S. Marvin Friedman

Mutation in the DNA gyrase A Gene of Escherichia coli that expands the quinolone resistance-determining region.

ABSTRACT In three Escherichia coli mutants, a change (Ala-51 to Val) in the gyrase A protein outside the standard quinolone resistance-determining region (QRDR) lowered the level of quinolone susceptibility more than changes at amino acids 67, 82, 84, and 106 did. Revision of the QRDR to include amino acid 51 is indicated.

Studies on the interaction between ribosomes and14CH3−F3 initation factor

SummaryInitiation factor F3 has been purified fromEscherichia coli and labelledin vitro by reductive alkylation. The14CH3−F3 so obtained had a specific activity of about 1 000 cpm/μg and was shown to have retained its biological activity. Labelled F3 binds to 30S ribosomal subunits ofEscherichia coli andBacillus stearothermophilus, but does not bind to either 70S ribosomes or 50S ribosomal subunits. The stoichiometry of the binding indicates that one molecule of14CH3−F3 is bound to each 30S ribosomal subunit. Several antibiotics, known to interact with 30S subunits, inhibit the binding. Functional studies indicate that F3 is released from 30S ribosomes as a result of the formation of the “70S initiation complex”.

Thermotolerant nalidixic acid-resistant mutants ofEscherichia coli

Nalidixic acid-resistant mutants ofEscherichia coli CGSC #6353 capable of growth at 48°C were obtained by mutagenesis withN-methyl-N′-nitro-N-nitrosoguanidine. Cotransductional analyses employing phage P1 indicated that the mutation resulting in the phenotype of growth at 48°C is an allele of thegyrA structural gene. Similar thermal inactivation kinetics were observed for ribosomes isolated from a thermotolerant (T/r) mutant grown at both 37°C and 48°C and from the parental strain grown at 37°C. Cell-free extracts prepared from the T/r mutant grown at 48°C exhibited a sharp increase in protein synthesis at 55°C, whereas this effect was not displayed by extracts from the mutant or parental strains grown at 37°C. In addition, preincubation at 55°C enhanced protein synthesis at 37°C up to 15-fold in an extract prepared from the T/r mutant grown at 48°C, whereas comparable values were 2.6- to 3.0-fold for extracts from the mutant and parental strains grown at 37°C.

Surface topography of the Bacillus stearothermophilus ribosome.

SummaryThe surface topography of the intact 70S ribosome and free 30S and 50S subunits from Bacillus stearothermophilus strain 2184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more 125I than did 50S subunits derived from 70S ribosomes, whereas free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of 125I. Iodinated 70S ribosomes and subunits retained 62–78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were S4, S7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins S2, S3, S7, S13, Sa, L5/9, L10, L11 and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including S5, S9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosome than in the free subunits. The locations of tyrosine residues in some homologus ribosomal proteins from B. stearothermophilus and E. coli are compared.The surface topography of the intact 70S ribosome and free 30S and 50S subunits from Bacillus stearothermophilus strain 2184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more 125I than did 50S subunits derived from 70S ribosomes, whereas free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of 125I. Iodinated 70S ribosomes and subunits retained 62–78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were S4, S7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins S2, S3, S7, S13, Sa, L5/9, L10, L11 and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including S5, S9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosome than in the free subunits. The locations of tyrosine residues in some homologus ribosomal proteins from B. stearothermophilus and E. coli are compared.

Gene expression profiling of intrinsic thermotolerance in Escherichia coli.

DNA microarrays were employed to compare gene expression in a thermotolerant, nalidixic acid–resistant mutant of Escherichia coli with that of the parental strain. When grown at 37°C, up-regulated genes in the mutant included those coding for multiple antibiotic resistance proteins and enzymes for the degradation of small molecules, whereas among the down-regulated genes were those coding for fimbrial, flagellar, and outer membrane proteins as well as sigma38. When the mutant grown at 42°C was compared to the mutant grown at 37°C, enhanced expression of several genes coding for flagellar proteins was detected. Reverse transcriptase-polymerase chain reaction analysis of selected genes confirmed results obtained with microarrays.

