- Research article
- Open Access
Sialic acid transport and catabolism are cooperatively regulated by SiaR and CRP in nontypeable Haemophilus influenzae
© Johnston et al; licensee BioMed Central Ltd. 2010
Received: 25 March 2010
Accepted: 15 September 2010
Published: 15 September 2010
The transport and catabolism of sialic acid, a critical virulence factor for nontypeable Haemophilus influenzae, is regulated by two transcription factors, SiaR and CRP.
Using a mutagenesis approach, glucosamine-6-phosphate (GlcN-6P) was identified as a co-activator for SiaR. Evidence for the cooperative regulation of both the sialic acid catabolic and transport operons suggested that cooperativity between SiaR and CRP is required for regulation. cAMP was unable to influence the expression of the catabolic operon in the absence of SiaR but was able to induce catabolic operon expression when both SiaR and GlcN-6P were present. Alteration of helical phasing supported this observation by uncoupling SiaR and CRP regulation. The insertion of one half-turn of DNA between the SiaR and CRP operators resulted in the loss of SiaR-mediated repression of the transport operon while eliminating cAMP-dependent induction of the catabolic operon when GlcN-6P was present. SiaR and CRP were found to bind to their respective operators simultaneously and GlcN-6P altered the interaction of SiaR with its operator.
These results suggest multiple novel features for the regulation of these two adjacent operons. SiaR functions as both a repressor and an activator and SiaR and CRP interact to regulate both operons from a single set of operators.
Sialic acid (5-N-acetylneuraminic acid, Neu5Ac) is used by nontypeable Haemophilus influenzae (NTHi) to assist in the evasion of the host innate immune response. Sialic acid is used to decorate the cell surface, primarily as the terminal non-reducing sugar on the lipooligosaccharride (LOS) and the biofilm matrix [1, 2]. The presence of sialic acid on the cell surface protects the cell from complement-mediated killing, although the precise mechanism of this protection is unknown and may even vary among strains of NTHi [3–5]. Regardless, the acquisition and utilization of sialic acid is a crucial factor in the virulence of the majority of NTHi [3, 4, 6–8].
Also present in the nan operon is the transcriptional regulator SiaR. SiaR is involved in the regulation of the nan and siaPT operons . SiaR was found to repress the expression of both the siaPT and nan operons, thus regulating both transport and catabolism. Binding of SiaR to the intergenic region between these two operons was demonstrated and the region of DNA protected by SiaR was identified. As expected, it was found that inactivation of siaR lead to a reduction in surface sialylation, demonstrating the need to control the expression of sialic acid catabolism. In addition to SiaR, the cAMP receptor protein (CRP) was identified as a regulator of the siaPT operon, however a role in the regulation of the nan operon was not observed [12, 14]. This is in part consistent with the observation that sialic acid is a cAMP-independent sugar . In H. influenzae, CRP has been shown to regulate utilization of galactose, ribose, xylose, and fucose , in addition to regulating the development of competence .
We now report on the role of intermediates in the Neu5Ac catabolic pathway in SiaR-mediated regulation. Also, the potential interaction between SiaR and CRP was investigated. SiaR was found to utilize glucosamine-6-phosphate (GlcN-6P) as a co-activator in the presence of the CRP-cAMP complex. SiaR and CRP were found to act in a cooperative manner to regulate the expression of the divergent transporter and catabolic operons. Our results reveal a unique mechanism of regulation of two divergent operons regulated by two transcription factors from a single location.
Promoter structure of the nan and siaPT operons
Glucosamine-6-phosphate is a co-activator for SiaR
Previous studies found limited activation of SiaR-regulated operons by sialic acid . The potential for intermediates in the sialic acid catabolic pathway to influence regulation by SiaR was explored. H. influenzae is unable to transport any of the intermediate sugars or phosphosugars of the sialic acid catabolic pathway [13, 18], therefore a mutagenesis strategy was necessary. Each gene encoding an enzyme in the catabolic pathway was deleted in an adenylate cyclase (cyaA) mutant strain, resulting in a series of double mutants. The ΔcyaA mutant strain was used to allow for CRP to be activated only by the addition of cAMP in subsequent experiments. In each mutant, sialic acid can be catabolized, but the sugar or phosphosugar immediately upstream of the inactivated enzyme should accumulate (Figure 1B).
