PCR analysis of E. coli strains.
A significant number of clinical E. coli isolates belonging to the most representative groups of pathogenic E. coli was analyzed by PCR with regard to the presence of the clyA gene, using primers designed according to clyAK-12 and flanking DNA sequences. The tested strains included 23 STEC, 7 EIEC, 8 EAEC, 7 ETEC, 17 EPEC, 14 UPEC, and 3 NMEC strains (Table ). Two additional strains which were isolated from the stool of healthy individuals, E. coli 764 and E. coli RS226, have not been assigned to a specific pathogroup but belong to serotypes that are frequently encountered among UPEC and NMEC strains, respectively.
By using several primer combinations, DNA fragments could be amplified from all STEC, EIEC, and EAEC strains, from 5 of the 7 ETEC strains (117/86, 147/1, G1253, 164/82, and ST3135B/01), and from 9 of the 17 EPEC strains (111/87, 212/87, 402/87, 227/63, 315/60, 12810, 16-2, 6447/89, and 6587/85) that were indistinguishable in size, when analyzed by agarose gel electrophoresis, from the PCR products obtained under the same conditions from E. coli K-12 (Fig. ). Slightly shorter DNA fragments were amplified from the ETEC strains 284/97 and 297/87, indicating the presence of small deletions in the clyA genes of these strains. DNA fragments about 0.7 kb shorter than expected were amplified from the remaining eight EPEC strains, from all UPEC and NMEC strains, and from E. coli 764 and E. coli RS226 when the PCR was conducted with primers binding more than 0.16 kb upstream and immediately downstream from clyAK-12. In addition, no PCR products were obtained from these strains when primers binding to the 5′-terminal two-thirds of clyAK-12 or to the DNA region preceding clyAK-12 were used, suggesting that the latter strains have chromosomal deletions of about 0.7 kb affecting clyA and the 5′-flanking DNA sequence.
FIG. 1. (A) Schematic presentation of the clyA gene from E. coli K-12. The position of the SlyA-controlled clyA promoter (p) (25) is indicated by an open box, and the binding sites of several primers used for PCR are shown by arrows (P1, 5′-GCCAGCAGATCAATACTG-3′; (more ...) Characterization of the clyA sequences of E. coli wild-type strains.
Sequencing of the PCR-amplified clyA-carrying or clyA-related DNA fragments from all 81 E. coli strains listed in Table yielded the following results.
(i) Identification of an intact clyA gene in STEC, EIEC, EAEC, and ETEC strains
A complete clyA gene encoding, like clyAK-12, a protein of 303 amino acid residues was found in 19 of the 23 tested STEC strains, in all 7 tested EIEC strains, in 6 of 8 EAEC strains, and in 4 of the 7 tested ETEC strains (Tables and ). The clyA genes of some of these strains (EAEC strains 17-2, 5477/94, and OPA065 and ETEC strain 117/86) were identical in sequence to clyAK-12, but in most cases several nucleotide substitutions (between 5 and 16) were detected. Some of these substitutions proved to be highly conserved in strains belonging to the same E. coli pathogroup or even in members of different pathogroups.
Characteristics of the clyA gene in E. coli wild-type strains analyzed in this study
The amino acid sequences predicted for the clyA gene products of the above strains were either identical to that of ClyAK-12 (EAEC strains 17-2, 5477/94, and OPA065; ETEC strain 117/86; serogroup O26 STEC strain ST2415/01) or contained between one and three amino acid exchanges, which corresponds to a sequence identity of >99%. In particular, compared to ClyAK-12 the following amino acid substitutions were found: a single N220→S exchange in the putative ClyA proteins of several EIEC (78-5, 107-11), EAEC (D4140-86, 4185/95), and ETEC strains (147/1, G1253, ST3135B/01); K75→N/A199→T in ClyA of the EIEC strains 76-5, 4608-58, and 309-36/85; L99→F/V185→I in ClyA of the EIEC strains 12860 and W7062 and of EAEC strain DEF40; D64→N/N220→S/K279→N in the ClyA proteins of all 18 serogroup O157 STEC strains harboring an intact clyA gene.
The DNA sequences flanking the intact clyA
genes of the different E. coli
strains proved to be very similar to those flanking clyAK-12
. In several cases (STEC strain ST2415/01; EIEC strains 12860, W7062, 76-5, 4608/58, and 309-36/85; EAEC strains 17-2, 5477/94, and OPA065; ETEC strains 117/86 and G1253) at least the first 185 bp preceding clyA
were the same as those in E. coli
K-12. This DNA region carries the SlyA-controlled promoter of clyA
). The remaining strains exhibited a few nucleotide substitutions in the clyA
5′-flanking sequence, but the −10 and −35 signals of the clyA
promoter (78 to 84 and 102 to 107 bp upstream from the translational start codon of clyA
) were generally not affected. Most of the latter strains, including all serogroup O157 STEC strains, exhibited a C→T and a G→T exchange in the spacer between the −10 and −35 signals and a T→C substitution 61 bp upstream from the clyA
(ii) Detection of frameshift mutations in clyA of several STEC, ETEC, and EPEC strains.
