Effect of lon Protease Overexpression on Endotoxin Production and Stress Resistance in Bacillus thuringiensis.

Lon protease, an intracellular protease, plays a key role in cell homeostasis in bacteria and is involved in numerous physiological processes. In this work, we aimed to study the impact of Lon on the production of endotoxins and stress response in Bacillus thuringiensis, which is an important bioinsecticide alternative for toxic chemicals. For this purpose, lon gene was cloned into a multi-copy vector with its original promoter and transcriptional terminator and expressed in B. thuringiensis serovar israelensis ATCC 35,646. Our results showed that the recombinant lon gene transcribed and translated efficiently and the resulting protein was active. Although the sporulation efficiency of the recombinant strain was found to be reduced and its mobility impaired, overexpression of the lon gene triggered the production of endotoxin. Together with increased biofilm formation, recombinant strain exhibited significantly better adaptation to osmotic and heat shock stresses and UV exposure compared to wild type and the control strain with empty plasmid. This study suggested a possible link between Lon protease and the production of insecticide and stress response in B. thuringiensis and provides a platform for future studies focusing on enhancing bio-insecticidal production using this bacterium.

Heterologous expression of 8-demethyl-tetracenomycin (8-dmtc) affected Streptomyces coelicolor life cycle.

Heterologous hosts are highly important to detect the expression of biosynthetic gene clusters that are cryptic or poorly expressed in their natural hosts. To investigate whether actinorhodin-overproducer Streptomyces coelicolor ∆ppk mutant strain could be a possible prototype as a heterologous expression host, a cosmid containing most of the elm gene cluster of Streptomyces olivaceus Tü2353 was integrated into chromosomes of both S. coelicolor A3(2) and ∆ppk strains. Interestingly, it was found that the production of tetracyclic polyketide 8-demethyl-tetracenomycin (8-DMTC) by recombinant strains caused significant changes in the morphology of cells. All the pellets and clumps were disentangled and mycelia were fragmented in the recombinant strains. Moreover, they produce neither pigmented antibiotics nor agarase and did not sporulate. By eliminating the elm biosynthesis genes from the cosmid, we showed that the morphological properties of recombinants were caused by the production of 8-DMTC. Extracellular application of 8-DMTC on S. coelicolor wild-type cells caused a similar phenotype with the 8-DMTC-producing recombinant strains. The results of this study may contribute to the understanding of the effect of 8-DMTC in Streptomyces since the morphological changes that we have observed have not been reported before. It is also valuable in that it provides useful information about the use of Streptomyces as hosts for the heterologous expression of 8-DMTC.

Higher nisin yield is reached with glutathione and pyruvate compared with heme in Lactococcus lactis N8.

There are different studies that aim to enhance the production of nisin by Lactococcus lactis since its chemical synthesis is not possible. In this study, glutathione (GSH) and pyruvate, which are known to reduce the oxidative stress of cells, have been shown to trigger the production of nisin at both transcriptional and translational levels in L. lactis cells grown under aerobic condition. Presence of GSH and pyruvate caused more nisin yield than the heme-supplemented medium. Moreover, the expression of genes that encode stress-related enzymes were apparently upregulated in the presence of GSH and pyruvate. It can be concluded that GSH and pyruvate contribute to the defense system of L. lactis cells and so that higher biomass was obtained which in turn enhance nisin production. Antioxidant effect of GSH and pyruvate was known; however, their stimulating effect on nisin production was shown for the first time in this study.

One Extra Copy of lon Gene Causes a Dramatic Increase in Actinorhodin Production by Streptomyces coelicolor A3(2).