Properties of the translational machinery from a sulfur-dependent archaebacterium

Summary Several characteristics of an in vitro polypeptide synthesizing system from the thermoacidophilic archaebacterium, Sulfolobus solfataricus , were examined. Poly A-directed lysine incorporation increased progressively as the incubation temperature was raised from 37 to 75°C. The stimulation of proline incorporation by poly C did not occur at 37 or 45°C and only a low level of incorporation was observed at the optimal temperature tested, namely 55°C. The addition of homologous unfractionated tRNA enhanced poly U-directed phenylalanine incorporation at 45°C more than two and one half-fold, whereas supplementation with tRNAs from Escherichia coli , bakers yeast, wheat germ and rat liver was without effect. Diphtheria toxin was a highly effective inhibitor of poly U-directed polyphenylalanine synthesis at 45°C. Although neomycin at a high concentration inhibited the poly U-phenylalanine reaction at 45°C, no inhibition was observed at 65°C. The antibiotic was not inactivated at 65 °C in the absence or in the presence of the extract. MS2 RNA stimulated phenylalanine incorporation at 15 mM ++ but not at 8 mM Mg ++ . Preincubation of ribosomes at 65°C for 40 min in the absence of spermine impaired their ability to carry out poly U-directed polyphenylalanine synthesis by 32%, while this value was only 3% if the ribosomes were preincubated in the presence of 3 mM spermine.

Protein Synthesis in Cell-Free Extracts from a Thermoacidophilic Archaebacterium

Summary An in vitro polypeptide synthesizing system was developed from the thermoacidophilic archaebacterium, Sulfolobus solfataricus . The stimulation of phenylalanine incorporation by poly U was entirely dependent upon the addition of polyamines or increasing the Mg ++ concentration. The largest stimulation was observed at 3 mM spermine in the presence of 10 mM Mg ++ . The optimal temperature for poly U-directed phenylalaline incorporation was 75 °C. The ability of poly G to code for glycine could be demonstrated in assays incubated at elevated temperatures. When tested at 45 °C, streptomycin and neomycin did not inhibit the poly U-phenylalanine reaction except in the case of neomycin at the highest concentration tested. Neither aminoglycoside induced significant miscoding for leucine at any concentration tested. Chloramphenicol inhibited poly UG-directed phenylalanine incorporation to a small extent. Tetracycline, virginiamycin and cycloheximide were ineffective in inhibiting polyphenylalanine synthesis, but this reaction was slightly inhibited by anisomycin at the highest concentration tested

Properties of cell-free extracts fromBacillus subtilis and its thermophilic mutant

After being heated at 65°C for 10 min, 51% of the protein in a cell-free extract fromBacillus subtilis BR151 was denatured, whereas the comparable value was 8% for the S-30 of a spontaneously occurring, temperature-resistant (T/r) mutant. Although ribosomes isolated from the T/r mutant retained 97% of their initial protein synthetic activity when preincubated at 60°C for 30 min, ribosomes prepared from the mesophilic parent were completely inactivated under these conditions. The optimum temperature for poly U-directed phenylalanine incorporation was 45°C for both parental and mutant extracts assayed in the absence of polyamines. The addition of spermidine to the S-30 from the mesophilic parent inhibited protein synthesis at each temperature tested, whereas this polyamine stimulated polyphenylalanine synthesis in the T/r extract at both 55°C and 65°C.

Overexpression of the cat-86 gene is associated with thermosensitivity in Bacillus subtilis

Abstract.Bacillus subtilis harboring the cat-86 constitutive plasmid pPL708C2 with an ochre mutation at the 9th codon (terc 9) was sensitive to chloramphenicol (Cms) and exhibited relative thermostability when heated at 47°C. Reversion to chloramphenicol resistance (Cmr) occurred at a frequency of 5.4 × 10−8. All of the plasmid Cmr revertants tested were thermosensitive. Similarly, wild-type pPL708C2 present in B. subtilis also rendered the bacterium thermosensitive. When a nonsense mutation is introduced at codon 141, however, this terc 141 variant of pPL708C2 failed to thermosensitize B. subtilis. Another variant of pPL708C2 that produces intact yet catalytically inactive CAT-86 has both His-16 and His-17 at the active site replaced by Pro. Nevertheless, cells of B. subtilis carrying this variant were thermosensitive. Plasmid-free and pPL708C2-bearing strains did not exhibit differences in major heat shock proteins. Electron micrographs revealed a threefold increase of inclusion bodies present in a strain harboring pPL708C2 when compared with those in an isogenic plasmid-free strain.