Department of Microbiology, University of
Texas Health Science Center at San Antonio, San Antonio, Texas
78284-7758
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TEXT |
The Mollicutes represent
a class of unique cell wall-less procaryotes that includes members of
the genus Mycoplasma, a distinct subgroup pathogenic for
humans and animals (3, 12). Gene structure and function
analyses of mycoplasmas and other genera taxonomically categorized
under the Mollicutes are limited by the lack of classical
genetic systems and the inability to express cloned genes in native
Mycoplasma hosts (7). A fundamental limitation of
Mycoplasma gene expression is the unusual codon usage
pattern displayed by mycoplasmas (24). Like mitochondria, Mycoplasma species utilize the UGA codon to encode
tryptophan (9) rather than to serve as a stop codon. In
general, Mycoplasma genes containing one or more UGA codons
will not be expressed in commonly used expression systems that strictly
adhere to the universal genetic code, due to premature termination. An
exception is the very limited readthrough of the UGA stop codon in
Escherichia coli and Salmonella enterica serovar
Typhimurium (18, 19). Interestingly, although UGA also
functions as a termination codon in Bacillus subtilis, the
efficiency of UGA readthrough in this gram-positive bacterium is high
due to the presence of a tRNA that reads the UGA termination codon as
tryptophan (13). Therefore, UGA readthrough in B. subtilis is substantially leakier than that in members of the
family Enterobacteriaceae (11). Furthermore, B. subtilis mutants with mutations in the structural gene
(prfB) for release factor 2 (RF2) demonstrated increased
readthrough levels by overcoming UGA-mediated termination
(11). These observations enabled us to use B. subtilis as a host to express specific UGA-containing Mycoplasma genes.
Mycoplasma strains and growth conditions.
Wild-type virulent Mycoplasma pneumoniae strain B9 and
Mycoplasma genitalium strain G37 were grown as described
earlier (4, 6). B. subtilis strains and plasmids
listed in Table 1 were grown in
Luria-Bertani (LB) medium at 37°C. Except for B. subtilis PY22, all other Bacillus strains were derivatives of
B. subtilis 168 strain BR151. E. coli strain
INV
F' (Invitrogen, Carlsbad, Calif.) was used for the transformation
and expression of the TGA-corrected p30 gene. Chromosomal
DNA from M. pneumoniae and M. genitalium was
isolated as described earlier (4, 22).
Expression of Mycoplasma genes in B. subtilis and E. coli.
The entire structural gene
(p30) of M. pneumoniae adhesin P30 is comprised
of an open reading frame of 825 nucleotides, encoding 275 amino acids
with a predicted molecular mass of 29,743 Da (4). The single
UGA codon that encodes tryptophan is located at amino acid position 16 (4). The p30 gene was amplified using primers listed in Table 2, and the resulting
864-bp DNA fragment was cloned into the PCR2.1 vector (Invitrogen) and
transformed into E. coli INVF' cells (20).
Plasmid PCR2.1-30 was digested with XbaI and
SalI. The excised 851-bp fragment was cloned downstream of
PSPAC into the E. coli and B. subtilis shuttle vector pDG28 to generate the recombinant plasmid
pDG28-30. Transformation in B. subtilis and E. coli was accomplished as reported earlier (20, 25), and
the transformants were selected on LB agar plates containing ampicillin
at 100 µg/ml for E. coli and erythromycin at 1 µg/ml for
B. subtilis.
The recombinant B. subtilis wild-type strain PY22 containing
the p30 gene under the control of
PSPAC (BS-P30) was grown in the presence or
absence of 1 mM IPTG
(isopropyl-
-D-thiogalactopyranoside). Bacterial cells
were pelleted and sonicated (40 W for 15 s three times with 45-s
intervals), and samples were analyzed by sodium dodecyl sulfate
(SDS)-12% polyacrylamide gel electrophoresis for visualization using
Coomassie blue staining and Western immunoblotting. No visible
differences were observed between the total protein profiles of the
induced and uninduced cultures in the Coomassie blue-stained SDS gels
(data not shown). The P30 protein, however, was detected by
immunoblotting with P30 monoclonal antibody in the induced B. subtilis strain BS-P30 but not in the uninduced strain (Fig.
1A) (5). This monoclonal
antibody recognizes an epitope in the middle region of the P30 protein.
As expected, P30 was absent in control cells (BS-28) carrying plasmid
pDG28 without p30. These data indicated that P30 synthesis
was under the control of the inducible PSPAC
promoter and that at least limited readthrough of the p30
gene is possible in B. subtilis. In contrast, no
immunologically detectable P30 was induced in recombinant E. coli (data not shown). Apparently, the efficiency of UGA
readthrough in members of the Enterobacteriaceae is very low.
