Malcolm E. Winkler

Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K‐12

The region immediately downstream from the miaA tRNA modification gene at 94.8 min contains the hfq gene and the hflA region, which are important in the bacteriophage Qβ and lambda life cycles. The roles of these genes in bacteria remain largely unknown. We report here the characterization of two chromosomal hfq insertion mutations. An omega (ω) cassette insertion near the end of hfq resulted in phenotypes only slightly different from the parent, although transcript mapping demonstrated that the insertion was completely polar on hfq expression. In contrast, an equally polar omega cassette insertion near the beginning of hfq caused pronounced pleiotropic phenotypes, including decreased growth rates and yields, decreased negative supercoiling of piasmids in stationary phase, increased cell size, osmosensitivity, increased oxidation of carbon sources, increased sensitivity to ultraviolet light, and suppression of bgl activation by hns mutations, hfq::ω mutant phenotypes were distinct from those caused by omega insertions early in the miaA tRNA modification gene. On the other hand, both hfq insertions interfered with lambda phage plaque formation, probably by means of polarity at the hflA region. Together, these results show that hfq function plays a fundamental role in Escherichia coli physiology and that hfq and the hflA region are in the amiB‐mutL‐miaA‐hfq‐hflX superoperon.

Solution Conformations of Unmodified and A37N6-dimethylallyl Modified Anticodon Stem-loops of Escherichia coli tRNAPhe

The modification of RNA nucleotide bases, a fundamental process in all cells, alters the chemical and physical properties of RNA molecules and broadly impacts the physiological properties of cells. tRNA molecules are by far the most diverse-modified RNA species within cells, containing as a group >80% of the known 96 chemically unique nucleic acid modifications. The greatest varieties of modifications are located on residue 37 and play a role in ensuring fidelity and efficiency of protein synthesis. The enzyme dimethylallyl (Delta(2)-isopentenyl) diphosphate:tRNA transferase catalyzes the addition of a dimethylallyl group to the exocyclic amine nitrogen (N6) of A(37) in several tRNA species. Using a 17 residue oligoribonucleotide corresponding to the anticodon arm of Escherichia coli tRNA(Phe), we have investigated the structural and dynamic changes introduced by the dimethylallyl group. The unmodified RNA molecule adopts stem-loop conformation composed of seven base-pairs and a compact three nucleotide loop. This conformation is distinctly different from the U-turn motif that characterizes the anticodon arm in the X-ray crystal structure of the fully modified yeast tRNA(Phe). The adoption of the tri-nucleotide loop by the purine-rich unmodified tRNA(Phe) anticodon arm suggests that other anticodon sequences, especially those containing pyrimidine bases, also may favor a tri-loop conformation. Introduction of the dimethylallyl modification increases the mobility of nucleotides of the loop region but does not dramatically alter the RNA conformation. The dimethylallyl modification may enhance ribosome binding through multiple mechanisms including destabilization of the closed anticodon loop and stabilization of the codon-anticodon helix.

