybridization of extracted DNA with the P. aureofaciens TX-1-specific primer-probe combination revealed that P. aureofaciens TX-1 not only became established in the rhizosphere and thatch but also was capable of overwintering. Separation of PCR-amplified partial 16S rRNA genes by DGGE showed that the repeated application of P. aureofaciens TX-1 in irrigation water resulted in transient displacement of a leaf surface bacterial community member. There was no obvious alteration of any dominant members of the thatch and rhizosphere microbial communities.

Thermophilic aerobic treatment of a synthetic wastewater in a membrane-coupled bioreactor.

Synthetic wastewater containing alpha-lactose and gelatin was treated in a thermophilic membrane-coupled bioreactor (MBR). Thermophilic (>45 degrees C) treatment represents a potentially advantageous process for high-temperature as well as high-strength industrial wastewaters susceptible to reactor autoheating. Thermophilic systems, however, generally support a nonflocculating biomass that resists conventional methods of cell separation from the treated wastewater. MBRs were applied to thermophilic treatment systems because bacterial cells can be retained regardless of cell aggregation. Thermophilic aerobic MBRs were successfully operated at high levels of biocatalyst and produced a better effluent quality than analogous thermophilic bioreactors without cell recycle. At a hydraulic residence time (HRT) of 13.1 h, the chemical oxygen demand (COD) of the membrane eluate improved from 760 mg l(-1) (without cell recycle) to 160 mg l(-1) (with cell recycle). Bacterial community shifts were detected by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) -amplified 16S rRNA gene fragments - 6 of 13 bands disappeared within 2 days of MBR operation. A concomitant 40-50% reduction in physiological indicators of cell reactivity (RNA:protein; ATP:protein) was also observed. The specific activity of beta-galactosidase and aminopeptidase, however, increased by 10-25%, indicating that there is a definite advantage to MBR operation at the highest biomass level possible. Nucleotide sequence analysis of 16S rDNA clones identified phylotypes from the low-G+C Gram-positive division and the beta- and gamma-subdivisions of Proteobacteria.

Aerobic biological treatment of a pharmaceutical wastewater: effect of temperature on cod removal and bacterial community development.

The effect of temperature was studied on the efficiency of soluble COD removal and bacterial community development during the aerobic biological treatment of a pharmaceutical wastewater. Using wastewater and bacterial inoculum obtained from the full-scale facility treating this wastewater, batch laboratory cultures were operated at 5 degrees C intervals from 30 degrees C to 70 C. Following four culture transfers to allow for bacterial acclimation, residual soluble COD levels were measured and bacterial community fingerprints were obtained by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments. Soluble COD removal efficiency declined as temperature increased from 30 degrees C (62%) to 60 degrees C (38%). Biological treatment of this wastewater failed to occur at temperatures higher than 60 C. Gradual shifts in bacterial community structure were detected as temperature increased, including a concomitant reduction in the number of different bacterial populations. The impact of temperature on a two-stage biological treatment process was also compared. Better soluble COD removal was achieved when both reactors were operated at 30 degrees C compared to a system where the two stages were consecutively operated at 55 degrees C and 30 degrees C. These results indicate that operation of aerobic biological wastewater treatment reactors at elevated temperatures can have adverse effects on process performance.

Phylogenetic analysis of bacterial communities in mesophilic and thermophilic bioreactors treating pharmaceutical wastewater.

The phylogenetic diversity of the bacterial communities supported by a seven-stage, full-scale biological wastewater treatment plant was studied. These reactors were operated at both mesophilic (28 to 32 degrees C) and thermophilic (50 to 58 degrees C) temperatures. Community fingerprint analysis by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of the 16S rRNA gene from the domain Bacteria revealed that these seven reactors supported three distinct microbial communities. A band-counting analysis of the PCR-DGGE results suggested that elevated reactor temperatures corresponded with reduced species richness. Cloning of nearly complete 16S rRNA genes also suggested a reduced species richness in the thermophilic reactors by comparing the number of clones with different nucleotide inserts versus the total number of clones screened. While these results imply that elevated temperature can reduce species richness, other factors also could have impacted the number of populations that were detected. Nearly complete 16S rDNA sequence analysis showed that the thermophilic reactors were dominated by members from the beta subdivision of the division Proteobacteria (beta-proteobacteria) in addition to anaerobic phylotypes from the low-G+C gram-positive and Synergistes divisions. The mesophilic reactors, however, included at least six bacterial divisions, including Cytophaga-Flavobacterium-Bacteroides, Synergistes, Planctomycetes, low-G+C gram-positives, Holophaga-Acidobacterium, and Proteobacteria (alpha-proteobacteria, beta-proteobacteria, gamma-proteobacteria and delta-proteobacteria subdivisions). The two PCR-based techniques detected the presence of similar bacterial populations but failed to coincide on the relative distribution of these phylotypes. This suggested that at least one of these methods is insufficiently quantitative to determine total community biodiversity-a function of both the total number of species present (richness) and their relative distribution (evenness).

