Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cell. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ‘Crabtree effect’, was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate hydrogenase was subject to glucose inactivation.     

Structural gene isolation and prepeptide sequence of gallidermin, a new lanthionine containing antibiotic

Peptide antibiotics containing lanthionine and 3-methyllanthionine bridges, named lantibiotics are of increasing interest. A new lantibiotic, gallidermin, has been isolated from Staphyloccus gallinarum. Here we report the isolation of its structural gene which we name gdmA. In all lantibiotics so far studied genetically, three peptides can be formally distinguished: (i) the primary translation product, which we call the prepeptide; (ii) the propeptide lacking the leader sequence and (iii) the mature lantibiotic. Unlike the plasmid-coded epidermin, gdmA is located on the chromosome. The gdmA locus codes for a 52 amino acid residue prepeptide, consisting of an alpha-helical leader sequence of hydrophilic character, which is separated from the C-terminus (propeptide) by a characteristic proteolytic processing site (Pro-2 Arg-1 Ile1). Although pro-gallidermin differs from pro-epidermin (a recently isolated lantibiotic) only by a single amino acid residue exchange. Leu instead of Ile, the N-terminus of the prepeptide differs by an additional two exchanges.     

Nisin, a peptide antibiotic: cloning and sequencing of the nisA gene and posttranslational processing of its peptide product.

Nisin produced by Streptococcus lactis is used as a food preservative and is the most important member of a group of antibiotics containing lanthionine bridges. To understand the genetic basis of these so-called lantibiotics (Schnell et al., Nature 333:276-278, 1988), we characterized the nisin structural gene, nisA, which is located on a plasmid and codes for a 57-amino-acid prepeptide. The prepeptide is processed posttranslationally to the pentacyclic antibiotic. Although nisin and the recently elucidated lantibiotic epidermin from Staphylococcus epidermidis are produced by different organisms, their gene organization is identical. As with epidermin, the nisin propeptide corresponds to the C-terminus of the prepeptide. The N-terminus of the prepeptide is cleaved at a characteristic splice site (Pro--2 Arg--1 Ile-+1). Remarkably, the N-terminus of prenisin shares 70% similarity with preepidermin, although the propeptide sequences are distinctly different. The structural similarities between these two lantibiotics are consistent with the fact that there is a common mechanism of biosynthesis of these lanthionine-containing antibiotics.     

Genetics of subtilin and nisin biosyntheses

Several peptide antibiotics have been described as potent inhibitors of bacterial growth. With respect to their biosynthesis, they can be devided into two classes: (i) those that are synthesized by a non-ribosomal mechanism and (ii) those that are ribosomally synthesized. Subtilin and nisin belong to the ribosomally synthesized peptide antibiotics. They contain the rare amino acids dehydroalanine, dehydrobutyrine, meso-lanthionine, and 3-methyl-lanthionine. They are derived from prepeptides which are post-translationally modiffied and have been termed lantibiotics because of their characteristic lanthionine bridges (Schnell et al. 1988). Nisin is the most prominent lantibiotic and is used as a food preservative due to its high potency against certain gram-positive bacteria (Mattick & Hirsch 1944, 1947; Rayman & Hurst 1984). It is produced by Lactococcus lactis strains belonging to serological group N. The potent bactericidal activities of nisin and other lantibiotics are based on depolarization of energized bacterial cytoplasmic membranes. Breakdown of the membrane potential is initiated by the formation of pores through which molecules of low molecular weight are released. A trans-negative membrane potential of 50 to 100 mV is necessary for pore formation by nisin (Ruhr & Sahl 1985; Sahl et al. 1987). Nisin occurs as a partially amphiphilic molecule (Van de Ven et al. 1991). Apart from the detergent-like effect of nisin on cytoplasmic membranes, an inhibition of murein synthesis has also been discussed as the primary effect (Reisinger et al. 1980). In several countries nisin is used to prevent the growth of clostridia in cheese and canned food. The nisin peptide structure was first described by Gross & Morall (1971), and its structural gene was isolated in 1988 (Buchman et al. 1988; Kaletta & Entian 1989). Nisin has two natural variants, nisin A and nisin Z, which differ in a single amino acid residue at position 27 (histidin in nisin A is replaced by asparagin in nisin Z (Mulders et al. 1991; De Vos et al. 1993). Subtilin is produced by Bacillus subtilis ATCC 6633. Its chemical structure was first unravelled by Gross & Kiltz (1973) and its structural gene was isolated in 1988 (Banerjee & Hansen 1988). Subtilin shares strong similarities to nisin with an identical organization of the lanthionine ring structures (Fig. 1), and both lantibiotics possess similar antibiotic activities. Due to its easy genetic analysis B. subtilis became a very suitable model organism for the identification and characterization of genes and proteins involved in lantibiotic biosynthesis. The pathway by which nisin is produced is very similar to that of subtilin, and the proteins involved share significant homologies over the entire proteins (for review see also De Vos et al. 1995b). The respective genes have been identified adjacent to the structural genes, and are organized in operon-like structures (Fig. 2). These genes are responsible for post-translational modification, transport of the modified prepeptide, proteolytic cleavage, and immunity which prevents toxic effects on the producing bacterium. In addition to this, biosynthesis of subtilin and nisin is strongly regulated by a two-component regulatory system which consists of a histidin kinase and a response regulator protein.     

