Regulation of alginate biosynthesis in Pseudomonas syringae pv. syringae

Abstract

Both Pseudomonas aeruginosa and the phytopathogen P. syringae produce the exopolysaccharide alginate. However, the environmental signals that trigger alginate gene expression in P. syringae are different from those in P. aeruginosa with copper being a major signal in P. syringae. In P. aeruginosa, the alternate sigma factor encoded by algT (sigma22) and the response regulator AlgR1 are required for transcription of algD, a gene which encodes a key enzyme in the alginate biosynthetic pathway. In the present study, we cloned and characterized the gene encoding AlgR1 from P. syringae. The deduced amino acid sequence of AlgR1 from P. syringae showed 86% identity to its P. aeruginosa counterpart. Sequence analysis of the region flanking algR1 in P. syringae revealed the presence of argH, algZ, and hemC in an arrangement virtually identical to that reported in P. aeruginosa. An algR1 mutant, P. syringae FF5.32, was defective in alginate production but could be complemented when algR1 was expressed in trans. The algD promoter region in P. syringae (PsalgD) was also characterized and shown to diverge significantly from the algD promoter in P. aeruginosa. Unlike P. aeruginosa, algR1 was not required for the transcription of algD in P. syringae, and PsalgD lacked the consensus sequence recognized by AlgR1. However, both the algD and algR1 upstream regions in P. syringae contained the consensus sequence recognized by sigma22, suggesting that algT is required for transcription of both genes.

FIG. 1

(A) Physical and functional map of the alginate structural gene cluster in Pseudomonas syringae pv. syringae FF5. The arrows within each open reading frame indicate the direction of translation. The locations of the primers (P1 and P2) used to amplify the algD promoter region are indicated. Abbreviations: F, algF; 44, alg44. (B) Expanded view of the region amplified with primers P1 and P2. The location and orientation of the coding region for algD are shown (horizontal arrow). The black boxes flanking the EcoRI site indicate the consensus sequence recognized by AlgT (ς²²). The location and orientation of the algD::uidA transcriptional fusions are indicated; GUS activity is shown in the column adjacent to each construct. Values followed by the same letter were not significantly different (P = 0.01). Abbreviations: E, EcoRI; H, HindIII; V, EcoRV.

FIG. 2

Constructs used for the cloning and sequencing of algR1 from P. syringae pv. syringae FF5. pAP32.1 is a subclone containing Tn5 (shaded region) and flanking DNA from P. syringae pv. syringae FF5.32 (hatched region). The HindIII-EcoRI fragment in pAP32.1 was used as a probe for algR1 in the current study. pMF6.1 and pMF6.2 are subclones derived from pMF6, a cosmid which complemented FF5.32 for alginate production. The 2.0-kb PstI fragment in pMF6.2 was sequenced on both strands and shown to contain an intact copy of algR1. Abbreviations: B, BamHI; E, EcoRI; H, HindIII; P, PstI.

FIG. 3

Alignment of the algD promoter sequences from P. syringae pv. syringae FF5 (Ps algD) and P. aeruginosa (Pa algD). The P. aeruginosa algD promoter was previously reported (24, 39); the nucleotides for this sequence are shown on the left, with +1 (asterisk) corresponding to the transcriptional start site. Nucleotides for the P. syringae pv. syringae algD promoter are shown on the right. The EcoRI site in the P. syringae sequence corresponds to the left border of EcoRI fragment 5 in Fig. 1A. Gaps (––) were used to maximize the alignment, and identical bases are shaded. The AlgR1 binding sites (ABS) in the P. aeruginosa algD promoter are shown in bold and double-underlined. The ς²² recognition sequence in both species is indicated in bold and single-underlined. The algD translational start site and coding region are shown in bold (algD–→).