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. Author manuscript; available in PMC: 2015 May 12.
Published in final edited form as: FEMS Microbiol Lett. 2009 May 8;296(2):248–256. doi: 10.1111/j.1574-6968.2009.01647.x

Pilin regulation in the pilT mutant of Neisseria gonorrhoeae strain MS11

Manuela Dietrich 1, Hans Mollenkopf 2, Magdalene So 3, Alexandra Friedrich 1,*
PMCID: PMC4428587  NIHMSID: NIHMS142395  PMID: 19486161

Abstract

The ATPase protein PilT mediates retraction of type IV pili (Tfp). Tfp retraction of Neisseria gonorrhoeae causes many signal transduction events and changes in gene expression in infected epithelial cells. To find out whether a pilT mutation and lack of Tfp retraction, respectively, lead also to gene regulation in bacteria we performed microarrays comparing the transcriptional profiles of the N. gonorrhoeae parent strain MS11 and its isogenic pilT mutant during growth in vitro. A loss-of-function-mutation in pilT led to altered transcript levels of 63 open reading frames. Levels of pilE transcripts and its deduced protein the major Tfp subunit pilin, were increased most markedly by a mutation in pilT. Further studies revealed that pilE expression was also controlled by two other genes encoding Tfp biogenesis proteins, pilD and pilF. Our studies strongly suggest that pilE expression is a finely-tuned process.

Keywords: Neisseria gonorrhoeae, pilT-responsive genes, pilE expression

INTRODUCTION

Type IV pili (Tfp) are fimbriate organelles that are widespread among Gram-negative bacteria (Mattick, 2002). For several pathogenic bacteria including the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae, Tfp play important roles in host cell interactions, including adhesion (Howie et al., 2005, Higashi et al., 2007). In addition, Tfp mediate twitching motility (a flagella-independent movement) and DNA uptake (Wall & Kaiser, 1999; Mattick, 2002; Chen & Dubnau, 2003). A Tfp fiber consists of repeating pilin subunits (PilE) and its assembly requires a nucleotide binding protein (PilF in N. gonorrhoeae), an inner membrane protein (PilG in N. gonorrhoeae), a prepilin peptidase (PilD) and the outer-membrane secretin PilQ through which the pilus fiber is extruded (for a review see Hansen & Forest, 2006). Several other proteins are also important for proper function and assembly of Tfp, and mutations in the corresponding pil genes lead to nonpiliation (Carbonnelle et al., 2005; Carbonnelle et al., 2006; Hansen & Forest, 2006). An important feature of Tfp is their ability to retract (Merz & So, 2000; Sun et al., 2000; Skerker & Berg, 2001). Cycles of fiber extension and retraction lead to the dynamics of Tfp and associated functions such as surface motility. Tfp retraction is driven by the hexameric ATPase protein PilT (Merz et al., 2000; Vale, 2000). PilT belongs to a highly conserved protein family homologous to AAA-type motor proteins and is proposed to cause Tfp to retract by disassembling the pilin subunits at the base of the fiber (Morand et al., 2004). A pilT mutant of N. gonorrhoeae strain MS11 is piliated and adheres to host cells. However, it cannot retract Tfp and is nonmotile and noncompetent for DNA uptake, and defective in many aspects of host cell interactions (Wolfgang et al., 1998; Howie et al., 2005; Lee et al., 2005; Higashi et al., 2007).

Even though many Tfp proteins have been identified, a function could be determined for only a few of these proteins. Based on the observation that the introduction of a pilT mutation in a collection of nonpiliated N. meningitidis mutants can lead to rescue of piliation and Tfp-associated phenotypes in some but not all double mutants, Carbonnelle et al. (2006) proposed a four-step model for Tfp biogenesis in N. meningitidis, which dissect the contribution of Tfp proteins to one of these four steps.

Because pilT is involved in N. gonorrhoeae signaling to epithelial cells (Howie et al., 2005; Lee et al., 2005; Higashi et al., 2007; Howie et al., 2008), we reasoned that it may also take part in regulating bacterial processes. We reported recently that pilT influences the expression of two neisserial ABC transporters (NGO0372-NGO0374, NGO2011-NGO2014), mtrF (multiple transferable resistance) and farR (fatty acid resistance) during epithelial cell infection (Friedrich et al., 2007). In the present study, we determined whether pilT regulation of N. gonorrhoeae gene expression occurs only in the context of an infection. We report here that pilT alters global gene expression in N. gonorrhoeae strain MS11 also in the absence of epithelial cells. We show that pilT dependent changes in transcript levels of the ABC transporter NGO0372-NGO0374 and farR does not require the presence of epithelial cells, while transcript levels of the ABC-transporter NGO2011-NGO2014 and mtrF were not altered under these in vitro conditions. Finally, we also show that pilT and other Tfp biogenesis factors affect the expression of pilE, encoding the major Tfp subunit protein.