Several E. coli wild-type strains were shown by DNA sequencing to harbor mutant clyA derivatives containing 1-bp frameshift mutations that cause premature truncation of the clyA open reading frame (ORF) (Tables and , Fig. ). In three serogroup O157 STEC strains (3817/96, 4299/96, and 4304/96) we found, for example, a clyA derivative exhibiting a unique 1-bp deletion in codon 248. Interestingly, this clyA derivative was otherwise identical in sequence to the intact clyA gene found in all other tested O157 STEC strains. In the clyA genes of the ETEC strains 284/97 and 297/87, which were already predicted from the PCR data to contain small deletions, we detected not only an in-frame deletion of the codons 179 to 182 but also an identical 1-bp insertion in codon 163. Furthermore, a unique 1-bp deletion was found in codon 15 of clyA from strain 297/87. All nine EPEC strains that did not exhibit noticeable clyA defects upon PCR analysis (111/87, 212/87, 402/87, 227/63, 315/60, 12810, 16-2, 6447/89, and 6587/85) were shown by DNA sequencing to harbor a mutant clyA gene exhibiting a specific 1-bp deletion in codon 165. The same deletion was also found in the EAEC strains DEF52 and DEF53, in ETEC strain 164/82, and in serogroup O128 STEC strain ST3494/03. In clyA of strain ST3494/03 we detected, in addition, a unique 1-bp deletion in codon 278. The clyA genes exhibiting the 1-bp deletion in codon 165 generally proved to be very similar or even identical in sequence. All of them encode an identical C-terminally truncated ClyA derivative with a predicted molecular mass of 19.03 kDa.
FIG. 2. Alignment of the amino acid sequences predicted for the truncated clyA gene products of several clinical E. coli isolates with the amino acid sequence of ClyA from E. coli K-12. Amino acid substitutions in the ClyA derivatives of the clinical E. coli (more ...)
The promoter regions of the mutant clyA genes from the above strains were either identical in sequence to that of clyAK-12 (ETEC strains 284/97, 297/87, and 164/82; all mentioned EPEC strains; EAEC strains DEF52 and DEF53; STEC strain ST3494/03) or corresponded to the clyA promoter regions of the O157 STEC strains harboring an intact clyA gene (STEC strains 3817/96, 4299/96, and 4304/96), suggesting that all these clyA derivatives may be expressed under appropriate conditions.
(iii) Detection of deletions at the clyA locus in UPEC, NMEC, and several EPEC strains.
Sequencing of the strikingly short clyA-related PCR products obtained from all tested UPEC and NMEC strains as well as from E. coli 764, E. coli RS226, and eight EPEC strains (700-36/85, 22CH, 273-4, 12-1, 1104/80, 3715/67, E2348/69, and 1083-36/91) demonstrated that all these strains harbor only DNA sequences corresponding to an internal fragment and to the 3′-terminal region of clyA. The sequence data further indicated that these clyA-related sequences are left from two deletions at the clyA locus which we refer to as deletion I and deletion II (Fig. , Tables and ). Deletion I generally comprised the 493-bp fragment spanning the 160 bp preceding clyA and the first 333 bp of clyA. Deletion II was found in two versions: in the UPEC strains 536, RZ460, and RZ485 it comprised the 217-bp fragment spanning the nucleotides 377 to 593 of clyA, while in all other strains it comprised the 204-bp fragment from nucleotides 382 to 585 of clyA (codons 128 to 195).
The residual clyA sequences of the above-mentioned E. coli strains were at least 96% identical to the corresponding fragments of clyAK-12. Several nucleotide substitutions were found in all or in most of these strains, whereas others could be detected only in strains belonging to the same pathotype. The following groups of strains harbored identical residual clyA sequences: (i) the EPEC strains 700-36/85, 22CH, 273-4, 1104/80, 3715/67, and E2348/69, UPEC strain RZ533, and E. coli 764; (ii) the three UPEC strains exhibiting the larger version of deletion II (536, RZ460, and RZ485); (iii) all tested UPEC strains containing the smaller version of deletion II, except J96 and RZ533; (iv) all tested NMEC strains (IHE3034, IHE3036, and RS218) and E. coli RS226. The clyA sequences of UPEC strain J96 differed from those of the NMEC strains only at a single nucleotide position.