The mutants were grown to early exponential phase and then either sialic acid, cAMP, or both were added. Expression levels of nanE and siaP, the first genes of the catabolic and transport operons, respectively, were compared using real time quantitative RT-PCR (qRT-PCR). RNA from a culture that received neither sialic acid nor cAMP served as a reference for each experiment. When both sialic acid and cAMP were added to cultures, expression of nanE was only moderately affected in strains 2019ΔcyaA, 2019ΔcyaA ΔnanK, 2019ΔcyaA ΔnanA, and 2019ΔcyaA ΔnagA (0.7- to 5-fold change). The most striking change in nanE expression occurred in 2019ΔcyaA ΔnagB, with expression elevated 83-fold (Fig, 3). This mutant would be unable to convert GlcN-6P to fructose-6P, thus accumulating GlcN-6P. These results suggest that GlcN-6P is a major co-activator in SiaR-mediated regulation.
SiaR and CRP interact to regulate the adjacent nan and siaPT operons
To demonstrate that SiaR and CRP interact to regulate the nan and siaPT operons, alteration of helical phasing was used. Alteration of helical phasing is accomplished by the insertion of one half turn to the helix between the SiaR and CRP operators. Briefly, 5 bp was inserted between the SiaR and CRP binding sites in strains 2019ΔcyaA and 2019ΔcyaA ΔnagB, resulting in strains 2019ΔcyaA+5 and 2019ΔcyaA ΔnagB+5, respectively. These strains were examined by qRT-PCR and the results were compared with those obtained from the parent strains.
In the 2019ΔcyaA ΔnagB background, altered helical phasing resulted in a steep reduction in nanE expression (from 83-fold in 2019ΔcyaA ΔnagB to 13-fold in 2019ΔcyaA ΔnagB+5) in the presence of both sialic acid and cAMP (Figures 5B and 5D). The induction level of nanE in the presence of sialic acid and cAMP was similar to the expression observed when sialic acid alone was added. The 5 bp insertion eliminated the cAMP-dependent activation of nanE that was observed in the 2019ΔcyaA ΔnagB strain.
In both the 2019ΔcyaA and 2019ΔcyaA ΔnagB backgrounds, altered helical phasing also resulted in the induction of siaP when cAMP was added (Figures 5A and 5C). In the 2019ΔcyaA+5 strain, the 5 bp insertion led to a 43-fold increase in siaP expression in the presence of cAMP (from 6-fold in 2019ΔcyaA) and a 29-fold increase (from 2-fold in 2019ΔcyaA) when both cAMP and sialic acid were present.
Taken together, these results indicate that altering the helical phasing succeeded in uncoupling SiaR- and CRP-mediated regulation of the nan and siaPT operons. It resulted in nanE expression becoming unresponsive to cAMP, much like it is in the 2019ΔcyaA ΔsiaR mutant. Altered helical phasing also prevented SiaR from exerting a negative influence on the expression of siaP. We conclude that the insertion eliminated the ability of SiaR and CRP to interact to regulate both the nan and siaPT operons.
SiaR and CRP bind to their respective operators simultaneously
GlcN-6P alters binding of SiaR to its operator
Many transcriptional regulators exhibit altered binding affinity for their operator sequences when a co-regulator is bound. To determine the effect of GlcN-6P on SiaR binding, EMSA was used. Serial dilutions of SiaR were incubated with DNA probes in the absence and presence of GlcN-6P (Figure 6B). In the presence of GlcN-6P, SiaR bound the probe and GlcN-6P slightly increased the binding affinity. While the presence of GlcN-6P did not result in a major change in the binding affinity of SiaR, the change in the shift does suggest that GlcN-6P is interacting with SiaR and impacting its ability to bind to its operator. Other phosphosugars of the sialic acid catabolic pathway (sialic acid, ManNAc, and GlcNAc-6P) nor GlcN-1P altered SiaR-binding (unpublished data) . Taken together with the expression data, this demonstrates that GlcN-6P interacts with SiaR and has an effect on its DNA-binding properties. SiaR is not displaced from the DNA, but instead functions as an activator with GlcN-6P as a co-activator.
