SUPPLEMENTARY DATA - MATERIALS AND METHODS
Construction of pSwtRlacZblueRz and pSwtRlacZwhiteRz plasmids: The
-complementation of E. coli’s -gal activity was used as the basis for phage strain
differentiation. The choice of the marker is based on a previous observation that the fulllength -gal still retains its activity while fused at the C-terminus of the  R protein
(WANG et al. 2003). For the current study, only the first 100 amino acid sequence (the fragment) of the -gal was fused at the C-terminus of the  R protein.
To construct pSwtRlacZblueRz, the wild-type (wt) lacZ fragment (the first 100
codons) was PCR-amplified from plasmid pZE1-pR'-tr3-lacZ, which contains the fulllength lacZ from E. coli (unpublished data), using the primer pair, RlacZ_For and
RlacZ_Rev, carrying complementary sequences to the 3’-end of 's R gene and the 5’end of the Rz gene, respectively. The resulting amplicon was used as a mega-primer for
site-directed mutagenesis PCR (GEISER et al. 2001), using plasmid pSwt (WANG 2006) as
the template. DNA sequencing was used to screen for the resulting plasmid,
pSwtRlacZblueRz. In this construct, the -fragment is fused between the last and
penultimate amino acid residues of  R protein, the same position as the previous study
(WANG et al. 2003).
To construct pSwtRlacZwhiteRz, site-directed mutagenesis PCR with the
QuickChange protocol (Startagene, La Jolla, CA) was performed, using
pSwtRlacZblueRz as the template and white__For and white__Rev, which contain
codons for five alanine substitutions at amino acid residues #14, 15, 16, 18 and 19 of the
E. coli wt lacZ sequence, as the primer pair. DNA sequencing was use to screen for
pSwtRlacZwhiteRz. The choice of these five amino acid residues is based on the
published structure of E. coli -gal (JUERS et al. 2001), in which these five amino acid
residues seem to contribute to the formation of dimer and tetramer of -gal, which is
essential for its activity. We predicted that replacement of these five residues would
result in only monomer formation, thus destroying the -gal activity. As predicted, no gal activity was detected when the -fragment, expressed by the XL1-Blue (Startagene,
La Jolla, CA) cells, was complemented by the mutant -fragment (data not shown).
Construction of  strains carrying the lacZ markers: Plasmid
pSwtRlacZblueRz was first transformed into  lysogen IN56 (WANG 2006). The
pSwtRlacZblueRz-harboring lysogen was then thermally induced (WANG 2006) to
generate recombinant phages. The R::lacZ+ phage was identified by the blue plaque it
made when plated on the XL1-Blue lawn containing  13 M IPTG and 0.05% X-gal in
3 mL top-agar. The R::lacZ+ phage was used to lysogenize IN25 (MC4100) to form
strain SYP045. To construct  strain carrying the lacZ- allele, pSwtRlacZwhiteRz was
transformed into SYP045. The R::lacZ- phage was screened with the same procedure
as described above, except that in this case the white plaque was picked. The R::lacZphage was used to lysogenize IN25 cells to form strain SYP049.
Construction of pZE1-J-Cam-stf and pZE1-J-stf+ plasmids, and stf+ 
strains: A  genomic region spanning from J to orf 314 (nt 17776 to 21417,
encompassing part of J, the entire lom and orf401, and part of orf314; GenBank accession
no. NC_001416) was PCR-amplified using primer pairs: J_to_stf_For and J_to_stf_Rev.
The amplicon was cloned into pZE12-luc (LUTZ and BUJARD 1997) between the AatII
and XbaI sites to replace the luc gene. The resulting plasmid, pZE1-J-stf, was used as the
template to perform a second PCR amplification using the primer pair: del_J_For and
del_stf_Rev. The resulting amplicon, which contains the backbone of pZE1-J-stf but
with a deletion spanning from part of J to orf401 (nt 18433 to 20913, GenBank accession
no. NC_001416), was ligated to the EcoRV fragment from plasmid pZA31-luc (LUTZ and
BUJARD 1997), which contains the cam gene, to form a new plasmid, pZE1-J-Cam-stf.
To construct the (J-stf)::cam phage, lysogen IN56 was transformed with pZE1J-Cam-stf and then thermally induced to generate recombinant phages as described
above. The resulting lysate was used to lysogenized IN25 and plated on LB agar plate
supplemented with 10 µg/ml chloromphenicol to select for the (J-stf)::cam lysogen,
SYP052. It is to be noted that in the lysate there is phenotypic mixing in the phage
population such that some phage virions, though bearing the tail fiber J, are actually
genetically J due to deletion in J.