REGULATION OF SUBSTRATE RECOGNITION BY THE MIAA TRNA PRENYLTRANSFERASE MODIFICATION ENZYME OF ESCHERICHIA COLI K-12

We purified polyhistidine (His6)-tagged and native Escherichia coliMiaA tRNA prenyltransferase, which uses dimethylallyl diphosphate (DMAPP) to isopentenylate A residues adjacent to the anticodons of most tRNA species that read codons starting with U residues. Kinetic and binding studies of purified MiaA were performed with several substrates, including synthetic wild-type tRNAPhe, the anticodon stem-loop (ACSLPhe) of tRNAPhe, and bulk tRNA isolated from a miaA mutant. Gel filtration shift and steady-state kinetic determinations showed that affinity-purified MiaA had the same properties as native MiaA and was completely active for tRNAPhe binding. MiaA had aK m app (tRNA substrates) ≈3 nm, which is orders of magnitude lower than that of other purified tRNA modification enzymes, aK m app (DMAPP) = 632 nm, and ak cat app = 0.44 s−1. MiaA activity was minimally affected by other modifications or nonsubstrate tRNA species present in bulk tRNA isolated from a miaA mutant. MiaA modified ACSLPhe with ak cat app/K m appsubstrate specificity about 17-fold lower than that for intact tRNAPhe, mostly due to a decrease in apparent substrate binding affinity. Quantitative Western immunoblotting showed that MiaA is an abundant protein in exponentially growing bacteria (660 monomers per cell; 1.0 μm concentration) and is present in a catalytic excess. However, MiaA activity was strongly competitively inhibited for DMAPP by ATP and ADP (K i app = 0.06 μm), suggesting that MiaA activity is inhibited substantially in vivo and that DMAPP may bind to a conserved P-loop motif in this class of prenyltransferases. Band shift, filter binding, and gel filtration shift experiments support a model in which MiaA tRNA substrates are recognized by binding tightly to MiaA multimers possibly in a positively cooperative way (K d app ≈0.07 μm).

Reduction of GC → TA Transversion Mutation by Overexpression of MutS in Escherichia coli K-12

Overexpression of the MutS repair protein significantly decreased the rate of lacZ GC --> TA transversion mutation in stationary-phase and exponentially growing bacteria and in mutY and mutM mutants, which accumulate mismatches between 8-oxoguanine (8-oxoG) and adenine residues in DNA. Conversely, GC --> TA transversion increased in mutL or mutS mutants in stationary phase. In contrast, overexpression of MutS did not appreciably reduce lacZ AT --> CG transversion mutation in a mutT mutant. These results suggest that MutS-dependent repair can correct 8-oxoG:A mismatches in Escherichia coli cells but may not be able to compete with mutation fixation by MutY in mutT mutants.

The mutL repair gene of Escherichia coli K‐12 forms a superoperon with a gene encoding a new cell‐wall amidase

We report a molecular genetic analysis of the region Immediately upstream from the Escherichia coli mutL DNA repair gene at 94.8 min. An open reading frame ending 9bp upstream from the start of mutL corresponds to a 48kDa polypeptide detected previously in minicells. The predicted amino acid sequence of this 48kDa polypeptide shows homology to the major N‐acetylmuramoyl‐L‐alanine amidase autolysin of Bacillus subtilis, a known amidase of Bacillus licheniformis, and the product of a Salmonella typhimurium gene that maps near SO min. Insertions in this upstream gene, which we named AmiB, or in mutL did not affect cell shape or viability; however, overexpression of the AmiB potypeptide caused ceil lysis, hypersensitivity to osmotic shock and treatment with water, and temporary autolysis by low levels of antibiotics, which are all consistent with AmiB acting as a cell‐wall hydrolase. Analysis of chromosomal transcription demonstrated that amiB forms a complex operon with mutL and two additional upstream genes. mutL transcripts also originated from an internal promoter, designated PmutL, located in amiB 312bp upstream from the translational start of mutL. Together, these results suggest that E. coli contains a second amidase possibly involved in cell‐wall hydrolysis, septation, or recycling, and that transcription of this amidase is directly linked to a gene central for DNA repair.

Overexpression, purification and characterization of two pyrimidine kinases involved in the biosynthesis of thiamin: 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase

Abstract The overexpression, purification and characterization of 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase (HMP kinase) and 4-amino-5-hydroxymethyl-2-methylpyrimidine monophosphate kinase (HMP-P kinase) are described. Surprisingly HMP-P kinase also shows HMP kinase activity. These enzymes are useful reagents for the preparation of intermediates on the thiamin biosynthetic pathway.

The miaA mutator phenotype of Escherichia coli K-12 requires recombination functions.

miaA mutants, which contain A-37 instead of the ms(2)i(6)A-37 hypermodification in their tRNA, show a moderate mutator phenotype leading to increased GC-->TA transversion. We show that the miaA mutator phenotype is dependent on recombination functions similar to, but not exactly the same as, those required for translation stress-induced mutagenesis.