Transport of hemolysin across the outer membrane of Escherichia coli ...

Vol. 154, No. 1

JOURNAL OF BACTERIOLOGY, Apr. 1983, p. 200-210 0021-9193/83/040200-11$02.00/0 Copyright C) 1983, American Society for Microbiology

Transport of Hemolysin Across the Outer Membrane of Escherichia colt Requires Two Functions WILMA WAGNER, MONIKA VOGEL, AND WERNER GOEBEL* Institut fur Genetik und Mikrobiolcgie, Universitat Wurzburg, Wurzburg, West Germany

Received 18 October 1982/Accepted 30 December 1982

Among a large collection of hemolysis-negative mutants obtained by mutagenesis of the Hly plasmid pHlyl52 with TnS, we have isolated two classes of mutants which are defective in the transport of hemolysin across the outer membrane. The two cistrons (hylBa and hlyBb) which are affected in these mutants are located adjacent to each other on the hly determinant but are transcribed from different promoters. Recombinant plasmids were constructed which carry the two functions as combined or separated cistrons. These were shown to complement the two types of transport mutants. Studies on the compartmentation of hemolysin in these two classes of mutants indicate that most hemolysin (>70%) in hlyBa mutants is located in the periplasmic space, whereas in hlyBb mutants a larger portion of hemolysin is associated with the outer membrane fraction. The phenotypic appearance of colonies from hlyBb mutants is that of beta-hemolytic Escherichia coli strains, indicating that a substantial portion of hemolysin has already reached the outside of the outer membrane without being released into the medium. Release was achieved readily when hlyBb mutants were complemented with a recombinant plasmid carrying hlyBb.

Hemolysin is an extracellular protein which is produced by some strains of Escherichia coli, particularly those that are isolated from extraintestinal infections in humans (12, 17). Its involvement in the pathogenesis of such infections has been extensively discussed, and recent in vivo experiments seem to confirm its importance (30; J. Hacker et al., submitted for publication). The alpha-hemolytic phenotype (27), i.e., the synthesis of active hemolysin and its secretion into the surrounding medium, is controlled by determinants which are located either on transmissible plasmids or on the chromosome (7, 9, 12, 17). These determinants are structurally and functionally very similar to one another (6, 18). Two cistrons, hlyA and hlyC, are involved in the synthesis of active hemolysin and its subsequent transport across the cytoplasmic membrane (8, 21). The transport across the outer membrane requires the function of another cistron, hlyB, which is also part of the hly determinant (19, 20). This transport occurs during the active growth phase and is not accompanied by lysis of the cells (28). We show here that hlyB consists of two genes, termed hlyBa and hlyBb, which were identified by transposon mutagenesis and complementation with recombinant plasmids carrying these two genes. hlyBa seems to be required for the translocation of hemolysin from the periplasm to

the outside of the cell, whereas hlyBb is necessary for the release of hemolysin from the outer membrane. 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 pANN202312 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. 200

