Lipid bilayer experiments were performed in the presence of hemolysin of Escherichia coli. The toxin had a rather low activity in membranes formed of pure lipids, such as phosphatidylcholine or phosphatidylserine. In membranes from asolectin, a crude lipid mixture from soybean, hemolysin was able to increase the conductance by many orders of magnitude in a steep concentration-dependent fashion, which suggested that several hemolysin molecules could be involved in the conductive unit. Furthermore, the much higher toxin activity in asolectin membranes would be consistent with the assumption that this lipid contains a receptor needed for membrane activity of the toxin. The results of single-channel records showed that the membrane activity of hemolysin is due to the formation of ion-permeable channels with a single-channel conductance of about 500 pS in 0.15 M KCl. The hemolysin channel seemed to be formed by a toxin oligomer which showed an association-dissociation reaction and had a mean lifetime of about 2 s at small transmembrane voltages. The conductance of the hemolysin channels was only moderately dependent on the salt concentration in the aqueous phase. Zero-current membrane potential experiments showed that the hemolysin channel is cation selective. The mobility sequence of the cations in the channel was similar to their mobility sequence in the aqueous phase, which was consistent with the assumption that the hemolysin channel is wide and that the interior field strength is not very high. From the single-channel conductance, a lower limit of about 1.0 nm for the effective channel diameter could be estimated.

MATERIALS AND METHODS Bacterial strains. E. coli K-12(pHlyl52) and the Tn3 mutants of pHlyl52 have been described previously (20, 21). E. coli 605 carrying TnS in the chromosome was given to us by A. Piihler. Plasmids. The recombinant plasmid pANN202-312, carrying the whole hly determinant of pHlyl52, and its derivatives have been recently described (8). The vector plasmid pUR222 was a gift from B. Muller Hill. Cleavage with restriction enzymes and in vitro recombination. Restriction enzyme cleavage, in vitro recombination, and transformation were carried out as described previously (8, 20). Transposon mutagenesis of pHlyl52 and pANN202-312 by Tn5. Plasmid pHlyl52 was transferred by conjugation from E. coli K-12(pHlyl52) into E. coli 605(TnS). In a second mating E. coli 605 (pHlyl52) was crossed with E. coli HB101 rpsL as described previously (9), and transconjugant colonies were selected on nutrient broth agar containing 25 1Lg of streptomycin and 30 ,ug of kanamycin per ml. Those colonies which did not exhibit the alpha-hemolytic phenotype and produced no external hemolysin in liquid medium but still showed production of internal hemolysin were taken for further analysis and designated Hly,j-/ Hlyin' mutants. Tn5 insertions into the hly determinant of pANN202-312 (8) were obtained by transforming pANN202-312 into E. coli 605 and growing this strain for more than 50 generations at 30°C.

The hemolytic phenotype in Escherichia coli is determined by four genes. Two (hlyC and hlyA) determine the synthesis of a hemolytically active protein which is transported across the cytoplasmic membrane. The other two genes (hlyBa and hlyBb) encode two proteins which are located in the outer membrane and seem to form a specific transport system for hemolysin across the outer membrane. The primary product of gene hlyA is a protein (protein A) of 106,000 daltons which is nonhemolytic and which is not transported. No signal peptide can be recognized at its N‐terminus. In the presence of the hlyC gene product (protein C), the 106,000‐dalton protein is processed to the major proteolytic product of 58,000 daltons, which is hemolytically active and is transported across the cytoplasmic membrane. Several other proteolytic fragments of the 106,000‐dalton protein are also generated. During the transport of the 58,000‐dalton fragment (and possible other proteolytic fragments of hlyA gene product), the C protein remains in the cytoplasm. In the absence of hlyBa and hlyBb the entire hemolytic activity (mainly associated with the 58,000‐dalton protein) is located in the periplasm: Studies on the location of hcmolysin in hlyBa and hlyBb mutants suggest that the gene product of hlyBa (protein Ba) binds hemolysin and leads it through the outer membrane whereas the gene product of hlyBb (protein Bb) releases hemolysin from the outer membrane. This transport system is specific for E coli hemoiysin. Other periplasmic enzymes of E coli and heterologous hemolysin (cereolysin) are not transported.

