Mu transposition complex mutagenesis in Lactococcus lactis--identification of genes affecting nisin production.

AIMS: This paper describes optimization of electrotransformation of Mu transposition complexes into Lactococcus lactis cells and identification of genes affecting nisin production. METHODS AND RESULTS: The highest transformation efficiency, 1.1 x 10(2) transformants microg(-1) of input transposon DNA, was achieved when cells were grown to an OD(600) of 0.5 in the presence of 1.5% of glycine and treated with 20 microg ml(-1) ampicillin for 60 min. Three insertions affecting nisin production, which were identified at nisB, fhuR, and rpiA genes, were screened from a library of approximately 2000 erythromycin-resistant transformants using a nisin bioassay method. NisB is part of the nisin biosynthetic machinery, explaining the loss of nisin production in nisB mutant. FhuR is a transcription regulator involved in sulphur acquisition. Inactivation of fhuR presumably results in a low cellular cystein level, which affects nisin biosynthesis that involves utilization of cystein. RpiA is involved in pentose phosphate pathway and carbon fixation. The rpiA mutant showed reduction in nisin production and slow growth rate. CONCLUSIONS: The results showed that Mu transposition complex mutagenesis can be used to identify genes in L. lactis. Three genes involved in nisin production were identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Expanding the Mu transposition-based mutagenesis to Lactococci adds a new tool for studies of industrially important bacteria.

Functional analysis of six ORFs from Saccharomyces cerevisiae chromosome IV: two-spored asci produced by disruptant of YDR027c and strain-dependent DNA heterogeneity around YDR036c.

Six S. cerevisiae FY1679 heterozygous deletion mutants were made by replacing six open reading frames (ORFs) of the chromosome IV right arm with kanMX4 selection marker. Haploid and homozygous diploid deletion mutants were obtained from sporulation, dissection and mating experiments. No essential genes were found. The basic phenotypic analysis showed that the haploid and homozygous deletants for the ORF YDR027c (LUV1, VSP54 or RKI1) grew slowly. The diploid homozygous deletants for this ORF had a low frequency of sporulation. They produced asci with no more than one or two haploid spores and the majority of these spores formed were not viable. The deletion of the other ORFs, YDR022c (CIS1), YDR030c (RAD28), YDR032c (PST2), YDR033w (MRH1) and YDR036c, did not change the phenotypes tested in strain FY1679 or the first four ORFs in strain CEN.PK2. This work showed some differences in the DNA sequences between FY1679 and CEN.PK2: the regions immediately 1 kb upstream from YDR036c in these two strains are too different to hybridize properly, preventing deletion of YDR036c in the CEN.PK2 background by recombination with a disruption cassette designed for FY1679. In addition, there are different sets of transposable elements on the other side of the ORF, the differences starting at about 3.5 kb downstream from YDR036c.

Expression and activity of matrix metalloproteinase-2 and -9 in experimental granulation tissue.

The restoration of functional connective tissue is a major goal of the wound healing process which is probably affected by matrix-modifying enzymes. To evaluate the spatial and temporal expression of matrix metalloproteinases (MMP) MMP-2 and MMP-9 and to study the regulation of MMP-2 in wound healing, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation for up to 3 months. MMP-2 mRNA expression was seen throughout the experiment and it was highest after 2 months. MMP-9 gene expression was low between days 8-21 and increased after 4 weeks of granulation tissue formation. Membrane-type 1 MMP (MT1-MMP) mRNA was upregulated early and tissue inhibitor 2 of MMP (TIMP-2) mRNA later during wound healing. In in situ hybridization the expression of MMP-2 mRNA was seen mostly in fibroblast-like cells and MMP-9 mRNA in macrophage-like cells. MMP-9 immunoreactivity was detected in the polymorphonuclear leukocytes and macrophage-like cells on days 3-8. MMP-9 proteolytic activity was observed only on days 3-8. The active form of the MMP-2 increased up to day 14, whereafter it remained at a constant level, whereas latent MMP-2 did not show any apparent changes during the experimental period. We conclude that MMP-2 is important during the prolonged remodelling phase, whereas the gelatinolytic activity of MMP-9 was demonstrated only in early wound healing, and the MMP-9 gene is upregulated when the granulation tissue matures.