MATERIALS AND METHODS

Growth conditions and strains

E. coli strains DH5α and TOP10 (Invitrogen) were used for all recombinant DNA manipulations and were grown in Luria broth supplemented as necessary with ampicillin (100 mg/l), kanamycin (50 mg/l) or erythromycin (300 mg/l). Neisseria gonorrhoeae parent strain MS11(wt) and its mutant strains were grown at 37°C in a humidified 5 % CO2 atmosphere on GCB medium with supplements. When necessary, kanamycin (50 mg/l) or erythromycin (10 mg/l) was added. Piliation and Opa phenotypes were monitored by colony morphology.

DNA manipulations

The molecular and genetic procedures used were standard techniques. Chromosomal DNA was isolated using the QIAamp DNA Mini Kit (Qiagen). In order to generate a nonpolar N. gonorrhoeae pilT mutant, 504 bp of pilT were deleted in-frame without inserting any antibiotic resistance gene. pilT plus flanking regions was amplified by PCR and the PCR product was subsequently cloned into pBluescript II. 504 bp of pilT were deleted by digestion with HincII and religation. The resultant fragment was cloned into pKC1 and a positive-negative selection method was used according to Cloud and Dillard (2002) to generate an unmarked pilT mutant of N. gonorrhoeae MS11. One such mutant, MS11ΔpilT, was selected for further studies. In order to create a pilD mutant, pilD plus flanking DNA was amplified as a 1.2 kb fragment and cloned into pKC1. A kanamycin resistance cassette was inserted into the single NAEI site of pilD and the resulting construct was used to transform N. gonorrhoeae MS11. One such kanamycin resistant mutant, MS11pilD, was selected for further studies. pilE sequences of all generated mutants were confirmed to correspond to the wt sequences.

RNA isolation

Total RNA was isolated from bacteria after 9 hours of growth on supplemented GCB agar plates and during growth in liquid cultures, respectively, using the RNeasy kit (Qiagen), according to the manufacturer’s instructions. Potential traces of DNA were removed by an additional on-column-digest with DNase I (Qiagen). RNA concentration was measured using NanoDrop 1000 (Thermo Fisher Scientific) and RNA quality was accessed using a 2100 Bioanalyzer (Agilent Technologies).

Microarray experiments

N. gonorrhoeae microarrays were designed with eArray (Agilent Technologies) as 8x15 K chips including all open reading frames (ORF) from the whole genome of N. gonorrhoeae strain FA1090 (accession number AE004969) plus all ORFs from the gonoccoccal genetic island of strain MS11 (accession number AY803022). Each ORF was covered by 6 specific oligonucleotides. Microarray experiments were carried out as two-color hybridizations. Equal amounts (10 µg) of total bacterial RNA were reverse transcribed and cDNA labeled with Cy3 or Cy5 using the CyScribe Postlabelling Kit (GE Healthcare). In order to compensate Cy-dye specific effects and to ensure statistically relevant data, a color-swap dye reversal setting with three biological experiments was used. Labeled samples were mixed and hybridized to the microarray according to the two-color microarray-based gene expression analysis protocol from Agilent Technologies. After washing and scanning of the hybridized microarrays data were analysed on the Rosetta Inpharmics platform Resolver Version 7.0. Ratio profiles were combined in an error-weighted fashion with Resolver to create ratio experiments. A two-fold change expression threshold for ratio experiments was applied together with anti-correlation of ratio profiles rendering the microarray analysis set highly significant (P-value < 0.05), robust and reproducible.

Real-time quantitative reverse transcription (RT)-PCR

Real-time quantitative RT-PCR (real-time qRT-PCR) was used to confirm microarray results, which was carried out as described recently (Friedrich, et al., 2007). Briefly, two micrograms of total RNA was reverse-transcribed to generate cDNA using the iScript cDNA Synthesis Kit (Bio-Rad) as recommended by the manufacturer. Oligonucleotides complementary to the genes of interest were designed using the primer express software (PE Applied Biosystems) to obtain amplicons of the same size. Oligonucleotides were purchased from Invitrogen and MWG, respectively. As a control, parallel samples were run in which reverse transcriptase was omitted from the reaction mixture. Real-Time PCR using SYBR GREEN PCR master mix (Applied Biosystems) was carried out on the ABI Prism 7000 Sequence Detector System (Applied Biosystems). Amplification plots were analyzed with the ABI Prism SDS Software package (version 1.0) and the data were processed using TaqMan Turbocrunch (96) v3. Relative quantification of gene transcription was performed by the comparative Ct (threshold cycle) method according to the manufacturer’s instruction. The relative amount of target cDNA was normalized using an internal reference standard, 16S rRNA gene as control. Four RT experiments were performed from four different biological experiments.