As in our previous studies , the binding of SiaR to the EMSA probe resulted in the appearance of two shifted bands (Figure 6). This was even more apparent when lower concentrations of SiaR were present in the binding reaction. The double shift is possibly caused by the binding of multiple SiaR proteins to the probe. This is a likely explanation, considering that the region protected by SiaR is large (53 bp) . Further work will be necessary to determine the exact cause for the double shift.
GlcN-6P accumulates in a nagB mutant
The importance of sialic acid in the protection of NTHi from the host immune response requires that most of the sialic acid transported into the cell is activated by SiaB and utilized for the decoration of the LOS and biofilm matrix. Therefore, it is important that the catabolism of sialic acid be tightly regulated. We previously identified SiaR as a repressor for these two operons, in addition to the role of CRP in activating the expression of the transporter . In this study, we present data that expands on our previous work, providing key details about the unique regulation of these adjacent operons. The two operons required for the transport and catabolism of sialic acid were found to be simultaneously regulated by SiaR and CRP in a novel mechanism for cooperative regulation. SiaR functions as both a repressor and activator, utilizes GlcN-6P as a co-activator, and interacts with CRP to regulate two adjacent and divergently transcribed promoters.
Since H. influenzae cannot transport the intermediates of the sialic acid catabolic pathway [13, 18], mutants in each gene of the pathway were used to examine the role of the sugar and phosphosugar intermediates in the expression of the SiaR-regulated operons. Increased expression of the nan operon in the 2019ΔcyaA ΔnagB double mutant suggested that GlcN-6P functions as a co-activator. This is unusual because catabolic pathways are typically regulated by the presence of the substrate. SiaR likely uses GlcN-6P as a co-activator because sialic acid is utilized rapidly after transport by H. influenzae, either by activation with SiaB or catabolism beginning with NanA. Thus, sialic acid never accumulates to levels that would allow for sufficient expression of the transporter. In contrast, using GlcN-6P allows for moderate activation of siaPT to provide for transport of sialic acid. Since GlcN-6P can also be synthesized by the cell, expression of the transporter is not reliant on the presence of high levels of sialic acid, while increased sialic acid and catabolism will elevate levels of GlcN-6P and increase expression of the nan and siaPT operons. Even though GlcN-6P is not an endpoint in the catabolic pathway, transient levels of the phosphosugar likely allow for sufficient expression of the two operons.
The interaction of CRP with another transcriptional regulator is not an unusual phenomenon, however the regulation of the adjacent nan and siaPT operons by CRP and SiaR appears to operate via a novel regulatory mechanism. What makes this regulatory region unique is that it appears that the two operons are regulated by one set of operators. Other examples of divergent operons regulated by CRP and additional regulators operate by distinctly different mechanisms. The most common mechanism is the formation of a repression loop. An example of this is in the glp regulon of E. coli . As with the siaPT operon of NTHi, only one of the divergent glp operons is induced by CRP . The difference between these two systems is that the repressor GlpR binds to four operators in the intergenic region and forms a repression loop . The two divergent operons of the L-rhamnose catabolic regulon of E. coli utilize yet another mechanism. In addition to having multiple CRP binding sites, the two rha operons are regulated by separate transcriptional regulators, RhaR and RhaS . RhaR and CRP interact to regulate the rhaSR operon while RhaS and CRP interact to regulate the rhaBAD operon [20, 21].
SiaR shares functional similarity to NagC, the regulator of N-acetylglucosamine catabolism in E. coli. Like SiaR, NagC regulates the expression of nagA and nagB, as well as a number of additional genes. Also, SiaR and NagC both regulate divergently transcribed operons and CRP is involved in regulation [22, 23]. There are a number of striking differences as well. GlcNAc-6P is the inducer of the NagC regulon. Just as inactivation of nagB causes induction of SiaR-regulated genes, the inactivation of nagA, and the subsequent accumulation of GlcNAc-6P, induces NagC-related genes . NagC is displaced from its binding site in the presence of GlcNAc-6P  while SiaR appears to always be bound to its operator. In E. coli, the alteration of phasing between NagC operator sequences results in derepression of both divergently transcribed operons. This is due to the inability of NagC to form a repression loop that is required for NagC-mediated repression . This differs significantly with what we observed in SiaR regulation. In our studies, the alteration of phasing did not result in derepression, but instead uncoupled SiaR- and CRP-mediated regulation of the nanE and siaP genes. The differences between SiaR and NagC suggest that, while some functional similarity exists between the two regulators, they both employ different mechanisms.