A polyphasic study on the taxonomic position of industrial sour dough yeasts.

The sour dough bread making process is extensively used to produce wholesome palatable rye bread. The process is traditionally done using a back-slopping procedure. Traditional sour doughs in Finland comprise of lactic acid bacteria and yeasts. The yeasts present in these doughs have been enriched in the doughs due to their metabolic activities, e.g. acid tolerance. We characterized the yeasts in five major sour bread bakeries in Finland. We found that most of the commercial sour doughs contained yeasts which were similar to Candida milleri on the basis of 18S rDNA and EF-3 PCR-RFLP patterns and metabolic activities. Some of the bakery yeasts exhibited extensive karyotype polymorphism. The minimum growth temperature was 8 degrees C for C. milleri and also for most of sour dough yeasts.

Cloning of cDNA for rat pro alpha1(V) collagen mRNA. Expression patterns of type I, type III and type V collagen genes in experimental granulation tissue.

A cDNA clone for rat pro alpha1(V) collagen mRNA was constructed using PCR amplification, with primers based on human and hamster COL5A1 gene sequences. The clone pRCVA1 is 560 nucleotides long and it encodes for the carboxy propeptide of type V procollagen. Homology shared with type I collagen sequence was 64%, with type II collagen 65% and with type III collagen 61%. To evaluate the spatial and temporal expression of type V collagen mRNA in wound healing model, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation. Analyses on granulation tissue were carried out on days 5, 8, 14, 21, 30, 59 and 84. Specific cDNA probes to pro alpha1(I), pro alpha1(III) and pro alpha1(V) collagen mRNA were used in slot blot, Northern and in situ hybridization. Type I collagen gene expression was upregulated at the initial stage of wound healing, type III collagen gene expression was constant and from the day 14 onwards type I and III collagen gene expressions were at the same level. Type V collagen gene expression was seen at every time point studied but at a considerably lower level than type I and III collagens. In situ hybridization showed that type V collagen was expressed in two different cell types. In conclusion, type V collagen was expressed in the wound healing model from at least day 5 onwards and it was synthesized by fibroblast-like and rounded cells.

Substrate specificities of alpha-galactosidases from yeasts.

Twenty-nine strains of yeasts, which are capable of using galactose, melibiose, or raffinose, were screened for alpha-galactosidase production. Among the strains, 5 produced intracellular and extracellular alpha-galactosidases, and 2 produced only intracellular enzyme. Substrate specificities of these enzymes were explored using 6(3)-alpha-D-galactosyl-1,4-beta-D-mannotriose and 6(3)-alpha-D-galactosyl-1,4-beta-D-mannotetraose. All enzymes liberated the terminal galactose from 6(3)-alpha-D-galactosyl-1,4-beta-D-mannotriose, but did not the stub galactose from 6(3)-alpha-D-galactosyl-1,4-beta-D-mannotetraose.

Superfamily of alpha-galactosidase MEL genes of the Saccharomyces sensu stricto species complex.

In order to study the molecular evolution of the yeasts grouped in the Saccharomyces sensu stricto species complex by analysis of the MEL gene family, we have cloned and sequenced two new species-specific MEL genes from Saccharomyces yeasts: S. paradoxus (MELp) and a Japanese Saccharomyces sp. (MELj). The clones were identified by sequence homology to the S. cerevisiae MEL1 gene. Both clones revealed an ORF of 1413 bp coding for a protein of 471 amino acids. The deduced molecular weights of the alpha-galactosidase enzymes were 52,767 for MELp and 52,378 for MELj. The nucleotide sequences of the MELp (EMBL accession no. X95505) and the MELj (EMBL accession no. X95506) genes showed 74.7% identity. The degree of identity of MELp to the MEL1 gene was 76.8% and to the S. pastorianus MELx gene, 75.7%. The MELj coding sequence was 75.1% identical to the MEL1 gene and 80.7% to the MELx gene. The data suggest that MEL1, MELj, MELp, and MELx genes are species-specific MEL genes. The strains studied each have only one MEL locus. The MELp gene is located on the S. paradoxus equivalent of S. cerevisiae chromosome X; the MELj gene was on the chromosome that comigrates with the S. cerevisiae chromosome VII/XV doublet and hybridizes to the S. cerevisiae chromosome XV marker HIS3.

