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ABSTRACT-Mutants of lactose permease of Escherichia coli with amino acid changes (Gly-24 -> Glu; Gly-24 --Arg; Pro-28 --Ser; Gly-24, Pro-28 -> Glu-Ser and Gly-24, -> Arg-Ser) within a putative membrane-spanning a-helix 12 24(Phe-Gly-Leu-Phe-Phe-Phe-Phe-Tyr-Phe-Phe-Ile-Met-GlyAla-Tyr-Phe-Pro-Phe-Phe-Pro-Ile) are incorporated into the cytoplasmic membrane. The mutant proteins retain the ability to bind galactosides, and the affinity for several substrates is actually increased. However, the rate of active transport is decreased to 0.01% of the wild-type rate in the mutants carrying Arg-24 or Arg-24, Ser-28. Kinetic analysis demonstrates that the two mutants require 10 min to cause occupied binding sites for galactoside and HI to change their exposure from the periplasm to the cytoplasm as compared to 50 ms in the wild type. The effect is less pronounced when these sites are unoccupied.

The two genes encoding the class IIS restriction-modification system MboII from Moraxella bovis were cloned separately in two compatible plasmids and expressed in E. coli RR1 delta M15. The nucleotide sequences of the MboII endonuclease (R.MboII) and methylase (M.MboII) genes were determined and the putative start codon of R.MboII was confirmed by amino acid sequence analysis. The mboIIR gene specifies a protein of 416 amino acids (MW: 48,617) while the mboIIM gene codes for a putative 260-residue polypeptide (MW: 30,077). Both genes are aligned in the same orientation. The coding region of the methylase gene ends 11 bp upstream of the start codon of the restrictase gene. Comparing the amino acid sequence of M.MboII with sequences of other N6-adenine methyltransferases reveals a significant homology to M.RsrI, M.HinfI and M.DpnA. Furthermore, M.MboII shows homology to the N4-cytosine methyltransferase BamHI.

The DNA sequence of several functionally interesting lac permease mutants of Escherichia coli has been determined. The phenotypes of the mutant permeases were described by Mieschendahl et al. (1981). The following exchanges are noteworthy: tyr to asp in codon 26 in Y-K MUB 7; thr to ile in codon 266 in Y-K AJ 33; gly to asp in codon 262 in Y-D 3 and in Y-D 4.

Mutations in the lacY gene of Escherichia coli have been used to analyze the functional organization of lactose permease. Deletions suggest that the NH2 terminus oflactose permease is not essential and can be replaced by residues of the cytoplasmic enzyme (3-galactosidase. Negative dominant mutations in the lacY gene can be explained by the assumption that membrane-associated lactose permease is active as a dimer or oligomer. The map positions of these mutations and other point mutations that lower or alter the sugar specificity define regions of lactose permease involved in sugar or proton binding and transport.