SDS-PAGE and immunoblotting

Samples were boiled for 5 to 10 min in a water bath prior to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% polyacrylamide gel as described previously (Laemmli, 1970). The separated proteins were transferred to Polyscreen PVDF transfer membranes (Perkin Elmer), which were blocked by incubation for 1 h at room temperature in TBST buffer (0.1 M Tris-HCl, 2.5 M NaCl, 0.05% Tween 20, pH 7.5) containing 3% bovine serum albumin. Subsequently, membranes were probed with specific antibodies, e.g. monoclonal anti-pilin antibody SM1 (24), polyclonal gonococcal PilC antiserum, polyclonal anti-PilT antibody (a generous gift from K.T. Forest, Madison, WI), polyclonal anti-Hfq antibody (a generous gift from Y. Pannekoek, Amsterdam, The Netherlands), polyclonal gonoccocal porin P.IB antiserum (a generous gift from T. Rudel, Würzburg, Germany) and polyclonal antibody against whole N. gonorrhoeae (α-GC-ab, US Biological, Swampscott, MA). Antigen detection was carried out using HRP-linked anti-rabbit and anti-mouse immunoglobulin G, respectively, and a Western Lightning Plus-ECL, Enhanced Chemiluminescence substrate (Perkin Elmer) as recommended by the manufacturer.

Cellular fractionation

To determine the subcellular localization of pilin (PilE) in N. gonorrhoeae, bacteria were collected by centrifugation and fractionated into soluble cytosolic proteins, inner and outer membranes as described elsewhere (Brossay et al., 1994). Prior to fractionation pili were removed following a modified protocol from Jonsson et al. (1991). Briefly, bacterial pellets were suspended in 0.15 M ethanolamine (pH 10.5) and pili were sheared off by vortexing for 2 min. After centrifugation pili-containing supernatant was removed and cell pellets were washed twice with PBS. Cells were disrupted by sonication in 10 mM HEPES buffer (pH 7.4) and centrifuged at 4000 × g for 10 min to remove cell debris. The supernatant was subjected to ultracentrifugation at 100000 × g for 1 h to separate cytoplasmic proteins (soluble fraction) from total membranes (insoluble fraction). From the total membrane pellet, inner and outer membranes were separated by treatment with sarcosyl and ultracentrifugation. Finally, Sarcosyl-insoluble outer membrane proteins were solubilized in RIPA buffer. Purity of the fractions was controlled in immunoblot analysis using antibodies specific for cytosolic proteins (anti-Hfq) and outer membrane proteins (anti-Porin).

RESULTS AND DISCUSSION

pilT affects global transcription of N. gonorrhoeae strain MS11 grown in vitro

Our previous work revealed four genes and gene clusters, respectively, that were differentially expressed in wild-type (wt) N. gonorrhoeae strain MS11 and its isogenic pilT mutant after infection of epithelial cells (Friedrich et al., 2007). These pilT-responsive genes comprise two putative ABC transporter gene clusters (NGO0372-NGO0374 and NGO2011-NGO2014), farR (fatty acid resistance), and mtrF (multiple transferable resistance) (Friedrich et al., 2007).

We have designed and constructed a new DNA microarray platform (see Materials and Methods) that increases the consistency of microarray signals. Using this new N. gonorrhoeae array, we sought to identify pilT-responsive genes whose regulation does not require the presence of epithelial cells. We compared the transcriptional profiles of N. gonorrhoeae strain MS11 and its isogenic pilT-derivative, MS11ΔpilT, during growth on agar plates. MS11ΔpilT contains an unmarked 504 bp in-frame deletion in pilT. Under these conditions no significant growth differences between wt and mutant strains were observed (data not shown). Total RNA was isolated from bacteria using the RNeasy kit (Qiagen) per manufacturer’s instruction. Microarray experiments were carried out as two-color hybridizations. In the context of micorarray data, we refer to changes in transcript levels as gene regulation, unless otherwise stated. Regulated genes were identified with a stringent threshold of 1.75-fold and P values <0.0001. For a summary of microarray results see Table 1 in supplemental material and Fig. 1A. Supporting microarray data have been deposited in the NCBI Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE12258. The microarray data on selected pilT-responsive genes were confirmed by real-time qRT-PCR (Fig. 1B).

Fig. 1. pilT-responsive genes in N. gonorrhoeae strain MS11.

Fig. 1

Fig. 1

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Transcriptional analysis (microarrays) revealed 63 ORFs that are differentially regulated in MS11ΔpilT (13 upregulated, 50 downregulated) compared to wt MS11.

(a) Grouping of pilT-responsive genes according to function or location.

(b) Real-Time qRT-PCR verification of microarray results. Each column represents the fold-change of transcript level of the indicated gene in MS11ΔpilT compared to that in wt MS11. Values and standard deviation are calculated from the results of four independent experiments. P has a value of <0.01.