Feeding inhibition and mortality in Reticulitermes flavipes (Isoptera: Rhinotermitidae) after exposure to imidacloprid-treated soils.

Feeding inhibition and mortality of Reticulitermes flavipes (Kollar) exposed to sand, sandy loam, loam, and silty clay loam soils treated with several concentrations of imidacloprid were studied using bioassay techniques under laboratory conditions. Termite workers stopped feeding after exposure to treated soils. Differences in feeding reduction varied among the soil types. Based on the magnitude of the F-statistics, the effect of imidacloprid on the reduction of termite feeding was greatest in sand followed by sandy loam, loam, and silty clay loam soils. Soil properties such as organic matter content, silt and clay proportions, pH, and cation exchange capacity were suggested to affect the bioavailability of imidacloprid. Similar soil effects on mortality were observed in termites continuously exposed to treated soil for 21 d. In three of four soils tested, susceptibility to imidacloprid was not affected by the source of the termites tested.

Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR.

Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10(2) to 10(3) gene copies, which was lowered to 10(0) to 10(1) gene copies by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2, 3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2, 3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.

Bacterioplankton Community Diversity in a Series of Thermally Stratified Lakes.

> Abstract The dominant members of the bacterioplankton community in a set of 10 small, thermally stratified lakes in northeastern Indiana were determined by denaturing gradient gel electrophoresis (DGGE) of a polymerase chain reaction amplified fragment of 16S rDNA. The variability in community composition was analyzed as function of vertical stratification (epilimnion vs metalimnion), time (July vs August samples), and geographical location. In 58 discrete samples, a range of 8-23 bands were detected (mean = 14, s.d. = 4). For all variables, sample pairs shared about 40-70% of bands. In comparisons between depth strata, pairs of oxic samples shared more bands than an oxic-anoxic pair. There was no obvious relationship between the geographical location of lakes (or their physical connection) and band sharing.http://link.springer-ny.com/link/service/journals/00248/bibs/38n2p126.html

Microbial biomass and activity in lead-contaminated soil

Microbial community diversity, potential microbial activity, and metal resistance were determined in three soils whose lead contents ranged from 0.00039 to 48 mmol of Pb kg of soil-1. Biomass levels were directly related to lead content. A molecular analysis of 16S rRNAs suggested that each soil contained a complex, diverse microbial community. A statistical analysis of the phospholipid fatty acids indicated that the community in the soil having the highest lead content was not related to the communities in the other soils. All of the soils contained active microbial populations that mineralized [14C]glucose. In all samples, 10 to 15% of the total culturable bacteria were Pb resistant and had MIC of Pb for growth of 100 to 150 &mgr;M.

Parallel and divergent genotypic evolution in experimental populations of Ralstonia sp.

Genetic rearrangements within a population of bacteria were analyzed to understand the degree of divergence occurring after experimental evolution. We used 18 replicate populations founded from Ralstonia sp. strain TFD41 that had been propagated for 1,000 generations with 2,4-dichlorophenoxyacetic acid (2,4-D) as the carbon source. Genetic divergence was examined by restriction fragment length polymorphism analysis of the incumbent plasmid that carries the 2,4-D catabolic genes and by amplification of random regions of the genome via PCR. In 18 evolved clones examined, we observed duplication within the plasmid, including the tfdA gene, which encodes a 2,4-D dioxygenase that catalyzes the first step in the 2,4-D catabolic pathway. In 71 of 72 evolved clones, a common 2.4-kb PCR product was lost when genomic fingerprints produced by PCR amplification using degenerate primers based on repetitive extragenic palindromic (REP) sequences (REP-PCR) were compared. The nucleotide sequence of the 2.4-kb PCR product has homology to the TRAP (tripartite ATP-independent periplasmic) solute transporter gene family. Hybridization of the 2. 4-kb REP-PCR product from the ancestor to genomic DNA from the evolved populations showed that the loss of the PCR product resulted from deletions in the genome. Deletions in the plasmid and presence and/or absence of other REP-PCR products were also found in these clones but at much lower frequencies. The common and uncommon genetic changes observed show that both parallel and divergent genotypic evolution occurred in replicate populations of this bacterium.

