Organization and differential regulation of a cluster of lignin peroxidase genes of Phanerochaete chrysosporium

Abstract

The lignin peroxidases of Phanerochaete chrysosporium are encoded by a minimum of 10 closely related genes. Physical and genetic mapping of a cluster of eight lip genes revealed six genes occurring in pairs and transcriptionally convergent, suggesting that portions of the lip family arose by gene duplication events. The completed sequence of lipG and lipJ, together with previously published sequences, allowed phylogenetic and intron/exon classifications, indicating two main branches within the lip family. Competitive reverse transcription-PCR was used to assess lip transcript levels in both carbon- and nitrogen-limited media. Transcript patterns showed differential regulation of lip genes in response to medium composition. No apparent correlation was observed between genomic organization and transcript levels. Both constitutive and upregulated transcripts, structurally unrelated to peroxidases, were identified within the lip cluster.

FIG. 1

Classification of lip genes by intron/exon structure. Introns and exons are represented by white and black bars, respectively. The positions of conserved amino acid residues coordinating the heme (49) the relative positions of signal cleavage sites and are indicated.

FIG. 2

Cladistic analysis of deduced amino acid sequences. The underlying scale shows the number of substitutions between sequences. Analysis was done with the Jotun Hein (24) algorithm on DNASTAR software.

FIG. 3

Transcript levels and genomic organization of the lip genes. Genomic organization is shown under the graph. Thickened arrows indicate transcriptional orientation. Physical distances are in kilobases, and genetic distances are in percent recombination. Two unlinked genes, lipD and lipF, are detached to the right. Transcript levels were determined by competitive RT-PCR and are given in picograms of cDNA.

FIG. 4

Competitive PCRs comparing four lip transcripts in samples harvested from nitrogen- or carbon-limited conditions. Transcript levels were estimated by determining the equivalence points (arrows) with competitive templates (46). The amount of plasmid competitive template added to each PCR is indicated below the gels and expressed in picograms. Ethidium bromide-stained gels were photographed with a Foto/Analyst Visionary system (Fotodyne, Hartland, Wis.) and scanned with a Microtek ScanMaker III and Adobe Photoshop 3.0.

FIG. 5

Southern blots of XL-PCR-amplified intergenic regions probed with ³²P-labeled cDNA from log-phase B3, C-limited B3, and N-limited B3 media.