Since the ptx and ptl genes are cotranscribed, PT and its transporter might be produced at comparable levels. Alternatively, the PT subunits and Ptl proteins could be produced in different amounts for a number of possible reasons. For example, regulatory signals, such as attenuators, might exist at points along the operon that could alter the transcriptional efficiency of specific regions of the operon. Or the levels of translation of specific regions of the mRNA might differ such that the quantity of the PT subunits produced would differ from that of the Ptl proteins.
If the ptx and ptl genes are transcribed and translated at comparable levels, then introduction of a large in-frame deletion of the DNA within the region should not affect the production of Ptl proteins encoded by genes located downstream of the deletion. If, on the other hand, the ptx and ptl genes are transcribed or translated at different levels because, for example, either specific regulatory signals exist along the DNA and/or mRNA, different portions of the mRNA exhibit different stabilities, or ribosomes are released from the mRNA after translation of a specific gene and reinitiation of translation at downstream genes fails to occur, then introduction of a large in-frame deletion within the operon should affect the production of Ptl proteins encoded by downstream genes. In order to test whether a large in-frame deletion in this region of the chromosome results in altered production of Ptl proteins from downstream genes, we generated a 7.1-kb deletion, from nucleotide 936 to nucleotide 8003 of the ptx-ptl region, that retained the reading frames of the two partial genes spanning the deletion (Fig. ). With this deletion, the 5′ end of ptxS1 was fused to the 3′ end of ptlD, resulting in a gene that encoded a fusion protein consisting of the N-terminal 50% of the S1 subunit of PT and the C-terminal 15% of PtlD. The seven genes between ptxS1 and ptlD (ptxS2, ptxS4, ptxS5, ptxS3, ptlA, ptlB, and ptlC) were deleted in their entirety. Such a deletion would move ptlF approximately 7.1 kb closer to the promoter region of the operon (Fig. ). We then compared the amount of PtlF produced by this strain (BP536Δptxptl936-8003) with the amount produced by the wild-type strain (BP536). As seen in Fig. , significantly more PtlF was produced by the strain with the 7.1-kb deletion than by the wild-type strain.
FIG. 2. Immunoblot analysis of PtlF in a wild-type strain and a deletion strain. Cell extracts from BP536 (lane 1) or BP536Δptxptl936-8003 (lane 2) were prepared as described in Materials and Methods. Samples (50 μl) were subjected to SDS-polyacrylamide (more ...)
In order to determine whether a single regulatory signal along the operon or message might account for the increase in the production of PtlF observed in the deletion strain described above, we constructed additional deletion strains. In each of these deletion strains, we also replaced the ptlF gene with the phoA gene so that protein production could easily be quantified simply by measuring alkaline phosphatase activity (Fig. ). In all of these strains phoA was fused to ptlF such that the fusion gene contained the initiation codon of ptlF followed by phoA devoid of its start codon. We compared the alkaline phosphatase activities of strains with no deletion or with a deletion from nucleotide 936 to 8003, from nucleotide 936 to 3941, or from nucleotide 3822 to 8003. As shown in Fig. , the level of alkaline phosphatase activity increased in proportion to the size of the deletion. Moreover, the two strains containing deletions from nucleotide 936 to 3941 or from nucleotide 3822 to 8003 exhibited similar alkaline phosphatase activities, neither of which reached the level of alkaline phosphatase activity produced by the strain with the largest deletion, spanning nucleotides 936 to 8003 (P < 0.05). These data are not compatible with the idea that only a single regulatory signal exists along the operon and/or message, deletion of which results in an increase in the production of proteins encoded by downstream genes.
FIG. 3. Alkaline phosphatase activities of strains containing in-frame deletions and ptlF′-phoA fusions. The strains containing the indicated deletions in the ptx-ptl region were analyzed for alkaline phosphatase activity. Values are averages from three (more ...)
In order to further examine the relative levels of production of proteins encoded by genes in the ptx-ptl operon, we constructed strains in which the phoA gene (devoid of its initiation codon) was fused to the initiation codon of either ptxS1, ptxS2, ptlA, ptlB, ptlD, or ptlF (Fig. ), and then we compared the alkaline phosphatase activities of these strains. As shown in Fig. , we found that alkaline phosphatase activity decreased in a relatively linear fashion as the phoA gene was moved from the 5′ to the 3′ end of the operon. The amounts of alkaline phosphatase activity produced by the strains containing the ptlD′-phoA fusion and the ptlF′-phoA fusion were significantly smaller than that produced by the strain containing the ptxS1′-phoA fusion (P < 0.05). These data suggest that production of the Ptl proteins located at the 3′ end of the operon is lower than that of the PT subunits. In fact, PT subunits may be produced in quantities as much as five times greater than those of Ptl proteins encoded by genes located at the 3′ end of the operon (Fig. ).
phoA gene fusions. phoA devoid of its initiation codon was fused to the ATG initiation codon of the indicated ptx and ptl genes as described in Materials and Methods. The ptx-ptl regions containing these fusions are depicted.
