The C-terminal sequence of the lambda holin constitutes a cytoplasmic regulatory domain

Abstract

The C-terminal domains of holins are highly hydrophilic and contain clusters of consecutive basic and acidic residues, with the overall net charge predicted to be positive. The C-terminal domain of lambda S was found to be cytoplasmic, as defined by protease accessibility in spheroplasts and inverted membrane vesicles. C-terminal nonsense mutations were constructed in S and found to be lysis proficient, as long as at least one basic residue is retained at the C terminus. In general, the normal intrinsic scheduling of S function is deranged, resulting in early lysis. However, the capacity of each truncated lytic allele for inhibition by the S107 inhibitor product of S is retained. The K97am allele, when incorporated into the phage context, confers a plaque-forming defect because its early lysis significantly reduces the burst size. Finally, a C-terminal frameshift mutation was isolated as a suppressor of the even more severe early lysis defect of the mutant SA52G, which causes lysis at or before the time when the first phage particle is assembled in the cell. This mutation scrambles the C-terminal sequence of S, resulting in a predicted net charge increase of +4, and retards lysis by about 30 min, thus permitting a viable quantity of progeny to accumulate. Thus, the C-terminal domain is not involved in the formation of the lethal membrane lesion nor in the "dual-start" regulation conserved in lambdoid holins. Instead, the C-terminal sequence defines a cytoplasmic regulatory domain which affects the timing of lysis. Comparison of the C-terminal sequences of within holin families suggests that these domains have little or no structure but act as reservoirs of charged residues that interact with the membrane to effect proper lysis timing.

FIG. 1

Features of the primary sequence of S. The predicted sequence of the λ S107 gene product is shown, with shaded bars over the three potential transmembrane domains and a hatched bar above the hydrophilic C-terminal domain characteristic of holins. Shown on the left is a blow-up of the amino-terminal sequences of the S107 and S105 products, including the corresponding mRNA sequence of the translational initiation region and the sdi stem-loop structure (31). Shown on the right is a blow-up of the C-terminal hydrophilic domain, including the corresponding mRNA sequence and, below that, the +1 frameshift mutation of λaj1, isolated as a suppressor of the plaque-forming defect of SA52G (also shown) (21). The time of lysis onset is shown in parentheses for both the SA52G early-lysis mutant and λaj1.

FIG. 2

The C-terminal hydrophilic domain of holin proteins from bacteriophages of gram-negative bacteria. The C-terminal sequence of the first member identified from each of the orthologous groups of holins is shown. Holin sequences are grouped into class I (three potential transmembrane domains) and class II (two potential transmembrane domains). Each holin is given with the total number of residues for the longest form of the predicted protein product. Class I orthologous groups: λ S (four orthologs, including P22 gp13), N15 (no orthologs), P2 Y (one ortholog, 186 Orf24), P1 LydA (no orthologs), and PRD1 OrfM (no orthologs). Class II orthologous groups: 21 S (six orthologs, including φ80 S); Hp1 Orf78 (one ortholog, Haemophilus somnus cryptic holin), T7 gp17.5 1 ortholog, and T3 Lys. If an ortholog has two or fewer residues changed, the changes are shown below the representative sequence. More-divergent termini within the orthologous group are shown in their entirety. Acidic and basic residues are shown in capital letters, and two or more consecutive charged residues are underlined. The H. somnus sequence comes from a cryptic prophage (29). A compilation of holin sequences can be found in Bläsi and Young (42) and in Young (41). The phage N15 holin sequence was obtained from R. Hendrix (19). Also shown are the C-terminal sequences of the predicted products of modified S alleles where a nucleotide sequence coding for an oligohistidine tag was inserted correctly (Sτ94) and incorrectly (Sτ94x, Sτ94z) after codon 94 (35).

FIG. 3

The C-terminal domain of the S protein is accessible to proteases in inverted membrane vesicles. E. coli MC4100(pBS112) cells were infected with λCE6 and labelled with [³⁵S]methionine at 25 min after infection. Spheroplasts and IMV, treated with proteinase K, with or without detergent, for various times as indicated. Each sample was subjected to immunoprecipitation with anti-S antibody (9), and the immunoprecipitate was analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Lanes: 1, in vitro translation products of S⁺ mRNA, as described previously (9); 2 to 5, spheroplast samples; 6 to 11, IMV samples.

FIG. 4

Sequence of S-R intergenic region. The sequence of the S-R intergenic region is shown, with the corresponding C-terminal sequences of S and N-terminal sequences of R. Also shown are the sequences of the D103am and E102am mutants. In each case, the presumptive Shine-Dalgarno sequence for R is underlined and both the wild-type start codon and the first downstream alternative start codon for R are shown in boldface. Asterisks denote the sequence alterations resulting from the creation of the corresponding amber codons in the S reading frame.

FIG. 5

Lysis profiles for induced lysogens with C-terminal alterations in S. (A) Lysogens of λb515 b519 Tn903 cI857 nin5 S105 carrying S alleles with different C-terminal amber mutations were thermally induced in logarithmic phase and monitored for lysis by measuring the A₅₅₀. Representative lysis profiles are shown for all alleles, including two profiles showing the variability of the K97am lysis kinetics. Symbols: ●, parental (no truncation); ■, D103am; ⧫, E102am; ▿ and ▾, K97am; ×, K92am. (B) Lysogens of λkan (●) and the isogenic phages λbj1 (SA52G [□]) and λaj1 (plaque-forming revertant of λbj1 [◊]) were thermally induced and monitored as in panel A.

FIG. 6

Possible membrane topologies of S. Depicted are putative membrane topologies for prototype class II (S from phage 21; left) and class I (S from phage λ; middle and right) holin proteins. The inner membrane is shown as shaded area with positively charged periplasmic and negatively charged cytoplasmic surfaces. Transmembrane helical domains are represented as white rectangles spanning the membrane. Basic residues in putative solvent-exposed domains are shown for both 21 and λ holins.