63 ORFs were differentially regulated in the pilT mutant MS11ΔpilT (13 were upregulated, 50 were downregulated). These pilT-responsive genes comprise several classes (Fig. 1A), with the largest classes encoding hypothetical and metabolic proteins. Among the upregulated loci was the ABC transporter gene cluster NGO0372-NGO0374, previously identified as pilT-responsive during infection (Friedrich et al., 2007). Our present data indicate that the influence of pilT on transcription levels of NGO0372-NGO0374 does not require host cells. In contrast, the other pilT-responsive ABC transporter gene cluster NGO2011-NGO2014 and mtrF, were not upregulated in the pilT mutant during growth in vitro, which suggests that pilT regulation of these genes may require infection conditions. Interestingly, transcript levels of pilE, which encodes the Tfp pilin subunit, were increased most markedly in MS11ΔpilT. Our previous microarray study suggested that pilE was also upregulated (~3-fold) in the pilT mutant during infection (unpublished data from previous study). Among the genes downregulated in MS11ΔpilT were three (putative) regulators, NGO0058 (farR), NGO1244 (marR) and NT03NG0080 (hpaR). farR was previously identified as pilT-responsive under infection conditions and shown to directly or indirectly regulate NGO0372-NGO0374, and also NGO2011-NGO2014 and mtrF to a minor extent (Friedrich et al., 2007).

The effect of pilT on the expression of these genes may be due to its function in Tfp retraction, though another activity of pilT in exerting these effects cannot be ruled out. In any case, its effect is unlikely to have resulted from nonspecific downstream effects of mutagenesis because the 504 bp deletion in pilT does not have a polar effect on the pilU gene downstream of pilT, as shown by real-time qRT-PCR (see Fig. 1B).

Upregulation of the Tfp subunit gene pilE in MS11ΔpilT

pilE, the pilin expression locus encoding a functional pilin subunit, was one of the most highly upregulated genes in MS11ΔpilT compared to wt MS11 (Table 1, supplemental material). This finding was validated by real-time qRT-PCR (Fig. 1B), which revealed a 2.8+/−0.7 fold (P value <0.005) increase in pilE transcript levels in MS11ΔpilT. By immunoblot analysis using the monoclonal anti-pilin antibody SM1 (Merz & So, 1997) pilin levels were also shown to be higher in MS11ΔpilT than in wt MS11 (Fig. 2, lane 1,2). The higher level of pilin expression in MS11ΔpilT is consistent with the hyperpiliation phenotype of the pilT mutant that we consistently observed by electron microscopy (data not shown), and with an earlier study showing that pilE transcription can alter the number of pili expressed per cell (Long et al., 2001).

Fig. 2. Amount of pilin in whole cell lysates from N. gonorrhoeae wt MS11 and different mutant strains.

Fig. 2

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Equal amounts of protein were separated by SDS-PAGE and transferred to Polyscreen PVDF transfer membranes for immunoblotting using the monoclonal anti-pilin antibody SM1 (Merz & So, 1997). The same filter was stripped and reprobed with a polyclonal antibody against whole N. gonorrhoeae (α-GC-ab purchased from US Biological) as a loading control. Molecular masses (in kDa) are indicated. Lane 1: MS11, lane 2: MS11ΔpilT, lane 3: MS11pilD, lane 4: MS11-307.

Hyperpiliation has been reported for pilT mutants in N. meningitidis, Pseudomonas aeruginosa and enteropathogenic Escherichia coli (Bradley, 1980; Bieber et al., 1998; Pujol et al., 1999). However, in these cases, the phenotype is suggested to result from non-retraction of Tfp and consequently from lack of disassembly of the Tfp fiber. Our data suggest that the hyperpiliation phenotype of MS11ΔpilT is due not only to lack of Tfp retraction but also to transcriptional upregulation of pilE in the pilT mutant background.

Interestingly, Wolfgang et al. (1998) observed neither hyperpiliation nor an increase in pilin levels in whole cell lysates of N. gonorrhoeae N400pilT compared to the N400 strain with a wt pilT. The disparity between our findings may be due to the differences in the genetic backgrounds of the two strains or to differences in the pilT mutation. N400 is a recA derivative of strain MS11 (Tonjum et al., 1995), in which pilE rearrangements are controlled by expressing recA under an inducible promoter (Koomey et al., 1987). Moreover, N400 contains only one pilE expression locus, pilE1, as pilE2 was deleted (Koomey et al., 1987), whereas MS11 has both pilE1 and pilE2 (Meyer et al., 1984). In contrast to the nonpolar mutation of pilT in our mutant, other pilT mutations can affect pilU transcription as both genes are transcribed from one promoter (Park et al., 2002). We transformed our pilT deletion construct into strain N400 to generate a nonpolar pilT mutant, N400ΔpilT. Transcription of pilU in N400ΔpilT was not affected by a polar effect as corroborated by real-time qRT-PCR (1.5+/−0.3 fold upregulation). As observed for other pilT mutations introduced into strain N400 (Wolfgang et al., 1998) PilE levels were not altered in N400ΔpilT (Fig. 1S in supplemental material). Since PilE levels were also independent of recA induction with IPTG (Fig. 1S in supplemental material) it is tempting to speculate that pilE2 (which is missing in strain N400ΔpilT) is indeed the regulated pilin gene in MS11ΔpilT.

To determine whether there is a reciprocal regulation of pilT by pilE, expression of pilT was analyzed in a pilE mutant of strain MS11, MS11-307, which is deleted of both pilin expression sites. As shown by real-time qRT-PCR transcription of pilT is not reciprocally regulated by pilE (1.3+/−0.2 fold downregulation, P value <0.005). Even though no pilus fibers are assembled, the amount and localization of PilT in the inner membrane appears to be unaltered in MS11-307 (data not shown).