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FIG. 1.
Expression of M. pneumoniae gene
p30 in B. subtilis and E. coli. B. subtilis strains BS-28 (control) and BS-P30 (p30) were
cultivated in LB medium and induced with 1 mM IPTG. E. coli
strain INVF' carrying the 4P3 plasmid (UGA-corrected p30)
was grown in LB medium under anaerobic conditions for the expression of
P30 from the nirB promoter. Sonicated protein extracts (120 µg) were fractionated by SDS-polyacrylamide gel electrophoresis and
analyzed by Western blotting with P30 antibodies. (A) Immunoblot
analysis of UGA readthrough of p30 gene in wild-type
B. subtilis PY22. Lanes: 1, BS-28 with no IPTG; 2, BS-28
with 1 mM IPTG; 3, BS-P30 with no IPTG; 4, BS-P30 with 1 mM IPTG. (B)
Immunoblot analysis of multiple P30-related proteins from the
expression of the UGA-corrected p30 gene in E. coli. Lanes: 1, M. pneumoniae; 2, E. coli
(4P3, UGA corrected); 3, E. coli (pDG28-30, not UGA
corrected).
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It is noteworthy that B. subtilis-expressed recombinant P30
protein appeared as a single band. Mycoplasma genes
previously expressed in E. coli generated multiple protein
bands (16). For example, the Spiroplasma gene
encoding spiralin contains no UGA codons but generates multiple
recombinant proteins in E. coli (15). Expression
of multiple proteins exceeding the coding capacity of a cloned genomic
fragment from Mycoplasma capricolum has also been described
for E. coli (1). Differences in UGA codon usage and initiation sites for translation in E. coli may account
for these variations in protein size (16). In order to
determine whether p30 underwent a similar mechanism of
recombinant expression in E. coli, we used the TGA-corrected
p30-carrying plasmid construct designated 4P3. Expression of
the TGA-corrected p30 gene under the nirB
promoter in E. coli (17) resulted in the
appearance of multiple P30-related proteins. One major protein band had
the mobility of authentic P30 (30 kDa), while a second major band had
the mobility of a larger protein exceeding the p30 coding capacity (45 kDa) (Fig. 1B).
In order to determine the efficiency of UGA readthrough of
p30 in B. subtilis (Fig. 1A), we compared the
level of expression of the wild-type p30 with that of a
p30 gene in which the TGA was changed to TGG by
site-directed mutagenesis. A recombinant plasmid carrying the
TGA-corrected p30 gene (strain BS-CP30 [Table 1]) was
constructed and transformed into B. subtilis. When the UGA-corrected p30 was expressed in Bacillus, the
protein was overexpressed, similar to the level in E. coli,
and without multiple proteins. Quantitation of translational products
by immunoblot analysis was performed on a Macintosh computer using the
public-domain NIH Image program (developed at the U.S. National
Institutes of Health and available on the Internet at
http://rsb.info.nih.gov/nih-image/). To calculate the level at which
the internal UGA codon of p30 was read as tryptophan, we
plotted a standard graph of P30 protein concentrations (the values
generated by the image analyzer on the immunoblot) versus total cell
protein (the amount of IPTG-induced BS-CP30 protein loaded on
SDS-polyacrylamide gels). Using 100 µg of IPTG-induced BS-CP30
protein concentration as 100%, we calculated the level of P30
expression in test samples by immunoblotting. By this method, the level
of UGA suppression for the wild-type strain (BS-P30) that carried the
UGA-containing p30 gene was calculated as 6%. This is
similar to the readthrough levels observed with cat genes
containing UGA codons in B. subtilis (13).
To further determine the capacity of B. subtilis to
translate through UGA codons, we selected the methionine sulfoxide
reductase (msr) structural gene of M. genitalium
with two UGA codons as a test gene. The Mycoplasma gene
msr has an open reading frame of 471 nucleotides, which
encodes a protein of 157 amino acids, with a calculated molecular mass
of 18,414 Da. The two UGA codons occur at amino acid positions 12 and
99 (8). Recently, we generated antibodies reactive against
the MSR (methionine sulfoxide reductase) protein of M. pneumoniae (S. Dhandayuthapani and J. B. Baseman, unpublished
results) that cross-react with the M. genitalium MSR protein. The M. genitalium DNA fragment encoding
msr was amplified using primers outlined in Table 2 and
cloned into the pDG28 expression vector to create pDG28-MSR.
Recombinant B. subtilis cells carrying the pDG28-MSR
(BS-MSR) were grown to mid-log phase and induced with 1 mM IPTG.
Immunoblot analysis revealed the synthesis of a full-length
msr gene product (Fig. 2B).
