The novel phosphatase RosC catalyzes the last unknown step of roseoflavin biosynthesis in Streptomyces davaonensis

The bacterium Streptomyces davaonensis produces the antibiotic roseoflavin, which is a riboflavin (vitamin B₂) analog. The key enzyme of roseoflavin biosynthesis is the 8-demethyl-8-amino-riboflavin-5ʹ-phosphate (AFP) synthase RosB which synthesizes AFP from riboflavin-5ʹ-phosphate. AFP is not a substrate for the last enzyme of roseoflavin biosynthesis the N, N-dimethyltransferase RosA, which generates roseoflavin from 8-demethyl-8-amino-riboflavin (AF). Consequently, the roseoflavin biosynthetic pathway depends on a phosphatase, which dephosphorylates AFP to AF. Here, we report on the identification and characterization of such an AFP phosphatase which we named RosC. The gene rosC is located immediately downstream of rosA and both genes are part of a cluster comprising 10 genes. Deletion of rosC from the chromosome of S. davaonensis led to reduced roseoflavin levels in the corresponding recombinant strain. In contrast to wild-type S. davaonensis, cell-free extracts of the rosC deletion strain did not catalyze dephosphorylation of AFP. RosC was purified from an overproducing Escherichia coli strain. RosC is the fastest enzyme of roseoflavin biosynthesis (k_cat 31.3 ± 1.4 min^–1). The apparent K_M for the substrate AFP was 34.5 µM. Roseoflavin biosynthesis is now completely understood––it takes three enzymes (RosB, RosC, and RosA) to convert the flavin cofactor riboflavin-5ʹ-phosphate into a potent antibiotic.     

A highly specialized flavin mononucleotide riboswitch responds differently to similar ligands and confers roseoflavin resistance to Streptomyces davawensis

Streptomyces davawensis is the only organism known to synthesize the antibiotic roseoflavin, a riboflavin (vitamin B(2)) analog. Roseoflavin is converted to roseoflavin mononucleotide (RoFMN) and roseoflavin adenine dinucleotide in the cytoplasm of target cells. (Ribo-)Flavin mononucleotide (FMN) riboswitches are genetic elements, which in many bacteria control genes responsible for the biosynthesis and transport of riboflavin. Streptomyces davawensis is roseoflavin resistant, and the closely related bacterium Streptomyces coelicolor is roseoflavin sensitive. The two bacteria served as models to investigate roseoflavin resistance of S. davawensis and to analyze the mode of action of roseoflavin in S. coelicolor. Our experiments demonstrate that the ribB FMN riboswitch of S. davawensis (in contrast to the corresponding riboswitch of S. coelicolor) is able to discriminate between the two very similar flavins FMN and RoFMN and shows opposite responses to the latter ligands.     

The bifunctional flavokinase/flavin adenine dinucleotide synthetase from Streptomyces davawensis produces inactive flavin cofactors and is not involved in resistance to the antibiotic roseoflavin

Streptomyces davawensis synthesizes the antibiotic roseoflavin, one of the few known natural riboflavin analogs, and is roseoflavin resistant. It is thought that the endogenous flavokinase (EC 2.7.1.26)/flavin adenine dinucleotide (FAD) synthetase (EC 2.7.7.2) activities of roseoflavin-sensitive organisms are responsible for the antibiotic effect of roseoflavin, producing the inactive cofactors roseoflavin-5'-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) from roseoflavin. To confirm this, the FAD-dependent Sus scrofa D-amino acid oxidase (EC 1.4.3.3) was tested with RoFAD as a cofactor and found to be inactive. It was hypothesized that a flavokinase/FAD synthetase (RibC) highly specific for riboflavin may be present in S. davawensis, which would not allow the formation of toxic RoFMN/RoFAD. The gene ribC from S. davawensis was cloned. RibC from S. davawensis was overproduced in Escherichia coli and purified. Analysis of the flavokinase activity of RibC revealed that the S. davawensis enzyme is not riboflavin specific (roseoflavin, kcat/Km = 1.7 10(-2) microM(-1) s(-1); riboflavin, kcat/Km = 7.5 10(-3) microM(-1) s(-1)). Similar results were obtained for RibC from the roseoflavin-sensitive bacterium Bacillus subtilis (roseoflavin, kcat/Km = 1.3 10(-2) microM(-1) s(-1); riboflavin, kcat/Km = 1.3 10(-2) microM(-1) s(-1)). Both RibC enzymes synthesized RoFAD and RoFMN. The functional expression of S. davawensis ribC did not confer roseoflavin resistance to a ribC-defective B. subtilis strain.     

