Enterotoxigenic Escherichia coli (ETEC) causes enterotoxin-induced diarrhoea and significant mortality. The molecular mechanisms underlying how the heat-labile enterotoxin (LT) is secreted during infection are poorly understood. ETEC produce outer-membrane vesicles (OMVs) containing LT that are endocytosed into host cells. Although OMV production and protein content may be a regulated component of ETEC pathogenesis, how LT loading into OMVs is regulated is unknown. The LeoA protein plays a role in secreting LT from the bacterial periplasm. To begin to understand the function of LeoA and its role in ETEC H10407 pathogenesis, a site-directed mutant lacking the putative GTP-binding domain was constructed. The ability of wild-type and mutant LeoA to hydrolyse GTP in vitro was quantified. This domain was found to be responsible for GTP binding; it is important to LeoA's function in LT secretion, and may play a modest role in the formation and protein content of OMVs. Deletion of leoA reduced the abundance of OmpX in outer-membrane protein preparations and of LT in OMVs. Immunoprecipitation experiments revealed that LeoA interacts directly with OmpA, but that the GTP-binding domain is nonessential for this interaction. Deletion of leoA rendered ETEC H10407 non-motile, through apparent periplasmic accumulation of FliC.

While wild-type Escherichia coZi K12 cannot grow with L-serine as carbon source, two types of mutants with altered methionine metabolism can. The first type, metJ mutants, in which the methionine biosynthetic enzymes are expressed constitutively, are able to grow with L-serine as carbon source. Furthermore, a plasmid carrying the metC gene confers ability to grow on L-serine. These observations suggest that in these mutants, L-serine deamination may be a result of a side-reaction of the metC gene product, cystathionine P-lyase. The second type is exemplified by two newly isolated strains carrying mutations mapping between 89-6 and 90 min. These mutants use L-wine as carbon source, and also require methionine for growth with glucose at 37 "C and above. The phenotypes of the new mutants resemble those of both met and his constitutive mutants in some respects, but have been differentiated from both of them.

Growth of human, but not porcine enterotoxigenic Escherichia coli (ETEC) isolates is inhibited during incubation with porcine intestinal epithelial cells and by a constitutively produced factor(s) present in unstimulated cell supernatants. The inhibitory factor(s) is heat stable, not produced by serum-starved cells, and is present in a diverse number of cultured epithelial cell lines of animal, but not of human origin. Susceptibility to porcine intestinal epithelial cells appears to be restricted to ETEC and not E. coli O157:H7 disease isolates.

While metabolism of para-aminobenzoate (P-AB) has been extensively investigated in the mammalian body( 1,2), its function and biochemical transformations in microorganisms are still obscure. Conversion of PAB into folic acid and the citrovorum factor occurs in some organisms (3,4) a1 though there is some question whether these are the active forms of PAB. A number of other routes of metabolism have been reported. Davis( 5 ,6 ) has implicated PAB in the metabolism by Escherichia C O Z~ of tryptophane, tyrosine and other aromatic compounds, as well as vit. BI2. Ratner et aZ.( 7 ) isolated a glutamic acid peptide of PAB from yeast, and Flynn et d ( 8 ) have found in 2 strains of streptococci an antibiotic containing PAB and cytosine among its components. Sloane et a2. (9) identified aniline and p-aminophenol as degradation products in Mycobacterium smegmatis. The present studies had their inception in the isolation by Lampen, Roepke, and Jones(l0) of an X-ray mutant of E. coZi which required exogenous PAB for growth but apparently did *Supported in part by grant from U. S. Atomic Energy Commission, Contract S o . .4T(30-1) -1107 and the C. S. -4ir Force, Contract No. -4F 18(600) -853. t Portions of these data were used in partial satisfaction of requirements for degree of Master of Science, George Washington University.not utilize it in producing folic acid. Since the wild strain of this organism normally has no requirement for preformed PAB. it seemed od interest to investigate the fate of this compound labeled with C14 in such a mutant.Materials and methods. PAB used was labeled in the carboxyl group with carbon 14 and had a specific activity of 0.037 mC/mg.$ The mutant of E . coli (ATCC S o . 9723a) was grown on basal medium modified from that of Cohen and Arbogast( 11) by use of 0.4% glucose. The medium of Lampen et aZ.( 10) contained asparagine as well, but this substance was not necessary for maximum growth, and was omitted to facilitate isolations. Organisms were grown in 3 liter batches with aeration for 24 hours at PAB concentration of 0.01 &ml.Experiments in which PAB level was 0.05 &ml gave similar results. Bacterial suspensions were filtered on Buechner funnel using Celite as filter aid. The filtrate contained less than 207% of total radioactivity. The cells, plus filster-aid, were suspended in water and made up to 60% acetone-water (v/v) by addition of acetone. The residue was filtered off, re-extracted, and extracts, containing approximately half the total cell radioactivity, were combined. Subsequent extractions of cells with hot acetone. chloroform, $ Purchased from Atomic Energy 'Commission, Los Alamos Laboratories.