An analysis of nucleotide sequences of two types of ligninase cDNAs isolated from the basidiomycete Phanerochaete chrysosporium, designated CLG4 and CLG5, are presented here. The amino acid sequences of the corresponding ligninase proteins, designated LG4 and LG5, respectively, have been deduced from the cDNA sequences. Mature ligninases LG4 and LG5 are preceded by leader sequences containing 28 and 27 amino acids (aa), respectively, and each contains 344 aa residues. The estimated Mrs of mature LG4 and LG5 are 36,540 and 36,607, respectively. Potential N-glycosylation site(s) with the general sequence Asn-X-Thr/Ser are found in both LG4 and LG5. Nucleotide sequence homology between the coding region of CLG4 and CLG5 is 71.5%, whereas the amino acid sequence homology between the two ligninases is 68.5%. The codon usage of ligninases is extremely biased in favor of codons rich in cytosine and guanine. Amino acid sequences of two tryptic peptides of ligninase H8 have exactly matching sequences in ligninase LG5. Also, the sequences of the oligodeoxynucleotide probes, which correspond to the sequences in the tryptic peptides of ligninase H8 and which were used in isolating the ligninase clones from the cDNA library, have exactly matching sequences in CLG5. The experimentally determined N-terminal sequence of purified ligninase H8 is found in the deduced N-terminal amino acid sequence of LG5. These results suggest that CLG5 encodes ligninase H8 and that CLG4 represents a related but different ligninase gene.

Ganoderma lucidum, a white rot basidiomycete widely distributed worldwide, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). Laccase levels observed in high-nitrogen (HN; 24 mM N) shaken cultures were much greater than those seen in low-nitrogen (2.4 mM N), malt extract, or wood-grown cultures and those reported for most other white rot fungi to date. Laccase production was readily seen in cultures grown with pine or poplar (100-mesh-size ground wood) as the sole carbon and energy source. Cultures containing both pine and poplar showed 5- to 10-fold-higher levels of laccase than cultures containing pine or poplar alone. Since syringyl units are structural components important in poplar lignin and other hardwoods but much less so in pine lignin and other softwoods, pine cultures were supplemented with syringic acid, and this resulted in laccase levels comparable to those seen in pine-plus-poplar cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of concentrated extracellular culture fluid from HN cultures showed two laccase activity bands (M(r) of 40,000 and 66, 000), whereas isoelectric focusing revealed five major laccase activity bands with estimated pIs of 3.0, 4.25, 4.5, 4.8, and 5.1. Low levels of MnP activity ( approximately 100 U/liter) were detected in poplar-grown cultures but not in cultures grown with pine, with pine plus syringic acid, or in HN medium. No LiP activity was seen in any of the media tested; however, probing the genomic DNA with the LiP cDNA (CLG4) from the white rot fungus Phanerochaete chrysosporium showed distinct hybridization bands suggesting the presence of lip-like sequences in G. lucidum.

The complete amino acid sequence of the human type IV collagenase preproenzyme was determined from cDNA and genomic clones. Primer extension and S1 nuclease analyses as well as nucleotide sequencing of a genomic clone indicate that the first exon has two closely spaced initiation sites for transcription and codes for 290 and 280 nt of a 5' untranslated region and a 29-residue signal peptide. The gene (CLG4) was localized to 16q21 using somatic cell hybrids and in situ hybridization.

Two methods allowing the analysis of expression of specific lignin peroxidase (LPO) genes from white rot fungi are presented. In the first method, degenerate oligonucleotide primers derived from amino acid sequence motifs held in common among all members of the LPO gene family are used to prime the polymerase chain reaction (PCR) amplification of LPO-related nucleotide sequences from cDNA prepared by using RNA from ligninolytic cultures. The PCR products are cloned and analyzed by restriction cleavage and DNA sequencing. This method was applied to the analysis of transcripts from carbon-limited cultures of Phanerochaete chrysosporium BKM-F-1767, revealing two major classes of PCR products. One class showed DNA sequences with a high degree of similarity to the previously described CLG4 cDNA sequence (H. A. De Boer, Y. Zhang, C. Collins, and C. A. Reddy, Gene 60:93-102, 1987), whereas the other harbored DNA sequences with similarities to the L18 cDNA sequence previously described for P. chrysosporium OGC101 (T. G. Ritch, Jr., V. J. Nipper, L. Akileswaran, A. J. Smith, D. G. Pribnow, and M. H. Gold, Gene 107:119-126, 1991). The second method is based on nuclease protection assays involving isoenzyme-specific RNA probes. By using this method, the L18-related gene of P. chrysosporium BKM-F-1767 was found to be expressed under conditions of carbon and of nitrogen limitation, although the transcript levels were found to be higher in carbon-limited cultures. Furthermore, it was found that omission of veratryl alcohol addition to the culture did not affect the levels of the L18-related transcripts in carbon-limited cultures.

