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. 2003 Nov;71(11):6279-91.
doi: 10.1128/iai.71.11.6279-6291.2003.

Low-level Pilin Expression Allows for Substantial DNA Transformation Competence in Neisseria Gonorrhoeae

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Low-level Pilin Expression Allows for Substantial DNA Transformation Competence in Neisseria Gonorrhoeae

Cynthia D Long et al. Infect Immun. .
Free PMC article

Abstract

The gonococcal pilus is a major virulence factor that has well-established roles in mediating epithelial cell adherence and DNA transformation. Gonococci expressing four gonococcal pilin variants with distinct piliation properties under control of the lac regulatory system were grown in different levels of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). These pilin variants expressed various levels of pilin message and pilin protein in response to the level of IPTG in the growth medium. Moreover, posttranslational modifications of the variant pilin proteins were detected, including S-pilin production and glycosylation. The ratio of the modified and unmodified pilin forms did not substantially change with different levels of pilin expression, showing that these modifications are not linked to pilin expression levels. DNA transformation competence was also influenced by IPTG levels in the growth medium. Substantial increases in transformation competence over an isogenic, nonpiliated mutant were observed when limited amounts of three of the pilin variants were expressed. Immunoelectron microscopy showed that when limited amounts of pilin are expressed, pili are rare and do not explain the pilin-dependent transformation competence. This pilin-dependent transformation competence required prepilin processing, the outer membrane secretin PilQ, and the twitching-motility-regulating protein PilT. These requirements show that a fully functional pilus assembly apparatus is required for DNA uptake when limited pilin is produced. We conclude that the pilus assembly apparatus functions to import DNA into the bacterial cell in a pilin-dependent manner but that extended pili are not required for transformation competence.

Figures

FIG. 1.
FIG. 1.
Schematic of the regulatable pilE construct and characteristics of regulatable pilE variants. (A) Cartoon of regulatable constructs. Shaded boxes indicate genes and are drawn to scale. Thick arrows indicate operators and promoters. The curved bracket indicates the inserted regulatory sequences. The triangle indicates the site of the cat gene insertion used to create regulatable RM21. (B) Characteristics of the four FA1090 pilin variants. S-pilin expression was determined by densitometric analysis of Coomassie blue-stained gradient gels (20). ++++, high level of S-pilin produced; +, low level of S-pilin produced. Variant pilin residue 63 is glycosylated when a serine is present at this position.
FIG. 2.
FIG. 2.
Q-RT-PCR analysis of pilE transcript levels. (A) The strains were grown on solid medium with the indicated level of IPTG. Total RNA was isolated with and reverse transcribed with specific primers. Transcript copies were determined by FRET hybridization probe detection in a Roche LightCycler compared to known amounts of cloned DNA standards. Values are the percentage of the wild-type RM11.2 strain value. Shown is an average of three replicates. (B) The strains were grown in liquid medium with the indicated level of IPTG. Q-RT-PCR was performed as described for panel A using por primers and probes, and RNA samples were normalized to contain similar levels of por transcript levels (data not shown). pilE transcript numbers for fully induced and uninduced regulatable (reg) pilE variants were determined and expressed as the percentage of wild-type (recA6) pilE transcript level for each variant.
FIG. 3.
FIG. 3.
Western analysis of regulatable pilE variants. Gonococcal strains were grown in medium containing the indicated amounts of IPTG. Cultures were separated into a cell pellet and culture supernatant, and the supernatant proteins were precipitated with trichloroacetic acid (see Materials and Methods). Pilin and S-pilin were detected with the MAb 1E8/G8. wt, wild-type pilE expressed in a recA6 background. Strains used are as follows: RM11 (A); RM11.2 (B); RM11.6 (C); RM21 (D).
FIG. 4.
FIG. 4.
IPTG dose response of DNA transformation competence in regulatable and control strains. Strains were induced as indicated during incubation with plasmid DNA that confers resistance to nalidixic acid (Nalr) when recombined into the chromosome. Efficiency was expressed as the number of Nalr transformants per CFU. The mean and standard error of 8 to 10 identical experiments are shown. Each variant with its wild-type promoter is shown without IPTG. The recA6 version of RM21 was used because the pilE sequence in wild-type RM21 was found to be unstable (data not shown).
FIG. 5.
FIG. 5.
Electron micrographs of immuno-gold-labeled pili. Regulatable RM11.2 recA6 was grown on solid medium in the presence of 0, 0.005, or 0.5 mM IPTG and lifted directly onto Formvar-coated grids. Pili were detected with an antipeptide polyclonal antiserum raised against the hypervariable loop sequence of the RM11.2 pilin and detected with gold-labeled secondary antibody. Representative electron micrographs are shown for each growth condition except for 0 mM IPTG, where the single detected pilus is shown. IPTG concentrations were as follows: 0 (A); 0.005 (B); 0.2 (C); 0.5 mM IPTG (D).
FIG. 6.
FIG. 6.
Effect of the prepilin processing Gly-1Ser mutation on DNA transformation competence in a regulatable strain. (A) Western blot analysis of pilin. Gonococci were grown overnight in liquid in the presence of 0, 0.02, or 0.5 mM IPTG as indicated. Pilin and S-pilin were detected with MAb 1E8/G8. wt, RM11.2 recA6. (B) Transformation efficiency was determined as described in the legend for Fig. 4. Values shown are the means and standard errors of three to nine experiments.
FIG. 7.
FIG. 7.
DNA transformation competence of pilQ and pilT loss-of-function mutants in regulatable RM11 and RM11.2. Transformation efficiency was determined as described in the legend for Fig. 4. Values shown are the mean and standard error of three to seven experiments. (A) Competence of pilQ mutants and control strains. (B) Competence of pilT mutants and control strains. The asterisk indicates a statistically significant difference from the ΔpilE strain, as determined by Student's independent t test (P < 0.05).
FIG. 8.
FIG. 8.
Models for pilin-dependent DNA transformation competence. (A) Hypothetical model of the pilus assembly apparatus. This cartoon shows production of the pilin and pilin-like subunits in the cytoplasm and processing by the PilD peptidase (D), followed by polymerization at an unknown location and extrusion through the PilQ secretin (Q), which is associated with PilP (P). PilT is shown interacting with unknown proteins in the cytoplasmic membrane, but this has not been demonstrated. The presence of pilin-like proteins in the exposed fiber is speculative. Not all proteins known to be required for pilus assembly or DNA transformation competence are shown. (B) The residual pilus model. When limited pilin is expressed in the regulatable strain grown without IPTG, a small amount of pilin is produced. These pilin subunits are transported into the periplasm, where the PilD peptidase cleaves the signal sequence and the mature pilin interacts with pilin-like proteins and other members of the assembly apparatus to produce a residual pilus. While the residual pilus is drawn as not being exposed on the cell surface, the residual pili would be predicted to be of different lengths with some protruding from the cell surface but too short to be detected frequently by negatively stained electron microscopy. (C) The pilus-independent transport model. In this model the pilus assembly apparatus assumes a transport-competent form which is dependent on pilin but does not require a polymerized pilus to be within the assembly apparatus that transports DNA. In this model, pilus assembly and DNA transport machinery are separate, but each requires the presence of pilin.

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