Supporting Information

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Fig. 6. Inhibition of sec-mediated secretion stabilizes Lyz in a dsbA mutant. Parallel cultures of RY8653 dsbA::kan pJFLyz were induced with isopropyl β-D-thiogalactoside. At the time of induction, one of the cultures was poisoned with 1 mM azide. After 60 min, the cells from a 1-ml aliquot of each culture were collected by centrifugation and boiled in SDS/PAGE sample buffer. The samples were then analyzed by SDS/PAGE and immunoblotting as described in Materials and Methods, in the main text. +, culture with azide; –, culture without azide.

Fig. 7. (A) Dual localization of Lyz is independent of expression level. lyz was cloned into the low-copy number vector pZA-32, carrying the p15A replicon (1). Soluble and membrane fractions were prepared from induced cells carrying pZA-Lyz and were analyzed by SDS/PAGE and immunoblotting as described in Materials and Methods in the main text. s, soluble fractions; m, membrane fractions. (B) lyz expression during induction of a P1 lysogen and from pJFLyz is comparable. Cultures of MC4100pJFLyz and MC4100 (P1tscam) were induced by the addition of isopropyl β-D-thiogalactoside or by shifting temperature from 30°C to 42°C. At the appropriate times, 1-ml aliquots were withdrawn from the induced cultures, the cells were collected by centrifugation, and the cell pellet was analyzed by SDS/PAGE and immunoblotting as described in Materials and Methods in the main text. Sample loads were adjusted so that protein from equivalent number of cells, based on culture turbidity, were applied to each lane. Lanes 1-4 are from the MC4100 pJFLyz culture harvested 10, 15, 20, and 25 min after induction, respectively. Because the expression of late genes of P1 commences approximately 20 min after induction of a P1 prophage (2, 3), corresponding samples from the induced MC4100 (P1tscam) were taken at 30, 35, 40, and 45 min after thermal induction and are displayed in lanes 5-8, respectively. In this experiment, lysis of the induced MC4100 (P1tscam) lysogen began 40-45 min after induction, so that some of the Lyz protein was lost when the cells were harvested by centrifugation.

1. Lutz, R. & Bujard, H. (1997) Nucleic Acids Res. 25, 1203-1210.

2. Guidolin, A., Zingg, J. M., Lehnherr, H. & Arber, W. (1989) J. Mol. Biol. 208, 615-622.

3. Lehnherr, H., Guidolin, A. & Arber, W. (1991) J. Bacteriol. 173, 6438-6445.

Strains. MC4100 (P1tscam) is a lysogen bearing the thermally inducible P1 prophage P1c1_clr100 with an insertion of the chloramphenicol-resistance element Tn9 (1).

Construction of l ::lyz. A lcIts (DSR) kanr lysogen (2) was transformed with a plasmid in which the lyz and lydAam genes were cloned between sequences encoding the lpR promoter and the l RzRz1 genes. To generate phage carrying the P1 lyz and lydAam genes at the position normally occupied by the lS and R genes, the transformant was thermally induced and the resulting lysate was plated on MC4100 at 37°C. Plaques produced by the desired recombinants, l::lyz, were purified and used to lysogenize MC4100 at 30°C by using kanamycin resistance as a selection.

1. Rosner, J. L. (1972) Virology 49, 679-689.

2. Raab, R., Neal, G., Sohaskey, C., Smith, J. & Young, R. (1988) J. Mol. Biol. 199, 95-105.

Fig. 8. Lyz produced from the induction of a l ::lyz prophage causes culture lysis and is found in both the membrane and soluble fractions. A culture of MC4100 (l ::lyz), constructed as described in Supporting Materials and Methods, was thermally induced and monitored for A550. (Inset) Forty minutes after induction, total (t), membrane (m), and soluble (s) fractions were prepared and were analyzed by SDS/PAGE and immunoblotting as described in Materials and Methods in the main text.

Fig. 9. A previously described signal sequence mutation of LamB may act as a SAR sequence. Shown are the first 25 residues of the unprocessed of wild-type LamB (wt), the A23D A25Y mutant. which is tethered to the inner membrane in an uncleaved form that is rapidly degraded, and the A23D A25Y R6L, which is localized to the outer membrane in a functional but unprocessed form (mutant residues are underlined) (1). The normal signal peptidase cleavage site (# sign) is to the carboxyl side of residue 25, the last residue shown. Asterisks indicate the extent of the putative SAR sequence created by the R6L suppressor mutation. Basic and acidic residues are shown in red and blue, respectively.

1. Duguay, A.R. & Silhavy, T. J. (2002) J. Bacteriol. 184, 6918-6928.