VOL. 154, 1983

Plasmid DNA was isolated as described previously (20) and run on a S to 20% sucrose gradient. Only those fractions sedimenting ahead of the band containing the supercoiled pANN202-312 DNA were taken for transformation of E. coli HB101 mod res recA. Transformants were selected on blood-agar plates (28) containing chloramphenicol (20 ijg/ml) and kanamychi (30 F.g/ ml). Since pANN202-312 carries the gene determining chlorarhphenicol acetyl transferase (cat gene), only pANN202-312 (TnS) derivatives will grow on these plates, and Tn5 mutants inserted into the hly determinant are easily recognized by the nonhemolytic phenotype. Complementation analyses. Complementation analyses were carried out in a recA background, either by transforming a pANN202-312 derivative into the recA strain HB101 carrying a pHlyl52::TnS mutant or by transferring the pHlyl52: :TnS or pHlyl52::Tn3 mutant irnto the recA strain already containing a pANN202-312 derivative. Cell fractionation. Cells were grown in brain heart infusion broth (Difco Laboratories) at 37°C to a density of 3 x 108 cells per ml. Cells were pelleted by centrifugation and resuspended either in "accumulation medium" as described previously (28) or in fresh brain heart infusion broth at a cell density of 3 x 109 cells per ml. After incubation at 37°C the culture was centrifuged again, and the supernatant was taken for the determination of external hemolysin. The cells were washed twice with 10 mM Tris-hydrochloride (pH 7.0) and then suspended into 1/10 of the original culture volume of buffer containing Tris-hydrochloride (pH 7.4, 10 mM), sucrose (25%), sodium EDTA (40 mM), and lysozyme (100 Fg/ml; Sigma Chemical Co.), as described by Koshland and Botstein (16). After 30 min on ice, the cells were pelleted, the supernatant (designated periplasmic) was removed, and the cell pellet was suspended in sodium phosphate buffer (pH 6.8, 10 mM) at a density of 3 x 109 cells per ml. Alternatively, cells were first osmotically shocked as described previously (19) and then resuspended in the same sodium phosphate buffer. Lysis of the cells was obtained by several (8 to 12) 15-s bursts with a Branson ultrasonifier. Unlysed cells and large cell debris were removed by low-speed centrifugation, and the supernatant containing the membranes and the cytoplasm was centrifuged at 35,000 rpm in a 50 Ti rotor for 2 h at 4°C. The supernatant (designated soluble fraction) was carefully removed, and the membrane pellet was suspended in 25% sucrose as described by Osborn et al. (22). The separation of the outer and inner membrane fractions was performed as described by Osborn et al. (22) by using a step gradient between 55 and 30%o sucrose. For the determination of hemolysin in the outer membrane, only the H band was used, and for the determination of hemolysin in the cytoplasmic membrane, both the Li and L2 bands were collected. The amount of hemolysin was normally low in the M band, which was therefore discarded. The membranes were pelleted by centrifugation and suspended in a small volume of sodium phosphate buffer (pH 6.8, 10 mM). Hemolysin assay. The hemolysin assay mixtures contained (in 0.8 ml) CaCl2 (20 mM), Tris-hydrochloride (pH 7.4, 10 mM) or sodium phosphate (pH 6.8, 10 mM), NaCl (160 mM), washed human erythrocytes (2%), and an appropriate volume of the various ex-

TRANSPORT OF HEMOLYSIN

201

tracts containing hemolysin. These were always adjusted in such a way that they were comparable (based on the cell number). The reaction mixtures were placed at 37°C, samples were removed at 1, 3, 5, and 10 min and chilled on ice, and the Uinlysed erythrocytes were removed by centrifugation for 1 min in an Eppendorf centrifuge. The absorbance of the supernatant at 530 nm was determined and plotted as a function of time. The values given are the relative rates of hemolysis, where the rate given by total internal hemolysin of E. coli 536 (used as a standard in all experiments described here) from cells of a 20-ml culture (density, 5 x 108 cells per ml) was set as 100.

RESULTS

Isolation of TnS-induced pHlyl52 mutants defective in transport of hemolysin across the outer membrane. The Hly plasmid pHlyl52 was transferred by conjugation into E. coli 605, which contains TnS integrated into the chromosome. This strain was used as the donor in a mating cross with E. coli HB101 rpsL. Transconjugants were selected which were hemolysin negative on erythrocyte plates containing streptomycin and kanamycin. We screened the transconjugants for mutants (Table 1) which still contained internal hemolysin, using the method described by Springer and Goebel (28). By propagating pANN202-312 in E. coli 605(TnS) we also isolated Tn5 mutants of this recombinant plasmid (Table 2), which contains the whole hemolysin (hly) determinant of pHlyl52 cloned into pACYC184 (8). Again, only those mutants, which formed hemolysis-negative colonies on erythrocyte plates but produced internal hemolysin were further analyzed. Similar mutants were previously obtained from pHlyl52 by transposon mutagenesis with Tn3 (20, 21) and were designated Hlyin+/Hlyex. To locate the site of the TnS insertions in the new Hlyin+/Hlyex mutants, plasmid DNA was isolated from several mutants and cleaved with EcoRI and HindlIl. TnS does not contain an EcoRI site but contains two symmetric IJindIII sites within the inverted repeats of TnS (15), thus allowing the precise location of Tn5 within the already known physical map of the hly determinant of pHlyl52 (8, 20). Figure 1 summarizes the data obtained from this analysis for some of the mutants used in this study. The distance between the left- and rightmost TnS insertions within the hly determinant leading to a Hlyin+/Hlyr - phenotype is almost 3 kilobase pairs (kb). The Tn3 insertions of Hlyin+/Hlye - mutants which we have previously mapped in pHlyl52 were all located within EcoRI-G (between coordinates 3.8 and 7 kb), whereas some of the new Hlyin+/Hlye. TnS mutants (e.g., mutant H in Fig. 1) analyzed here are more than 1 kb to the right of EcoRI-G, indicating that the affected region leading to a

