Mineralization of the dibenzothiophene biodegradation products 3-hydroxy-2-formyl benzothiophene and dibenzothiophene sulfone.

Dibenzothiophene is degraded to 3-hydroxy-2-formyl benzothiophene by various bacteria, including a strain of Pseudomonas putida that also forms dibenzothiophene sulfone via an alternate pathway. By using these end products as substrates, mixed enrichment cultures that could degrade 3-hydroxy-2-formyl benzothiophene and dibenzothiophene sulfone with the formation of CO2 were established.     

DNA amplification to enhance detection of genetically engineered bacteria in environmental samples.

The polymerase chain reaction (PCR) was performed to amplify a 1.0-kilobase (kb) probe-specific region of DNA from the herbicide-degrading bacterium Pseudomonas cepacia AC1100 in order to increase the sensitivity of detecting the organism by dot-blot analysis. The 1.0-kb region was an integral portion of a larger 1.3-kb repeat sequence which is present as 15 to 20 copies on the P. cepacia AC1100 genome. PCR was performed by melting the target DNA, annealing 24-base oligonucleotide primers to unique sequences flanking the 1.0-kb region, and performing extension reactions with DNA polymerase. After extension, the DNA was again melted, and the procedure was repeated for a total of 25 to 30 cycles. After amplification the reaction mixture was transferred to nylon filters and hybridized against radiolabeled 1.0-kb fragment probe DNA. Amplified target DNA was detectable in samples initially containing as little as 0.3 pg of target. The addition of 20 micrograms of nonspecific DNA isolated from sediment samples did not hinder amplification or detection of the target DNA. The detection of 0.3 pg of target DNA was at least a 10(3)-fold increase in the sensitivity of detecting gene sequences compared with dot-blot analysis of nonamplified samples. PCR performed after bacterial DNA was isolated from sediment samples permitted the detection of as few as 100 cells of P. cepacia AC1100 per 100 g of sediment sample against a background of 10(11) diverse nontarget organisms; that is, P. cepacia AC1100 was positively detected at a concentration of 1 cell per g of sediment. This represented a 10(3)-fold increase in sensitivity compared with nonamplified samples.     

Plasmid Frequency Fluctuations in Bacterial Populations from Chemically Stressed Soil Communities

The frequency of plasmids in chemically stressed bacterial populations was investigated by individually adding various concentration of kanamycin, ampicillin, and mercuric chloride to soil samples. Viable bacterial populations were enumerated, soil respiration was monitored for up to 6 weeks as an indicator of physiological stress, and bacterial isolates from stressed and control soils were screened for the presence of plasmids. Low levels of the chemical stress factors did not for the most part significantly alter population viability, soil respiration, or plasmid frequency. Exposure to high stress levels of mercury and ampicillin, however, resulted in altered numbers of viable organisms, soil respiration, and plasmid frequency. Plasmid frequency increased in response to ampicillin exposure but was not significantly changed after exposure to kanamycin. In mercuric chloride-stressed soils, there was a decrease in plasmid frequency despite an increase in overall mercury resistance of the isolates, suggesting that mercury resistance in these populations is largely, if not completely, chromosome encoded. Chemical stress did not cause an increase in plasmid-mediated multiple resistance. A genetic response (change in plasmid frequency) was not found unless a physiological (phenotypic) response (change in viable cells and respiratory activity) was also observed. The results indicate that a change in plasmid frequency is dependent on both the amount and type of chemical stress.     

Chimpanzee fetal G gamma and A gamma globin gene nucleotide sequences provide further evidence of gene conversions in hominine evolution

The fetal globin genes G gamma and A gamma from one chromosome of a chimpanzee (Pan troglodytes) were sequenced and found to be closely similar to the corresponding genes of man and the gorilla. These genes contain identical promoter and termination signals and have exons 1 and 2 separated by the conserved short intron 1 (122 bp) and exons 2 and 3 separated by the more rapidly evolving, larger intron 2 (893 bp and 887 bp in chimpanzee G gamma and A gamma, respectively). Each intron 2 has a stretch of simple sequence DNA (TG)n serving possibly as a "hot spot" for recombination. The two chimpanzee genes encode polypeptide chains that differ only at position 136 (glycine in G gamma and alanine in A gamma) and that are identical to the corresponding human chains, which have aspartic acid at position 73 and lysine at 104 in contrast to glycine and arginine at these respective positions of the gorilla A gamma chain. Phylogenetic analysis by the parsimony method revealed four silent (synonymous) base substitutions in evolutionary descent of the chimpanzee G gamma and A gamma codons and none in the human and gorilla codons. These Homininae (Pan, Homo, Gorilla) coding sequences evolved at one-tenth the average mammalian rate for nonsynonymous and one-fourth that for synonymous substitutions. Three sequence regions that were affected by gene conversions between chimpanzee G gamma and A gamma loci were identified: one extended 3' of the hot spot with G gamma replaced by the A gamma sequence, another extended 5' of the hot spot with A gamma replaced by G gamma, and the third conversion extended from the 5' flanking to the 5' end of intron 2, with G gamma replaced here by the A gamma sequence. A conversion similar to this third one has occurred independently in the descent of the gorilla genes. The four previously identified conversions, labeled C1-C4 (Scott et al. 1984), were substantiated with the addition of the chimpanzee genes to our analysis (C1 being shared by all three hominines and C2, C3, and C4 being found only in humans). Thus, the fetal genes from all three of these hominine species have been active in gene conversions during the descent of each species.      

Legionella contamination of dental-unit waters.