Identification of the alpha-galactosidase MEL genes in some populations of Saccharomyces cerevisiae: a new gene MEL11.

In this report we mapped a new MEL11 gene and summarize our population studies of the alpha-galactosidase MEL genes of S. cerevisiae. The unique family of structural MEL genes has undergone rapid translocations to the telomeres of most chromosomes in some specific Saccharomyces cerevisiae populations inhabiting olive oil processing waste (alpechin) and animal intestines. A comparative study of MEL genes in wine, pathogenic and alpechin populations of S. cerevisiae was conducted using genetic hybridization analysis, molecular karyotyping and Southern hybridization with the MEL1 probe. Five polymeric genes for the fermentation of melibiose, MEL3, MEL4, MEL6, MEL7, MEL11, were identified in an alpechin strain CBS 3081. The new MEL11 gene was mapped by tetrad analysis to the left telomeric region of chromosome I. In contrast, in wine and pathogenic populations of S. cerevisiae, MEL genes have been apparently eliminated. Their rare Mel+ strains carry only one of the MEL1, MEL2, or MEL8 genes. One clinical strain YJM273 was heterozygotic on the MEL1 gene; its mel1(0) allele did not have a sequence of the gene.

Consideration of the evolution of the Saccharomyces cerevisiae MEL gene family on the basis of the nucleotide sequences of the genes and their flanking regions.

Analysis of the DNA sequences of new members of the Saccharomyces cerevisiae MEL1-MEL10 gene family showed high homology between the members. The MEL gene family, alpha-galactosidase-coding sequences, have diverged into two groups; one consisting of MEL1 and MEL2 and the other of MEL3-MEL10. In two S. cerevisiae strains containing five or seven MEL genes each, all the genes are nearly identical, suggesting very rapid distribution of the gene to separate chromosomes. The sequence homology and the abrupt change to sequence heterogeneity at the centromere-proximal 3' end of the MEL genes suggest that the distribution of the genes to new chromosomal locations has occurred partly by reciprocal recombination at solo delta sequences. We identified a new open reading frame sufficient to code for a 554 amino acid long protein of unknown function. The new open reading frame (Accession number Z37509) is located in the 3' non-coding region of MEL3-MEL10 genes in opposite orientation to the MEL genes (Accession numbers Z37508, Z37510, Z37511). Northern analysis of total RNA showed no hybridization to a homologous probe, suggesting that the gene is not expressed efficiently if at all.

Characterization of MEL genes in the genus Zygosaccharomyces.

We cloned and sequenced a Zygosaccharomyces cidri MEL gene with a view to investigating the structure and regulation of yeast MEL genes. The amino acid sequence deduced from the nucleotide sequence showed 78.6% and 78.2% similarity to Saccharomyces cerevisiae and Saccharomyces pastorianus alpha-galactosidases, respectively. The expression of the MEL gene in several Zygosaccharomyces strains was induced by galactose. An electrophoretic karyotype of several Zygosaccharomyces species was obtained using contour-clamped electric field gel electrophoresis. The minimum number of chromosomes was five for Z. cidri, six for Z. fermentati, three for Z. florentinus, and four for Z. microellipsoides. The sizes of the chromosomes were generally larger than those of S. cerevisiae, the smallest containing approximately 0.4 megabase. The MEL gene was located, using the Z. cidri MEL gene as a probe, on the largest chromosome of the Z. cidri strains. In addition, a smaller chromosome (600 kb) in Z. cidri strain CBS4575 showed hybridization to the homologous MEL probe. This chromosome was absent in Z. cidri strain CBS5666. The probe hybridized to the largest chromosome of Mel+ Z. fermentati strains but failed to hybridize to any chromosome of Mel+ Z. mrakii or Z. florentinus strains. These results suggest the existence of a polymorphic MEL gene family in the yeast Zygosaccharomyces.