202

WAGNER, VOGEL, AND GOEBEL

J. BACTERIOL.

Hlyin+/Hly,j-

TABLE 1. Hemolysin activity in mutants of E. coli K-12(pHlylS2) and complementation by recombinant plasmids carrying hlyB cistrons Complementationb to Hly,x+/Hlyin+ by: Class of mutant

Plasmid carried

Hly..a

Hly.na

pANN205-222 (hlyBa+

pANN250-222 (hlyB8+)

pANN260 (hIyBb+)

hlyBb+)

Control (wild type)

pHlyl52

Ic (hlyBa)

Ild (hlyBb)

75

65

pHlyl52::TnS-1 pHlyl52::Tn5-2 pHlyl52::TnS-12 pHlyl52::TnS-15 pHlyl52::TnS-23

0 0 0 0 0

92 95 97 93 98

+ + + + +

pHlyl52::TnS-5

0 0 0 0 0

55 58 62 65 51

+ + + + +

pHlyl52::TnS-6 pHlyl52::TnS-13 pHlyl52::Tn5-18 pHlyl52::TnS-19

+ + + + +

-

-

-

+ + + + +

-

-

IIIe (hlyA)

pHlyl52::TnS-4 0 33 pHlyl52::TnS-11 0 31 a Determinations of external (cell-free) and internal hemolysin activity (Hly,x and Hlyin, respectively) were performed as previously described (8). The amount of hemolysin activity is given relative to that of a standard strain (E. coli 536) which carries a chromosomal hly determinant with an hlyBb mutation. This strain was grown under the same conditions as the test strains, and its total hemolysin activity (entirely internal) was set at 100. b Complementation was always performed in a recA background. c The TnS insertions of these pHlyl52 mutants were mapped as described in the legend to Fig. 1 and found to be located between 5.5 and 6.5 kb. d The TnO insertions of these pHlylS2 mutants map between 7.1 and 8.3 kb. e Mapping of the TnS insertions of these pHlylS2 mutants is shown in Fig. 1.

defect in the secretion of hemolysin is larger than we previously assumed (8, 20). Complementation analysis of Hlymn+/Hlyex. mutants by recombinant plasmids. It was previously shown (20) that the recombinant plasmid pANN250, which contains the EcoRI-G fragment inserted into pACYC184, is capable of complementing Tn3 mutants which are phenotypically HlYin+/Hlyex-. The external hemolysin was, however, never restored to the wild-type

level by this complementation. We therefore tried to increase the complementation activity located on this fragments by inserting the EcoRI-G fragment behind the lac OP region into the single EcoRI site of pUR222 (26). As expected from previous results (20, 21), full complementation activity to the Hly/Hly phenotype was only observed in one orientation (orientation 3.8 to 7 kb [Fig. 2]). The level of excreted hemolysin was considerably higher

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FIG. 1. Physical map of the hly determinant of pHlylS2 and location of Tn5 insertions leading to hemolysisnegative mutants. The symbols indicate the site where Tn5 is inserted as determined by EcoRI and HindIII cleavages of the isolated plasmid DNA (see text). A, pANN202-312::TnS-4 (Table 3); B and C, pHlylS2::TnS-4 and pHlylS2::TnS-11 (Table 1), respectively; D, pHlyl52::TnS-1; E, pANN202-312::TnS-2; F, pHlylS2::Tn5-23; G, pHlylS2::TnS-13; H, pHlylS2::TnS-19. The coordinates of the hly determinant have been previously described (8). Symbols: + HindIII; A BamHI; PstI; A, EcoRI; 4 BglII. ,

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VOL. 154, 1983

TRANSPORT OF HEMOLYSIN

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204

WAGNER, VOGEL, AND GOEBEL hlyA

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FIG. 2. Restriction map of the hly determinant and structure of deletions, inversions, and recombinant plasmids derived from pANN202-312. The 10-kb segment of the hly determinant within pANN202-312 is shown with the hly cistrons placed according to the functional analysis of these derivatives and the described transposon mutants of pHlylS2 (see text). The deletion mutant pANN202-3129 was obtained by digesting pANN202-312 partially with PstI and joining the fragments by treatment with T4 DNA ligase. The deletion mutant pANN2023128 was obtained by Sall cleavage and religation of the DNA of a Tn5 insertion mutant of pANN202-312 (pANN202-312::Tn5-2, indicated by the bar with the single Sail site, at coordinate 6.2 kb). The inversion in pANN202-3127 ( I ) was performed by cleaving pANN202-312 completely with BamHI and partially with BglII and religating the fragments. pANN205-222 is a recombinant plasmid carrying the BgIII fragment from coordinates 3.9 to 9.8 kb inserted into the BamHI site of pUR222 (26), whereas in pANN25S-222 the EcoRI fragment from coordinates 3.7 to 7 kb was inserted into the EcoRI site of pUR222. pANN260 carries the BglII fragment from the BglIH site at the right part of TnS (the TnS insertion of pANN202-312 at coordinate 6.2 kb) to the BgiII site of the hly determinant at 9.8 kb, inserted into the BamHI site of pBR322. The symbols for restriction sites are as described in the legend to Fig. 1. o, Sall.

