Genetic analysis of the requirement for flp-2, tadV, and rcpB in Actinobacillus actinomycetemcomitans biofilm formation

Abstract

The tad locus of Actinobacillus actinomycetemcomitans encodes a molecular transport system required for tenacious, nonspecific adherence to surfaces and formation of extremely strong biofilms. This locus is dedicated to the biogenesis of Flp pili, which are required for colonization and virulence. We have previously shown that 11 of the 14 tad locus genes are required for adherence and Flp pilus production. Here, we present genetic and phylogenetic analyses of flp-2, tadV, and rcpB genes in biofilm formation. We show that tadV, predicted to encode prepilin peptidase, is required for adherence. In contrast, targeted insertional inactivation of flp-2, a gene closely related to the prepillin gene flp-1, did not abrogate biofilm formation. Expression studies did not detect Flp2-T7 protein under standard laboratory conditions. We present phylogenetic data showing that there is no significant evidence for natural selection in the available flp-2 sequences from A. actinomycetemcomitans, suggesting that flp-2 does not play a significant role in the biology of this organism. Mutants with insertions at the 3' end of rcpB formed biofilms equivalent to wild-type A. actinomycetemcomitans. Surprisingly, 5' end chromosomal insertion mutants in rcpB were obtained only when a wild-type copy of the rcpB gene was provided in trans or when the Tad secretion system was inactivated. Together, our results strongly suggest that A. actinomycetemcomitans rcpB is essential in the context of a functional tad locus. These data show three different phenotypes for the three genes.

FIG. 1.

Arrangement of the tad genes in A. actinomycetemcomitans. The location of the EZ::TN<KAN-2> insertions in specific plasmids and mutant strains (in parentheses) is shown by vertical arrows.

FIG. 2.

Phenotypes and genetic complementation of tadV mutants. tadV mutant strains Aa3074 (A to C) and Aa3072 (D to F), containing the vector pJAK17, and tadV mutant strains Aa3074 (G to J) and Aa3072 (J to L), containing pJAK17 with cloned tadV ORF from strain CU1000N, are shown (bar, 1.0 mm). Images from stereomicroscopy of 3-day-old colonies are shown in panels A, D, G, and J. Images from light microscopy of bacterial cells at magnification of ×100 are shown in panels B, E, H, and K. Images from TEM of negatively stained cells are shown in panels C, F, I, and L (bar, 0.2 μm).

FIG. 3.

Phenotypes of flp2 mutants. Wild-type rough strain DF2200N (A to C) and flp-2 mutant strain Aa3237 (D to F) as described in the legend of Fig. 2. In panels C and F, the bars are 0.2 μm and 0.1 μm, respectively.

FIG. 4.

Adherence by flp-2 and tadV mutants. Shown are the adherent properties of wild-type A. actinomycetemcomitans strains CU1000N and DF2200N, the flp-2 mutant strain, the tadV mutant strains, and the complemented tadV mutant strains. Adherence was measured in the wells of 96-well microtiter plates. The amount of crystal violet (CV) released is proportional to biofilm formation (25).

FIG. 5.

Sequence comparison of the upstream regions of flp-1 and flp-2 from A. actinomycetemcomitans strains CU1000N, DF2200N, and IDH781N. (A) The underlined sequence represents the ribosome binding site or Shine-Dalgarno of A. actinomycetemcomitans (http://www.oralgen.lanl.gov/oralgen/bacteria/aact/). (B) Analysis of flp-2-T7 and flp-1-T7 expression. Whole-cell extracts of E. coli strains SK108, SK363, BP039 were prepared and analyzed by α-T7 immunoblotting. Cultures were induced with 1 mM IPTG, as discussed in Materials and Methods, for the indicated amount of time. (C) Analysis of flp-2_SD-T7 and flp-2-T7 expression. Whole-cell extracts of E. coli strains BP039 and BP038 were immunoblotted using the α-T7 antibody. Cultures were induced with 1 mM IPTG for the indicated amount of time. The black arrow indicates the position of Flp2-T7. The predicted molecular sizes for immature flp-1 and flp-2 are ∼8 kDa and ∼8.3 kDa, respectively.

FIG. 6.

Evolution of flp-1 and flp-2 in the γ-proteobacteria. (A) Shown is the strict consensus of the most parsimonious trees found using a concatenated alignment of the amino acid and nucleotide sequences of flp genes. Support for branches is shown on each branch as Bremer decay index/bootstrap percentages. Bremer support values are preceded with the letter “d.” Nodes that did not exist in the bootstrap tree are labeled N. Probable duplication events are indicated with black ovals. Note the duplication that separates flp-1 and flp-2 clades. (B and C) Shown are plots of the normalized value of dN-dS (y axis) along the nucleotide alignment/sequence length (x axis) as computed by the SLAC program (29, 30) for the flp-1 clade and the flp-2 clade, respectively. In this analysis, natural selection was calculated as a difference (dN=dS). The flp-1 analysis (B) also includes the three flp gene sequences from H. ducreyi (24). Positive deflections indicate selection pressure to change, and negative deflections represent selection pressure not to change. Gray lines indicate values with nonsignificant P values (>0.1), and black lines are values with significant P values (<0.1). Codons with significant P values and those in the Flp prepilin motif are designated with their single-letter amino acid codes. The numbers in both panels refer to the position in flp-1 and flp-2, respectively, of A. actinomycetemcomitans HK1651. Asterisks indicate residues with statistically significant dN-dS values when the analysis was restricted to either flp-1 or flp-2 genes from A. actinomycetemcomitans only. Note the lack of asterisks in panel C as no selection was demonstrated for flp-2 genes from A. actinomycetemcomitans. Daggers in panel C indicate residues in flp-2 genes from the Pasteurellaceae shown to have statistically significant selection values even with the exclusion of all A. actinomycetemcomitans flp-2 genes except for flp-2 from HK1651.

FIG. 7.

Phenotypes of 3′ end rcpB mutants. Shown are images from mutant strain Aa3245 (A to D), mutant strain Aa3235 (E to G), mutant strain Aa3250 (H to J), mutant strain Aa3234 (K to M), mutant strain Aa3229 (N to P), and mutant strain Aa3232 (Q to S). Note the emergence of spontaneous, smooth mutants (D). These figures were done as described in the legend of Fig. 2. Images from TEM of negatively stained cells have bars of 0.1 μm (C and G) and 0.2 μm (J, M, P and S).

FIG. 8.

Adherence by 3′-end rcpB mutants. Adherent properties of wild-type A. actinomycetemcomitans strains DF2200N, the tadZ mutant strain, and the rcpB mutant strains. Adherence was measured in the wells of 96-well microtiter plates, as described in the legend of Fig. 4.