Given the nature of regulation by SiaR and CRP, the nan and siaPT operons will never be maximally expressed when H. influenzae is in its natural environment. This is due to a number of factors, including the low abundance of sialic acid in the host and the rapid utilization of intracellular sialic acid. Instead, regulation acts to subtly modulate expression of the operons, keeping expression under constant control so that catabolism does not outpace utilization and the expression of the transporter is appropriate for the availability of the ligand. These requirements are also in balance with the need to prevent the accumulation of inhibitory amounts of sialic acid, however, this need is likely minimal considering the factors of sialic acid availablity and utilization discussed above.
The role of CRP in the regulation of sialic acid transport and catabolism suggests that sialic acid is utilized as an emergency carbon source in the host. H. influenzae can use sialic acid as a sole carbon source as efficiently as glucose . Sialic acid catabolism is not required for virulence as a nanA mutant exhibits increased fitness in multiple infection models . However, the fact that catabolism is present and conserved among H. influenzae strains suggests that it provides some advantage to the organism. The previous study examining virulence of a nanA mutant was performed using an encapsulated, invasive type B strain rather than a non-typeable strain and did not test all possible environments within the host . Additionally, intranasal mixed-challenge experiments did not reveal an advantage for either the wild-type or nanA mutant strain . Therefore, it is possible that sialic acid catabolism is advantageous in certain conditions or has increased importance for non-typeable strains. The need for cell surface sialylation has been well established in multiple infection models for NTHi, but it is unclear at this time if and when catabolism is required.
GlcN-6P, an intermediate in the catabolism of sialic acid, was found to function as a co-activator of SiaR in the regulation of the catabolic and transport operons for sialic acid in NTHi. SiaR functions as both a repressor and an activator, depending on conditions, and is required for CRP-dependent activation of the catabolic operon. Direct interactions between SiaR and CRP are likely involved in regulation.
Bacterial strains, media and growth
Strains and plasmids
Strain or plasmid
Genotype, relevant phenotype or selection marker
Source or reference
E. coli DH5α
E. coli BL21 Star
Clinical respiratory isolate
NTHi 2019ΔcyaA mutant
NTHi 2019ΔcyaA ΔsiaR mutant, kanamycin resistant
NTHi 2019ΔcyaA ΔnanA mutant
NTHi 2019ΔcyaA ΔnagA mutant
NTHi 2019ΔcyaA ΔnagB mutant
NTHi 2019ΔcyaA ΔnanK mutant
NTHi 2019ΔcyaA ΔnanE mutant
NTHi 2019ΔcyaA mutant with 5 bp insertion between SiaR and Crp operators
NTHi 2019ΔcyaA ΔnagB mutant with 5 bp insertion between SiaR and Crp operators
pCR2.1 with PCR fragment spanning
General cloning vector
New England Biolabs
nanA deletion construct
siaR knockout vector
C-terminal his-tagged SiaR expression vector
tetR-sacB/kan R cassette
C-terminal his-tagged CRP expression vector
cyaA deletion construct
nagA deletion construct
nagB deletion construct
nanK deletion construct
nanE deletion construct
pCR2.1_443 with 5 bp deletion
pJJ321 with tetR-sacB/kan R
Construction of mutants
gaa ttc CTG CTT CTT CAT TAA GTT CTC GC
ccc ggg CAT ATT CTG TTC CTA ATA TCA ACA TCA GTT
ccc ggg TAA TAG TAA ACA CTT AAA TAG TTA ATT GAT TTA AAA ATC
gca tgc TCA AAA ACA GCA ACA CGG TGC
