Relative amounts of PcsB and MreC in mutants used in the studya

StrainRelevant genotypeRelative PcsB amt (%)Relative MreC amt (%)
EL59bR6 parent≡100 (4,400 ± 1,400 monomers bound/cell)≡100
IU1979R6 ΔpcsB<>ermAM ΔbgaA′::Pc-pcsB+14 ± 668 ± 21
IU2801cR6 ΔpcsB<>ermAM ΔbgaA′::PfcsK -pcsB++fucose: 110 ± 2691 ± 9
IU1744R6 Pc-pcsB+ (native locus)21 ± 538 ± 9
IU2608cR6 IU1744 (pLS1RGFP; vector)+maltose: 24 ± 5+maltose: 43 ± 2
IU2564cR6 IU1744 (pLS1RGFP::Pmal-pcsB+)+maltose: 256 ± 105+maltose: 62 ± 11
IU1545c,eR6 PfcsK-pcsB+ (native locus)+fucose: 84 ± 28; −fucose: < 8+fucose: 22 ± 1; −fucose: 21 ± 7
IU1983dR6 ΔmreCD::aad998 ± 5None detected
IU1690bD39 parent≡100≡100
IU2537eD39 ΔpcsB<>ermAM ΔbgaA′::Pc-pcsB+<355 ± 8
IU2547D39 ΔbgaA′::aad9 (control)76 ± 1595 ± 11
IU1807D39 Pc-pcsB+ (native locus)29 ± 1453 ± 17
IU2091c,eD39 PfcsK-pcsB+ (native locus)+fucose: 140 ± 35; −fucose: <3+fucose: 25 ± 3; −fucose: 40 ± 13
IU2102dD39 ΔmreCD<>aad985 ± 5None detected
IU1945bD39 Δcps≡100 (5,400 ± 3000 monomers bound/cell)≡100
IU2336D39 Δcps Pc-pcsB+ (native locus)13 ± 349 ± 20
  • a Relative PcsB and MreC amounts were determined by quantitative Western blotting normalized to total protein amounts (see Fig. 2 and Materials and Methods). Amounts are relative to R6, D39, and D39 Δcps parent strains run on the same immunoblots. Cell-associated and secreted PcsB were added to give total amounts, and the fraction of secreted PcsB (see Results) did not change in R6 and D39 strains underexpressing PcsB (data not shown). All amounts represent at least three biological replicates, except for IU2608, IU2564, and IU2102, which were done twice. Standard errors of the means are indicated.

  • b The average number of cells per chain used in calculations was 3 or 2 for EL59 or IU1945, respectively, based on counting fields of several hundred cells by phase microscopy. The amounts of PcsB and MreC in IU1690 were comparable to those in strains EL59 and IU1945 when run on the same Western blots (data not shown). We quantitated PcsB amount per cell in IU1945 instead of IU1690, because IU1945 was predominantly diplococcal whereas IU1690 formed chains of various lengths (Fig. 5 and data not shown).

  • c 1.2 % (wt/vol) fucose (IU2801), 0.2% (wt/vol) fucose (IU1545 and IU2091), or 1% (wt/vol) maltose (IU2608 and IU2564) was added to cultures. Shifts to medium lacking fucose to deplete PcsB amounts were performed as described in Materials and Methods.

  • d No MreC was detected in autoradiograms or by an IVIS imaging system (see Materials and Methods).

  • e The amounts of PcsB in IU1545, IU2537, and IU2091 (−fucose) were below the limit of reliable quantitation, although very faint bands could be detected on autoradiograms exposed for long periods of time.