It is unlikely that these residual clyA sequences are expressed, because they lack a translational start codon and a fortuitous TAA stop codon is present 22 bp upstream from deletion I, in frame with the clyA coding sequence. Furthermore, the clyA promoter region is completely removed by deletion I.
Analysis of the stability of clyA in E. coli wild-type strains.
In order to test the stability of the clyA sequence in E. coli wild-type strains, four randomly selected clyA+ strains (STEC 3232/96, EIEC 4608-58, EAEC 5477/94, and ETEC G1253) were grown for 7 days in 2×YT broth with daily dilution of the cultures (1:100) into fresh medium. Subsequently, the clyA gene was amplified by PCR, using in each case the bacteria from 1 μl of the final culture as template. Sequencing of the PCR products yielded definite clyA sequences identical to those originally determined for the corresponding strains, indicating that the clyA genes of these strains are quite stable upon prolonged subculturing.
Expression of clyA from clinical E. coli isolates in E. coli K-12.
genes of several clinical E. coli
isolates were cloned into pUC18 and pUC19 as described in Materials and Methods. Four of the resulting plasmids, pCLYA3232/96, pCLYA12860, pCLYA5477/94, and pCLYAG1253, carrying the functional clyA
genes from STEC 3232/96, EIEC 12860, EAEC 5477/94, and ETEC G1253 under control of their native promoter regions, caused a hemolytic phenotype when introduced into the E. coli
K-12 strain DH5α. The hemolytic activity on blood agar resembled in each case that of DH5α carrying clyAK-12
on plasmid pAL202 (Fig. ). As shown in Fig. , these recombinant DH5α clones also produced amounts of the 34-kDa ClyA protein similar to amounts produced by E. coli
DH5α/pAL202. Transformation of a slyAK-12
-carrying plasmid (pAL108) into the DH5α clones harboring pAL202, pCLYA3232/96, pCLYA12860, pCLYA5477/94, and pCLYAG1253 resulted in each case in enhanced production of ClyA and in a significantly stronger hemolytic phenotype on blood agar, demonstrating that the clyA
genes of the corresponding clinical E. coli
isolates are positively controlled by SlyA, like clyAK-12
(Fig. and ). It should be pointed out that the stronger hemolytic phenotype of the DH5α double transformants does not completely reflect the enhancement of clyA
expression, because ClyA overproduced in E. coli
accumulates in the periplasmic space and only small amounts of it are released from the bacteria (25
and data not shown).
FIG. 3. Hemolytic phenotypes of E. coli DH5α clones harboring the following plasmids: pAL202 (1a), pCLYA3232/96 (1b), pCLYA12860 (1c), pCLYA5477/94 (1d), pCLYAG1253 (1e), pCLYA212/87 (2a), pCLYA284/97 (2b), pCLYA297/87 (2c), pAL202 and pAL108 (3a), pCLYA3232/96 (more ...)
FIG. 4. Western blot analysis of the expression of cloned clyA genes from clinical E. coli isolates in E. coli DH5α, using a polyclonal anti-ClyA antiserum. The production of ClyA was determined in E. coli DH5α clones carrying the following plasmids: (more ...)
By using the method of quantitative real-time reverse transcription-PCR we recently observed that transcription of clyA in exponentially growing E. coli DH5α/pAL202/pAL108 is 5- to 10-fold stronger than in E. coli DH5α carrying pAL202 only in combination with the vector pACYC184 (C. von Rhein and A. Ludwig, unpublished data). Similar results would be expected for corresponding experiments performed with isogenic E. coli DH5α clones carrying pCLYA3232/96, pCLYA12860, pCLYA5477/94, or pCLYAG1253 instead of pAL202.
The plasmids pCLYA212/87, pCLYA284/97, and pCLYA297/87, carrying the mutant clyA genes of EPEC 212/87 (clyA212/87), ETEC 284/97 (clyA284/97), and ETEC 297/87 (clyA297/87) under control of their own promoter sequences, did not cause a hemolytic phenotype when introduced into E. coli DH5α. Furthermore, transformation of pAL108 into the DH5α clones harboring these plasmids caused in each case only very weak hemolytic activity on blood agar due to the SlyA-mediated induction of the chromosomal clyAK-12 gene (Fig. and ). Proteins corresponding in size to the predicted products of clyA212/87 (19.03 kDa) and clyA284/97 (19.28 kDa) were specifically detected by Western blot analysis in cell lysates of E. coli DH5α harboring pCLYA212/87 and pCLYA284/97, respectively. The corresponding DH5α double transformants carrying pAL108 as well produced markedly larger amounts of these ClyA derivatives, confirming that clyA212/87 and clyA284/97 are positively controlled by SlyA (Fig. ). Nevertheless, in the absence as well as in the presence of pAL108 the cellular levels of ClyA212/87 and ClyA284/97 were significantly lower than those of complete, functional ClyA proteins expressed under identical conditions, which suggests that these truncated ClyA derivatives are more unstable. E. coli DH5α transformed with pCLYA284/97A, a pUC18 derivative carrying clyA284/97 under control of the lacZ promoter, produced rather large amounts of ClyA284/97 (Fig. ) but was also nonhemolytic on blood agar. In addition, no significant hemolytic activity could be detected in cell lysates of this strain by a quantitative hemolytic activity assay. The product of clyA297/87 (predicted molecular mass, 2.63 kDa) could be detected neither in lysates of E. coli DH5α/pCLYA297/87 nor in those of DH5α carrying both pCLYA297/87 and pAL108.
