Spatial and Temporal Variation of Enterobacter Genotypes in Sediments and the Underlying Hyporheic Zone of an Agricultural Stream.

Population studies of enteric bacteria in an agriculturally impacted stream (Ledbetter Creek, Murray, Kentucky, USA) were conducted over a period of 2 years. Total number of bacteria, cultivated heterotrophic aerobic bacteria, and enteric bacteria showed significant differences between winter and summer. The cultivated numbers of heterotrophic aerobic bacteria and enteric bacteria were significantly more abundant in summer than in winter. The abundance of enteric bacteria was 12.9% in an upwelling zone and 9.8% in a downwelling zone in summer. Most of the enteric bacterial strains isolated on MacConkey agar were assigned to Enterobacter cloacae and E. agglomerans by API 20E and an analysis of the restriction patterns produced by amplified DNA coding for 16S rRNA (ARDRA) with the enzyme Hpa II. E. cloacae and E. agglomerans genotypes isolated from three hyporheic and gravel bar depth intervals (0-10 cm, 15-25 cm, and 30-40 cm) in summer and fall showed significant spatial variation and were heterogeneously distributed along the stream. Temperature, inorganic nutrients, and occurrence of anoxic zones affected the distribution of enteric bacteria. These techniques can be used as a model to monitor shifts among different species in the stream ecosystem.

Diversity of culturable heterotrophic aerobic bacteria in pristine stream bed sediments.

More than 900 culturable, heterotrophic aerobic isolates were obtained from the sediments of a forested, pristine stream and analyzed using three classical microbiological tests: API 20E, amplified ribosomal DNA restriction analysis (ARDRA), and fatty acid analysis. Gram-negative bacteria comprised most of the heterotrophic aerobic isolates (66.7%), similar to other oligotrophic environments. The isolates were assigned to the genus level as Pseudomonas, Flavobacterium, Micrococcus, Bacillus, Chromobacterium, Acinetobacter, Alcaligenes, Aeromonas, Methylobacterium, Enterobacter, Corynebacterium, and Sporolactobacillus. Genotypic analysis by ARDRA facilitated the comparison among strains within Pseudomonas, Bacillus, and Enterobacter groups. Temperature and predation may influence the survival of bacteria during seasons, as shown previously by others. Our results showed that the number of heterotrophic aerobic bacteria, especially Enterobacter, Alcaligenes, and Aeromonas, and Gram-positive bacteria, decreased in winter compared to summer conditions.

The nucleotide sequence of the luxA and luxB genes of Xenorhabdus luminescens HM and a comparison of the amino acid sequences of luciferases from four species of bioluminescent bacteria.

The luxA and luxB genes of bioluminescent bacteria encode the alpha and beta subunits of luciferase, respectively. Sequences of the luxA and luxB genes of Xenorhabdus luminescens, the only terrestrial bioluminescent bacterium known, were determined and the amino acid sequence of luciferase deduced. The alpha subunit was found to contain 360 amino acids and has a calculated molecular weight of 41,005 Da, while the beta subunit contains 327 amino acids and has a calculated molecular weight of 37,684 Da. Alignment of this luciferase with the luciferases of three marine bacteria showed 196 (or 55%) conserved residues in the alpha subunit and 114 (or 35%) conserved residues in the beta subunit. The highest degree of homology between any two species was between the luciferases of X. luminescens and Vibrio harveyi with 84% identity in the alpha subunits and 59% identity in the beta subunits.

The nucleotide sequence of the luxE gene of Vibrio harveyi and a comparison of the amino acid sequences of the acyl-protein synthetases from V. harveyi and V. fischeri.

The luxE gene of bioluminescent bacteria encodes the acyl-protein synthetase component of the fatty acid reductase complex. The complex is responsible for converting tetradecanoic acid to the aldehyde which serves as a substrate in the luciferase-catalyzed reaction. The nucleotide sequence of the luxE gene of Vibrio harveyi was determined and the amino acid sequence of the acyl-protein synthetase deduced. The protein consists of 378 amino acid residues and has a molecular weight of 42,965 daltons. Alignment of the V. harveyi enzyme with the V. fischeri acyl-protein synthetase showed 62% identity.

Polypeptide folding and dimerization in bacterial luciferase occur by a concerted mechanism in vivo.

Bacterial luciferase is a heterodimeric enzyme comprising two nonidentical but homologous subunits, alpha and beta, encoded by adjacent genes, luxA and luxB. The two genes from Vibrio harveyi were separated and expressed from separate plasmids in Escherichia coli. If both plasmids were present within the same E. coli cell, the level of accumulation of active dimeric luciferase was not dramatically less than within cells containing the intact luxAB sequences. Cells carrying the individual plasmids accumulated large amounts of individual subunits, as evidenced by two-dimensional polyacrylamide gel electrophoresis. Mixing of a lysate of cells carrying the luxA gene with a lysate of cells carrying the luxB gene resulted in formation of very low levels of active heterodimeric luciferase. However, denaturation of the mixed lysates with urea followed by renaturation resulted in formation of large amounts of active luciferase. These observations demonstrate that the two subunits, alpha and beta, if allowed to fold independently in vivo, fold into structures that do not interact to form active heterodimeric luciferase. The encounter complex formed between the two subunits must be an intermediate structure on the pathway to formation of active heterodimeric luciferase.

Nucleotide sequence of the luxB gene of Vibrio harveyi and the complete amino acid sequence of the beta subunit of bacterial luciferase.