Identification of new chromosomes of Saccharomyces bayanus using gene probes from S. cerevisiae.

The Saccharomyces cerevisiae genes HIS3 (chr. XV), LYS2 (chr. II), ARG4 (chr. VIII), MF alpha 1 (chr. XVI) and MEL1 (chr. II) and the S. pastorianus gene MELx were used for identification of the homeologous chromosomes in S. bayanus by using pulsed-field gel electrophoresis and Southern blot hybridization. The results showed differences in hybridization intensities and mobilities between the homeologous chromosomes of the two sibling yeasts S. cerevisiae and S. bayanus. The probes HIS3, LYS2, MEL1, and MF alpha 1 identified four new homeologous chromosomes of S. bayanus. The ARG4 probe confirmed the previously identified location of chr. VIII (NAUMOV et al. 1992b). Using the MF alpha 1 gene, another chromosome besides chr. XVI was identified in some S. bayanus strains, suggesting translocation of this sequence to the S. bayanus chromosome that normally migrates to the position of chr. XI of S. cerevisiae.

Physical mapping of the MEL gene family in Saccharomyces cerevisiae.

Nine members, MEL2-MEL10, of the MEL gene family coding for alpha-galactosidase were physically mapped to the ends of the chromosomes by chromosome fragmentation. Genetic mapping of the genes supported the location of all the MEL genes in the left arm of their resident chromosomes.

Characterization of an intronless collagen gene family in the marine sponge Microciona prolifera.

Two independent clones from the genomic DNA of a marine sponge Microciona prolifera were isolated by hybridization to the Caenorhabditis elegans Col-1 gene and one clone was obtained from genomic DNA by PCR. They contain open reading frames (MpCol1, MpCol2, MpCol3, MpCol4) capable of coding for a family of collagens different from those previously found in sponges. Southern blotting of genomic DNA suggested the presence of several other homologous genes. cDNA clones covering most of the triple-helical coding domain and the 3' untranslated region of MpCol1 were isolated by specific primers and reverse PCR. Two cDNA clones end in the middle of an AATAAA sequence 170 bp downstream from the translation stop codon of MpCol1. The putative NH2-terminal noncollagenous peptide is composed of only seven amino acid residues. The 1074-bp triple-helical coding region is not interrupted by intervening sequences. It codes for a polypeptide of 120 Gly-Xaa-Yaa triplets with only one short interruption near the COOH terminus. A putative N-glycosylation sequence (Asn-Gly-Ser), three Arg-Gly-Asp triplets known as cell recognition peptides, frequent Lys residues in the Yaa position (which are templates for hydroxylation), several Lys-Gly-Asn/Xaa-Arg peptides known as the lysyl oxidase recognition site, and long stretches without imino acids could be found within the triple-helical domain. The short COOH-terminal noncollagenous domain closely resembles that of nematode cuticular collagens and vertebrate nonfibrillar collagens. Our results strongly support the idea that the diversity of collagen genes and gene families found in higher organisms already existed in sponge.

MEL gene polymorphism in the genus Saccharomyces.