ATP-dependent Lon protease plays important roles in different physiological processes, including cellular differentiation of the bacteria and is a part of an important stress response regulon (HspR/HAIR). In Streptomyces, biosynthesis of secondary metabolites starts with cellular differentiation and stress is one of the factor that affect metabolite production. To clarify the effect of Lon protease on secondary metabolite production, we constructed a recombinant strain of Streptomyces coelicolor A3(2) that has one extra copy of lon gene with its own promoter and transcriptional terminator in its genome. Expression of lon gene in the recombinant strain was determined by quantitative real time (RT-qPCR). Actinorhodin and undecylprodigiosin production of the recombinant cell was measured in liquid R2YE and it was found to produce about 34 times more actinorhodin and 9 times more undecylprodigiosin than the wild-type at 168 h of growth. Development of stable Streptomyces strains capable of producing high amounts of secondary metabolites is valuable for biotechnology industry. One extra copy of lon gene is enough to boost antibiotic production by S. coelicolor A3(2) and this change do not cause any metabolic burden in the cell.

Two overlapping antiparallel genes encoding the iron regulator DmdR1 and the Adm proteins control siderophore [correction of sedephore] and antibiotic biosynthesis in Streptomyces coelicolor A3(2).

The dmdR1 gene of Streptomyces coelicolor encodes an important regulator of iron metabolism. An antiparallel gene (adm) homologous to a development-regulated gene of Streptomyces aureofaciens has been found to overlap with dmdR1. Both proteins DmdR1 and Adm are formed in solid and liquid cultures of S. coelicolor A3(2). The purpose of this study was to assess possible interaction between the products of these two antiparallel genes. Two mutants with stop codons resulting in arrested translation of either DmdR1 or Adm were obtained by gene replacement and compared with a deletion mutant (DeltadmdR1/adm) that was defective in both genes. The deletion mutant was unable to form either protein, did not sporulate and lacked desferrioxamine, actinorhodin and undecylprodigiosin biosynthesis; biosynthesis of these compounds was recovered by complementation with dmdR1/adm genes. The mutant in which formation of Adm protein was arrested showed normal levels of DmdR1, lacked Adm and over-produced the antibiotics undecylprodigiosin and actinorhodin (in MS medium), suggesting that Adm plays an important role in secondary metabolism. The mutant in which DmdR1 formation was arrested synthesized desferrioxamines in a constitutive (deregulated) manner, and produced relatively normal levels of antibiotics. In conclusion, our results suggest that there is a fine interplay of expression of these antiparallel genes, as observed for other genes that encode lethal proteins such as the toxin/antitoxin systems. The Adm protein seems to have a major effect on the control of secondary metabolism, and its formation is probably tightly controlled, as expected for a key regulator.

Transcriptional regulation of the desferrioxamine gene cluster of Streptomyces coelicolor is mediated by binding of DmdR1 to an iron box in the promoter of the desA gene.

Streptomyces coelicolor and Streptomyces pilosus produce desferrioxamine siderophores which are encoded by the desABCD gene cluster. S. pilosus is used for the production of desferrioxamine B which is utilized in human medicine. We report the deletion of the desA gene encoding a lysine decarboxylase in Streptomyces coelicolor A3(2). The DeltadesA mutant was able to grow on lysine as the only carbon and nitrogen source but its desferrioxamine production was blocked, confirming that the L-lysine decarboxylase encoded by desA is a dedicated enzyme committing L-lysine to desferrioxamine biosynthesis. Production of desferrioxamine was restored by complementation with the whole wild-type desABCD cluster, but not by desA alone, because of a polar effect of the desA gene replacement on expression of the downstream des genes. The transcription pattern of the desABCD cluster in S. coelicolor showed that all four genes were coordinately induced under conditions of iron deprivation. The transcription start point of the desA gene was identified by primer extension analysis at a thymine located 62 nucleotides upstream of the translation start codon. The -10 region of the desA promoter overlaps the 19-nucleotide palindromic iron box sequence known to be involved in iron regulation in Streptomyces. Binding of DmdR1 divalent metal-dependent regulatory protein to the desA promoter region of both S. coelicolor and S. pilosus was shown using electrophoretic mobility-shift assays, validating the conclusion that iron regulation of the desABCD cluster is mediated by the regulatory protein DmdR1. We conclude that the genes involved in desferrioxamine production are under transcriptional control exerted by the DmdR1 regulator in the presence of iron and are expressed under conditions of iron limitation.