Location of pilin in the retraction-deficient pilT mutant MS11ΔpilT

The amount and location of pilin in wt MS11 and its pilT derivative, MS11ΔpilT, was next analyzed by immunoblotting of cell fractions (Fig. 3). After removal of Tfp fibers (modified from Jonsson et al. (1991)), the bacterial cells were disrupted and fractionated into soluble cytosolic and total membrane fractions. The membrane fraction was further separated into inner and outer-membrane as described previously (Brossay et al., 1994). Samples were blotted using the monoclonal anti-pilin antibody SM1 (Merz & So, 1997). Purity of fractions was confirmed using antibodies specific for outer membrane and cystosolic proteins. Our data show that higher amounts of pilin are measured in MS11ΔpilT than wt MS11 in the cytosolic, total membrane and inner membrane fractions (Fig. 3). Because Tfp were sheared off prior to fractionation, no pilin was detected in the outer-membrane fraction of either MS11 or MS11ΔpilT (Fig. 3, lane 9,10). The pilus fiber is thought to be disassembled into the inner membrane during Tfp retraction (Morand et al., 2004). This model predicts that in a Tfp-retraction deficient pilT mutant less pilin would be present in the inner membrane compared to the wt strain. However, our data show the opposite: in MS11ΔpilT pilin is found in higher amounts than wt in all cell fractions including the inner membrane (Fig. 3). Since pilin in the inner membrane of the retraction-deficient MS11ΔpilT cannot result from Tfp retraction, it probably derives from higher levels of pilin produced in this mutant. These data give additional support to our hypothesis that hyperpiliation of MS11ΔpilT is not only due to a defect in Tfp disassembly but results additionally from a higher expression of pilE in the MS11ΔpilT mutant.

Fig. 3. Localization of pilin in cell fractions of N. gonorrhoeae wt MS11 and MS11ΔpilT.

Fig. 3

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Cytosolic fractions, inner and outer membranes were separated according to Brossay et al. (Brossay et al., 1994) after external pili were removed by shearing. The purity of fractions was confirmed by using specific antibodies for each fraction (data now shown). Equivalent amounts of the different cell fractions were separated by SDS-PAGE and the proteins transferred to Polyscreen PVDF transfer membranes for immunoblotting with the monoclonal anti-pilin antibody SM1 (Merz & So, 1997). The same filter was stripped and reprobed with a polyclonal antibody against whole N. gonorrhoeae (α-GC-ab purchased from US Biological) as a loading control. Lane 1: whole cell lysate MS11, lane 2: whole cell lysate MS11ΔpilT, lane 3: cytosolic fraction MS11, lane 4: cytosolic fraction MS11ΔpilT, lane 5: total membranes MS11, lane 6: total membranes MS11ΔpilT, lane 7: inner membrane MS11, lane 8: inner membrane MS11ΔpilT, lane 9: outer membrane MS11, lane 10: outer membrane MS11ΔpilT.

pilT mutation does not affect expression of other Tfp biogenesis genes

The pilT mutation did not affect the expression of other Tfp biogenesis genes, as shown by microarray experiments (see Table 1 in supplemental material, GEO series accession number GSE12258). A recent study found that the N. meningitidis pilC1, which encodes a protein essential for Tfp biogenesis and adhesion (Nassif et al., 1994), is upregulated 2.5–3 fold in the pilT mutant during exponential growth in the absence of epithelial cells (Yasukawa et al., 2006). In our study, the N. gonorrhoeae pilC1 was not pilT-responsive, as shown by microarrays, real-time qRT-PCR and western blot analysis (Table 1 and Fig. 2S in supplemental material, GSE12258). The disparity in results may simply reflect different growth conditions used, or may be due to differential regulation of pilC1 expression in N. gonorrhoeae and N. meningitidis; indeed, PilC1 protein function is not identical in these two pathogens (Nassif et al., 1994).

Several silent pilin (pilS) genes appeared also to be upregulated in MS11ΔpilT compared to wt MS11 (Table 1, supplemental material), which is likely due to partial cross-hybridization of the pilE/pilS 60-mer oligonucleotides in the microarray. Real-time qRT-PCR using pilS-specific 20-mer oligonucleotides with no homology to pilE confirmed that pilS loci were not transcribed in either MS11 or MS11ΔpilT (data not shown). Taken together, our data strongly indicate that the pilT mutation unbalances pilE expression without influencing expression of other Tfp genes.

