The Aeromonas hydrophila wb*O34 gene cluster: genetics and temperature regulation

Abstract

The Aeromonas hydrophila wb*(O34) gene cluster of strain AH-3 (serotype O34) was cloned and sequenced. This cluster contains genes necessary for the production of O34-antigen lipopolysaccharide (LPS) in A. hydrophila. We determined, using either mutation or sequence homology, roles for the majority of genes in the cluster by using the chemical O34-antigen LPS structure obtained for strain AH-3. The O34-antigen LPS export system has been shown to be a Wzy-dependent pathway typical of heteropolysaccharide pathways. Furthermore, the production of A. hydrophila O34-antigen LPS in Escherichia coli K-12 strains is dependent on incorporation of the Gne enzyme (UDP-N-acetylgalactosamine 4-epimerase) necessary for the formation of UDP-galactosamine in these strains. By using rapid amplification of cDNA ends we were able to identify a transcription start site upstream of the terminal wzz gene, which showed differential transcription depending on the growth temperature of the strain. The Wzz protein is able to regulate the O34-antigen LPS chain length. The differential expression of this protein at different temperatures, which was substantially greater at 20 degrees C than at 37 degrees C, explains the previously observed differential production of O34-antigen LPS and its correlation with the virulence of A. hydrophila serotype O34 strains.

FIG. 1.

Chemical structures of the O34-antigen LPS (A) and the LPS core (B) from A. hydrophila strain AH-3 (27, 28). The O34-antigen LPS is linked to the Gal residue (indicated by italics) of the LPS core (28).

FIG. 2.

(A) LPS from A. hydrophila AH-3 (wild type) (lane 1), mutants AH-901, AH-902, AH-405ΔwecA, and AH-405Δwzy (lanes 2, 3, 4, and 5, respectively), mutants AH-901 and AH-902 complemented with plasmids COS-O34 and AH-405ΔwecA, respectively (lanes 6 and 7, respectively), and mutant AH-405Δwzy complemented with pBAD33-wecA and pBAD33-wzy (lanes 8 and 9, respectively). LPS was extracted and analyzed by 12% SDS-PAGE as described by Darveau and Hancock (14). (B) SDS-Tricine-PAGE gels for LPS core of mesophilic Aeromonas strains. The lanes contained the same strains as the lanes in panel A. (C) LPS from A. hydrophila AH-3 (wild type) (lane 1), E. coli K-12 strain CLM4 (lane 2), strain CLM4 with the COS-O34 plasmid (lane 3), and strain CLM4 with the COS-O34 and pGEMT-Gne plasmids (lane 4). All the strains were grown at 20°C, and strains with pBAD33 plasmids were grown under induced conditions.

FIG. 3.

(A) Genetic organization of the A. hydrophila AH-3 wb*_O34 region from the COS-O34 plasmid. The transcription direction and stops (lollipop symbols) are indicated. The positions of mini-Tn5 (AH-901 and AH-902) mutations are indicated. (B) Comparison of A. hydrophila PPD134/91 (wb*_O18 cluster) with A. hydrophila AH-3 (wb*_O34 cluster) from ORF1 to ORF17. aa, amino acids.

FIG. 4.

Electrophoresis of LPS from A. hydrophila AH-3 (wild type) (lanes 1 and 4), mutant AH-405Δwzz (lanes 2 and 5), and mutant AH-405Δwzz complemented with plasmid pBAD33-wzz (lanes 3 and 6). The strains in lanes 1, 2, and 3 were grown at 20°C, while the strains in lanes 4, 5, and 6 were grown at 37°C. The arrow in lane 4 indicates the small amount of large O34-antigen LPS repeating units observed in the wild-type strain grown at 37°C. The strain with the pBAD33-wzz plasmid was grown under induced conditions.

FIG. 5.

(A) RT-PCR DNA fragments obtained from the rmlB-rmlC region and wzz internal region of A. hydrophila AH-3 genomic DNA isolated when the strain was grown at 20 and 37°C. (B) β-Galactosidase activities of E. coli DH5α with the pRS-PWZZA (wzz_AH-3 promoter-lacZ fusion) and pRS-PWZZP (wzz_PPD134/91 promoter-lacZ fusion) plasmids and of A. hydrophila AH-405 with the pACYC-PWZZA (wzz_AH-3 promoter-lacZ fusion) and pACYC-PWZZP (wzz_PPD134/91 promoter-lacZ fusion) plasmids were determined for cultures grown at 20°C (open bars) and 37°C (shaded bars) (19, 36). As a control we also determined the activities of E. coli DH5α with the pRS550 plasmid vector and A. hydrophila AH-405 with pACYC-lacZ at both growth temperatures.

FIG. 6.

Amplification of the A. hydrophila AH-3 wzz cDNA 5′ end performed using the 5′ RACE system (version 2.0; Invitrogen). (A) RACE scheme showing the primers and products described in Materials and Methods. (B) Amplicon obtained by nested PCR using primers AUAP and A3-ROLF. Lane 1, primary PCR template; lane 2, PCR negative control; lane 3, molecular weight standard (Invitrogen). (C) Location of the transcriptional start site (GGGGT) for A. hydrophila AH-3 wzz (indicated by bold type). RBS, ribosome binding site.