The Linear Plasmid SCP1 of Streptomyces coelicolor A3(2) Possesses a Centrally Located Replication Origin and Shows Significant Homology to the Transposon Tn4811

The linear plasmid SCP1 of Streptomyces coelicolor A3(2) is one of the genetically more studied linear streptomycete replicons. Although the genetics of SCP1 and its interaction with the host chromosome have been analyzed for nearly three decades no information exists on its replication. With the help of an ordered cosmid contig for the complete 360-kb element, we have localized a 5439-bp fragment from the central region that confers autonomous replication in Streptomyces lividans. The minimal origin contains two overlapping ORFs which are separated from an AT-rich region which might correspond to the replication start point. ORF1 revealed intensive similarity to a class of DNA-primase/helicases of actinophages and archael plasmids. In addition, we have identified a region in both terminal inverted repeats of SCP1 that shows significant homology to the transposable element Tn4811 located near the ends of the S. lividans 66 chromosome.     

Riboflavin-overproducing strains of Lactobacillus fermentum for riboflavin-enriched bread

Lactobacillus fermentum isolated from sourdough was able to produce riboflavin. Spontaneous roseoflavin-resistant mutants were obtained by exposing the wild strain (named L. fermentum PBCC11) to increasing concentrations of roseoflavin. Fifteen spontaneous roseoflavin-resistant mutants were isolated, and the level of vitamin B₂ was quantified by HPLC. Seven mutant strains produced concentrations of vitamin B₂ higher than 1 mg L^?1. Interestingly, three mutants were unable to overproduce riboflavin even though they were able to withstand the selective pressure of roseoflavin. Alignment of the rib leader region of PBCC11 and its derivatives showed only point mutations at two neighboring locations of the RFN element. In particular, the highest riboflavin-producing isolates possess an A to G mutation at position 240, while the lowest riboflavin producer carries a T to A substitution at position 236. No mutations were detected in the derivative strains that did not have an overproducing phenotype. The best riboflavin overproducing strain, named L. fermentum PBCC11.5, and its parental strain were used to fortify bread. The effect of two different periods of fermentation on the riboflavin level was compared. Bread produced using the coinoculum yeast and L. fermentum PBCC11.5 led to an approximately twofold increase of final vitamin B₂ content.     

Formation of roseoflavin from guanine through riboflavin.

A synthesis of roseoflavin by Streptomyces davawensis from guanine through riboflavin was demonstrated. The lines of evidence are (1)incorporations of 14C of [2-and U-14C] guanine and [2-14C] riboflavin into roseoflavin, (2) no incorporation of 14C of [8-14C] guanine into roseoflavin, (3) localizations of 14C in roseoflavin, and (4) a decrease of specific radioactivity of roseoflavin formed from [2-14C]guanine on addition of riboflavin to the culture. The 14C atoms in roseoflavin formed were localized by radioactivity analysis of the NaOH-hydrolysis products, i.e., urea and 1,2-dihydro-6-methyl-7-dimethylamino-2-keto-1-D-ribityl-3-quinox-alinecarboxylic acid (QC), a new substance. These hydrolysis products were identified by the isolation of dixanthylures, decomposition with urease, and from the properties of QC and QC tetraacetate isolated. These finding suggest that the pyrimidine ring of guanine is conserved in the formation of roseoflavin from guanine through riboflavin.     

Characterization of two glycosyltransferases involved in early glycosylation steps during biosynthesis of the antitumor polyketide mithramycin by <Emphasis Type="Italic">Streptomyces argillaceus</Emphasis>

A 2580-bp region of the chromosome of Streptomyces argillaceus, the producer of the antitumor polyketide mithramycin, was sequenced. Analysis of the nucleotide sequence revealed the presence of two genes (mtmGIII and mtmGIV?) encoding proteins that showed a high degree of similarity to glycosyltransferases involved in the biosynthesis of various antibiotics and antitumor drugs. Independent insertional inactivation of both genes produced mutants that did not synthesize mithramycin but accumulated several mithramycin intermediates. Both mutants accumulated premithramycinone, a non-glycosylated intermediate in mithramycin biosynthesis. The mutant affected in the mtmGIII gene also accumulated premithramycin A1, which contains premithramycinone as the aglycon unit and a D-olivose attached at C-12a-O. These experiments demonstrate that the glycosyltransferases MtmGIV and MtmGIII catalyze the first two glycosylation steps in mithramycin biosynthesis. A model is proposed for the glycosylation steps in mithramycin biosynthesis.     

