Isolation and characterization of additional hypercompetence mutations in sxy
The original sxy-1
hypercompetent mutant was isolated from a pool of EMS-mutagenized H. influenzae
cells created in a search for genes that regulate competence development (3
). The present study began by using the same strategy to select additional hypercompetence mutations from the same population. Wild-type cells growing at low cell density in rich medium do not express competence genes or take up DNA, so an EMS-mutagenized culture was incubated with DNA carrying a novobiocin-resistance allele and NovR
transformants were selected. Screening of the rare transformants identified four additional strains with mutations that mapped to sxy
; the alleles were named sxy-2, sxy-3, sxy-4
. As shown in , all four mutants demonstrated the same 50-fold to 500-fold increased transformation frequencies as the sxy-1
mutant, during both log phase growth (A600
0.2) and late-log phase growth (A600
1.0). All mutants grew normally in rich medium (sBHI). In MIV starvation medium, mutants and wild-type cells survived equally well and transformed at equally high frequencies (right panel of ).
Figure 1. Natural competence assayed by transformation to novobiocin resistance. Transformation frequencies of wild-type (black) and sxy-1-5 mutants (gray) under non-inducing conditions (log phase: sBHI at A600 0.2), partially inducing conditions (late log: sBHI (more ...)
Sequencing revealed that each strain carried a distinct single-point mutation in sxy; these are shown in A. The sxy-2 mutation (G102A) is a silent substitution in the coding region, 4 bp upstream of the site of the sxy-1 mutation (G106A, V19I). The other three mutations are clustered outside the coding region, near the 5′ end of the 51 nt UTR (sxy-3: C14T; sxy-4: T15C; sxy-5: G16T).
Figure 2. Analysis of Sxy levels in wild-type and mutant cells under different growth conditions. (A) Locations of key features and mutations in the sxy gene. Regulatory elements (−10, −35) are shown relative to the transcription start site (16 (more ...)
Because these mutations did not alter the Sxy protein sequence, site-directed mutagenesis was used to confirm that no other mutations, either in sxy
or elsewhere in the genome, were responsible for the hypercompetence phenotypes. As had been done for sxy-1
), each of the four mutations was re-created in a H. influenzae sxy
plasmid cloned in E. coli
, and introduced into wild-type H. influenzae
chromosomes by transformation; these mutants all had phenotypes identical to the originals and were used in the experiments described here. This confirmed that all of the four new hypercompetence mutations increased competence without changing the sequence of Sxy or any other protein. We thus hypothesized that all five mutations acted by altering control of sxy
expression rather than by changing Sxy function. As Sxy is an activator of competence genes, and as we observed elevated expression of all CRP-S regulon genes in microarray analysis of the sxy-1
mutant in rich medium (data not shown), we predicted that the mutations would cause hypercompetence by increasing rather than decreasing sxy
Hypercompetence mutations lead to elevated Sxy under non-inducing and semi-inducing conditions
To compare Sxy levels between wild-type and mutant cells, we generated polyclonal anti-Sxy antibodies and used western blot analysis to quantify protein levels. In exponential growth (A600 0.2), all hypercompetence mutants (sxy-1-5) had elevated Sxy levels, with 7- to 16-fold more protein than wild-type cells (B; light gray bars). In late-log phase (A600 1.0), the difference was even more striking, with mutants having 13- to 25-fold more Sxy protein than wild-type cells (B, dark gray bars). C graphs transformation frequencies as a function of Sxy protein levels for wild-type and mutant cells in the log and late-log phases of growth. The strong positive correlation between Sxy abundance and transformation frequencies confirms that Sxy levels limit competence development during growth in rich medium, and that changes in the amount of Sxy are responsible for the hypercompetence of the sxy mutants.
How do the sxy-1-5 mutations cause increased Sxy production? Their locations rule out several possible modes of action. The mutations do not improve the affinity of the core promoter elements (−10 and −35 sequences), nor create a more efficient start codon or Shine–Dalgarno (SD) sequence, so they are unlikely to act by changing factors that determine baseline expression. The transformation frequencies and Sxy levels of the sxy-1-5 mutants increase as the culture medium becomes exhausted, so the mutations must change the sensitivity of the inducing mechanism rather than ablating repression. The mutations are unlikely to act by changing the binding site for a transcription factor, as they are outside of the promoter and spread over 94 bp of transcript sequence.
