Transition from cometabolic to growth-linked biodegradation of vinyl chloride by a Pseudomonas sp. isolated on ethene.

Pseudomonas aeruginosa strain DL1 was isolated on ethene as a sole carbon and energy source. When ethene-grown DL1 was first exposed to vinyl chloride (VC), the rate of VC consumption was very rapid and then declined sharply, indicative of a cometabolic process. A lack of growth and significant release of soluble products during this interval also indicates that the initial activity on VC was cometabolic. Following the rapid initial rate of VC cometabolism, a slow rate of VC utilization continued. After an extended period of incubation (>40 days), a transition occurred that allowed DL1 to begin using VC as a primary growth substrate, with an observed yield, maximum growth rate, and Monod half saturation coefficient of 0.21 mg of total suspended solids/mg VC, 0.046 d(-1), and 1.17 microM VC, respectively, at 22 degrees C. Acetylene inhibits consumption of ethene and VC by ethene-grown cells, suggesting a monooxygenase is responsible for initiating metabolism of these alkenes. Resting cells grown on ethene cometabolized VC with an observed transformation capacity of 9.1 micromol VC/mg total suspended solids and a transformation yield of 0.22 mol VC/mol ethene. The presence of 40 microM ethene increased the rate and amount of VC cometabolized. However, consumption of higher concentrations of ethene decreased the total amount of VC consumed, and VC inhibited ethene utilization. A kinetic model was developed that describes substrate interactions during batch depletion of ethene and VC for a range of initial concentrations. The results suggest that ethene may stimulate in situ biodegradation of VC either by functioning as a primary substrate to support cometabolism of VC or by selecting for organisms that can utilize VC as a primary substrate.

A rapid procedure for isolating chromosomal DNA from Lactobacillus species and other Gram-positive bacteria.

This study describes a rapid procedure for the isolation of genomic DNA from various Gram-positive bacteria. Species tested included Lactobacillus delbrueckii subsp. lactis ATCC 4797, Lact. acidophilus N2, Staphylococcus aureus, Staph. epidermidis, Propionibacterium jensenii P126, Bacillus pumilus and Enterococcus faecalis. Our technique for chromosomal DNA isolation circumvents the need for phenol-chloroform extractions and caesium chloride gradients. Isolated DNA is consistently greater than 25 kb in size and can be used directly for subcloning, polymerase chain reaction amplification, restriction digestions and library construction.

Cloning and expression analysis of the 28 kDa protein from Lactobacillus delbrueckii subsp. lactis ATCC 4797 hypothesized to influence lactacin B production.

AIMS: A cell wall-associated lactacin B inducer protein (IP) was purified from Lactobacillus delbrueckii subsp. lactis ATCC 4797 (Lact. lactis) by chromatofocusing and gel filtration HPLC (Barefoot et al. 1994). METHODS AND RESULTS: N-terminal sequence of the purified IP was used to design an oligonucleotide (24-mer) for gene identification by Southern and colony hybridizations. Southern hybridization on Lact. lactis chromosomal DNA digested with EcoRI and PstI produced a single 4-5 kbp DNA fragment. Colony hybridizations with 6250 clones produced four positive recombinants for the proposed IP. Sequence of the DNA isolated from RU43e9 revealed a 4623 bp DNA fragment containing three open reading frames (ORF) potentially encoding enzymes that function in glycolysis. One ORF, coding for an active triosephosphate isomerase (Tpi), showed 98% homology to the N-terminal domain of the HPLC purified IP. PCR primers were designed to amplify the ORF encoding the proposed IP for subcloning, protein expression, purification and bacteriocin enhancing assays on pure cultures of Lactobacillus acidophilus N2. CONCLUSIONS: The regions flanking the Tpi gene (data not shown) were also sequenced and it is concluded that the proposed IP reported by Barefoot et al. (1994) is located on an operon containing several glycolytic enzymes that function in glycolysis. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this study do not support previously published research (Barefoot et al. 1994) hypothesizing that a purified IP from Lact. lactis, homologous to a Bacillus stearothermophilus Tpi, is capable of enhancing bacteriocin synthesis in Lact. acidophilus N2.

Characterization of an isolate that uses vinyl chloride as a growth substrate under aerobic conditions.

An aerobic enrichment culture was developed by using vinyl chloride (VC) as the sole organic carbon and electron donor source. VC concentrations as high as 7.3 mM were biodegraded without apparent inhibition. VC use did not occur when nitrate was provided as the electron acceptor. A gram-negative, rod-shaped, motile isolate was obtained from the enrichment culture and identified based on biochemical characteristics and the sequence of its 16S rRNA gene as Pseudomonas aeruginosa, designated strain MF1. The observed yield of MF1 when it was grown on VC was 0.20 mg of total suspended solids (TSS)/mg of VC. Ethene, acetate, glyoxylate, and glycolate also served as growth substrates, while ethane, chloroacetate, glycolaldehyde, and phenol did not. Stoichiometric release of chloride and minimal accumulation of soluble metabolites following VC consumption indicated that the predominant fate for VC is mineralization and incorporation into cell material. MF1 resumed consumption of VC after at least 24 days when none was provided, unlike various mycobacteria that lost their VC-degrading ability after brief periods in the absence of VC. When deprived of oxygen for 2.5 days, MF1 did not regain the ability to grow on VC, and a portion of the VC was transformed into VC-epoxide. Acetylene inhibited VC consumption by MF1, suggesting the involvement of a monooxygenase in the initial step of VC metabolism. The maximum specific VC utilization rate for MF1 was 0.41 micromol of VC/mg of TSS/day, the maximum specific growth rate was 0.0048/day, and the Monod half-saturation coefficient was 0.26 microM. A higher yield and faster kinetics occurred when MF1 grew on ethene. When grown on ethene, MF1 was able to switch to VC as a substrate without a lag. It therefore appears feasible to grow MF1 on a nontoxic substrate and then apply it to environments that do not exhibit a capacity for aerobic biodegradation of VC.

Comparison of mean skin temperature using 'covered' versus 'uncovered' contact thermistors.

The purpose of this study was to compare the mean skin temperature (Tsk) obtained using 'covered' versus 'uncovered' contact thermistors in a variety of environmental conditions. Ten male subjects walked on a treadmill at 6.4 kph for 30 min in three different environments: thermoneutral (23 degrees C/50% RH), hot-humid (35 degrees C/80%), and hot-dry (42 degrees C/20%). Tsk was measured using three 'uncovered' contact thermistors, and three 'covered' thermistors. The 'uncovered' probes were attached using acrylic rings that allowed the outer surface of the thermistors to have free exchange with the environment. The 'covered' probes were affixed to the skin using foam patches and tape. The latter procedure is commonly used in many research and clinical laboratories. The 'covered' probes resulted in a significantly (p < 0.05) higher Tsk under all three environmental conditions. The largest difference occurred in the thermoneutral condition with the 'covered' Tsk being 1.3 degrees C higher than the 'uncovered' Tsk. These results suggest that covering a contact skin thermistor hinders heat loss and results in an artificially high Tsk. It is hoped that the results of this study will encourage the adoption of 'uncovered' contact skin thermistors in the future.