The nucleotide sequence of the 1.30-kilobase EcoRI/BglII fragment from Vibrio harveyi carrying the majority of the luciferase beta subunit coding region (luxB gene) has been determined. The EcoRI/BglII fragment was derived from a 4.0-kilobase HindIII fragment carrying both luxA and luxB which was detected in a genomic clone bank based on the expression of bioluminescence from colonies of Escherichia coli carrying V. harveyi HindIII fragments in plasmid pBR322 (Baldwin, T. O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., and Ziegler, M. M. (1984) Biochemistry 23, 3663-3667). The entire alpha subunit coding sequence (luxA gene) and the amino-terminal 13 codons of the beta subunit sequence (luxB gene) were contained on a 1.85-kilobase EcoRI fragment, the sequence of which has been reported (Cohn, D. H., Mileham, A. J., Simon, M. I., Nealson, K. H., Rausch, S. K., Bonam, D., and Baldwin, T. O. (1985) J. Biol. Chem. 260, 6139-6146). The beta subunit coding sequence was found to terminate 972 bases past the start of the luxB coding sequence. The beta subunit had a calculated molecular weight of 36,349 and comprised a total of 324 amino acid residues; the alpha beta dimer had a molecular weight (alpha + beta) of 76,457. There were 27 base pairs separating the stop codon of the beta subunit structural gene and a 340-base open reading frame extending to (and beyond) the distal BglII site. Approximately two-thirds of the beta subunit was sequenced by protein chemical techniques. The amino acid sequence predicted from the DNA sequence, with few exceptions, confirmed the chemically determined sequence, and the measured amino acid composition was in excellent agreement with the composition implied from the DNA sequence.

Encephalic microvascular abnormalities in hypertension. A light and X-ray microscopic study.

Brain and cervical spinal cord removed at autopsy from hypertensive humans have been subjected to 4 levels of microscopic study, 3 being first histochemically prepared so as to stain the arterial and capillary system. Thick (100 microns) sections for light microscopy and thicker (500-1000 microns) for x-ray microscopy facilitate the study of long, complex anatomic units. Arterial and arteriolar microaneurysms have not been found. In addition to the usual age related vascular changes in normal people we have found: moniliform capillary dilatations, focal segments of arteriolar narrowing, état pre-criblé, and lacunar infarcts. Few changes have been encountered in the corpus callosum. While the segmental arteriolar narrowing occurs in the cortex as well as deep white matter, the effects of resultant poor perfusion might be expected to be more profound in the deep white matter.

Streptomycin-induced, third-position misreading of the genetic code.

Streptomycin was used to increase the frequency of errors in protein synthesis in vivo. In the system under study two misreading errors were observed. Both involved the erroneous insertion of lysine at asparagine codons, because of misreading of a pyrimidine as a purine at the 3' position of the codon. Streptomycin increased the errors at the two codons AAU and AAC to the same extent, thereby maintaining the error ratio found for basal level mistranslation.

Codon specificity of starvation induced misreading.

Mistranslated derivatives of the coat protein of the bacteriophage MS2 were isolated from infected cells starved for asparagine. This protein contains a high level of lysine for asparagine substitutions. By peptide analysis and amino acid sequencing we show that there is a six-fold greater frequency of errors at AAU codons than at AAC codons. This ratio is the same as that found in unstarved cells where the overall error frequency is 100-fold less. We also demonstrate that, at least for AAC codons, context affects error frequency.

Codon usage and mistranslation. In vivo basal level misreading of the MS2 coat protein message.

The coat protein of the small RNA virus MS2 shows charge heterogeneity in vivo. In most strains there is a basic satellite of the native protein. We have shown that this basic satellite is greatly diminished or absent in strains with the streptomycin-resistant allele, rpsL, a mutation which leads to increased translational accuracy. Further, the satellite is present in cells where the coat protein is encoded by duplex DNA. Tryptic digests of the satellite show that it contains new lysine-containing peptides which appear to be the same as those found in derivatives of coat protein which have a lysine for asparagine substitution. Sequencing of the NH2-terminal 19 amino acids of the satellite protein shows that the asparagine codon AAU at amino acid 12 is misread approximately 8 times more frequently than the AAC at amino acid 3. We conclude that the satellite species is the result of basal level lysine for asparagine substitution. These substitutions are most likely caused by preferential misreading of AAU codons at a frequency of approximately 5 X 10(-3), 10-fold higher than the average error frequency.

Mistranslation in cells infected with the bacteriophage MS2: direct evidence of Lys for Asn substitution.

The coat protein of the bacteriophage MS2 was found to show an increased level of charge heterogeneity when synthesized in Escherichia coli starved for Asn or Lys. No such increase was found when the host was starved for Arg, His Ile or Pro. This is the pattern predicted by "two-out-of-three" codon misreading in the coat protein gene. In the case of Asn starvation, direct measurements of the relative incorporation of Lys demonstrate that the observed charge heterogeneity is the result of mistranslation. Asn starvation increased the error frequency in coat protein to over 0.3 mistake per asparagine codon. The small amount of charge heterogeneity seen in unstarved cells seems also to be the result of misreading Asn codons.

Studies on the mechanism of estradiol uptake by rat uterine cells and on estradiol binding to uterine plasma membranes.

A method for the preparation of viable uterine cell suspensions is described. Using this system the kinetics of estradiol uptake were studied in order to asses whether the steroid enters uterine cells by passive diffusion (1) or protein mediated diffusion (2). The data presented show that a) the rates of estradiol entry are nonsaturable; b) temperature dependence of uptake kinetics gives a linear Arrhenius plot; c) E2-6-CMO-BSA does not inhibit 3H-E2 uptake; d) uterine plasma membranes do not contain specific estrogen binding sites. Thus, estrogen uptake occures by passive diffusion.