Escherichia coli B cells were sensitized to ionophore A23187 by polymyxin B nonapeptide, and the induced magnesium and potassium ion fluxes were studied. Combined ionophore treatment permeabilized the cytoplasmic membrane of E. coli in an ion-specific manner and allowed the manipulation of intracellular Mg2+ content from the outside. A23187-induced Mg2+ efflux or influx was dependent on the free Mg2+ concentration gradient between the outside and inside of the cytoplasmic membrane and on the pH gradient. Most of the intracellular Mg2+ was bound, whereas only 1 to 2 mM was free in solution in the cellular sap.

Leader peptidase cleaves the leader sequence from the amino terminus of newly made membrane and secreted proteins after they have translocated across the membrane. Analysis of a large number of leader sequences has shown that there is a characteristic pattern of small apolar residues at -1 and -3 (with respect to the cleavage site) and a helix-breaking residue adjacent to the central apolar core in the region -4 to -6. The conserved sequence pattern of small amino acids at -1 and -3 around the cleavage site most likely represents the substrate specificity of leader peptidase. We have tested this by generating 60 different mutations in the +1 to -6 domain of the M13 procoat protein. These mutants were analyzed for in vivo and in vitro processing, as well as for protein insertion into the cytoplasmic membrane. We find that in vivo leader peptidase was able to process procoat with an alanine, a serine, a glycine, or a proline residue at -1 and with a serine, a glycine, a threonine, a valine, or a leucine residue at -3. All other alterations at these sites were not processed, in accordance with predictions based on the conserved features of leader peptides. Except for proline and threonine at +1, all other residues at this position were processed by leader peptidase. None of the mutations at -2, -4, or -5 of procoat (apart from proline at -4) completely abolished leader peptidase cleavage in vivo although there were large effects on the kinetics of processing.(ABSTRACT TRUNCATED AT 250 WORDS)

We have characterized a novel mutant of EcoDXXI, a type IC DNA restriction and modification (R‐M) system, in which the specificity has been altered due to a Tn5 insertion into the middle of hsdS, the gene which encodes the polypeptide that confers DNA sequence specificity to both the restriction and the modification reactions. Like other type I enzymes, the wild type EcoDXXI recognizes a sequence composed of two asymmetrical half sites separated by a spacer region: TCA(N7)RTTC. Purification of the EcoDXXI mutant methylase and subsequent in vitro DNA methylation assays identified the mutant recognition sequence as an interrupted palindrome, TCA(N8)TGA, in which the 5′ half site of the wild type site is repeated in inverse orientation. The additional base pair in the non‐specific spacer of the mutant recognition sequence maintains the proper spacing between the two methylatable adenine groups. Sequencing of both the wild type and mutant EcoDXXI hsdS genes showed that the Tn5 insertion occurred at nucleotide 673 of the 1221 bp gene. This effectively deletes the entire carboxyl‐terminal DNA binding domain which recognizes the 3′ half of the EcoDXXI binding site. The truncated hsdS gene still encodes both the amino‐terminal DNA binding domain and the conserved repeated sequence that defines the length of the recognition site spacer region. We propose that the EcoDXXI mutant methylase utilizes two truncated hsdS subunits to recognize its binding site. The implications of this finding in terms of subunit interactions and the malleability of the type I R‐M systems will be discussed.

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SUMMARYPhage LL-H-induced cation (K +, Na +, Mg 2÷, Ca z÷, Cd 2+) movements in Lactobacillus lactis bacteria have been studied. The effects of the m.o.i, and external cation concentration have been quantified. LL-H-induced effluxes showed cation specificity: K+ but practically no Mg 2÷ was lost during LL-H infection at low and moderate m.o.i. (up to about 100). Simultaneously to K + efflux, divalent cation influxes were observed. These were dependent on the m.o.i, and on concentrations of external divalent cations and were concomitant with phage DNA transport, as concluded from the timing of the first phage-promoted biochemical changes in host cell metabolism and from electron microscopical observations. Host energy was not mobilized with phage-induced divalent cation influx. Several features of divalent cation influxes support the view that divalent cations have to be cotransported into the cell as counterions of LL-H DNA. Phage DNA associated with divalent cations may be the basic feature of the divalent cation dependence of LL-H infection.