gaa ttc CAT CAT CGC TGA AAC AGG C
ccc ggg CAT ATT AGC CTT CCT TTA TTA TTG ACC G
ccc ggg TAG AGA TCT ATT CTT CAT CTT TAT GTA GGG
gca tgc GGT TTC AAC GCT AGT TTG GTC G
gaa ttc CCG TCC TTT TGT GAA TGT CC
ccc ggg CAT AAC TTA TCC TTA TAG TGT AAA GTC TTT TCT CAC
ccc ggg AAG CCT GAA GGA ACA ATT TAT GGC TAA
gca tgc GCC GTT TCA GCA GAA TAA CCA G
gaa ttc CGC TCC TGT GTG AAC TTA TG
ccc ggg CAT ATA ACA CCC CTC ATT TAA ATC TGA AT
ccc ggg TAG TCG TAA GAC GTG TGA GAA AAG ACT T
gca tgc CGA ACG CAA AAT CGT ATC GGC
gag ctc CAT TTT GCT GAC GAG GAA CTG
ccc ggg CAT TAC ATA AAC ACC TAA AAT TGG TGG
ccc ggg TAA TAT TTT CCT GTG GTT GAT AGG TTA CC
aag ctt AAA GCA ATG GAG TGG ACC ACA ATT
CCG CAG CAA TTT TTG TCC
TTT ATG AAA AAA CAC TTC AAA AAT
ATT TTT GAA GTG TTT TTT CAT AAA TTA AA T GTG ATC AAC TTC TC
CCA TTA CGG CAC ACT AAA GAG G
aag ctt AAT AAA ACG GAA TTT TTG AAA CAG G
ctc gag TCT TGC GCC ATA TAC AAC GAT TGT
ACA CCC CTC ATT TAA ATC TGA ATA AAT CAC
CCC CCA AAA TAG GAT TCG
CGA CAG GTT GGC AAG AAG AAA TAA GAC C
ATC AGC GGC AAG AAC AGC AG
All mutants used in this study were constructed as non-polar deletions using a counter-selectable cassette. The cassette used was a variation of sacB cassettes that have been described previously [27, 28]. In this cassette, which is described in more detail in work to be submitted elsewhere, sacB is under the control of the tetracycline promoter and Tet repressor. The cassette also contains genes for the repressor, tetR, and nptII, a kanamycin resistance marker. This allows for inducible expression of sacB in the presence of the tetracycline analog chlortetracycline. Constructs for mutagenesis were prepared for each gene using the design detailed as follows. Regions flanking the target gene were amplified by PCR using primers that had restriction sites added to the 5'-end. These primers were designed to contain the start codon for the upstream fragment and stop codon for the downstream fragment. These products were cloned into pGEM-T (Promega, Madison, WI) and sequentially subcloned into pUC19 using the primer-encoded restriction sites. The resulting plasmid contained the flanking regions ligated to form an open reading frame consisting of a start codon, SmaI site, and stop codon. This plasmid would serve as the deletion construct. SmaI was then used to open the plasmid and the sacB-KanR cassette was inserted. The resulting plasmid was transformed into the desired NTHi strain, selecting for resistance to ribostamycin. A RibR, SucS isolate was then transformed with the deletion construct and transformants were selected on LB agar supplemented with 5% sucrose, chlortetracycline (1 μg/ml), hemin, and NAD. Deletions were confirmed by PCR. Confirmed mutants were then able to be transformed with the sacB-KanR cassette to delete additional genes.
PCR SOEing and mutagenesis
PCR splicing by overlap extension (PCR SOEing) was used to insert 5 bp between SiaR and CRP operators. Primers were designed to insert 5 bp between the operators of SiaR and CRP while conserving the 3 bp that are shared between the two (Table 2). The junction primers contained a 24 bp overlap to allow for splicing. Fragments were amplified by PCR with primer pairs 145R8/145M2 and 145M3/146R2 and products were purified using the QiaQuick PCR Clean Up Kit (Qiagen). PCR products were quantified with NanoDrop and mixed to yield a final concentration of 5 ng/μl of each and this mixture was used as the template in the SOEing reaction with primers 145R8 and 146R2. The product from the splicing reaction was cleaned up and used for transformation.