Growth phase-dependent regulation and membrane localization of SpaB, a protein involved in biosynthesis of the lantibiotic subtilin.

The information responsible for biosynthesis of the lantibiotic subtilin is organized in an operon-like structure that starts with the spaB gene. The spaB gene encodes an open reading frame consisting of 1,030 amino acid residues, and it was calculated that a protein having a theoretical molecular mass of 120.5 kDa could be produced from this gene. This is consistent with the apparent molecular weight for SpaB of 115,000 which was estimated after sodium dodecyl sulfate-gel electrophoresis and identification with SpaB-specific antibodies. The SpaB protein is very similar to proteins EpiB and NisB, which were identified previously as being involved in epidermin and nisin biosynthesis. Upstream from SpaB a characteristic sigma A promoter sequence was identified. An immunoblot analysis revealed that SpaB expression was strongly regulated. No SpaB protein was detected in the early logarithmic growth phase, and maximum SpaB expression was observed in the early stationary growth phase. The expression of SpaB was strongly correlated with subtilin biosynthesis. Deletion mutations in either of two recently identified regulatory genes, spaR and spaK, which act as a "two-component" regulatory system necessary for growth phase-dependent induction of subtilin biosynthesis (C. Klein, C. Kaletta, and K. D. Entian, Appl. Environ. Microbiol. 59:296-303, 1993), also resulted in failure of SpaB expression. To investigate the intracellular localization of SpaB, vesicles of Bacillus subtilis were prepared. The SpaB protein cosedimented with the vesicle fraction and was released only after vigorous resuspension of the vesicles. Our results suggest that SpaB is membrane associated and that subtilin biosynthesis occurs at the cytoplasmic membrane of B. subtilis.     

Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis.

Epidermin is produced by Staphylococcus epidermidis Tü3298 which harbors the 54-kb plasmid, pTü32. The plasmid contains not only the epidermin structural gene epiA, but also a flanking DNA region which is necessary for epidermin biosynthesis. The DNA sequence of this region revealed, in addition to epiA, five additional open reading frames, epiB, C, D, Q and P [Schnell, N., Engelke, G., Augustin J., Rosenstein, R., Ungermann, V., G?tz, F. & Entian, K.-D. (1992) Eur. J. Biochem. 204, 57-68]. We isolated a number of stable mutants from strain Tü3298 which are unable to produce biologically active epidermin. Complementation studies using the newly constructed staphylococcal plasmid vectors pT181mcs and pCU1 led to their classification as epiA, epiB, epiC or epiD mutants. Furthermore, evidence is presented that epiB lacks its own promoter and is co-transcribed from the epiA promoter. There is evidence that epiC and D possess their own promoters. Although epiQ and epiP mutants were not isolated, it could be shown by heterologous gene expression in S. carnosus and S. xylosus that the corresponding DNA region is involved in epidermin biosynthesis. We can not exclude the possibility that, in addition to the four open reading frames, epiA, B, C, D, and the DNA region comprising epiQ and P, host-encoded functions are necessary for epidermin production. Thus, the genetic information for epidermin biosynthesis in S. carnosus and S. xylosus is located on an 8-kb DNA fragment of pTü32. A further characterization of the two epiA mutants revealed that in both mutants, the preepidermin nucleotide sequence was changed. In one mutant, the mutation led to a substitution of Ser3 by Asn; in the other of Gly10 by Glu.     

Two different lantibiotic-like peptides originate from the ericin gene cluster of Bacillus subtilis A1/3

A lantibiotic gene cluster was identified in Bacillus subtilis A1/3 showing a high degree of homology to the subtilin gene cluster and occupying the same genetic locus as the spa genes in B. subtilis ATCC 6633. The gene cluster exhibits diversity with respect to duplication of two subtilin-like genes which are separated by a sequence similar to a portion of a lanC gene. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analyses of B. subtilis A1/3 culture extracts confirmed the presence of two lantibiotic-like peptides, ericin S (3,442 Da) and ericin A (2,986 Da). Disruption of the lanB-homologous gene eriB resulted in loss of production of both peptides, demonstrating that they are processed in an eriB-dependent manner. Although precursors of ericins S and A show only 75% of identity, the matured lantibiotic-like peptides reveal highly similar physical properties; separation was only achieved after multistep, reversed-phase high-performance liquid chromatography. Based on Edman and peptidase degradation in combination with MALDI-TOF MS, for ericin S a subtilin-like, lanthionine-bridging pattern is supposed. For ericin A two C-terminal rings are different from the lanthionine pattern of subtilin. Due to only four amino acid exchanges, ericin S and subtilin revealed similar antibiotic activities as well as similar properties in response to heat and protease treatment. For ericin A only minor antibiotic activity was found.