with this new recombinant plasmid (pANN250222) than with pANN250 (Table 3). Of the newly isolated Hlyinl/Hlycx- mutants, only those which contained the TnS insertion between coordinates 5.3 and 6.5 kb (mutants D to F in Fig. 1) could be complemented by pANN250-222 to hemolysin secretion (Hlyin+/Hly,x+). None of the Hlyin+/Hly.,j mutants with TnS insertions located further upstream (mutants B and C at coordinates 4.7 and 4.8 kb) or further downstream (mutants G and H at coordinates 7.2 and 8.3 kb) from this region were complemented by pANN250-222 (Table 1). The BglII fragment between coordinates 3.9 and 9.8 kb comprises the whole region of the hly determinant where Tn5 insertions lead to Hlyi,+/ Hly,.- mutants. This fragment was inserted into the BamHI site of pUR222 in both orientations. The obtained recombinant plasmid, pANN205-222 (Fig. 2), was able to complement in both orientations all Hlyi.+/Hlyex- mutants with Tn5 insertions between coordinates 7.0 and 8.3 kb (Table 1) which were not comple-

TABLE 3. Complementation of hIyBa mutants by recombinant plasmids carrying the hlyBa function under the control of lac OP Hemolysin activitya after Recombinant

plasmid

None pANN250

pANN250-222b pANN250-222c pANN205-222d

complementation of: pANN202312: :TnS-2

pHlyl52::Tn5-2

Hlyi.

HlYex

Hlyiy

115 98 65 120 69

0 6

125 105 58 117 73

55

20 62

Hly.. 0 13 55

15 55

aThe hemolysin activity was determined as described in Table 1. b The orientation of the EcoRI-G fragment was from 3.7 to 7.0 kb of the hly determinant of pHlyl52 (Fig. 1) in the EcoRI site of pUR222. c The orientation of EcoRI-G was from 7.0 to 3.7 kb in pUR222. dThe orientation of the BglIl fragment was from 9.8 to 3.9 kb in the BamHI site of pUR222.

VOL. 154, 1983

mented by pANN250-222. Again, the two Hlyi,+/Hlye,- mutants at coordinates 4.7 and 4.8 kb were not complemented by pANN205-222 in either orientation. An unexpected result was obtained when the complementation of Hlyi,+/ Hlyex- mutants with TnS insertions between 5.3 and 6.5 kb was carried out with pANN205-222. From the previous results with pANN250-222, we expected that the BgIII fragment would complement these mutants only when inserted in orientation from coordinate 3.9 to 9.8 kb into the BamHI site of pUR222 behind the lac OP region. Yet, when pANN205-222 in this orientation was transformed into class I mutants, tiny hemolytic colonies were obtained which grew very poorly. These colonies could not be restreaked on fresh plates, indicating that they contained only nonviable cells. The other orientation, however, which was expected to be inactive, yielded hemolytic colonies which grew normally and produced almost normal levels of hemolysin (Table 3). We interpret this result as indicating that either the overproduction of the complementing activity or a protein formed by this recombinant DNA which consists of the first amino acids from ,-galactosidase and the Cterminal end of hlyA is lethal to this class of Hlyin+/Hlye,- mutants. The induction of hlyA has been previously shown to be lethal to the cells (8). The latter interpretation seems to be more likely since pANN250-222, which expresses, in orientation 3.8 to 7 kb, this activity under the same promoter but which may not synthesize such a fused protein, forms hemolytic viable cells after complementation of class I mutants. The complementation activity in the other orientation of pANN205-222 could be the result of transcription of the gene determining this complementation activity from the P-lactamase promoter of the vector. A weak complementing activity for class I mutants was also observed with pANN250-222 in this orientation (Table 3), which would be in agreement with this latter assumption. The two HlYin+/Hly,x, mutants with Tn5 insertions at coordinates 4.7 and 4.8 kb could not be complemented by one of the two recombinant plasmids (Table 1) and do not seem to belong to one of the two classes of Hlyin+/Hly,j- mutants. The TnS insertions of the latter mutants are located at the right end of a region of the hly determinant which has been identified in a previous study as cistron hlyA (8), determining a protein of 106,000 daltons which is presumably a precursor of hemolysin (8, and unpublished data). We therefore interpret this class of Hlyin+/Hlyex- mutants as hlyA mutants which produce a hemolysin with an altered C-terminal end. We-assume that this slightly truncated hlyA protein still contains the active center for the

