Substrate specificity of the acyl transferase domains of EpoC from the epothilone polyketide synthase.

The production of epothilone mixtures is a direct consequence of the substrate tolerance of the module 3 acyltransferase (AT) domain of the epothilone polyketide synthase (PKS) which utilises both malonyl- and methylmalonyl-CoA extender units. Particular amino acid motifs in the active site of AT domains influence substrate selection for methylmalonyl-CoA (YASH) or malonyl-CoA (HAFH). This motif appears in hybrid form (HASH) in epoAT3 and may represent the molecular basis for the relaxed specificity of the domain. To investigate this possibility the AT domains from modules 2 and 3 of the epothilone PKS were examined in the heterologous DEBS1-TE model PKS. Substitution of AT1 of DEBS1-TE by epoAT2 and epoAT3 both resulted in functional PKSs, although lower yields of total products were observed when compared to DEBS1-TE (2% and 11.5% respectively). As expected, epoAT3 was significantly more promiscuous in keeping with its nature during epothilone biosynthesis. When the mixed motif (HASH) of epoAT3 within the hybrid PKS was mutated to HAFH (indicative of malonyl-CoA selection) it resulted in a non-productive PKS. When this mixed motif was converted to YASH (indicative of methylmalonyl-CoA selection) the selectivity of the hybrid PKS for methylmalonyl-CoA showed no statistically significant increase, and was associated with a loss of productivity.

Gene expression analysis of Corynebacterium glutamicum subjected to long-term lactic acid adaptation.

Corynebacteria form an important part of the red smear cheese microbial surface consortium. To gain a better understanding of molecular adaptation due to low pH induced by lactose fermentation, the global gene expression profile of Corynebacterium glutamicum adapted to pH 5.7 with lactic acid under continuous growth in a chemostat was characterized by DNA microarray analysis. Expression of a total of 116 genes was increased and that of 90 genes was decreased compared to pH 7.5 without lactic acid, representing 7% of the genes in the genome. The up-regulated genes encode mainly transcriptional regulators, proteins responsible for export, import, and metabolism, and several proteins of unknown function. As much as 45% of the up-regulated open reading frames code for hypothetical proteins. These results were validated using real-time reverse transcription-PCR. To characterize the functions of 38 up-regulated genes, 36 single-crossover disruption mutants were generated and analyzed for their lactic acid sensitivities. However, only a sigB knockout mutant showed a highly significant negative effect on growth at low pH, suggesting a function in organic-acid adaptation. A sigE mutant already displayed growth retardation at neutral pH but grew better at acidic pH than the sigB mutant. The lack of acid-sensitive phenotypes in 34 out of 36 disrupted genes suggests either a considerable redundancy in acid adaptation response or coincidental effects. Other up-regulated genes included genes for ion transporters and metabolic pathways, including carbohydrate and respiratory metabolism. The enhanced expression of the nrd (ribonucleotide reductase) operon and a DNA ATPase repair protein implies a cellular response to combat acid-induced DNA damage. Surprisingly, multiple iron uptake systems (totaling 15% of the genes induced >or=2-fold) were induced at low pH. This induction was shown to be coincidental and could be attributed to iron-sequestering effects in complex media at low pH.

Inhibition of Listeria monocytogenes by food-borne yeasts.

Many bacteria are known to inhibit food pathogens, such as Listeria monocytogenes, by secreting a variety of bactericidal and bacteriostatic substances. In sharp contrast, it is unknown whether yeast has an inhibitory potential for the growth of pathogenic bacteria in food. A total of 404 yeasts were screened for inhibitory activity against five Listeria monocytogenes strains. Three hundred and four of these yeasts were isolated from smear-ripened cheeses. Most of the yeasts were identified by Fourier transform infrared spectroscopy. Using an agar-membrane screening assay, a fraction of approximately 4% of the 304 red smear cheese isolates clearly inhibited growth of L. monocytogenes. Furthermore, 14 out of these 304 cheese yeasts were cocultivated with L. monocytogenes WSLC 1364 on solid medium to test the antilisterial activity of yeast in direct cell contact with Listeria. All yeasts inhibited L. monocytogenes to a low degree, which is most probably due to competition for nutrients. However, one Candida intermedia strain was able to reduce the listerial cell count by 4 log units. Another four yeasts, assigned to C. intermedia (three strains) and Kluyveromyces marxianus (one strain), repressed growth of L. monocytogenes by 3 log units. Inhibition of L. monocytogenes was clearly pronounced in the cocultivation assay, which simulates the conditions and contamination rates present on smear cheese surfaces. We found no evidence that the unknown inhibitory molecule is able to diffuse through soft agar.