Analysis of the expression of clyA in clinical E. coli isolates.
Several E. coli strains possessing a functional clyA gene, such as STEC (EHEC) 3232/96, EIEC 12860, EIEC 4608-58, and ETEC G1253 showed a nonhemolytic phenotype when grown overnight on blood agar containing horse erythrocytes (the weak enterohemolytic phenotype of STEC strain 3232/96 caused by the production of EHEC-HlyA was visible only on sheep blood agar). Nevertheless, the colonies of EIEC strain 12860 developed a hemolytic phenotype on horse blood agar when the agar plate was stored for several days at 4°C after the initial overnight incubation at 37°C. A clyA knockout mutant of strain 12860 (E. coli 12860ΔclyA) remained nonhemolytic under the same conditions, demonstrating that this hemolytic phenotype is clyA dependent (Fig. ).
FIG. 5. (A) Phenotypes of the EIEC strains 12860 and 4608-58 and of derivatives of these strains on blood agar. Shown are E. coli 12860 (1a), E. coli 12860/pAL115 (1b), E. coli12860ΔclyA (1c), E. coli 12860ΔclyA/pAL115 (1d), E. coli 4608-58 (2a), (more ...)
To further study the expression of clyA in E. coli strains possessing a functional chromosomal clyA gene, we analyzed the cellular ClyA levels in stationary-phase cultures by immunoblotting with a polyclonal anti-ClyA antiserum. When a highly sensitive Western blotting detection system was employed, a protein of about 34 kDa corresponding to ClyA could be specifically detected in cell lysates of all tested E. coli strains harboring an intact clyA gene, such as DH5α, 4608-58, and 12860, but not in lysates of E. coli 12860ΔclyA (Fig. ). The amounts of ClyA found in the different clyA+ strains were very similar to each other, indicating that all these strains expressed clyA at similar low, basal levels. According to these data, the hemolytic phenotype of older colonies of EIEC strain 12860 is apparently not due to a stronger expression of clyA in this strain compared to that in the other strains but most likely is due to enhanced release of the toxin from the bacteria.
Introduction of a slyAK-12
-carrying plasmid (pAL105 or pAL115) into the E. coli
strains 3232/96, 12860, 4608-58, and G1253 by electroporation caused in each case a hemolytic phenotype, in line with the finding that the functional clyA
genes of these strains are positively controlled by SlyA. Consistent with this, it was recently shown at the protein level that overexpression of SlyA in EIEC strain 12860 causes enhanced production of ClyA (41
). As shown in Fig. , E. coli
12860/pAL115 exhibited clearly stronger hemolytic activity on blood agar than E. coli
4608-58/pAL115, again suggesting that strain 12860 releases ClyA more readily than other clyA+ E. coli
knockout mutants of E. coli
12860 and E. coli
) remained nonhemolytic after introduction of pAL115, confirming that the hemolytic activity of the SlyA-overproducing wild-type strains is dependent on clyA
ETEC strain 297/87 and EPEC strain 212/87 (Ampr) were nonhemolytic on blood agar and retained this phenotype after introduction of slyAK-12-carrying plasmids (pAL105 and pAL108, respectively), consistent with the finding that the clyA genes of both strains encode only truncated, obviously nonhemolytic ClyA derivatives. ETEC strain 284/97 (Cmr), also harboring a defective clyA gene (see above), exhibited a strongly hemolytic phenotype that was not affected by introduction of pAL105. Southern blot analysis of genomic DNA from E. coli 284/97 using an E. coli α-hemolysin-specific DNA probe isolated from plasmid pANN202-812 revealed a single DNA fragment that hybridized with this probe. In addition, a protein possessing a molecular mass similar to that of HlyA (approximately 110 kDa) was specifically detected in culture supernatants of E. coli 284/97 by Western blot analysis using a polyclonal anti-HlyA antiserum (data not shown). These findings indicated that the hemolytic activity of E. coli 284/97 is most likely due to the production and secretion of α-hemolysin or of a closely related toxin.