A method of sex identification using the polymerase chain reaction technique is described. Using a pair of nucleotide primers from an X-Y homologous region, both the X and the Y sequences can be amplified simultaneously, and more importantly, they result in fragments of different lengths. The success of the procedure is therefore monitored by the presence of a X-specific band while sex is identified by the presence or absence of a Y-specific band.

Mutants of Escherichia coli with much decreased activity of D-alanine carboxypeptidase (peptidyl-D alanine hydrolase, EC 3.4.12.1-1) were found among E. coli K12 extensively mutagenized with nitrosoguanidine treatment by assaying individual colonies for the enzyme activity. One such mutant with only 10-12% residual activity was characterized extensively. The D-Alanine carboxypeptidase (peptidyl-D alanine hydrolase, EC 3.4.12.11) (CPase) is one of a few enzymes known to be inhibited by penicillin and its derivatives (2-4). The enzyme activity in vitro is assayed (2) by the release of the terminal D-alanyl residue from uridine disphospho-N-acetylmuramyl-pentapeptide (UDP-MurNAc-L-Ala-D-Glu-meso-A2pm-D-Ala-DAla), a precursor molecule in cell wall synthesis (5). The activity in tvo and function of CPase is not known. It has been speculated that it might be regulating the degree of crosslinkage between two peptidoglycan molecules by converting MurNAc-pentapeptide into MurNAc-tetrapeptide; the latter is not a substrate for the transpeptidatimn reaction (6). Recently it was reported that the CPase can function as a "pseudo" transpeptidase enzyme in vitro (3,(7)(8)(9) and it has been suggested. (3, 7) that it functions in this way in vivo as well as in vitro. At least one of these carboxypeptidases is also able to catalyze the hydrolysis of cell walls, i.e., it has endopeptidase activity (8).The primary objective in the present study was to use genetic studies to probe the physiology of the multiple penicillin binding components of Escherichia coli, and also in this way to identify which one(s) had transpeptidase activity in vivo, and which ones were lethal target sites for penicillin. The lethal target of penicillin action has long been believed to be an enzyme participating in cell wall synthesis, presumably the transpeptidase (6), and the inhibitory activity of penicillin on transpeptidation has been shown in Staphylococcus aureus in vivo (10), and in E. coli (5), Bacillus stearothermophilus (11), Sporosarcina ureae (12) and many other organisms in vitro. In Abbreviations: CPase, D-alanine carboxypeptidase; A2pm, a, ediaminopimelate; DYABT, DYAB medium supplemented with thymine, 20 ,g/ml (see ref. 1); PBC, penicillin-binding component.

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Mutants of Escherichia coli capable of growing in the presence of 10 ,g of mecillinam per ml were selected after intensive mutagenesis. Of these mutants, 1.4% formed normal, rod-shaped cells at 300C but grew as spherical cells at 420C. The phenotype of one of these rod(Ts) mutants was 88% cotransducible with lip (14.3 min), and all lip' rod(Ts) transductants of a lip recipient had the following characteristics: (i) growth was relatively sensitive to mecillinam at 300C but relatively resistant to mecillinam at 420C; (ii) penicillin-binding protein 2 was present in membranes of cells grown at 300C in reduced amounts and was undetectable in the membranes of cells grown at 420C. The mecillinam resistance, penicillin-binding protein 2 defect, and rod phenotypes all cotransduced with lip with high frequency. Thus the mutation [rodA(Ts)] is most likely in the gene for penicillin-binding protein 2 and causes the organism to grow as a sphere at 420C, although it grows with normal rodlike morphology at 300C. At 420C, cells of this strain were round with many wrinkles on their surfaces, as revealed by scanning electron microscopy. In these round cells, chromosomes were dispersed or distributed peripherally, in contrast to normal rod-shaped cells which had centrally located, more condensed chromosomes. The round cells divided asymmetrically on solid agar, and it seemed that the plane of each successive division was perpendicular to the preceding one. On temperature shift-down in liquid medium many cells with abnormal morphology appeared before normal rod-shaped cells developed. Few abnormal cells were seen when cells were placed on solid medium during temperature shift-down. These pleiotropic effects are presumably caused by one or more mutations in the rodA gene.

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Genes responsible for round morphology in mecillinam-resistant, round morphological mutants of Escherichia coli have been mapped. Three mutants, called rodX, mapped at around 14 min, and two, called rodY, mapped at around 70 min by P1 transduction. These are either the same or very close to the loci reported, respectively, for the rodA (H. Matsuzawa, K. Hayakawa, T. Sato, and K. Imahori, J. Bacteriol. 115:436-442, 1973) and envB genes (B. Westling-Häggström and S. Normark, J. Bacteriol. 123:75-82, 1975). This suggests that mecillinam can be used very efficiently to select for found morphological mutants of rodA and envB after nitrosoguanidine treatment.

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