No abstract

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.

Human growth hormone (hGH), a 191 residue protein containing two disulfide bonds, was fused to the carboxyl-terminal domain of the gene III protein, a minor coat protein exposed at one end of the filamentous phage M13. The gene fusion was cloned into a plasmid containing origins of replication for Escherichia coli and filamentous phage and was packaged into phagemid particles upon infection by an M13KO7 helper phage. Transcription of the hGH-gene III fusion was controlled so that usually no more than one copy of the fusion protein was displayed along with the four copies of the wild-type gene III protein. The hGH-gene III fusion protein was properly folded, as judged by reactivity with six hGH monoclonal antibodies whose epitopes are sensitive to the folded conformation of hGH. Moreover, the hGH-gene III phagemid particles were enriched over 5000-fold from non-hGH phage, and 8-fold from a mutant hGH phagemid following a single hGH-specific elution step from hGH receptor-coated beads. The hGH phagemid should be useful for isolating new receptor binding mutants of hGH. More generally, this expression system may allow other large proteins with discontinuous binding epitopes to be displayed, and binding selections applied to their mutated gene III fusions on filamentous phage.

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.

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.