The two-component phoR-phoP system of Streptomyces natalensis: Inactivation or deletion of phoP reduces the negative phosphate regulation of pimaricin biosynthesis.

The biosynthesis of the antifungal pimaricin in Streptomyces natalensis is very sensitive to phosphate regulation. Concentrations of inorganic phosphate above 1mM drastically reduced pimaricin production. At 10mM phosphate, expression of all the pimaricin biosynthesis (pim) genes including the pathway-specific positive regulator pimR is fully repressed. The phoU-phoR-phoP cluster of S. natalensis encoding two-component Pho system was cloned and sequenced. Binding of the response regulator PhoP to the consensus PHO boxes in the phoU-phoRP intergenic promoter region was observed. A phoP-disrupted mutant and a phoR-phoP deletion mutant were obtained. Production of pimaricin in these two mutants increased up to 80% in complex yeast extract-malt extract (YEME) or NBG media and showed reduced sensitivity to phosphate control. Four of the pim genes, pimS1, pimS4, pimC and pimG showed increased expression in the phoP-disrupted mutant. However, no consensus PHO boxes were found in the promoter regions of any of the pim genes, suggesting that phosphate control of these genes is mediated indirectly by PhoR-PhoP involving modification of pathway-specific regulators.

An efficient gene transfer system for the pimaricin producer Streptomyces natalensis.

Streptomyces natalensis produces the antifungal polyene macrolide pimaricin. Genetic manipulation of its biosynthetic genes has been hampered by the lack of efficient gene transfer systems. We have developed a gene transfer system based on intergeneric conjugation from Escherichia coli. Using this approach, we managed to attain transformation efficiencies of 1 x 10(-4) exconjugants per recipient when using self-replicating vectors such as pHZ1358. The use of integrative vectors such as pSET152 or pSOK804 resulted in significantly lower efficiencies. Site-specific integration or the use of self-replicating plasmids did not affect pimaricin production or the essential functions of S. natalensis. Use of DNA methylation proficient E. coli donor strains resulted in no transformants, indicating the presence of methyl-specific restriction systems in S. natalensis. This methodology will enable easier manipulation of the genes responsible for pimaricin biosynthesis, and could prove valuable for the generation of new designer polyene macrolides with better antifungal activity and pharmacological properties. As an example of the validity of the method, we describe the introduction of Supercos-1-derived cosmid vectors into S. natalensis in order to promote gene replacements by double crossover recombination.

Cloning, characterization and heterologous expression of the aspartokinase and aspartate semialdehyde dehydrogenase genes of cephamycin C-producer Streptomyces clavuligerus.

Carbon flow through the lysine branch of the aspartate biosynthetic pathway is a rate-limiting step in the formation of cephamycin C, a broad spectrum beta-lactam antibiotic produced by Streptomyces clavuligerus. In this study, genes which encode the enzymes catalyzing the first two steps of the aspartate pathway, ask (aspartokinase) and asd (aspartate semialdehyde dehydrogenase), in S. clavuligerus NRRL 3585 were cloned and sequenced. Nucleotide sequencing and codon preference analysis revealed three complete open reading frames (ORFs). ORF2 starts within ORF1 and terminates by utilizing the same stop codon as ORF1, an arrangement typical of many ask genes. ORF3 is located 2 nucleotides downstream of ORF1,2. Database comparisons with these proteins identified ORF1 as the large (alpha) subunit of aspartokinase, ORF2 as the small (beta) subunit of aspartokinase and ORF3 as the aspartate semialdehyde dehydrogenase. The cloned genes were functionally expressed in auxotrophic Escherichia coli strains, CGSC5074 (ask(-)) and E. coli CGSC5080 (asd(-)), the two enzymes were partially purified from E. coli cell extracts and their kinetic parameters were determined. The effects of end product amino acids and diaminopimelic acid on the activity of Ask and Asd enzymes were also described.