TRANSPORT OF HEMOLYSIN

205

hemolytic activity but cannot be properly transported across the inner membrane (see below). The two other classes of Hlyi0+/Hlyex- mutants which are defective in secreting hemolysin can be complemented to the wild-type Hlyin+/ Hlyex+ phenotype by the recombinant plasmid carrying the BglII fragment. But in class I mutants complementation seems to be orientation dependent, and complementation (again orientation dependent) occurs also with a recombinant plasmid carrying EcoRI-G alone, whereas in the other class (class II) complementation by the recombinant plasmid carrying the BglII fragment is independent of the orientation and does not occur with EcoRI-G. It thus appears that two distinct functions are affected in these two classes of Hlyin+/HlYex- TnS mutants. This is also suggested by the appearance of the mutant colonies on erythrocyte plates. Fresh colonies of class I mutants were completely nonhemolytic, and a diffuse zone of hemolysis around the colony appeared only after several days, probably due to partial lysis of the cells. In contrast, fresh colonies of class II mutants formed small hemolysis zones, indicating that at least some hemolysin is exposed to the outside of the cell but probably can not diffuse into the surrounding medium. To clone the complementing activity for class II mutants, we started from the DNA of a Tn5 mutant of pANN202-312 which carries the TnS insertion at coordinate 6.2 kb of the hly determinant (Fig. 2). This DNA was cleaved with BglII, and the BglII fragments were cloned into the BamHI site of pBR322. Among the isolated recombinant plasmids, we obtained a few which could complement class II mutants. The recombinant plasmid (pANN260) of these clones carried the BglII fragment starting from the right BglII site of TnS to the BglII site at coordinate 9.8 kb of the hly determinant (Fig. 2). The complementating activity of this recombinant plasmid for class II mutants was independent of the orientation of the fragment within the vector, which indicates that this activity is expressed by its own promoter, in contrast to the complementing activity for hlyBa, which seems to be expressed under the control of the same promoter as hlyA and hlyC (see below). Below, we designate the complementing activity for Hlyin+/ Hlye,x mutants of class I as HlyBa and the cistron determining this activity as hlyBa, and the complementing activity for HlYin+/HlYex mutants of class II as HlyBb and its cistron as hlyBb. Cistron hyB. is not part of cistron hlyA. The statement that the HlyBa activity is really determined by an independent cistron (hlyBa) and that the complementation of Hlyin+/Hly,x- mutants of class I by pANN205-222 and pANN250-222

206

WAGNER, VOGEL, AND GOEBEL

J. BACTERIOL.

TABLE 4. Complementation of TnS- and Tn3-induced mutants of pHlyl52 by hlyA and hlyBa mutants of pANN202-312 Complementation by:' Mutant plasmid

pANN202-312::Tn5-2

pANN202-3127

(hlyB0)

(hlyA)

pANN202-3129 (hIyA)

+

+

+ +

+ +

+ +

+ +

pHlyl52::Tn5-22 (hly-) + pHlyl52::Tn3-4 (hlyC) pHlyl52::TnS-25 (hlyA) + pHlyl52::Tn3-21 (hlyA) pHlyl52::TrLS-1 (hlyBa) pHlyl52::Tn3-8 (hlyB,.) + pHlyl52::TnS-19 (hlyBb) + pHlyl52::Tn3-14 (hlyBb) a These mutant derivatives of pANN202-312 are described in Fig. 2.