In Saccharomyces spp. the ability to use melibiose depends on the presence of a MEL gene encoding alpha-galactosidase. We used two cloned MEL genes as probes to characterize the physical structure and chromosomal location of the MEL genes in several industrial and natural Mel+ strains of Saccharomyces cerevisiae, Saccharomyces pastorianus, and Saccharomyces bayanus. Electrokaryotyping showed that all of the S. pastorianus strains and most of the S. bayanus strains studied had one MEL locus. The MEL gene in S. bayanus strains was similar but not identical to the S. pastorianus MEL gene. Mel+ S. cerevisiae strains had one to seven loci containing MEL sequences. The MEL genes of these strains could be divided into two categories on the basis of hybridization to MEL1, one group exhibiting strong hybridization to MEL1 and the other group exhibiting weak hybridization to MEL1. In S. pastorianus and S. bayanus strains, the MEL gene was expressed as a single 1.5-kb transcript, and the expression was galactose inducible. In some S. cerevisiae strains, the MEL genes were expressed even without induction at fairly high levels. Expression was usually further induced by galactose. In two strains, CBS 5378 and CBS 4903, expression of the MEL genes was at the same level without induction as it was in most other strains with induction. In all S. cerevisiae strains, irrespective of the number of MEL genes, mRNA of only one size (1.6 kb) was observed.

A new family of polymorphic metallothionein-encoding genes MTH1 (CUP1) and MTH2 in Saccharomyces cerevisiae.

By pulsed-field gel electrophoresis of chromosomal DNA and hybridization with a cloned MTH1 (CUP1) gene, we determined the locations of metallothionein-encoding gene sequences on chromosomes in monosporic cultures of 76 natural strains of Saccharomyces cerevisiae. Most of the strains (68) exhibited a previously known location for the MTH sequence on chromosome (chr.) VIII. Seven strains (resistant or sensitive to Cu2+) showed a MTH sequence in a new locus, MTH2, on chr. XVI. One strain carried an MTH locus on both chromosomes VIII and XVI. Restriction fragment and Southern blot analyses showed that the two MTH loci were very closely related. The strains displayed heterogeneity in the size and structure of their MTH2 locus. The length of the repeat unit of MTH2 varied: a 1.9-kb or 1.7-kb unit was found, instead of the 2.0-kb unit of the MTH1 locus. The most resistant strain (resistant to 1.2 mM CuSO4) contained a 0.9-kb repeat unit in addition to those of 1.9 kb and 1.7 kb. All three sensitive (to over 0.3 mM CuSO4) strains with an mth2 locus had a repeat unit of 1.9 kb or 1.7 kb, suggesting the presence of at least two copies of the MTH2 gene, with one always being in the junction area outside of the repeat unit. A monogenic tetrad segregation of 2:2 was usually found in crosses of resistant MTH2 and sensitive mth2 strains. Hybrids between strains with different MTH loci in all combinations showed low ascospore viability, suggesting that the complete lack of an MTH locus may lead to the death of segregants on YPD medium. The MTH1 and MTH2 loci were exchangeable. Strains with a high level of Cu2+ resistance were also resistant to Cd2+. However, these two properties did not cosegregate in heterozygotic hybrids.

Polymeric genes MEL8, MEL9 and MEL10--new members of alpha-galactosidase gene family in Saccharomyces cerevisiae.

We used a combination of genetic hybridization analysis and electrokaryotyping with radioactively labelled MEL1 gene probe hybridization to isolate and identify seven polymeric genes for the fermentation of melibiose in strain CBS 5378 of Saccharomyces cerevisiae (syn. norbensis). Four of the MEL genes, i.e. MEL3, MEL4, MEL6 and MEL7, were allelic to those found in S. cerevisiae strain CBS 4411 (syn. S. oleaginosus) whereas three genes, i.e. MEL8, MEL9 and MEL10 occupied new loci. Electrokaryotyping showed that all seven MEL genes in CBS 5378 were located on different chromosomes. The new MEL8, MEL9 and MEL10 genes were found on chromosomes XV, X/XIV and XII, respectively.

Cloning, sequence and chromosomal location of a MEL gene from Saccharomyces carlsbergensis NCYC396.