A Phanerochaete chrysosporium BKMF1767 genomic library, constructed in the BamHI site of vector YRp12, was screened with the lignin peroxidase(LIP)-encoding cDNAs, CLG4 and CLG5, that have been shown to encode LIP2 (previously H2) and LIP6 (previously H10), respectively. Six distinct LIP genomic clones, designated pGLG1, pGLG2, pGLG3, pGLG4, pGLG5, and pGLG6, were isolated in this study. Probe CLG4 hybridized only to pGLG1. Probe CLG5 gave intense hybridization to pGLG2 and weaker hybridization to clones pGLG3 through pGLG6, but showed little or no hybridization to pGLG1. These results, in agreement with previous biochemical results, indicate the existence of LIP gene subfamilies. The limits and transcriptional orientation of the LIP gene in each clone were determined. The sequence data showed that pGLG2 contains the LIP6 gene, which encodes a protein identical in amino acid (aa) composition to that encoded by CLG5. It contains a leader sequence of 27 aa and a mature protein of 344 aa (Mr 36,607). Archetypal TATA-box-like and CAAT-box-like sequences in the 5'-noncoding region are located 51 and 97 nt upstream from the cDNA start point, respectively. S1 nuclease analysis of the 5' region of LIP6 revealed two transcription start points 8 nt apart downstream from the TATA box. Comparison of the sequence of LIP6 with its corresponding cDNA CLG5 showed that the gene contains nine small introns which range in size from 50 to 62 bp. These introns contained consensus splice junction sequences similar to those reported in other fungal and yeast introns.

Probiotics represent an alternative to chemotherapy and vaccination to control fish diseases, including lactococcosis caused by Lactococcus garvieae. The aims of this study were (i) to determine the in vitro probiotic properties of three bacteriocinogenic Lactococcus lactis subsp. cremoris of aquatic origin, (ii) to evaluate in vivo the ability of L. cremoris WA2-67 to protect rainbow trout (Oncorhynchus mykiss, Walbaum) against infection by L. garvieae, and (iii) to demonstrate the role of nisin Z (NisZ) production as an anti-infective mechanism. The three L. cremoris strains survived in freshwater at 18 °C for 7 days, withstood exposure to pH 3.0 and 10 % (v/v) rainbow trout bile, and showed different cell surface hydrophobicity (37.93-58.52 %). The wild-type NisZ-producer L. cremoris WA2-67 and its non-bacteriocinogenic mutant L. cremoris WA2-67 ∆nisZ were administered orally (10(6) CFU/g) to rainbow trout for 21 days and, subsequently, fish were challenged with L. garvieae CLG4 by the cohabitation method. The fish fed with the bacteriocinogenic strain L. cremoris WA2-67 reduced significantly (p < 0.01) the mortality (20 %) compared to the fish treated with its non-bacteriocinogenic knockout isogenic mutant (50 %) and the control (72.5 %). We demonstrated the effectiveness of L. cremoris WA2-67 to protect rainbow trout against infection with the invasive pathogen L. garvieae and the relevance of NisZ production as an anti-infective mechanism. This is the first report demonstrating the effective in vivo role of LAB bacteriocin (NisZ) production as a mechanism to protect fish against bacterial infection. Our results suggest that the wild-type NisZ-producer strain L. cremoris WA2-67 could be used in fish farming to prevent lactococcosis in rainbow trout.