thus locally boosting their biomass in natural habi- 
tats above levels that are critical for their continued 
survival. From a medical and public health perspec- 
tive, the genetic mechanisms that underlie both the 
intestinal and the environmental phases of their life- 
styles are of interest, because an understanding of 
the former may lead to new vaccines, while an ap- 
preciation of the latter could lead to new strategies 
for their eradication from the biosphere. 
In the preceding year, Dr. Schoolnik's laboratory 
has studied biochemical and genetic events in en- 
teropathogenic E. coli (EPEC) that appear to tran- 
spire within minutes of the organism's infection of 
the small intestine; the evolution of virulence genes 
of V. cholerae was also examined. 
Adaptation of EPEC 
to an Intraintestinal Life-Style 
EPEC are a significant cause of childhood diar- 
rhea, affecting millions of children each year. The 
principal reservoir in nature of EPEC is contami- 
nated water, where it grows as free-living, isolated 
bacteria. However, small bowel biopsies of children 
infected with EPEC show discrete colonies of the 
organism attached to intestinal mucous membranes. 
Thus on entering the intestinal environment, EPEC 
adopt a colonial mode of growth. This process has 
been studied experimentally and shown to occur 
within 60 min of the organism's contact with cul- 
tured human cells. Ultrastructural studies show that 
the organism produces new surface structures dur- 
ing this transition that appear to mediate colony 
formation. 
These structures are composed of tens to 
hundreds of small filaments (termed bundle- 
forming pili [BFP]) that emanate from the surface of 
each bacterium. The filaments of adjacent organ- 
isms become entwined to form bundles of filaments 
within which are embedded the bacteria of the col- 
ony. Each BFP is composed of a principal, repeating 
19.5-kDa subunit that is structurally related to other 
members of the type 4 family of pilins, a widely 
disseminated gene found in pathogenic Neisseriae, 
Pseudomonads, Vibrios, Bacteroides, and Morax- 
ellae. The structural gene of the BFP subunit, bfp, is 
located on a 60-MDa plasmid, known to be impor- 
tant for virulence and for the colony-forming pheno- 
type. Other genes carried by this plasmid are re- 
quired for the biogenesis of mature BFP, including a 
gene that directs the cleavage of the BFP precursor 
at an unusual signal peptidase recognition site; an- 
other gene appears to be required for the assembly 
of the subunits into a filamentous polymer. The spe- 
cific physicochemical signal within the intestinal 
milieu and how this signal effects induction of the 
BFP operon are under investigation. 
Evolutionary Source of V. cholerae 
Virulence Genes 
Most cholera epidemics originate in the Ganges 
delta of India and Bangladesh, an esturine region 
where the salinity and the availability of other sol- 
utes varies according to the season. The cholera case 
rate also is seasonally determined, and there is now 
compelling evidence that in the interepidemic pe- 
riod, V. cholerae persists in the environment as a 
marine organism. To determine if this capacity may 
have originated with primitive nonpathogenic ma- 
rine vibrios. Dr. Schoolnik and his colleagues 
sought homologues of two V. cholerae virulence 
genes in V. fisheri, a bioluminescent commensal of 
the light organs of cenain fish and squid. The epithe- 
lia of these light organs and their interaction with V. 
fisheri resemble histologically the interaction of V. 
cholerae with mammalian intestinal surfaces. By us- 
ing ctxA and ctxB of V. cholerae as probes, low- 
stringency hybridization experiments revealed ho- 
mologues of each in a V. fisheri genomic library. 
Similarly, a homologue of the V. cholerae ToxR pro- 
tein, which serves to regulate the expression of chol- 
era toxin in response to changes in pH and osmolal- 
ity, was immunologically identified in V. fisheri 
through the use of ToxR-specific antisera. The enzy- 
matically active fragment of cholera toxin (CtxA) 
ADP-ribosylates the regulatory protein-guanosine 
triphosphate complex of adenyl cyclase leading to 
the production of cAMP. 
Consistent with the DNA hybridization experi- 
ments described above, V. fisheri lysates were found 
to have ADP-ribosylating activity, and the organism 
was able to subsist on cAMP as its sole carbon 
source. These findings indicate that the cholera 
toxin ancestral genes in V. fisheri may have been 
regulated by varying degrees of salinity in the ma- 
rine environment, where they originally functioned 
to support the commensalism of this species for its 
nonmammalian hosts through the production of 
cAMP. If so, the ToxR/cholera toxin precursors, 
once installed in V. cholerae, may have acquired a 
new role as the principal virulence determinants of 
the organism, while retaining their original ability 
to permit its persistence in the brackish water of 
estuaries between epidemics of human disease. 
Dr. Schoolnik is also Associate Professor of Med- 
icine and of Microbiology and Immunology at 
Stanford University School of Medicine. 
CELL BIOLOGY AND REGULATION 
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