Water samples collected from 28 dental facilities in six U.S. states were examined for the presence of Legionella pneumophila and other Legionella spp. by the PCR-gene probe, fluorescent-antibody microscopic, and viable-plate-count detection methods. The PCR and fluorescent-antibody detection methods, which detect both viable and viable nonculturable Legionella spp., gave higher counts and rates of detection than the plate count method. By the PCR-gene probe detection method, Legionella spp. were detected in 68% of the dental-unit water samples and L. pneumophila was detected in 8%. Concentrations of Legionella spp. in dental-unit water reached 1,000 organisms per ml or more in 36% of the samples, and 19% of the samples were in the category of 10,000/ml or above. L. pneumophila, when present in dental-unit water, never reached concentrations of 1,000/ml or more. Microscopic examination with fluorescent-antibody staining indicated that the contamination was in the dental-unit water lines rather than in the handpieces. Legionella spp. were present in 61% of potable water samples collected for comparative analysis from domestic and institutional faucets and drinking fountains; this percentage was not significantly different from the rate of detection of Legionella spp. in dental-unit water. However, in only 4% of the potable water samples did Legionella spp. reach concentrations of 1,000 organisms per ml, and none was in the 10,000 organisms-per-ml category, and so health-threatening levels of Legionella spp. in potable water were significantly lower than in dental-unit water. L. pneumophila was found in 2% of the potable water samples, but only at the lowest detectable level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monitoring genetically engineered microorganisms in freshwater microcosms

Summary The effectiveness of gene probe methods for tracking genetically engineered microorganisms (GEMs) in the environment was tested by inoculating nutrient-supplemented freshwater microcosms withAlcaligenes A5 (a naturally occurring 4-chlorobiphenyl degrader) orPseudomonas cepacia AC1100 (a genetically engineered 2, 4, 5 T-degrader) and following the fates of the introduced bacterial populations. Colony hybridization of the viable heterotrophic bacterial populations and dot blot hybridization of DNA recovered from the total microcosm microbial communities showed persistence of bothAlcaligenes A5 andP. cepacia AC1100 in the microcosms in the presence and absence of the xenobiotic substrates that these organisms biodegrade. Although there was a gradual decline in the added populations, both of the bacterial populatins were still detected in the microcosms two months after their introduction into the microcosms. Addition of 2, 4, 5-T enhanced the survival ofP. cepacia AC1100 — and 4-chlorobiphenyl addition resulted in increased levels ofAlcaligenes A5. The results indicate that both organisms may persist for very long periods in freshwater habitats.     

On the active site of elastase: Partial mapping by means of specific peptide substrates

RNase-S peptide as well as some related octa- and hexapeptides were found to be highly, reactive substrates of porcine elastase (e.g. Ala(4)-Lys-Phe: K(m) = 4500 M(-1), k(cat) = 32 sec(-1), C = 1.4 x 10(5) M(-1) sec(-1)). Comparison of the various peptides led to the conclusion that the active site of porcine elastase is composed of 6-7 subsites (c.f. [1]). Preliminary mapping shows that subsites S(2), S'(1) and S'(2) have hydrophobic character. Occupation of subsite S(4) by the substrate is important for efficient hydrolysis. Binding at this subsite was found to be stereospecific.     

Role of increased glomerular filtration rate in atrial natriuretic factor-induced natriuresis in the rat

One of the major renal hemodynamic actions of atrial natriuretic factor (ANF) is to increase glomerular filtration rate (GFR). To assess the role of this effect on ANF-induced natriuresis (UNaV), diuresis (V) and kaliuresis (UKV) we performed late clamp experiments in six rats. After control periods (C), synthetic ANF (auriculin A) was infused i.v. (2 micrograms X min-1/kg body wt) throughout the experiment (150 min). After pre-clamp periods, the perfusion pressure of the left kidney (LK) was reduced to 75-80 mmHg. The right kidney (RK) served as a time control. In LK, before the late clamp, ANF increased (p less than 0.01) GFR from 1.5 +/- 0.1 to 1.8 +/- 0.1 ml/min, V from 17 +/- 5 to 53 +/- 5 microliters/min, and UNaV from 2.1 +/- 0.6 to 10.0 +/- 0.9 microEq/min. Almost identical increases occurred in the RK. The late clamp returned all parameters in LK to C values (p greater than 0.05): GFR to 1.4 +/- 0.1 ml/min, V to 6.3 +/- 1.2 microliter/min, and UNaV to 1.0 +/- 0.3 microEq/min. The late clamp also reversed the ANF-induced increase in UKV. In the RK, GFR (1.8 +/- 0.1 ml/min), V (38 +/- 4 microliter/min) and UNaV (7.8 +/- 0.8 microEq/min) remained elevated (p less than 0.01 vs. C) to the end of the experiment. These data demonstrate that upon return of GFR to control levels, the ANF-induced diuresis, natriuresis and kaliuresis is abolished. The results support our previous view that the increase in GFR together with a decrease in inner-medullary hypertonicity account wholly or in great part for the natriuretic action of ANF.     

Denitrification and Nitrogen Fixation in Alaskan Continental Shelf Sediments

Rates of nitrogen fixation and denitrification were measured in Alaskan continental shelf sediments. In some regions, rates of nitrogen fixation and denitrification appeared to be equal; in other areas, rates were significantly different. Potential rates of denitrification were found to be limited primarily by the available nitrate substrate. Major regional differences in rates of denitrification were not statistically significant, but significant differences were found for nitrogen fixation rates in different regions of the Alaskan continental shelf. Estimated net losses of nitrogen from Bering Sea sediments were calculated as 1.8 × 10¹² g of N/yr. Experimental exposure of continental shelf sediments to petroleum hydrocarbons reduced rates of nitrogen fixation and denitrification in some cases but not others. Long-term exposure was necessary before a reduction in nitrogen fixation rates was observed; unamended rates of denitrification but not potential denitrification rates (NO₃⁻ added) were depressed after exposure to hydrocarbons.