TABLE 3.

Complementation of morphology by mosaic and mutant PBPs^a

Protein or plasmid	Sequences around KTG motif			Complementationb
	197-203	204-219	220-228
Proteins
PBP 5	NRNGLLW	DNSLNVDGIKTGHTDK	AGYNLVASA
PBP 6	NRNRLLW	SSNLNVDGMKTGTTAG	AGYNLVASA
DacD	NRNGLLW	DKTMNVDGLKTGHTSG	AGFNLIASA
pAG656-11 derivativesc
pAG656-11	NRNGLLW	DNSLNVDGIKTGHTDK	AGYNLVASA	+
pAG656-X2A	NRNRLLW	DNSLNVDGIKTGHTDK		+
pAG656-M1A		DSSLNVDGIKTGHTDK		+
pAG656-M2A		DNNLNVDGIKTGHTDK		+
pAG656-M3A		DNSLNVDGMKTGHTDK		+
pAG656-H		DNSLNVDGIKTGTTDK		+
pAG656-M4A		DNSLNVDGIKTGHTAK		−
pAG656-M11A		DNSLNVDGIKTGHTDG		−
pAG656-M5A		DNSLNVDGIKTGHTAG		−
pAG565-3 derivativesd
pAG565-3	NRNRLLW	SSNLNVDGMKTGTTAG	AGYNLVASA	−
pAG565-X1A	NRNGLLW	SSNLNVDGMKTGTTAG		−
pAG565-M6Ae		SNNLNEDGMKTGTTAG		−
pAG565-M7Ae		SSSLNEDGIKTGTTAG		−
pAG565-T		SSNLNVDGMKTGHTAG		−
pAG565-M9A		SSNLNVDGMKTGTTDG		+
pAG565-M10A		SSNLNVDGMKTGTTAK		+
pAG565-X5B	NRNRLLW	SSNLNVDGMKTGTTAG		−
pAG565-X6A	NRNGLLW	SSNLNVDGMKTGTTAG		−
pPJ5 derivativesf (full-length PBP 5 and mutants):
pPJ5	NRNGLLW	DNSLNVDGIKTGHTDK	AGYNLVASA	+
pPJ5-DK/AG		DNSLNVDGIKTGHTAG		+
pAG5-X3A	NRNRLLW	DNSLNVDGIKTGHTDK		+
pAG5-X4A	NRNRLLW	DNSLNVDGIKTGHTAG		+
pPJ5-T217A		DNSLNVDGIKTGHADK		+
pPJ5-K213E		DNSLNVDGIETGHTDK		−
pPJ5-K213R		DNSLNVDGIRTGHTDK		−

^aResidue numbers refer to the mature protein and correspond to the numbering system used in the crystal structure of PBP 5 (9). Amino acids that differ from the sequence of PBP 5 are boldfaced and double underlined. The KTG active site motif is underlined once in the first sequence. Residues that are boldfaced and double underlined indicate differences from the first sequence shown in each section. For clarity, the sequence of residues 197 to 203 and 220 to 228 were omitted from the table when these were identical to the first sequence in each section. The inserted genes in each plasmid were sequenced to confirm that no other alterations were present. The sequences of the primer pairs used to create each mutant can be inferred from the amino acid alterations, but these are also available on request.

^bEffect of plasmid-encoded proteins on the cellular morphology of E. coli CS701-1 or CS703-1: +, complete or virtually complete restoration of uniform wild-type rod-shaped morphology; −, little or no restoration of uniform wild-type rod-shaped morphology.

^cThese derivatives had PBP 5 residues 200 to 219 inserted into PBP 6, and thus the residues listed in the 204-219 column are from PBP 5.

^dThese derivatives had PBP 6 residues 200 to 219 inserted into PBP 5, and thus the residues listed in the 204-219 column are from PBP 6. The residue at position 141 was D in all these derivatives except the last two, in which it was G. The G141D mutation was present in plasmid pAG565-3 and several of its derivatives. However, this mutation did not affect the complementation properties of the PBP mosaic proteins (compare the results for pAG565-3 with those for pAG565-X5B and pAG565-X6A).

^eThe glutamic acid residue (E) at position 209 in pAG565-M6A and pAG565-M7A is present in the E. coli K-12 genomic sequence (6), but a valine residue (V) is present in E. coli O157:H7 (15) and in the parental E. coli strain CS109 used in this work. Thus, PBP 5 with either glutamic acid or valine in this position has wild-type properties.

^fThese derivatives had full-length PBP 5 with various mutations.