Assay of Escherichia coli enterotoxins by in vivo perfusion in rats was evaluated by examining the effects of variously prepared fractions of heat-labile (LT) and heat-stable (ST) toxins on water transport in this system. The assay was found to respond equally well, in a dose-related manner, to both LT and ST; it was sufficiently sensitive to detect the toxigenic effect of concentrations as small as 1 ng/ml. With the assay, it was found that LT is produced in cultures grown under aerobic, but not anaerobic, conditions; in contrast, ST is elaborated in stationary aerobic and anaerobic broth cultures but not in those grown under agitated aerobic conditions. Both toxins can be precipitated by either ammonium sulfate or acetone. The two toxin forms were completely separated from each other by sequential ultrafiltration. LT alone (thermolabile after exposure to 100°C for 30 min) was retained by a PM-30 membrane, and ST alone was present in UM-2 retentates; ST was retained more effectively by a UM-05 membrane, with a 1,000-fold increase in activity over that of the UM-2 retentate. Washed ultrafiltration retentates containing either LT or ST derived from the proper culture conditions all induced water secretion at concentrations of 100 ng or less per ml. These results indicate that in vivo perfusion in rats is a sensitive, duplicable assay for both the LT and ST forms of E. coli enterotoxin.Enterotoxigenic strains of Escherichia coli produce two forms of enterotoxin: a low-molecular-weight, heat-stable toxin (ST) either alone or along with a large-molecular-weight, heatlabile toxin (LT) (11,22,32,35). These two forms are generally considered to be separate, distinct entities based on dissimilarities in their physical (4, 14, 15, 23), antigenic (7, 11, 12, 35), and biological properties (3,7,8, 9). Whereas the techniques for producing LT alone are well recognized (13,35), and sensitive, quantitative in vitro tests are available for its assay (3,5, 9), such is not the case for ST. Cellfree broth filtrates of strains of E. coli that produce LT and ST contain both forms of toxin, 9.

The Escherichia coli dnaZ gene, a deoxyribonucleic acid (DNA) polymerization gene, is located 1.2 min counterclockwise from purE, at approximately min 10.5 on the E. coli map. From a lysogen with XcI857 integrated at a secondary attachment site near purE, transducing phages (XdnaZ+) that transduced a dnaZts (A+) recipient to temperature insensitivity (TS+) were discovered. Three different plaque-forming transducing phages were isolated from seven primary heterogenotes. Genetic tests and heteroduplex mapping were used to determine the length and position of E. coli DNA within the X DNA. Complementation tests demonstrated that the deletions in all three strains removed both att P and the int gene, i.e., DNA from both prophage ends. Heteroduplex mapping confirmed this result by demonstrating that all three strains had deletions of X DNA that covered the b2 to red region, thereby removing both prophage ends. Specifically, the deletions removed A DNA between the points 39.3 to 66.5% of X length (measured in percent length from the left end of A phage DNA) in all three strains. The three strains are distinct, however, because they had differing lengths of host DNA insertions. These phages must have been formed by an anomalous procedure, because standard X transducing phages are deleted for one prophage end only. In Xgal and Xbio strains, the deletions of A DNA begin at the union of prophage ends (i.e., position 57.3% of A length) and extend leftward or rightward, respectively (Davidson and Szybalski, in A. D. Hershey [ed.], The Bacteriophage Lambda, p. 1971). Models for formation of the AdnaZ+ phages are discussed.The Escherichia coli dnaZ gene is one of the four known genes that code for products necessary for deoxyribonucleic acid (DNA) polymerization (10). In addition to participating in E. coli chromosome replication, the dnaZ product also is required for growth of phages M13, OX174, and A, but not for T7 or T4 growth (30,32). In the cases of M13 and kX174, the dnaZ product participates in vivo in parental replicative-form formation and replication and in single-strand synthesis (17; W. G. Haldenwang and J. R. Walker, unpublished data). Haldenwang and Walker (17) suggested that the dnaZ product participates in the polymerization phase of M13 replicative-form synthesis, rather than in initiating synthesis. Wickner and Hurwitz (36) demonstrated that the dnaZ protein participates in vitro in elongation of DNA during 4X174, M13, and ST-1 replicative-form synthesis from primed single-strand DNAs. Mapping studies demonstrated that dnaZ is cotransducible with purE, which is located near min 12 on the E. coli chromosome map (10).Shimada et al. (25) described X lysogens that contain prophages at secondary attachment sites. One such lysogen contained A integrated nearpurE (i.e., near dnaZ). The availability of this lysogen and the usefulness of transducing phages for host mapping and studies on host gene expression encouraged a search for AdnaZ+ phages. This paper describes the isolation and characterization of AdnaZ+ trans...