Structure and biosynthesis of myxochromides S1-3 in Stigmatella aurantiaca: evidence for an iterative bacterial type I polyketide synthase and for module skipping in nonribosomal peptide biosynthesis.

The myxobacterium Stigmatella aurantiaca DW4/3-1 harbours an astonishing variety of secondary metabolic gene clusters, at least two of which were found by gene inactivation experiments to be connected to the biosynthesis of previously unknown metabolites. In this study, we elucidate the structures of myxochromides S1-3, novel cyclic pentapeptide natural products possessing unsaturated polyketide side chains, and identify the corresponding biosynthetic gene locus, made up of six nonribosomal peptide synthetase modules. By analyzing the deduced substrate specificities of the adenylation domains, it is shown that module 4 is most probably skipped during the biosynthetic process. The polyketide synthase MchA harbours only one module and is presumably responsible for the formation of the variable complete polyketide side chains. These data indicate that MchA is responsible for an unusual iterative polyketide chain assembly.

Import of mitochondrial transcription factor A (TFAM) into rat liver mitochondria stimulates transcription of mitochondrial DNA.

Mitochondrial transcription factor A (TFAM) has been shown to stimulate transcription from mitochondrial DNA promoters in vitro. In order to determine whether changes in TFAM levels also regulate RNA synthesis in situ, recombinant human precursor proteins were imported into the matrix of rat liver mitochondria. After uptake of wt-TFAM, incorporation of [alpha-32P]UTP into mitochondrial mRNAs as well as rRNAs was increased 2-fold (P < 0.05), whereas import of truncated TFAM lacking 25 amino acids at the C-terminus had no effect. Import of wt-TFAM into liver mitochondria from hypothyroid rats stimulated RNA synthesis up to 4-fold. We conclude that the rate of transcription is submaximal in freshly isolated rat liver mitochondria and that increasing intra-mitochondrial TFAM levels is sufficient for stimulation. The low transcription rate associated with the hypothyroid state observed in vivo as well as in organello seems to be a result of low TFAM levels, which can be recovered by treating animals with T3 in vivo or by importing TFAM in organello. Thus, this protein meets the criteria for being a key factor in regulating mitochondrial gene expression in vivo.

Steroid biosynthesis in prokaryotes: identification of myxobacterial steroids and cloning of the first bacterial 2,3(S)-oxidosqualene cyclase from the myxobacterium Stigmatella aurantiaca.

Steroids, such as cholesterol, are synthesized in almost all eukaryotic cells, which use these triterpenoid lipids to control the fluidity and flexibility of their cell membranes. Bacteria rarely synthesize such tetracyclic compounds but frequently replace them with a different class of triterpenoids, the pentacyclic hopanoids. The intriguing mechanisms involved in triterpene biosynthesis have attracted much attention, resulting in extensive studies of squalene-hopene cyclase in bacteria and (S)-2,3-oxidosqualene cyclases in eukarya. Nevertheless, almost nothing is known about steroid biosynthesis in bacteria. Only three steroid-synthesizing bacterial species have been identified before this study. Here, we report on a variety of sterol-producing myxobacteria. Stigmatella aurantiaca is shown to produce cycloartenol, the well-known first cyclization product of steroid biosynthesis in plants and algae. Additionally, we describe the cloning of the first bacterial steroid biosynthesis gene, cas, encoding the cycloartenol synthase (Cas) of S. aurantiaca. Mutants of cas generated via site-directed mutagenesis do not produce the compound. They show neither growth retardation in comparison with wild type nor any increase in ethanol sensitivity. The protein encoded by cas is most similar to the Cas proteins from several plant species, indicating a close evolutionary relationship between myxobacterial and eukaryotic steroid biosynthesis.

