UV absorption complicates PCR decontamination.

UV irradiation is widely used to inactivate contaminating DNA in PCR. Highly UV-absorbent deoxyribonucleoside triphosphates in PCR mixtures reduce the efficiency of UV decontamination. Optimal decontamination may be achieved by irradiating the PCR mixture without the deoxyribonucleoside triphosphates.

Hyperamylasemia and its relationship to binge-purge episodes: development of a clinically relevant laboratory test.

Hyperamylasemia and parotid hypertrophy are conditions found in bulimic patients. The authors studied serum amylase levels in 56 underweight anorectics, 24 weight-recovered anorectics, 23 normal-weight bulimics, and 31 volunteer women. Normal-weight bulimic patients had significantly higher admission serum amylase values (mean +/- SE = 73.4 +/- 8.0 IU/L) than controls (40.8 +/- 2.4 IU/L). Additionally, the serum amylase test distinguished between restrictor anorectics (N = 31, 44.7 +/- 4.7 IU/L) and bulimic anorectics (N = 25, 68.8 +/- 8.6 IU/L, p less than .05) with a high degree of specificity and a fair degree of sensitivity. A subsample (N = 7) of bulimics showed twofold to fourfold increases in serum amylase values after a controlled period of binge eating and vomiting, whereas normal volunteers showed no change in serum amylase values (p less than .001) after ingesting a large meal. Serum amylase values declined significantly within 6 to 15 days of admission. However, after passes off the unit, serum amylase values returned to admission values, presumably as a result of binge-vomit episodes. The authors observe that modest increases of serum amylase values appear to be a consequence of binge-vomit behavior and suggest that serial serum amylase determination may be useful in monitoring the degree of patient abstinence in therapeutic programs.

Methionine limitation in Escherichia coli K-12 by growth on the sulfoxides of D-methionine.

The synthesis and resolution of the diastereoisomers of d-methionine sulfoxide in high yield is described. Growth of two methionine auxotrophs (strains HfrC and AB1932) on the d-methionine sulfoxides is slower than on l-methionine, and the resultant cells are markedly derepressed for three enzymes of the methionine regulon (cystathionine synthetase, cystathionase, and S-adenosyl-l-methionine synthetase). Strain HfrC grows more rapidly on the sulfoxides and shows less derepression than strain AB1932. Although growth on d-methionine-d-sulfoxide is much slower than on d-methionine-l-sulfoxide (two- to threefold increase in division times), cells grown on d-methionine-l-sulfoxide generally have higher enzyme activities. The sulfoxides of d-methionine appear to provide a useful supplement to obtain methionine-limited growth in Escherichia coli.

Regulation of methionine biosynthesis in Escherichia coli: mapping of the metJ locus and properties of a metJ plus-metJ minus diploid.

MetJ(-) mutants of Escherichia coli have elevated nonrepressible levels of the enzymes of methionine biosynthesis and S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6). In E. coli, as in Salmonella typhimurium, the metJ locus is close to metB (95% cotransduction of metB and metJ markers), but in E. coli the order is reversed, with metJ mapping clockwise to metB. A stable merodiploid, heterozygous for metJ, is subject to repression by methionine. Thus, metJ functions via a diffusible product. MetJ could either be a regulatory locus or could code for an enzyme required for the synthesis of a methionine metabolite that functions in the control system.

Role of methionine in the regulation of serine hydroxymethyltransferase in Eschericia coli.

Significant derepression of serine hydroxymethyltransferase is observed when metE or metF mutants of Escherichia coli K-12 are grown on D-methionine sulfoxide instead of L-methionine. The derepression is not prevented by addition of glycine, adenosine, guanosine, guanosine, and thymidine to the growth medium of methionine-limited metF cells showing that the effect is not due to a secondary deficiency of these nutrients. On the other hand, methionine-limited growth of a metA mutant leads to derepression of met regulon enzymes, but only a marginal increase in serine hydroxymethyltransferase activity. A prototrophic metJ strain grown on minimal medium has about the same serine hydroxymethyltransferase as the wild type. The enzyme activity of the metJ strain is not influenced by methionine, but it is partially repressed by glycine, adenosine, and thymidine. metK strains have about twice as much serine hydroxymethyltransferase activity as wild-type cells when grown on minimal medium; but when both types of cells are grown on medium supplemented with glycine, adenosine, guanosine, and thymidine, their enzyme activities are about the same. The results show that methionine limitation can lead to depression of serine hydroxymethyltransferase, but that the regulatory system is different from the one which controls the methionine regulon.