does not represent intracistronic complementation by an active polypeptide, made from the Cterminal end of hlyA, which may be essential for the transport of hemolysin across the outer membrane is based on the two following experimental observations. (i) Hly- mutants of pHlyl52 which carry TnS in hlyC or hlyA did not show HlyB. activity when complemented with an hIyBa mutant of pANN202-312 (e.g., mutant H in Fig. 2; TnS was inserted at coordinate 6.3 kb into the cloned hly determinant of this recombinant plasmid). However, Hly- mutants of pHlyl52 with Tn3 insertions in hlyC or hlyA showed complementation activity with the same recombinant hlyBa mutant (Table 4). Likewise, a Tn3 mutant of pHlyl52 with a defect in hIyBa could not be complemented by an Hly- mutant of pANN202-312 with a TnS insertion in hlyC (mutant A in Fig. 1). Yet, when the TnS insertion was removed by cleavage with XhoI (J. Collins, personal communication), which leads to the removal of TnS and a small deletion in hlyC of pANN202-312, complementation with the Tn3 mutant of pHlyl52 was observed (Table 2). Since Tn5 is strongly polar in both orientations (1) but Tn3 is not, this suggests that hlyBa is transcribed from the same promoter as hlyC and hlyA but that the message for hlyBa is translated independently of that of hlyA. (ii) Two derivatives of pANN202-312 were constructed (Fig. 2). The first was the plasmid pANN202-3127 (Fig. 2), which carries the Bam-BglII fragment from coordinates 1.2 to 3.9 kb in an inverted orientation compared with pANN202-312. This plasmid does not exhibit hlyA activity but does exhibit hlyBa activity, as shown by complementation of Tn3 or TnS mutants of pHly152 which are blocked in hlyBa (Table 4). The second derivative was plasmid pANN202-3129, which has a deletion extending from coordinates 1.4 to 5.1 kb (Fig. 2). This deletion removes hlyA but still retains the hlyBa activity (Table 4). Of course, hlyC and hlyBb activities are also unaffected by this deletion.

Compartmentation of hemolysin in the hemolytic wild-type strain and its transport mutants. The data presented above indicate that both cistrons, hlyBa and hlyBb, are required for the secretion of hemolysin into the surrounding medium. Yet, they do not necessarily indicate that both functions are involved in the transport of hemolysin across the outer membrane, since the procedure used to test for internal hemolysin does not properly discriminate between cytoplasmic and periplasmic hemolysin. We therefore fractionated the cells into cytoplasm, periplasm, and membranes, which were further subdivided into inner and outer membrane fractions by the procedure of Osborn et al. (22). The active hemolysin was tested in each pool (Table 5). Under our conditions, the hemolytic wildtype strains excreted about 30%o of the total hemolysin into the medium; more than 50% was found in the periplasm as judged from the amount of hemolysin released during the formation of spheroplasts (8, 16, 19). The residual 20%o of the total hemolysin was about equally distributed in the membrane fraction and in the supernatant after high-speed centrifugation of the sonicated spheroplasted cells. The supernatant fraction contained cytoplasmic hemolysin or hemolysin which is only loosely bound to the membranes and is released during sonication or centrifugation. It may also contain residual periplasmic hemolysin which is not released during the spheroplast formation. The separation of the total membrane pellet into inner and outer membrane fractions according to the method of Osborn et al. (22) leads to a high loss of hemolytic activity (>90%o), probably due to proteolytic inactivation. The distribution of hemolysin between the two membranes has to be taken with caution, therefore, since we do not know whether hemolysin activity is lost in both membranes with the same efficiency. Within this limitation, the results indicate that most membrane-bound hemolysin of the wild-type strain is located in the outer membrane (data not shown).

TRANSPORT OF HEMOLYSIN

VOL. 154, 1983

Type of strain

Wild type

TABLE 5. Compartmentation of hemolysin in Hlyn+/Hlyex- mutants % of total hemolysin in: Membrane Total Peiplasmic Plasmid carried serplasic hemolysina fraction Supernatant space (total) 50 10 30 140 pHlylS2 32 52 8 130 pANN202-312

hlyBb mutant

hlyA mutant

a

Determined

Soluble fraction

fraction

10 8

135

31

51

9

9

pHlyl52::Tn5-1 pHlyl52::Tn5-3 pHlylS2::Tn5-15 pHlyl52::TnS-23 pANN202-312::TnS-2 pANN202-312::TnS-7

92 95 93 98 82 85

0 0 0 0 0 0

62 65 71 71 65 63

12 17 12 9 13 12

26 18 17 20 22 19

Avg

91

66

13

21

pHlyl52::TnS-5 pHlylS2::TnS-6 pHlylS2::TnS-13 pHlylS2::TnS-18 pHlylS2::TnS-19

55 58 62 65 51

48 42 40 45 25

36 32 38 40 43

16 26 22 15 32

Avg

58

40

38

22

pHlylS2::TnS4 pHlyl52::TnS-11

33 31