Transformation of NTHi strains was performed as detailed above. JWJ091 and JWJ116 were transformed with the plasmid pJJ331, a construct that spans from within the nan operon and into the siaPT operon and has the sacB-KanR cassette inserted near the insertion target. pJJ331 had an unintentional mutation in the CRP binding site that allowed for the plasmid to be maintained in E. coli. KanR, SucS transformants were then transformed with the PCR SOEing product and selected for growth on sucrose. Transformants were then screened by PCR and sequenced to confirm the presence of the 5 bp insertion and the absence of additional mutations. The resultant strains, JWJ159 (2019cyaA+5 bp) and JWJ160 (2019cyaAnagB+5 bp) were used for subsequent analysis.
RNA extraction and transcriptional analysis
RNA was extracted using the hot acid phenol method as described previously . DNA was removed from extracted RNA by digestion with DNase I (New England Biolabs) and cleaned up with the RNeasy Mini Kit (Qiagen, Valencia, CA). RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA) and the concentration was determined using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). For real time RT-PCR analysis, primer/probe sets were obtained using the Custom TaqMan Gene Expression Service (Applied Biosystems, Foster City, CA). Primer/probe sets were designed using the sequence of HI0145 and HI0146 from H. influenzae 2019. A primer/probe set for the 16S rRNA of H. influenzae was designed and used as a control. The TaqMan RNA-To-CT 1-Step Kit (Applied Biosystems) was used following the manufacturer's protocol. Reactions were set up in triplicate using 20 ng of RNA. Reactions were carried out using the StepOnePlus Real Time PCR System (Applied Biosystems) with StepOne analysis software. Results were calculated using the comparative CT method to determine the relative expression ratio between RNA samples. The primer and probe set for HI16S rRNA was used as the endogenous reference to normalize the results. Two independent sets of RNA samples were used for each experiment and the mean fold change is reported. Data are expressed as mean +/- SD.
Protein expression and purification
SiaR was expressed and purified as described previously , with modified buffers to enhance stability of the purified protein and an additional purification step. Cells were resuspended in the SiaR lysis and equilibration buffer (10 mM Tris, pH 8.0, 300 mM NaCl, 0.1% CHAPS) prior to lysis by French press. After protein binding, the resin was washed with the SiaR wash buffer (10 mM Tris, pH 8.0, 1,150 mM NaCl, 10% glycerol, 0.1% CHAPS, 5 mM imidazole) and protein was eluted with the SiaR elution buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, 0.1% CHAPS, 500 mM imidazole). The purified protein was concentrated using an Amicon Ultra centrifugation filter (Millipore, Billerica, MA) with a 10 kDa molecular weight cutoff. The protein sample was then desalted into the SiaR storage buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, 0.1% CHAPS) using FPLC through a 10 ml (2-5 ml) HiTrap Desalting Column (GE Healthcare, Piscataway, NJ). Protein concentration was determined using the NanoDrop ND-1000 Spectrophotometer and an extinction coefficient of 7,575 M-1 cm-1. Aliquots of the purified protein were frozen in liquid nitrogen and stored at -80°C until needed.
CRP was expressed and purified in a similar manner. Primers were used to amplify crp with the restriction sites HindIII and XhoI on the 5' and 3' ends, respectively (Table 2). The 41 bases immediately upstream of crp were included to ensure that the native bacterial translation signals were present. The downstream primer included the last codon of the crp open reading frame, excluding the stop codon, to allow for the fusion of a multiple-histidine tag. The PCR product was cloned into pGEM-T and subsequently subcloned into pET-24(+) (Novagen, Madison, WI) using the HindIII and XhoI sites. The resulting plasmid, pJJ276, was expected to express CRP with a carboxy-terminal His•Tag.
Protein expression was induced using the Overnight Express Autoinduction System 1 (Novagen) grown at 37°C overnight. Expressed protein was purified using the BD TALON Metal Affinity Resin (BD Biosciences, Palo Alto, CA). Purification was performed in native conditions following the manufacturer's protocol and using the suggested TALON buffers. Eluted fractions were examined by SDS-PAGE and fractions containing CRP were pooled. Protein was concentrated using an Amicon Ultra centrifugation filter and desalted as described above. The protein concentration was determined using the NanoDrop ND-1000 Spectrophotometer and an extinction coefficient of 21,555 M-1 cm-1. Purified protein was stored at 4°C.