pilE regulation in a pilD and a pilF mutant

In contrast to pilT, other pil mutants are not piliated (reviewed by (Fussenegger et al., 1997; Tonjum & Koomey, 1997). We next determined whether other Tfp assembly proteins also regulate pilE expression. PilD is a peptidase that removes the signal sequence from Tfp prepilins. pilD mutants lack the prepilin peptidase and produce unprocessed, assembly-deficient pilins (Freitag et al., 1995). To our knowledge, the effect of the pilD mutation on pilE expression levels in N. gonorrhoeae strain MS11 has not been examined. pilE transcript and pilin protein levels were next analyzed in MS11pilD, which contains a kanamycin resistance cassette inserted into pilD. This mutation does not affect expression of the upstream or downstream pilF and pilG genes, respectively. Real-time qRT-PCR revealed a 2.2-fold (+/−0.25, P value of <0.005) reduction of the pilE transcript in MS11pilD compared to wt MS11. This suggests that the pilD mutation has negatively affected pilE transcription. Immunoblot analysis showed that decreased transcription of pilE in MS11pilD is accompanied by reduced pilin levels (Fig. 2, lane 1,3). As anticipated (Nunn & Lory, 1991; Freitag et al., 1995), the MS11pilD pilin migrates more slowly in SDS-PAGE than wt MS11 pilin, strongly suggesting that it is unprocessed in the mutant strain (Fig. 2, lane 1,3). This very low level of prepilin in the pilD mutant (Fig. 2, lane 3) likely not only reflects the 2.2-fold reduction of pilE transcript in MS11pilD but also suggests that the unprocessed pilins in MS11pilD have been degraded more quickly.

PilF is a cytosolic pilus assembly factor with a putative ATP-binding motif and other similarities to PilT (Freitag et al., 1995). In contrast to pilT mutants, pilF mutants produce processed pilins that are not assembled into Tfp fibers (Freitag et al., 1995). It is suggested that PilF and PilT work antagonistically to control Tfp fiber assembly and disassembly, respectively (Wolfgang et al., 2000). To find out whether a pilF mutation also alters pilE expression, we compared pilE expression levels in wt MS11 and its pilF mutant derivative MS11pilF. MS11pilF was constructed by transforming pPilF2 (a gift from M. Koomey; Freitag et al., 1995) into MS11. Real-time qRT-PCR revealed a 2.3-fold (+/−0.4) reduction of pilE transcript in MS11pilF, which was accompanied by decreased pilin levels (Fig. 4). When Tfp were sheared off prior to fractionation, highest pilin levels were found in the wt pili fraction; in contrast this fraction did not contain any pilin in the nonpiliated mutant MS11pilF (Fig. 4, lane 3,4). Interestingly, in MS11pilF pilin levels were higher in total membranes than in the corresponding wt fraction after Tfp were removed (Fig. 4, lane 7,8), suggesting that pilin is incorporated into the inner membrane of MS11pilF but is not assembled any further.

Fig. 4. Amount and localization of pilin in wt MS11 and the pilF mutant MS11pilF.

Fig. 4

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Cytosolic fractions and total membrane fractions were separated according to Brossay et al. (Brossay et al., 1994) after external pili were removed by shearing. The purity of fractions was confirmed by using specific antibodies for each fraction (data not shown). Equal amounts of protein and equivalent amounts of the different cell fractions, respectively, were separated by SDS-PAGE and proteins transferred to Polyscreen PVDF transfer membranes for immunoblotting with the monoclonal antibody SM1. The same filter was stripped and reprobed with a polyclonal antibody against whole N. gonorrhoeae (α-GC-ab purchased from US Biological) as a loading control. Lane 1: whole cell lysate MS11, lane 2: whole cell lysate MS11pilF, lane 3: Tfp fraction of MS11 (Tfp-containing supernatant after treatment for removal of Tfp), lane 4: “pili fraction” (supernatant after treatment for removal of Tfp ) of MS11pilF, lane 5: cytosolic fraction MS11, lane 6: cytosolic fraction MS11pilF, lane 7: total membrane fraction MS11, lane 8: total membrane fraction MS11pilF.

Consistent with the influence of a pilT mutation on pilin expression in N. gonorrhoeae strain MS11, but not in strain N400 (Wolfgang et al., 1998; our study), Freitag et al. (1995) did not observe a decrease in pilin levels in whole cell lysates of pilD and pilF mutants of N. gonorrhoeae strain N400.

Taken together, our data indicate that pilT, pilD and pilF can affect pilE expression in N. gonorrhoeae strain MS11, which suggests that these Tfp biogenesis genes may form part of a feedback loop(s) that controls pilin expression.

Upregulation of pilE in MS11ΔpilT is not mediated through FarR

Our previous work revealed that the known transcriptional repressor FarR (Lee et al., 2003) regulates directly or indirectly pilT-reponsive genes, such as NGO0372-NGO0374 (Friedrich et al., 2007). Here we found farR to be more than two-fold downregulated in MS11ΔpilT after growth in vitro (Table 1 in supplemental material, Fig. 1B); therefore we investigated whether FarR regulates pilE. pilE transcription levels were compared in MS11 and in the isogenic farR null mutant MS11farR (Friedrich et al., 2007) after growth on agar plates. Real-time qRT-PCR revealed no significant differences in pilE transcript levels between wt and farR mutant (1.2 +/− 0.1 fold downregulation, P value <0.005). Consistent with the unaltered transcript levels of pilE in MS11farR, western blot analysis using the monoclonal SM1 antibody revealed comparable pilin levels in MS11 and MS11farR (Fig. 3S in supplemental material). Hence, we conclude that FarR is not involved in pilE regulation. However, it is tempting to speculate that pilT-dependent pilE expression is regulated by another pilT-responsive regulator, such as NGO1244 (MarR) and NT03NG0080 (HpaR).