The clustering of the mutations into two regions suggested that mRNA secondary structure might play a role in regulation. Examination of sxy mRNA for possible base pairing between these regions revealed a long stretch of potential base pairing between positions 9–26 of the UTR and positions 94–111 of the coding region, with only a single 2 × 2 bubble. All five hypercompetence mutations fall within this predicted stem. Moreover, each of the mutations eliminates a base pair within this stem, so that each is expected to destabilize the secondary structure. Analysis of this region with the RNA-folding program Mfold supported this folding model, and also predicted pairing between segments internal to this stem, creating two additional stems and three loops, as shown in A.
Mfold analysis predicted the same optimal topology for sxy-1-5 mRNAs as for wild-type mRNAs, but the sxy-1-5 structures were not as thermodynamically stable and were predicted to have higher frequencies of single strandedness throughout. Supplementary Figure 2A plots the stability of Stem 1 in hypercompetent mutants, calculated by Mfold, showing that each point mutation is predicted to reduce the stability of Stem 1. In addition, this analysis predicts that Stem 1 will be the most important contributor to the overall stability of the mRNA secondary structure, and thus may help explain why all five hypercompetence mutations were found in this region. Supplementary Figure 2B presents the analysis of single-strandedness across multiple thermodynamically stable folding predictions for the bases in Stem 1. The sxy point mutations are predicted to cause 2- to 3-fold increases in single-strandedness.
Nuclease mapping confirms the predicted sxy mRNA secondary structure
Nuclease mapping was used to test whether sxy mRNA folds into the predicted secondary structure in vitro, and to test whether hypercompetence mutations alter RNA folding. We first examined cleavage of wild-type sxy RNA by the structure-specific ribonucleases RNase T1 and RNase A, using a cloned sxy fragment extending from base 1 to base 323. RNase T1 cuts specifically at single-stranded Gs, while RNase A cuts single-stranded Cs and Us. B shows the cleavage intensities of all scorable positions between positions 1 and 122, normalized to positions 78 and 80, which were consistently cleaved. (Data for some Cs and Us are not shown because they were not cut by RNase A even when the RNA was denatured.) The strong cleavages between positions 42 and 48, and between positions 72 and 80, confirm that Loops B and C form in vitro, and that they are separated by segments that are protected by pairing. The moderate cleavage at position 29 is consistent with the presence of Loop A. Only three positions in the upstream side of Stem 1 are informative; positions 12 and 16 are protected but position 24 is moderately cleaved, suggesting that Stem 1b may be weak. (Cleavage of position 24 could also be explained by a slight modification to the predicted pairing, with nucleotide 24 bulged out and the 3 nt below it shifted up one in their pairings.) The segment that forms the downstream side of Stem 1 (94–111) has more informative positions; these are consistently protected.
The nuclease-assay support for Stems 2 and 3 and Loops B and C suggests that the sxy SD site and start codon may by sequestered within a small loop and stem, respectively, likely preventing the initiation of translation. The biochemical evidence for Stem 1 is fairly strong, with most of the informative positions protected from cleavage. Importantly, of the sites of the five hypercompetence mutations, the three that are scorable in these assays are all strongly protected, supporting the hypothesis that they normally are paired.
We then examined mutant sxy-1 RNA for changes in secondary structure; C (upper panel) shows the effect of this single mutation. Note that this figure shows ratios of sxy-1 cleavages to cleavages of wild-type sxy RNAs, not absolute intensities, so that bars above the line represent positions with increased nuclease sensitivity. The expected moderate destabilization of Stem 1a by the loss of base pairing between positions 14 and 106 is confirmed by the increased cleavage of positions 12 and 16 and positions 102–110. Modest increases in nuclease sensitivity were also seen in Stem 1b (position 24) and Stem 2 (positions 57 and 59), and position 115 was very strongly cleaved.