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FIG. 2.
Comparison of UGA readthrough of Mycoplasma
genes p30 and msr among the different
prfB mutant strains of B. subtilis. The B. subtilis strains were cultivated in LB medium with 0.5% glucose
and induced with IPTG (1 mM). (A) Immunoblot analysis of the P30
protein induction. Lanes: 1, BS5-P30 (prfB5); 2, BS3-P30
(prfB3); 3, BS2-P30 (prfB2); 4, BS4-P30
(prfB4). (B) Immunoblot analysis of MSR protein induction.
Lanes: 1, BS5-MSR (prfB5) uninduced; 2, BS5-MSR
(prfB5); 3, BS2-MSR (prfB2); 4, BS4-MSR
(pfrB4); 5, BS3-MSR (prfB3); 6, BS-MSR (wild
type); 7, BS-MSR (wild type) uninduced.
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Expression of Mycoplasma genes in prfB
mutants of B. subtilis.
Mutations in RF2 genes
(prfB) fail to terminate translation efficiently and allow
misreading of UGA (2). The combination of tRNA UGA
suppression and RF2 mutation has moderately increased UGA readthrough
in E. coli. However, the expression of Mycoplasma genes under these conditions was limited due to the synthesis of
multiple-sized, related Mycoplasma proteins for unknown
reasons (21). Since B. subtilis permits
readthrough of UGA, we used B. subtilis RF2 mutants
(prfB) in an attempt to increase the level of UGA
readthrough of Mycoplasma genes. A recent study describing prfB mutants of B. subtilis demonstrated that
such mutants could suppress a catA86-TGA mutation between 19 and 54%, compared to the 6% level detected in wild-type
Bacillus strains (11).
Specific B. subtilis prfB mutant strains with impaired RF2
function and enhanced UGA suppression were selected (11).
Plasmids pDG28-30 and pDG28-MSR were transformed individually into
strains with novel prfB alleles (mutations prfB2
and prfB4 affect ribosome-binding site, mutation
prfB5 affects frameshifting, and mutation prfB3 has an unknown activity due to an alteration from Glu to Lys at amino
acid residue 2). The recombinant RF2 strains were then induced with
IPTG. The level of expression of p30 was increased two- to ninefold in these mutants compared to wild-type B. subtilis
(Fig. 2A and Table 3). The maximum level
of UGA suppression was observed with the BS5-P30 (prfB5)
strain. Bacillus strains BS2-P30 (prfB2), BS4-P30
(prfB4), and BS3-P30 (prfB3) permitted
readthrough of p30 in decreasing order (Table 3). A similar
pattern of Mycoplasma protein expression was observed with
msr readthrough (Fig. 2B). In this case, the level of
msr expression was increased from two- to sevenfold in the
RF2 mutants compared to wild-type B. subtilis (data not
shown). Interestingly, no truncated MSR-related protein of 98 amino
acids was observed, suggesting that the truncated peptide was not
stable. A possible explanation for the synthesis of full-length MSR by
B. subtilis, with no truncated peptides, could be the
presence of adenine next to the UGA (at amino acid 99) of
msr. It has been observed that the nucleotide 3' to the UGA
triplet increases UGA suppression (increased readthrough) in E. coli in the order A > G > C > U (14).
In p30, the nucleotide 3' to the UGA triplet (i.e., at amino
acid 16) is adenine; in msr, the nucleotide next to amino
acid 12 is guanine (i.e., UGAG) and at amino acid 99 is UGAA. Thus, the
synthesis of intact P30 and MSR proteins is favored.
Again, the expected level of msr expression in
prfB was consistent with p30 expression for all
Bacillus mutants, except for BS5-(prfB5), in
which expression of p30 was significantly higher. The
difference in readthrough of p30 and msr could be
due to the influence of factors like codon bias, the position of UGA in
the coding sequence, and the size of the open reading frame. It may be
possible to increase UGA readthrough more than the level observed (54%), although this may be detrimental to cell growth or even result
in lethality (10). However, further enhancement of
Mycoplasma gene expression might be accomplished by cloning
selected UGA-containing Mycoplasma genes under a strong
Bacillus promoter to heighten mRNA levels. Based on our
study, we predict that Mycoplasma open reading frames
containing up to three TGA codons can be readily expressed in RF2
mutants of B. subtilis, suggesting that the obstacle of
readthrough of Mycoplasma genes containing UGAs can be
overcome using this expression host.
We are grateful to W. Haldenwang (pDG28 and PY22 and manuscript
review), J. Glass (4P3), and J. Piggot (prfB strains of
B. subtilis) for providing plasmids and strains.
This work was supported by grant AI 41010 from the National Institute
of Allergy and Infectious Diseases.
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