Adhesion to solid surfaces impacts pilT-dependent regulation

In the present study N. gonorrhoeae strain MS11 and MS11ΔpilT were grown in the absence of epithelial cells on agar plates. Nevertheless, bacteria grown under these in vitro conditions might be subject to similar physical stresses as bacteria attached to epithelial cells; pilus retraction may lead to a counterforce only when bacteria are attached to solid surfaces. If true, differences in gene expression between wt and its retraction-deficient pilT mutant will become noticeable only in bacteria grown on solid surfaces. Therefore, MS11 and MS11ΔpilT grown in liquid cultures were also checked for altered transcript levels of known pilT-responsive genes. Samples were taken in the early and late logarithmic growth phase and subjected to real-time qRT-PCR and western blot analysis, respectively. As observed after growth on agar plates (Fig. 1B), pilE transcript levels were significantly increased (2.7 +/−0.3 in early and 2.6 +/−0.6 in late logarithmic growth phase) in MS11ΔpilT compared to wt. Pilin levels were also higher in the pilT mutant under these in vitro growth conditions (see Fig. 4S in supplemental material). These data support our hypothesis that upregulation of pilE in MS11ΔpilT is due to an intrinsic difference between MS11 and the pilT mutant irrespective of growth conditions used. To complete this study, pilin levels were also measured in pilD and pilF mutants of strain MS11 and were found to be decreased (see Fig. 4S in supplemental material), as observed during growth on agar plates.

In contrast to pilE, other pilT-responsive genes, farR, marR, NGO2093 and NGO1973 were regulated neither in early nor in late logarithmic growth phase. NGO0372 was found to be slightly upregulated (1.9+/–0.5) only in late logarithmic phase (compared to 3.3 +/−0.8 fold upregulation on agar plates), suggesting that expression of these genes is regulated by pilus retraction and retraction force, respectively, on solid surfaces, such as on infected human epithelial cells or agar plates.

In summary, our results provide evidence that pilT has a global effect on transcription in N. gonorrhoeae regardless of the presence or absence of epithelial cells. In particular, our data demonstrate an effect of pilT, pilD and pilF on pilE expression in N. gonorrhoeae strain MS11, suggesting that Tfp biogenesis is a finely-tuned process in the sense that a mutation in one pil gene could unbalance the expression of another. Our new microarray platform will allow us to conduct detailed time-course studies to determine which of these genes are regulated by pilT in the context of infection.

Supplementary Material

Supp Fig 1. Supporting Information.

Additional Supporting Information may be found in the online version of this article:

Fig. S1. Amount of pilin in whole cell lysates from N. gonorrhoeae strain N400 and its isogenic pilT mutant N400ΔpilT with and without recA-induction.

Equal amounts of protein were separated by SDS-PAGE and transferred to Polyscreen PVDF transfer membranes for immunoblotting using the monoclonal anti-pilin antibody SM1 (Merz & So, 1997). The same filter was stripped and reprobed with a polyclonal antibody against whole N. gonorrhoeae (α-GC-ab purchased from US Biological) as a loading control. Molecular masses (in kDa) are indicated. Lane 1: N400, lane 2: N400ΔpilT, lane 3: N400, lane 4: N400ΔpilT.

Supp Fig 2

Fig. S2. Amount of PilC in whole cell lysates from N. gonorrhoeae wt MS11 and MS11ΔpilT.

Equal amounts of protein were separated by SDS-PAGE and transferred to Polyscreen PVDF transfer membranes for immunoblotting using the polyclonal anti-PilC antibody. The same filter was stripped and reprobed with a polyclonal antibody against whole N. gonorrhoeae (α-GC-ab purchased from US Biological) as a loading control. Molecular masses (in kDa) are indicated. Lane 1: MS11, lane 2: MS11ΔpilT.

Supp Fig 3

Fig. S3. Amount of pilin in whole cell lysates from N. gonorrhoeae wt MS11 and MS11farR.

Equal amounts of protein were separated by SDS-PAGE and transferred to Polyscreen PVDF transfer membranes for immunoblotting using the monoclonal anti-pilin antibody SM1 (Merz & So, 1997). The same filter was stripped and reprobed with a polyclonal antibody against whole N. gonorrhoeae (α-GC-ab purchased from US Biological) as a loading control. Molecular masses (in kDa) are indicated. Lane 1: MS11, lane 2: MS11ΔpilT, lane 3: MS11farR.

Supp Fig 4

Fig. S4. Amount of pilin in whole cell lysates from N. gonorrhoeae wt MS11 and different mutant strains. Bacteria were grown in liquid cultures and samples taken from early and late logarithmic phase.