Mutations that strengthen Stem I reduce sxy expression
The definitive test of whether a mutant phenotype results from disruption of base pairing is creation of compensatory mutations that restore the hypothesized base pairing. The test is especially clear here, as the sxy-1 and sxy-3 mutations make complementary substitutions disrupting the same proposed base pair. If both do increase sxy expression by destabilizing the secondary structure, then a double mutant carrying both substitutions will have base pairing restored and thus will have a more normal phenotype (lower competence) than either single mutant, rather than the more extreme phenotype expected if the mutations increase expression in some other way. The desired double mutant, sxy-6, was created by site-directed mutagenesis in E. coli, followed by transformation into the H. influenzae chromosome. This combined the sxy-1 and sxy-3 mutations to generate an A:U pair where wild-type sxy has a G:C pair (A). B shows that sxy-6 cells produced wild-type levels of Sxy protein, much less than either parent mutant, confirming that the sxy-1 and sxy-3 mutations act by disrupting base pairing. The altered sequence but wild-type phenotype of the sxy-6 mutant is strong evidence that the single mutant phenotypes are not due to sequence-dependent interactions with a regulatory protein or RNA.
To further characterize the ability of base pairing to limit sxy expression, a second mutant with enhanced base pairing was constructed. In sxy-7, two adjacent substitutions (C20G and U21A) create two new base pairings at the site of the 2 × 2 bubble separating Stems 1a and 1b (A), so Stem 1 of sxy-7 has 18 contiguous base pairings. C (lower panel) shows that this change strongly reduced RNase cleavages at positions 16 and 102–107 (Stem 1b), 37 and 54 (Stem 2), 69 and 86 (Stem 3) of the RNA (again the values are relative to those in B); the generally decreased cleavage in the entire region indicates stronger base pairing throughout. As expected, C100, the predicted pairing partner of what is now G20, was not cleaved. Sxy protein was barely detectable in this mutant (B) and cells could not be transformed even after transfer to MIV (data not shown). Together the sxy-6 and sxy-7 mutations confirm that base pairing in Stem 1 limits sxy expression and competence development.
How does mRNA secondary structure regulate sxy expression?
In principle, the secondary structure of sxy mRNA could limit production of Sxy protein by interfering with elongation of transcription or by reducing the resulting mRNA's stability or translation efficiency. As described below, two independent methods (measurements of β-galactosidase production from sxy::lacZ fusions and direct measurements of sxy RNA and protein levels) both showed that the structure affects both accumulation and translation of sxy mRNA.
The relative impacts of the sxy
secondary structure on transcription and translation were first investigated using transcriptional and translational fusions of positions 83 and 317 of the wild-type sxy
sequence to the E. coli lacZ
gene (A). The position 317 fusions maintain all of the secondary structure shown in A. The position 83 fusions eliminate the downstream strands of Stems 1 and 3 and thus eliminate these stems. Full characterization of these and related lacZ
fusions is provided by reference 14
Figure 4. Effect of sxy mutations on sxy mRNA levels and translation efficiency. (A) Expression from sxy::lacZ transcriptional and translational fusions. Triangles, fusions to sxy position 317; circles, fusions to sxy position 83. Filled symbols, transcriptional (more ...)
Expression from the sxy317 transcriptional fusion (black triangles) revealed that transcription from the sxy promoter is quite stable during exponential growth and early stationary phase in rich medium. The absence of the lacZ translation start site in the corresponding translational fusion (white triangles) did not significantly change the amount of β-galactosidase activity produced, indicating that the sxy and lac translation start sites have comparable activities. Elimination of Stems 1 and 3 in the sxy83 transcriptional fusion increased expression 2-fold (black circles). The 5-fold increase in expression from the corresponding translational fusion (white circles) therefore represents a 2.5-fold increase in the amount of protein produced from each mRNA.
A parallel experiment directly tested whether the increased Sxy protein in hypercompetence mutants results from changes in accumulation and/or translation of sxy mRNA. B plots mRNA abundance measured by quantitative PCR (upper panel), and the corresponding translational efficiencies (lower panel) calculated for wild-type cells and hypercompetent mutants from the protein levels in B. Although hypercompetent mutants in early log produced 7- to 16-fold more protein than wild-type cells, they produced only slightly more mRNA, indicating that mutant translational efficiencies were elevated 3.5- to 6.5-fold. When wild-type cells entered late-log growth, the amount of sxy transcript and Sxy protein both doubled, so the translational efficiency did not change. However, when the hypercompetent mutants entered late log they showed an even greater disproportion between mRNA and protein than in early log, implying a greater increase in translational efficiency. Transfer of exponentially growing wild-type cells to MIV resulted in a 2.5-fold increase in the amount of sxy mRNA and a 9-fold increase in protein after 90 min, implying a 3.5-fold increase in translational efficiency. These results suggest that transfer to MIV maximizes competence in wild-type cells because it releases the translation limitation caused by mRNA secondary structure in rich medium.