Insertion mutagenesis of the metJBLF gene cluster of Escherichia coli K-12: evidence for an metBL operon.

The effects of Mu or transposon 5 insertions on the expression of genes of the metJBLF cluster show that metB and metL form an operon, transcribed from metB to metL, and that metF and metJ are independently transcribed.

Cloning of the methionine regulatory gene, metJ, of Escherichia coli K12 and identification of its product.

Both wild-type and mutant forms of the methionine regulatory gene, metJ, of Escherichia coli K12 have been cloned in derivatives of pBR322. In cells carrying plasmids with a functional copy of metJ, the methionine regulon appears to be repressed even under conditions of methionine limitation. Maxicell labeling experiments show that the plasmids code for a small peptide (12 kilodaltons) only when they carry a functional copy of metJ. The lesions in five independently isolated metJ mutants are located in, or slightly upstream from, a coding sequence proposed to be metJ by Saint-Girons, I., Duchange, N., Cohen, G. N., and Zakin, M. M. [1984) J. Biol. Chem. 259, 14282-14285).

Regulation of in vivo transcription of the Escherichia coli K-12 metJBLF gene cluster.

We subcloned DNA of the intercistronic region between the divergently transcribed metJ and metB genes of Escherichia coli into the transcription-fusion vector pK01 and localized the metJ promoters by deletion analysis. The plasmid-borne promoters of both genes were repressed by chromosomal metJ. In addition, S1 nuclease mapping of chromosomally derived mRNA from a derepressed strain revealed the start sites of transcription for metBL, metF, and metJ. The metBL and metF genes each had a single transcript which was repressed by metJ, while the metJ gene had three transcripts, of which the first was strongly repressed by metJ, the second was less strongly repressed, and the third was not repressed.

Purification and properties of cystathionine gamma-synthase from overproducing strains of Escherichia coli.

To characterize the methionine biosynthetic enzyme cystathionine gamma-synthase from Escherichia coli, we have constructed high copy number plasmids containing the metB structural gene but lacking the closely linked metJ regulatory gene. When cloned into an appropriate strain, these plasmids can direct the overproduction of cystathionine gamma-synthase such that about 10% of the soluble protein is this enzyme. An efficient purification scheme has been developed that has allowed us to obtain gram quantities of enzyme. The active form is a tetramer with subunits of about 40,000 daltons and one pyridoxal phosphate cofactor per monomer. The kinetic constants for several enzyme-catalyzed reactions were determined at 25 degrees C. The Km value for the elimination reaction with O-succinyl-L-homoserine was calculated to be 0.33 mM with maximal velocity of 460 min-1. The Km for the elimination (deamination) reaction with vinylglycine was 5.6 mM with maximal velocity of 900 min-1. The Km values for the replacement reaction were calculated to be 1.0 mM for O-succinyl-L-homoserine and 0.05 mM for L-cysteine with maximal velocity of 700 min-1. The enzyme shows an absorption band at 422 nm (epsilon = 8463 M-1 cm-1) attributable to the Schiff base form of the pyridoxal phosphate cofactor. Steady-state spectra of reaction complexes show appearance of new longer wavelength absorbing materials during reaction with O-succinyl-L-homoserine, vinylglycine, or vinylglycine and L-cysteine. Reaction with O-succinyl-L-homoserine and L-cysteine produces only a red shift and slight reduction of the band at 422 nm.

Properties of metK mutants of Escherichia coli K-12.

Some of the properties of three metK mutants of Escherichia coli K-12 have been examined. All three strains have lower than normal levels of SAM (S-adenosyl-l-methionine) synthetase and elevated levels of cystathionine synthetase and cystathionase. One strain (RG73) appears to have an unstable SAM synthetase, suggesting that it carries a structural gene mutation. The two strains (RG62 and RG109) which have the lowest levels of SAM synthetase when grown on minimal medium have appreciably higher levels of enzyme when grown on complete medium. Growth on defined media supplemented with leucine or methionine causes a several-fold increase in the specific activity of SAM synthetase with associated decreases in cystathionine synthetase and cystathionase, but the changes are not as large as those seen in cells grown on LB broth. The SAM pools of strains RG62 and RG109 are markedly lower than normal while that of strain RG73 is slightly below normal. The methionine pools of all three strains are elevated several-fold. The metK strains are able to synthesize cyclopropane fatty acids, but the rate of their formation is slowed. Modification and restriction of phage 21 appears to be normal, suggesting that these strains are able to methylate DNA.

Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction.