Equal amounts of protein were separated by SDS-PAGE and transferred to Polyscreen PVDF transfer membranes for immunoblotting using the monoclonal anti-pilin antibody SM1 (Merz & So, 1997). The same filter was stripped and reprobed with a polyclonal antibody against whole N. gonorrhoeae (α-GC-ab purchased from US Biological) as a loading control. Molecular masses (in kDa) are indicated. Lane 1: MS11, lane 2: MS11ΔpilT, lane 3: MS11pilD, lane 4: MS11pilF, lane 5: MS11, lane 6: MS11ΔpilT, lane 7: MS11pilD, lane 8: MS11pilF.

Supp Tab

Table S1: Regulated ORFs in response to the pilT mutation in MS11ΔpilT (in vitro after growth on agar plates)

ACKNOWLEDGMENTS

This work was funded in part by National Institutes of Health grant R01AI068033 to M. So and by a grant from the Fonds der Chemischen Industrie (FCI) to A. Friedrich. M. Dietrich and A. Friedrich also were supported by fellowships from the Liebig-Programme of the Fonds der Chemischen Industrie. The authors thank K. T. Forest for the PilT antibody, T. Rudel for the porin antibody and Y. Pannekoek for the Hfq antibody. We thank W. H. Shafer for the farR strain, M. Koomey for plasmid pPilF2, and J. Dillard for kindly providing plasmid pKC1. We would like to thank Marion Gottschald for help with the N400 mutagenesis and Lesley Ann-Ogilvie for critically reading the manuscript. We thank T.F. Meyer for fruitful discussions.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Fig 1. Supporting Information.

Additional Supporting Information may be found in the online version of this article:

Fig. S1. Amount of pilin in whole cell lysates from N. gonorrhoeae strain N400 and its isogenic pilT mutant N400ΔpilT with and without recA-induction.

Equal amounts of protein were separated by SDS-PAGE and transferred to Polyscreen PVDF transfer membranes for immunoblotting using the monoclonal anti-pilin antibody SM1 (Merz & So, 1997). The same filter was stripped and reprobed with a polyclonal antibody against whole N. gonorrhoeae (α-GC-ab purchased from US Biological) as a loading control. Molecular masses (in kDa) are indicated. Lane 1: N400, lane 2: N400ΔpilT, lane 3: N400, lane 4: N400ΔpilT.

NIHMS142395-supplement-Supp_Fig_1.pdf (139.7KB, pdf)
Supp Fig 2

Fig. S2. Amount of PilC in whole cell lysates from N. gonorrhoeae wt MS11 and MS11ΔpilT.

Equal amounts of protein were separated by SDS-PAGE and transferred to Polyscreen PVDF transfer membranes for immunoblotting using the polyclonal anti-PilC antibody. The same filter was stripped and reprobed with a polyclonal antibody against whole N. gonorrhoeae (α-GC-ab purchased from US Biological) as a loading control. Molecular masses (in kDa) are indicated. Lane 1: MS11, lane 2: MS11ΔpilT.

NIHMS142395-supplement-Supp_Fig_2.pdf (57.6KB, pdf)
Supp Fig 3

Fig. S3. Amount of pilin in whole cell lysates from N. gonorrhoeae wt MS11 and MS11farR.

Equal amounts of protein were separated by SDS-PAGE and transferred to Polyscreen PVDF transfer membranes for immunoblotting using the monoclonal anti-pilin antibody SM1 (Merz & So, 1997). The same filter was stripped and reprobed with a polyclonal antibody against whole N. gonorrhoeae (α-GC-ab purchased from US Biological) as a loading control. Molecular masses (in kDa) are indicated. Lane 1: MS11, lane 2: MS11ΔpilT, lane 3: MS11farR.

NIHMS142395-supplement-Supp_Fig_3.pdf (151.3KB, pdf)
Supp Fig 4

Fig. S4. Amount of pilin in whole cell lysates from N. gonorrhoeae wt MS11 and different mutant strains. Bacteria were grown in liquid cultures and samples taken from early and late logarithmic phase.

Equal amounts of protein were separated by SDS-PAGE and transferred to Polyscreen PVDF transfer membranes for immunoblotting using the monoclonal anti-pilin antibody SM1 (Merz & So, 1997). The same filter was stripped and reprobed with a polyclonal antibody against whole N. gonorrhoeae (α-GC-ab purchased from US Biological) as a loading control. Molecular masses (in kDa) are indicated. Lane 1: MS11, lane 2: MS11ΔpilT, lane 3: MS11pilD, lane 4: MS11pilF, lane 5: MS11, lane 6: MS11ΔpilT, lane 7: MS11pilD, lane 8: MS11pilF.

NIHMS142395-supplement-Supp_Fig_4.pdf (205.3KB, pdf)
Supp Tab

Table S1: Regulated ORFs in response to the pilT mutation in MS11ΔpilT (in vitro after growth on agar plates)

NIHMS142395-supplement-Supp_Tab.pdf (90.7KB, pdf)

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