mRNA secondary structure impedes translation in vitro
We used an in vitro
coupled transcription/translation system to directly test the influence of mRNA secondary structure on the translation efficiency of sxy
transcripts. Reactions were carried out at 25°C because this temperature reduces the transcription rate of T7 RNAP to a rate comparable to bacterial RNAP at 37°C, allowing nascent RNAs to fold correctly (15
). All input DNAs (wild-type, sxy-1
alleles) produced indistinguishable amounts of mRNA from the T7 promoter (data not shown), but wild-type and mutant mRNAs produced very different amounts of Sxy protein (A). Spot-blot quantification of Sxy protein levels revealed that sxy-1
mRNA was translated 3-fold more efficiently than wild-type mRNA, whereas sxy-7
mRNA was translated about 4-fold less efficiently than wild-type mRNA.
Figure 5. Sxy protein generated by in vitro transcription/translation. (A) Protein production from wild-type (WT), sxy-1 and sxy-7 DNA templates. The average and standard deviation of three independent time courses are shown. (B) Protein production in reactions (more ...)
We also tested whether a ssDNA oligonucleotide complementary to nucleotides 2–19 in Stem 1a (see inset of B) could compete with formation of sxy mRNA secondary structure and thus improve translation efficiency. The oligonucleotide was designed to hybridize with the 5′-terminus of sxy, sxy-1 and sxy-7 mRNAs, allowing it to compete with formation of Stem 1a (the location of all 5 hypercompetence mutations) without interfering with ribosome binding to the SD (A). B shows that inclusion of the oligonucleotide in reaction mixtures allowed wild-type mRNA to be translated as efficiently as sxy-1 mRNA, but had no significant effect on the translatability of sxy-1 or sxy-7 mRNA. These results suggest that, in vitro, secondary structure does not inhibit translation of sxy-1 mRNA. Conversely, the high stability of the sxy-7 mRNA secondary structure not only prevents translation but also prevents competition of Stem 1a by the complementary oligonucleotide. The ability of this oligonucleotide to increase translation of wild-type sxy mRNA confirms that the wild-type secondary structure is strong enough to inhibit translation but sufficiently labile to respond to changing conditions.
CRP and cAMP strongly induce sxy transcription
Zulty and Barcak reported that the sxy
promoter contained two CRP-binding sites, one centered at −5.5 and one at −61.5 relative to the transcription start point, and that sxy
expression in rich medium was CRP-dependent (16
). However the site they reported at −5.5 (overlapping the transcription start site) was created by an A → G substitution at position −12, and the authentic sequence of this site (17
) (also confirmed in our lab) has no significant resemblance to known H. influenzae
CRP sites (2
). The site centered at −61.5 scored as an excellent CRP site when tested for goodness-of-fit with 58 experimentally determined H. influenzae
CRP sites, as previously described (1
), and is at the optimal position to activate transcription through a class I mechanism (18
To test whether CRP induces and/or represses sxy in starvation medium (MIV), we measured sxy transcript levels in a cyaA− mutant that cannot synthesize CRP's allosteric effector cAMP (). Transcription was induced only slightly in a cyaA− mutant. Adding 1 mM cAMP resulted in very strong induction of sxy, indicating that CRP does activate the sxy promoter. The promoter could be induced by cAMP added after 20 min in MIV, but the amount of sxy transcript still fell back to pre-induction levels after 40 min in MIV. Thus, CRP is a strong inducer of sxy expression, but continuing expression appears to be overridden by a repressing mechanism after 40 min. This mechanism is unlikely to be auto-repression of the crp gene by CRP, because sxy repression did not depend on when cAMP was added.
Control of sxy transcription by cAMP–CRP. RR668 cells (cyaA−) at A600 0.2 in sBHI were transferred to MIV, with 1 mM cAMP added at t = 0′ or t = 20′. sxy transcript was measured using real-time PCR.