FIG. 5. Alkaline phosphatase activities of strains containing ptx′- or ptl′-phoA gene fusions. Alkaline phosphatase activities of strains containing the indicated phoA fusions and grown in liquid culture were measured as described in Materials (more ...)
Since our data indicate that certain Ptl proteins might be produced in quantities smaller than those of the PT subunits, we wanted to determine whether the Ptl proteins might be limiting in the secretion of the toxin from the bacteria. We first attempted to introduce a broad-host-range vector, pAMC111, containing the ptx
promoter followed by the ptl
region, into a wild-type strain. However, we were unable to isolate a strain in which the plasmid containing the ptl
region replicated extrachromosomally. Such a finding could be explained if overproduction of Ptl proteins were toxic to the cell. However, we were able to isolate a strain in which the plasmid had integrated into the chromosome (see Materials and Methods for details) such that the strain contained a single extra copy of the ptl
region. We examined the production of PtlF in this strain and found that more PtlF was produced in this strain than in the wild-type strain (data not shown), as would be expected if both copies of the ptlF
gene within the chromosome of this strain were expressed. We then compared the secretion of PT in this strain containing 2 copies of the ptl
region to that in the wild-type strain. As shown in Fig. , we observed that secretion of PT from the wild-type strain (BP536) was inefficient, as reported previously (9
). When we examined the secretion of PT from the strain that contains 2 copies of the ptl
region (BP536::pAMC111), we found that significantly more of the S1 subunit was observed in the supernatant of BP536::pAMC111 than in that of BP536. BP536::pAMC111 and BP536 produced approximately the same amount of PT, as determined by densitometry of the amount of PT found in the culture supernatant and the amount that remained associated with the cell (data not shown). These results suggest that the Ptl proteins are limiting in the secretion of the toxin.
FIG. 6. Secretion of PT from BP536 and BP536::pAMC111. Samples of culture supernatants (100 μl) and cell extracts (100 μl) of the indicated strains were prepared as described in Materials and Methods. Samples were subjected to SDS-polyacrylamide (more ...)
As a first step in determining which Ptl proteins might be limiting in the secretion of the toxin, we introduced plasmids containing either ptlA-ptlC controlled by the ptx-ptl promoter, ptlD-ptlF controlled by the lac promoter, or ptlI-ptlH controlled by the lac promoter into BP536 to yield strains BP536(pAMC147), BP536(pTC11), and BP536(pAMC151), respectively. As shown in Fig. , an increase in the gene dosage of ptlA-ptlC resulted in a significant increase in the proportion of S1 secreted over that for the wild-type strain. The total amount of PT produced by BP536(pAMC147) was similar to that produced by BP536, as determined by densitometry of the amount of PT found in the supernatant and that associated with the cell (data not shown). Overexpression of either ptlD-ptlF or ptlI-ptlH did not increase secretion. We examined the amounts of PtlF produced by BP536(pTC11) and BP536(pAMC151) and found that they were greater than that produced by the wild-type strain, thus ensuring that the ptl genes on the plasmids of both strains were being expressed (Fig. ).
FIG. 7. Secretion of PT from strains overexpressing subsets of the ptl genes. (A) Samples of culture supernatants (100 μl) and cell extracts (100 μl) of the indicated strains were prepared as described in Materials and Methods. Samples were subjected (more ...)
Since an increase in the gene dosage of ptlA-ptlC increased the secretion of PT, we next wanted to determine which of these genes might be responsible for the increased secretion. Therefore, we introduced plasmids containing either ptlA controlled by the ptx-ptl promoter or ptlB-ptlC controlled by the ptx-ptl promoter into BP536 to yield strains BP536(pAMC171) and BP536(pAMC176), respectively. We found that neither of these strains exhibited increased secretion of PT relative to that of the wild-type strain, indicating that a combination of ptlA, ptlB, and/or ptlC is needed for increased secretion (data not shown).