Regulation of pga operon expression and biofilm formation in Actinobacillus pleuropneumoniae by sigmaE and H-NS

Abstract

Clinical isolates of the porcine pathogen Actinobacillus pleuropneumoniae often form adherent colonies on agar plates due to expression of an operon, pgaABCD, encoding a poly-beta-1,6-N-acetyl-D-glucosamine (PGA) extracellular matrix. The adherent colony phenotype, which correlates with the ability to form biofilms on the surfaces of polystyrene plates, is lost following serial passage in broth culture, and repeated passage of the nonadherent variants on solid media does not result in reversion to the adherent colony phenotype. In order to investigate the regulation of PGA expression and biofilm formation in A. pleuropneumoniae, we screened a bank of transposon mutants of the nonadherent serovar 1 strain S4074(T) and identified mutations in two genes, rseA and hns, which resulted in the formation of the adherent colony phenotype. In other bacteria, including the Enterobacteriaceae, H-NS acts as a global gene regulator, and RseA is a negative regulator of the extracytoplasmic stress response sigma factor sigma(E). Transcription profiling of A. pleuropneumoniae rseA and hns mutants revealed that both sigma(E) and H-NS independently regulate expression of the pga operon. Transcription of the pga operon is initiated from a sigma(E) promoter site in the absence of H-NS, and upregulation of sigma(E) is sufficient to displace H-NS, allowing transcription to proceed. In A. pleuropneumoniae, H-NS does not act as a global gene regulator but rather specifically regulates biofilm formation via repression of the pga operon. Positive regulation of the pga operon by sigma(E) indicates that biofilm formation is part of the extracytoplasmic stress response in A. pleuropneumoniae.

FIG. 1.

Alignment of A. pleuropneumoniae (App) H-NS and E. coli H-NS protein sequences. Asterisks show identical amino acids, and dots show conservative substitutions. The locations and nucleotide sequences of the 5′-end (mutants 5B3 and 24D3) and 3′-end (mutant 3A3) Tn10 insertion sites are shown above the A. pleuropneumoniae H-NS, as is the core DNA-binding domain (19).

FIG. 2.

Complementation of E. coli MG1655Δhns with pMCAphns. Expression of cloned A. pleuropneumoniae hns (pMCAphns) in an E. coli hns mutant (MG1655Δhns) resulted in restoration of motility (A) and repression of mucoidy (B), similar to those seen in the wild type (MG1665). For the motility assay, bacteria were plated on 0.4% agar (Bacto). For detection of mucoidy, strains were plated on LB agar.

FIG. 3.

Expression levels of pgaA, hns, and rpoE, as determined by qRT-PCR. The relative levels of pgaA, hns, and rpoE expression in 19B10 (rseA::Km) and 5B3 (hns::Km) compared to those of the WT (S4074^T) samples showed congruency between the results from the microarray and those from the real-time RT-PCR. The adherent clinical isolate (M2000) showed increased expression of rpoE and pgaA, whereas hns expression was only slightly lower, compared to those levels of S4074^T. Results were obtained using three independent biological samples and four technical replicates. Standard errors of the means are indicated.

FIG. 4.

Determination of in vitro biofilm formation by strains of A. pleuropneumoniae. The absorbance at 600 nm was measured, following ethanol solubilization of crystal violet-stained adherent bacterial cells grown for 6 h in wells of polystyrene plates in BHI-NAD broth with and without addition of dispersin B (DspB), an enzyme that specifically degrades the PGA matrix. The double mutants (rseA::Km ΔpgaC and hns::Km ΔpgaC) were not tested in the presence of dispersin B, as there was no detectable binding in the absence of dispersin B. Bars represent the mean results from two independent experiments performed in quadruplicate. Error bars represent the standard deviations.

FIG. 5.

Analysis of the sequence upstream of pgaA. (A) Plot of GC content of the 1,473-bp region amplified for use in the gel shift assay; highest and lowest percent GC are indicated to the right, the average GC content of the A. pleuropneumoniae genome is 41%, and the average GC content of the 750 bases upstream of pgaA is 27%. The locations of the σ^E promoter site (gray arrowhead) and three potential high-affinity H-NS binding sites (black arrowheads) are indicated. The AanI restriction map for this region is shown at the bottom. (B) Competitive gel shift assay with H-NS. AanI-digested DNA (0.5 μg) was incubated with a range of concentrations of H-NS for 20 min at 37°C. The fragments were subjected to electrophoresis through 1.5% agarose at 75 V for 5.5 h at 37°C. The 436-bp fragment containing the three predicted high-affinity H-NS binding sites shows the greatest mobility shift, despite the higher concentration and lower AT content compared to those of the 189-bp fragment. The gel shift assay was also run at 25°C, with similar results (data not shown).