Regulation of pga Operon Expression and Bioﬁlm Formation in Actinobacillus pleuropneumoniae by (cid:1) E and H-NS (cid:1)

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- (cid:1) -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 (cid:2) E . Transcription proﬁling of A. pleuropneumoniae rseA and hns mutants revealed that both (cid:2) E and H-NS independently regulate expression of the pga operon. Transcription of the pga operon is initiated from a (cid:2) E promoter site in the absence of H-NS, and upregulation of (cid:2) 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 (cid:2) E indicates that bioﬁlm formation is part of the extracytoplasmic stress response in A. pleuropneumoniae . The presence of gene-speciﬁc cDNA was conﬁrmed by PCR using primers pgaA_GSP2 and pgaA_GSP3. A homopolymeric tail was then added to the 3 (cid:7) end of the cDNA using terminal deoxynucleotidyltransferase (TdT) and dCTP. Initially, this tailing reaction was performed at 37°C for 10 min, according to the manufacturer’s instructions. However, attempts to PCR amplify gene-speciﬁc product from these samples using the abridged anchor primer (AAP) and the nested gene-speciﬁc primer pgaA_GSP2 failed. Subsequently, the tailing reac- tion was performed on ice for 60 min, as recommended for samples with strong secondary structure in the 3 (cid:7) end of the cDNA. PCR ampliﬁcation of these samples yielded speciﬁc product of approximately 800 bp for both 5B3 and 19B10. These PCR products were sequenced on both strands using pgaA_GSP4 and the nested anchor primer AUAP, and the transcriptional start site was identiﬁed. Gel shift assay. A fragment of DNA containing 1,150 bases upstream, as well as the ﬁrst 323 bases of pgaA , was PCR ampliﬁed using pgaEMSA_F and pgaEMSA_R. The PCR product was digested with AanI (Fermentas Life Sciences) and puriﬁed using a QIAquick PCR puriﬁcation kit (Qiagen). The DNA fragments (0.5 (cid:3) g) were combined with different concentrations of puriﬁed E. coli H-NS (kindly provided by M. Goldberg, Birmingham, United Kingdom) for 20 min at 37°C, as described by Ono et al. (41). The samples were subjected to electrophoresis through 1.5% agarose at 75 V for 5.5 h at 37°C. The gel was then stained with ethidium bromide to reveal the DNA- protein complexes.