The sequence of small-subunit rRNA varies in an orderly manner across phylogenetic lines and contains segments that are conserved at the species, genus, or kingdom level. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, we amplified bacterial 16S ribosomal DNA sequences with the polymerase chain reaction. Priming sites were located at the extreme 5' end, the extreme 3' end, and the center of 16S ribosomal DNA. The isolates tested with these primers included members of the genera Staphylococcus, Coxiella, Rickettsia, Clostridium, Neisseria, Mycobacterium, Bilophila, Eubacterium, Fusobacterium, and Lactobacillus and the family Enterobacteriaceae. Initially, the yields from the reactions were erratic because the primers were self-complementary at the 3' ends. Revised primers that were not self-complementary gave more reproducible results. With the latter primers, 0.4 pg of Escherichia coli DNA consistently gave a visible band after amplification. This method should be useful for increasing the amounts of bacterial 16S ribosomal DNA sequences for the purposes of sequencing and probing. It should have a broad range of applications, including the detection and identification of known pathogens that are difficult to culture. This approach may make it possible to identify new, nonculturable bacterial pathogens.

Regulation of S-adenosylmethionine synthetase in Escherichia coli.

Addition of methionine to the growth medium of Escherichia coli K-12 leads to a reduction in the specific activity of S-adenosylmethionine (SAM) synthetase. Thus the enzyme appears to be repressible rather than inducible. Mutant strains (probably metJ(-)) are constitutive for SAM synthetase as well as for the methionine biosynthetic enzymes, suggesting that the regulatory systems for these enzymes have at least some elements in common. Cells grown to stationary phase in complete medium, which have low specific activities of the enzymes, were routinely used for derepression experiments. The lag in growth and derepression when these cells are incubated in minimal medium is shortened by threonine. Ethionine, norleucine, and alpha-methylmethionine are poor substrates or nonsubstrates for SAM synthetase and are ineffective repressors. Selenomethionine, a better substrate for SAM synthetase than methionine, is also slightly more effective at repression than methionine. Although SAM is considered to be a likely candidate for the corepressor in the control of the methionine biosynthetic enzymes, addition of SAM to the growth medium does not cause repression. Measurement of SAM uptake shows that too little is taken into the cells to have a significant effect, even if it were active in the control system.

Isolation and characterization of specialized lambda transducing bacteriophage carrying the metBJF methionine gene cluster.

Secondary attachment site lysogens of Deltaatt(lambda)Deltappc-argECBH strains of Escherichia coli with lambdacI857 integrated into the bfe gene (88 min) were isolated. Of 20 such lysogens examined, 2 produce lysates with transducing phage containing the metBJF gene cluster (87 min). Reintroduction of the ppc-argECBH chromosome segment (which lies between the bfe and met genes) into these strains virtually abolishes the production of met transducing phage. All of the phage examined have lost essential genes from the left arm of the lambda chromosome. Approximately 85% of the phage appear to have the same genetic composition, containing the metBJF gene cluster, but not the closely linked gene cytR, and having lost phage genes G and J. Analytical CsCl density gradient centrifugation of five representatives of this major class of phage shows four of them to have identical densities (lighter than lambda), while the fifth cannot be resolved from lambda. The four apparently identical phage were isolated from three separate lysates, which suggests the existence of preferred sites for illegitimate recombination on the bacterial and phage chromosomes. Three specialized transducing phage that carry cytR in addition to metB, metJ, and metF have also been studied. Each of these viruses has a different amount of phage deoxyribonucleic acid. Two of them have less deoxyribonucleic acid than lambda, whereas the third has about the same amount. The metB, metF, and cytR genes of the transducing phage have been shown to function in vivo. The phage-borne metB and metF genes are subject to metJ-mediated repression.

In vitro synthesis of cystathionine gamma-synthetase in Escherichia coli K-12.

Synthesis of cystathionine gamma-synthetase directed by DNA from a lambdadmet transducing phage has been achieved in cell extracts from Escherichia coli K-12. Enzyme synthesis was stimulated two- to threefold by the addition of guanosine 3'-diphosphate 5'-diphosphate to the incubation mixtures. Kinetic studies showed a 1.5- to 2.0-min lag between initiation of transcription and completion of a translatable message. This lag is shorter than that observed for beta-galactosidase synthesis with DNA from a lac transducing phage known to initiate transcription at the lac promoter. This result, together with information on the structure of the transducing phage, shows that pL is not used for initiation of in vitro metB transcription. Attempts to demonstrate repression were not successful, and unexpectedly, extracts from metJ+ strains were found to be more effective at enzyme synthesis than those from their metJ derivatives.