The cis-diol dehydrogenase cbaC gene of Tn5271 is required for growth on 3-chlorobenzoate but not 3,4-dichlorobenzoate.

The nucleotide sequence of cbaC, the 1-carboxy-3-chloro-4,5-dihydroxycyclohexa-2,6-diene (cis-diol) dehydrogenase gene from the 3-chlorobenzoate (3-Cba) catabolic transposon Tn5271 was determined. The functional significance of the deduced open reading frame was evaluated by deletion of an internal BstEII restriction site in cbaC and by the creation of nested deletions using exonuclease III. Expression studies were carried out with Alcaligenes sp. strain BR6024, a chloramphenicol-resistant, tryptophan auxotroph derived from the wild-type isolate BR60. BR6024 hosts carrying complete cbaAB (3-Cba 3,4-(4,5)-dioxygenase and reductase) genes, with deletions of cbaC, metabolized 3Cba to the cis-4,5-diol metabolite. These mutants failed to grow on 3-Cba; however, they grew on 3,4-dichlorobenzoate, accumulating 5-chloroprotocatechuate transiently. These results indicated the cbaC dehydrogenase was not required for re-aromatization of the unstable 3,4-dCba cis-4,5-diol metabolite. Spontaneous elimination of HCl from this metabolite is proposed to generate 5-chloroprotocatechuate, which is a substrate for the protocatechuate metaring fission pathway in Alcaligenes sp. BR60. The relationship of the deduced amino acid sequence of cbaC with 15 other oxidoreductases and sequences of unknown function from bacteria, plants and animals revealed a conserved N-terminal GxxGxG dinucleotide-binding domain and a conserved region with a H(x11)KHVLxEKPxA consensus flanked by alpha-helical domains. o-Phthalate cis-diol dehydrogenase (Pseudomonas putida), glucose-fructose oxidoreductase (Zymomonas mobilis), myo-inositol-2-dehydrogenase (Bacillus subtilis) and D-galactose-1-dehydrogenase (Pseudomonas fluorescens) are related proteins. These dehydrogenases are unrelated to the Type I, II and III dehydrogenase superfamilies that include the cis-diol dehydrogenases involved in benzoate, toluene, biphenyl and naphthalene catabolism (Type II) and benzene catabolism (Type III).

Evidence for Acquisition in Nature of a Chromosomal 2,4-Dichlorophenoxyacetic Acid/(alpha)-Ketoglutarate Dioxygenase Gene by Different Burkholderia spp.

We characterized the gene required to initiate the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by the soil bacterium Burkholderia sp. strain TFD6, which hybridized to the tfdA gene of the canonical 2,4-D catabolic plasmid pJP4 under low-stringency conditions. Cleavage of the ether bond of 2,4-D by cell extracts of TFD6 proceeded by an (alpha)-ketoglutarate-dependent reaction, characteristic of TfdA (F. Fukumori and R. P. Hausinger, J. Bacteriol. 175:2083-2086, 1993). The TFD6 tfdA gene was identified in a recombinant plasmid which complemented a tfdA transposon mutant of TFD6 created by chromosomal insertion of Tn5. The plasmid also expressed TfdA activity in Escherichia coli DH5(alpha), as evidenced by enzyme assays with cell extracts. Sequence analysis of the tfdA gene and flanking regions from strain TFD6 showed 99.5% similarity to a tfdA gene cloned from the chromosome of a different Burkholderia species (strain RASC) isolated from a widely separated geographical area. This chromosomal gene has 77.2% sequence identity to tfdA from plasmid pJP4 (Y. Suwa, W. E. Holben, and L. J. Forney, abstr. Q-403, in Abstracts of the 94th General Meeting of the American Society for Microbiology 1994.). The tfdA homologs cloned from strains TFD6 and RASC are the first chromosomally encoded 2,4-D catabolic genes to be reported. The occurrence of highly similar tfdA genes in different bacterial species suggests that this chromosomal gene can be horizontally transferred.