Yeast strains producing alpha-galactosidase (alpha Gal) are able to use melibiose as a carbon source during growth or fermentation. We cloned a MEL gene from Saccharomyces carlsbergensis NCYC396 through hybridization to the MEL1 gene cloned earlier from Saccharomyces cerevisiae var. uvarum. The alpha Gal encoded by the newly cloned gene was galactose-inducible as is the alpha Gal encoded by MEL1. A probable GAL4-protein recognition sequence was found in the upstream region of the NCYC396 MEL gene. The gene was transcribed to a 1.5-kb mRNA which, according to the nucleotide sequence, encodes a protein of 471 amino acids (aa) with an Mr of 52,006. The first 18 aa fulfilled the criteria for the signal sequence, but lacked positively charged aa residues, except the initiating methionine. The enzyme activity was found exclusively in the cellular fraction of the cultures. The deduced aa sequence was compared to the aa sequences of other alpha Gal enzymes. It showed 83% identity with the S. cerevisiae enzyme, but only 35% with the plant enzyme, 30% with the human enzyme and 17% with the Escherichia coli enzyme. With pulsed-field electrophoresis, the MEL gene was located on chromosome X of S. carlsbergensis, whereas the S. cerevisiae var. uvarum MEL1 gene is located on chromosome II.

A new family of polymorphic genes in Saccharomyces cerevisiae: alpha-galactosidase genes MEL1-MEL7.

Using genetic hybridization analysis we identified seven polymorphic genes for the fermentation of melibiose in different Mel+ strains of Saccharomyces cerevisiae. Four laboratory strains (1453-3A, 303-49, N2, C.B.11) contained only the MEL1 gene and a wild strain (VKM Y-1830) had only the MEL2 gene. Another wild strain (CBS 4411) contained five genes: MEL3, MEL4, MEL5, MEL6 and MEL7. MEL3-MEL7 were isolated and identified by backcrosses with Mel- parents (X2180-1A, S288C). A cloned MEL1 gene was used as a probe to investigate the physical structure and chromosomal location of the MEL gene family and to check the segregation of MEL genes from CBS 4411 in six complete tetrads. Restriction and Southern hybridization analyses showed that all seven genes are physically very similar. By electrokaryotyping we found that all seven genes are located on different chromosomes: MEL1 on chromosome II as shown previously by Voll-rath et al. (1988), MEL2 on VII, MEL3 on XVI, MEL4 on XI, MEL5 on IV. MEL6 on XIII, and MEL7 on VI. Molecular analysis of the segregation of MEL genes from strain CBS 4411 gave results identical to those from the genetic analyses. The homology in the physical structure of this MEL gene family suggests that the MEL loci have evolved by transposition of an ancestral gene to specific locations within the genome.

Synthesis of glycosaminoglycans and collagen in skin fibroblasts cultured from a patient with lichen myxedematosus.

A patient is described with typical skin lesions of lichen myxedematosus and IgG-type lambda paraproteinemia. Fibroblasts cultured from the skin of the patient and from the skin of control persons were used to study glycosaminoglycan and collagen synthesis; the cultures were labelled with 3H-glucosamine and 3H-proline, respectively. Fibroblasts from the patient grew to a cell density which was lower than that of the control fibroblasts. The production of glycosaminoglycans was increased in lichen myxedematosus cultures, so that the ratio of hyaluronic acid to sulphated glucosaminoglycans was higher in the patient's cultures than in control cultures. Collagen production in the patient's cultures was about half of that in control cultures, whereas the ratio of type III to type I collagen was normal.

Effect of Mycoplasma pulmonis infection on protein and glycosaminoglycan synthesis of cultured connective tissue cells.

Human synovial cells, fetal skin fibroblasts and rat granulation tissue fibroblasts were experimentally infected with Mycoplasma pulmonis, a species identified as a contamination of cell cultures, and studied for collagen, total protein and glycosaminoglycan synthesis. Hyaluronic acid and sulfated glycosaminoglycan synthesis were stimulated in cultures where the infection reduced cell density, while they were retarded in cultures which had proliferated into higher density than the controls. An extra polypeptide with molecular weight of 20 kD was seen in [35S]methionine-labelled cells. Media of rat granulation tissue cells showed a shift of a 39-42 kD polypeptide to 33-36 kD position in [35S]methionine and [3H]proline labellings. Other minor changes were also noticed. Collagen synthesis or procollagen conversion to collagen were, however, not altered.