Genetic characterization of the metK locus in Escherichia coli K-12.

Three independently isolated metK mutants have been shown to have leisions lying between speB and glc near 57 min on the Escherichia coli chromosome. Two deletions result in a lack of the metC gene product but neither extends into the metK glc region. The three metK mutations are recessive to the wild-type allele carried on the KLF16 episome.

Localization of the metJBLF gene cluster of Escherichia coli in lambda met transducing phage.

The position of the metJBLF gene cluster in the transducing phage lambda met102 was determined by ligation of its leftmost EcoRI fragment (102-1) to the lambda BCDEF (nin5) EcoRI fragment of lambda gtl (lambda BC) and characterization of the resultant recombinant phage. The new transducing phage carries about 6kb of bacterial DNA which contains the entire met gene cluster including the promoter of its rightmost member metF. Reasonable estimates of the coding capacity required for the four genes indicate that most of the bacterial DNA of the recombinant phage is occupied by the met gene cluster.

Species-specific oligonucleotide probes for rRNA of Clostridium difficile and related species.

The large copy number of rRNA makes it an appealing target for oligonucleotide probes designed to identify microorganisms. Given that nucleotide sequences in rRNA are known to reflect phylogeny, species-specific rRNA probes should be feasible if the sequences found in closely related species are different. We sequenced portions of the 16S rRNA of three closely related clostridia found in the human colonic microflora: Clostridium bifermentans, C. sordellii, and C. difficile. The rRNAs of these three species showed 97 to 98% sequence similarity. Five oligonucleotide probes complementary to unique segments of the sequences were end labeled with 32P and hybridized on a nylon filter to the immobilized rRNA of each clostridium. Each probe efficiently hybridized only to the rRNA of the species to which it was directed. Complementary probes emitted a signal that exceeded by a factor of 100 to 1,000 the signal of probes that mismatched the target rRNA by 2 to 5 bases. Even a 1-base difference in rRNA sequence allowed a clear distinction between species. A systematic approach can efficiently yield taxon-specific oligonucleotide probes directed at rRNA.

Reaction mechanism of Escherichia coli cystathionine gamma-synthase: direct evidence for a pyridoxamine derivative of vinylglyoxylate as a key intermediate in pyridoxal phosphate dependent gamma-elimination and gamma-replacement reactions.

Cystathionine gamma-synthase catalyzes a pyridoxal phosphate dependent synthesis of cystathionine from O-succinyl-L-homoserine (OSHS) and L-cysteine via a gamma-replacement reaction. In the absence of L-cysteine, OSHS undergoes an enzyme-catalyzed, gamma-elimination reaction to form succinate, alpha-ketobutyrate, and ammonia. Since elimination of the gamma-substituent is necessary for both reactions, it is reasonable to assume that the replacement and elimination reaction pathways diverge from a common intermediate. Previously, this partitioning intermediate has been assigned to a highly conjugated alpha-iminovinylglycine quininoid (Johnston et al., 1979a). The experiments reported herein support an alternative assignment for the partitioning intermediate. We have examined the gamma-replacement and gamma-elimination reactions of cystathionine gamma-synthase via rapid-scanning stopped-flow and single-wavelength stopped-flow UV-visible spectroscopy. The gamma-elimination reaction is characterized by a rapid decrease in the amplitude of the enzyme internal aldimine spectral band at 422 nm with a concomitant appearance of a new species which absorbs in the 300-nm region. A 485-nm species subsequently accumulates in a much slower relaxation. The gamma-replacement reaction shows a red shift of the 422-nm peak to 425 nm which occurs in the experiment dead time (approximately 3 ms). This relaxation is followed by a decrease in absorbance at 425 nm that is tightly coupled to the appearance of a species which absorbs in the 300-nm region. Reaction of the substrate analogues L-alanine and L-allylglycine with cystathionine gamma-synthase results in bleaching of the 422-nm absorbance and the appearance of a 300-nm species. In the absence of L-cysteine, L-allylglycine undergoes facile proton exchange; in the presence of L-cysteine, L-allylglycine undergoes a gamma-replacement reaction to form a new amino acid, gamma-methylcystathionine. No long-wavelength-absorbing species accumulate during either of these reactions. These results establish that the partitioning intermediate is an alpha-imino beta,gamma-unsaturated pyridoxamine derivative with lambda max congruent to 300 nm and that the 485-nm species which accumulates in the elimination reaction is not on the replacement pathway.