To construct stf+ strains, plasmid pZE1-J-stf+ was first generated by using the
QuickChange protocol (Stratagene, La Jolla, CA) for site-directed mutagenesis PCR,
with pZE1-J-stf as the template and Lam_Ur_For/Lam_Ur_Rev as the primer pair, which
would result in an insertion of a C (cytosine) in orf401 (between nt 20834 and 20835,
GenBank accession no. NC_001416), thus restoring the frameshift mutation present in wt
laboratory  (PaPa) (HENDRIX and DUDA 1992). SYP052 was transformed with pZE1J-stf+ and the transformant thermally induced to generate recombinant phages as
described above. Since (J-stf)::cam phage from SYP052 cannot form plaques due to
lack of tail fiber J, any plaque formed must be the result of recombination between (J-
stf)::cam on the prophage and stf+ on pZE1-J-stf+. The stf+ phage was used to
lysogenize IN25 to form strain SYP053.
Construction of the "blue" and "white" stf+  strains: The “blue” and “white”
stf+  phages were generated similarly as described above, except that SYP053, instead
of IN56, was used for the transformation by the corresponding plasmids. The resulting
“blue” stf+ phage and ‘white stf+ phage were then used to lysogenize IN25 to form strain
SYP046 and SYP056, respectively.
Construction of pSwtdelS_Cam plasmid and  strains carrying S::cam: A
PCR reaction was performed using pSwt as the template and delS16-92_For/delS1692_Rev as the primer pair. The resulting DNA fragment, which contains the backbone of
pSwt with an internal deletion in S gene (between codons 16-92), was ligated with
EcoRV fragment from pZE31-luc (containing the cam gene) (LUTZ and BUJARD 1997) to
form plasmid pSwtdelS_Cam.
Plasmid pSwtdelS_Cam was then transformed into strain SYP045 (LacZ+, Stf-),
SYP046 (LacZ+, Stf+), and SYP056 (LacZ-, Stf+). The transformants were thermally
induced to generate recombinant phages as described above. IN25 was lysogenized by
each lysate separately and plated on LB agar plates supplemented with 10 µg/ml
chloramphenicol to select for S::cam lysogens. The MC4100(cI857 S::cam
R::lacZ+ stf-) lysogen strain is named as SYP047, the MC4100(cI857 S::cam
R::lacZ+ stf+) strain SYP061, and the MC4100(cI857 S::cam R::lacZ- stf+) strain
SYP062.
Construction of isogenic  strains carrying different S alleles: A detailed
protocol has been described previously (WANG 2006). In this study, five plasmids each
with different S alleles (pS105, pSwtS68C, pS105/C51S, pS105/C51S/S76C and
pS105/V77G) were individually transformed into either SYP047, SYP061, or SYP062.
The transformants were thermally induced to generate recombinant phages as described
above. Since the parental S::cam can not form plaques, the phages forming the plaques
are recombinants carrying different S alleles. Lysogens carrying different combinations
of S, stf, and lacZ alleles were individually lysogenized into IN25. The bacterial strains
carrying these prophages are listed in Table 1.
LITERATURE CITED
GEISER, M., R. CEBÈ, D. DREWELLO and R. SCHMITZ, 2001 Integration of PCR fragments
at any specific site within cloning vectors without the use of restriction enzymes
and DNA ligase. Biotechniques 31: 88-90, 92.
HENDRIX, R. W., and R. L. DUDA, 1992 Bacteriophage λPaPa: not the mother of all λ
phages. Science 258: 1145-1148.
JUERS, D. H., T. D. HEIGHTMAN, A. VASELLA, J. D. MCCARTER, L. MACKENZIE et al.,
2001 A structural view of the action of Escherichia coli (lacZ) β-galactosidase.
Biochemistry 40: 14781-14794.
LUTZ, R., and H. BUJARD, 1997 Independent and tight regulation of transcriptional units
in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory
elements. Nucleic Acids Res 25: 1203-1210.
WANG, I.-N., J. DEATON and R. YOUNG, 2003 Sizing the holin lesion with an endolysinβ-galactosidase fusion. J Bacteriol 185: 779-787.
WANG, I. N., 2006 Lysis timing and bacteriophage fitness. Genetics 172: 17-26.