A novel biosynthetic pathway providing precursors for fatty acid biosynthesis and secondary metabolite formation in myxobacteria.

Short chain carboxylic acids are well known as the precursors of fatty acid and polyketide biosynthesis. Iso-fatty acids, which are important for the control of membrane fluidity, are formed from branched chain starter units (isovaleryl-CoA and isobutyryl-CoA), which in turn are derived from the degradation of leucine and valine, respectively. Branched chain carboxylic acids are also employed as starter molecules for the biosynthesis of secondary metabolites, e.g. the therapeutically important anthelmintic agent avermectin or the electron transport inhibitor myxothiazol. During our studies on myxothiazol biosynthesis in the myxobacterium, Stigmatella aurantiaca, we detected a novel biosynthetic route to isovaleric acid. After cloning and inactivation of the branched chain keto acid dehydrogenase complex, which is responsible for the degradation of branched chain amino acids, the strain is still able to produce iso-fatty acids and myxothiazol. Incorporation studies employing deuterated leucine show that it can only serve as precursor in the wild type strain but not in the esg mutant. Feeding experiments using (13)C-labeled precursors show that isovalerate is efficiently made from acetate, giving rise to a labeling pattern in myxothiazol that provides evidence for a novel branch of the mevalonate pathway involving the intermediate 3,3-dimethylacryloyl-CoA. 3,3-Dimethylacrylic acid was synthesized in deuterated form and fed to the esg mutant, resulting in strong incorporation into myxothiazol and iso-fatty acids. Similar experiments employing Myxococcus xanthus revealed that the discovered biosynthetic route described is present in other myxobacteria as well.

The biosynthesis of the aromatic myxobacterial electron transport inhibitor stigmatellin is directed by a novel type of modular polyketide synthase.

Deductions from the molecular analysis of the 65,000-bp stigmatellin biosynthetic gene cluster are reported. The biosynthetic genes (stiA-J) encode an unusual bacterial modular type I polyketide synthase (PKS) responsible for the formation of this aromatic electron transport inhibitor produced by the myxobacterium Stigmatella aurantiaca. Involvement of the PKS gene cluster in stigmatellin biosynthesis is shown using site-directed mutagenesis. One module of the PKS is assumed to be used iteratively during the biosynthetic process, which seems to involve an unusual transacylation of the biosynthetic intermediate from an acyl carrier protein domain back to the preceding ketosynthase domain. Finally, the polyketide chain which is presumably catalyzed by a novel C-terminal domain in StiJ that does not resemble thioesterases, is cyclized and aromatized. The presented results of feeding experiments are in good agreement with the proposed biosynthetic scheme. In contrast to all other PKS type I systems reported to date, each module of StiA-J is encoded on a separate gene. The gene cluster contains a "stand alone" O-methyltransferase and two unusual O-methyltransferase domains embedded in the PKS. In addition, inactivation of a cytochrome P450 monooxygenase-encoding gene involved in post-PKS hydroxylation of the aromatic ring leads to the formation of two novel stigmatellin derivatives.

SigB, an alternative sigma factor of the myxobacterium Stigmatella aurantiaca, is synthesized during development and heat shock.

Alternative sigma factors have been detected in the myxobacterium Stigmatella aurantiaca during indole-induced sporulation, fruiting body formation and heat shock using an antiserum raised against sigma factor SigB. The time course of sigB gene expression was analysed by RT-PCR and by determining beta-galactosidase activity during development in a merodiploid strain that harboured a sigB-lacZ fusion gene. Inactivation of the sigB gene by insertion of the neo gene resulted in the loss of one sigma factor as shown by Western analysis. Neither fruiting body formation nor sporulation, nor the production of possible SigB targets, such as DnaK, GroEL or HspA, were affected.