Actinobacillus actinomycetemcomitans strains showing a 530-bp deletion in the promoter region of the leukotoxin gene operon elaborate high amounts of leukotoxin that may play a role in the pathogenesis of periodontal disease. This study used polymerase chain reaction detection to determine the occurrence of the 530-bp deletion in 94 A. actinomycetemcomitans strains from individuals of various ethnic backgrounds. Eleven blacks and one Hispanic subject but no Caucasian or Asian subjects showed the 530-bp deletion in the leukotoxin promoter region, suggesting that the deletion is mainly a characteristic of individuals of African descent. A. actinomycetemcomitans strains exhibiting a deletion in the leukotoxin promoter region occurred both in individuals having severe periodontitis and in adolescents revealing no evidence of destructive periodontal disease.

No abstract

The HlyA protein (Mr 110 kDa) which is the gene product of the hlyA gene encoded by the hemolysin determinant of Escherichia coli (Goebel, W. & Hedgpeth, J. (1982) J. Bacteriol. 151, 1290-1298) was observed to accumulate in the culture supernatant (in the presence of the three other Hly proteins HlyC, B and D) throughout the active growth cycle. However, the amount of extracellular HlyA protein did not correlate with the external hemolytic activity, which declined when the cells entered the stationary phase. External hemolytic activity was highly sensitive to phospholipase C and to ultrasonication. The size of the HlyA protein on SDS-PAGE was not changed by these treatments although the hemolytic activity was entirely abolished. On a polyacrylamide gel containing 2M urea but only 0.1% SDS hemolytically active HlyA migrated slightly ahead of the inactive HlyA suggesting that HlyA is more negatively charged than HlyA. Active hemolysin from unconcentrated hemolytic supernatants migrated on Sephacryl S-400 and on glycerol gradients as large complexes. Analysis of the hemolytically active fractions on SDS-PAGE yielded in both cases only HlyA (110 kDA) as major protein. An internal hemolytic activity appeared in most Escherichia coli K-12 strains in the stationary phase which was independent of the presence of HlyA or any other Hly gene product. This hemolytic activity which reached in some strains about 10% of the level determined by the hly genes was sensitive to proteinase K and disappeared upon shift of the cells to the logarithmic phase.

By using improved pulsed field gel conditions, the karyotypes of several strains of the protozoan parasite Trypanosoma cruzi were analyzed and compared with those of Leishmania major and two other members of the genus Trypanosoma. There was no difference in chromosome migration patterns between different life cycle stages of the T. cruzi strains analyzed. However, the sizes and numbers of chromosomal bands varied considerably among T. cruzi strains. This karyotype variation among T. cruzi strains was analyzed further at the chromosomal level by using multicopy genes as probes in Southern hybridizations. The chromosomal location of the genes encoding a-and 13-tubulin, ubiquitin, rRNA, spliced leader RNA, and an 85-kilodalton protein remained stable during developmental conversion of the parasite. The sizes and numbers of chromosomes containing these sequences varied among the different strains analyzed, implying multiple rearrangements of these genes during evolution of the parasites. During continuous in vitro cultivation of T. cruzi Y, the chromosomal location of the spliced leader gene shifted spontaneously. The spliced leader gene encodes a 35-nucleotide RNA that is spliced in trans from a 105-nucleotide donor RNA onto all mRNAs in T. cruzi. The spliced leader sequences changed in their physical location in both the cloned and uncloned Y strains. Associated with the complex changes was an increase in the infectivity of the rearranged variant for tissue culture cells. Our results indicate that the spliced leader gene clusters in T. cruzi undergo high-frequency genomic rearrangements.

We describe the first example of unstable gene amplification consisting of linear extrachromosomal DNAs in drug-resistant eukaryotic cells. cx-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an amplified ornithine decarboxylase (ODC) gene copy number contained two new extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a preexisting wild-type chromosome, probably by a conservative amplification mechanism. Both elements were unstable in the absence of DFMO, and their disappearance coincided with a decrease in ODC activity and an increase in DFMO growth sensitivity. These results suggest the possibility that ODC70-C may play a role in DFMO resistance. These data expand the diversity of known amplification mechanisms in eukaryotes to include the simultaneous unstable amplification of both linear and circular DNAs. Further characterization of these molecules will provide insights into the molecular mechanisms underlying gene amplification, including the ability of linear amplified DNAs to acquire telomeres and the determinants of chromosomal stability.