Electrophoretic mobility shift assay
Electrophoretic mobility shift assay (EMSA) was used to study the binding of SiaR and CRP to potential promoter sequences as done previously . The probe for EMSA was amplified by PCR using primer pairs P146F1 and P146R4 (Table 2), resulting in a probe that spans the region from the nanE start codon to +18 of the siaPT transcript. Binding reactions were prepared using the EMSA Kit (Molecular Probes, Eugene, OR) following the manufacturer's directions with some modifications. Binding reactions consisted of the binding buffer (150 mM KCl, 0.1 mM DTT, 0.1 mM EDTA, 10 mM Tris, pH 7.4), the DNA probe (15 nM), and 1 μM SiaR and/or CRP. Control reactions without protein were set up for each probe. Reactions were incubated at room temperature for 20 minutes. After incubation, 6× EMSA gel-loading solution was added and reactions were loaded onto a 6% DNA Retardation Gel (Invitrogen) with prechilled 0.5× TBE buffer and run at 200 V for 60 minutes. After electrophoresis, the gel was stained with SYBR Green EMSA gel stain and bands were visualized by UV transillumination. Images were captured using a Kodak EDAS 120 camera with an EDAS 590 mm filter (Eastman Kodak Company, Rochester, NY). cAMP was added to reactions when indicated to a final concentration of 100 μM.
Primer extension analysis
Primer extension analysis was used to identify the transcriptional start sites for both nan and siaPT operons. Primers 145R7 (nan) and 146R1 (siaPT) were labeled with 32P using T4 polynucleotide kinase (New England Biolabs) and γ-[32P]-ATP (GE Healthcare). Illustra Microspin G-25 columns (GE Healthcare) were used to remove unincorporated 32P. The primer extension reaction was performed using SuperScript III First-Strand Synthesis SuperMix (Invitrogen) following the supplied protocol. After first-strand synthesis RNA was degraded by incubation with RNase A (New England Biolabs) at 37°C for 15 min. Nucleic acids were precipitated by the addition of 300 μl of chilled ethanol, incubation in a dry ice bath for 15 min, and centrifugation at 4°C. Dried samples were dissolved in loading buffer (98% deionized formamide, 10 mM EDTA, 0.025% xylene cyanol FF, 0.025% bromophenol blue) prior to loading on sequencing gel. Sequencing reactions were set up for each labeled primer using the SequiTherm EXCEL II DNA Sequencing Kit (Epicentre Technologies, Madison, WI). A PCR fragment amplified with the primers 145R7 and 146R1 was used as a template. Sequencing and primer extension reactions were loaded onto an 8% sequencing gel. After electrophoresis, the gel was dried and exposed to film at -80°C.
Strains 2019 wild-type and the 2019ΔcyaA ΔnagB mutant were grown in 100 ml cultures of sRPMI without Neu5Ac to early exponential phase. Neu5Ac, cAMP, or both were added and cultures were incubated for 20 min. Cells were pelleted and resuspended in 0.5 ml of MOPS buffer (40 mM MOPS, pH 7.3, with 50 μl D2O). Phosphorus NMR spectra were acquired at 162 MHz on a 400 MHz Varian Inova spectrometer in a 5 mm probe. Spectra were obtained upon excitation with at 45° pulse and digitization of 0.8 s followed by a delay of 1.7 s for recovery between scans. Spectra 20 kHz wide were collected and processed with gaussian line-broadening of 0.1 s prior to Fourier transformation. Samples were maintained at 15°C, 2048 transients were averaged in an experiment lasting 1.5 hours. For each sample, two such spectra were collected one after the other. These were not significantly different, indicating that relatively minor changes take place on the time scale of data collection. However a third spectrum collected some 13 hours later indicated significant change in some cases. Chemical shifts were referenced relative to external 85% phosphoric acid at 0 ppm.
This work was supported by funding from NIAID Grants AI024616 and AI30040 and NIH grant GM085302.
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