Complete Genome Sequence of Klebsiella pneumoniae 1084, a Hypermucoviscosity-Negative K1 Clinical Strain

We report the complete genome sequence of Klebsiella pneumoniae 1084, a hypermucoviscosity-negative K1 clinical strain. Sequencing and annotation revealed a 5,386,705-bp circular chromosome (57.4% G+C content), which contains 4,962 protein-coding genes, 80 tRNA genes, and 25 rRNA genes.     

Physical mapping of 5S and 18S-26S ribosomal RNA gene families inAllium victorialis var.platyphyllum

A physical map of the 5S and 18S–26S rRNA genes was determined using bi-color fluorescencein situ hybridization technique inA. victorialis var.platyphyllum. 5S rRNA genes were positioned in the intercalary regions of the short arms in homologous chromosomes 6. Two major loci of the 18S-26S rRNA genes were detected in the secondary constrictions flanking with a pair of satellite and terminal region of short arm in chromosome 4. And two additional minor loci were heterotype, representing one signal on the terminal region of the short arm in one homolog of chromsome 2, and other on one homolog of chromosome 6 with linked 5S rRNA loci. In addition chromomycin A₃ (CMA,) fluorescent banding method was used to identify the relation between Nucleolus Organizer Region (NOR) sites and CMA, positive heterochromatin sites. In homologous chromosome 4 showing 18S–26S rDNA hybridization signals revealed also distinct CMA, positive band.     

Genome sequence and description of Timonella senegalensis gen. nov., sp. nov., a new member of the suborder Micrococcinae

Timonella senegalensis strain JC301^T gen. nov., sp. nov. is the type strain of T. senegalensis gen. nov., sp. nov., a new species within the newly proposed genus Timonella. This bacterial strain was isolated from the fecal flora of a healthy Senegalese patient. In this report, we detail the features of this organism, together with the complete genome sequence and annotation. Timonella senegalensis strain JC301^T exhibits the highest 16S rRNA similarity (95%) with Sanguibacter marinus, the closest validly published bacterial species. The genome of T. senegalensis strain JC301^T is 3,010,102-bp long, with one chromosome and no plasmid. The genome contains 2,721 protein-coding genes and 72 RNA genes, including 5 rRNA genes. The genomic annotation revealed that T. senegalensis strain JC301^T possesses the complete complement of enzymes necessary for the de novo biosynthesis of amino acids and vitamins (except for riboflavin and biotin), as well as the enzymes involved in the metabolism of various carbon sources, chaperone genes, and genes involved in the regulation of polyphosphate and glycogen levels.     

Introduction of the foreign transposon Tn4560 in Streptomyces coelicolor leads to genetic instability near the native insertion sequence IS1649

We report an altered pattern of genetic instability for Streptomyces coelicolor when the bacterium harbored a foreign transposon, Tn4560. Deletions, amplifications, and circularizations of the linear 8.7-Mb chromosome occurred more frequently at sites adjacent to native insertion elements, notably IS1649. In contrast, deletions, amplifications, and circularizations of a wild-type strain happened at heterogeneous sites within the chromosome. In 50 strains examined, structural changes removed or duplicated hundreds of contiguous S. coelicolor genes, altering up to 33% of the chromosome. S. coelicolor shows a bias toward one type of genetic instability during this particular assault from the environment, the invasion of foreign DNA.     

Engineering of Synechococcus sp. strain PCC 7002 for the photoautotrophic production of light-sensitive riboflavin (vitamin B2)

Due to their capability of photosynthesis and autotrophic growth, cyanobacteria are currently investigated with regard to the sustainable production of a wide variety of chemicals. So far, however, no attempt has been undertaken to engineer cyanobacteria for the biotechnological production of vitamins, which is probably due to the light-sensitivity of many of these compounds. We now describe a photoautotrophic bioprocess to synthesize riboflavin, a vitamin used as a supplement in the feed and food industry. By overexpressing the riboflavin biosynthesis genes ribDGEABHT from Bacillus subtilis in the marine cyanobacterium Synechococcus sp. PCC 7002 riboflavin levels in the supernatant of the corresponding recombinant strain increased 56-fold compared to the wild-type. Introduction of a second promoter region upstream of the heterologous ribAB gene – coding for rate-limiting enzymatic functions in the riboflavin biosynthesis pathway – led to a further increase of riboflavin levels (211-fold compared to the wild-type). Degradation of the light-sensitive product riboflavin was prevented by culturing the genetically engineered Synechococcus sp. PCC 7002 strains in the presence of dichromatic light generated by red light-emitting diodes (λ = 630 and 700 nm). Synechococcus sp. PCC 7002 naturally is resistant to the toxic riboflavin analog roseoflavin. Expression of the flavin transporter pnuX from Corynebacterium glutamicum in Synechococcus sp. PCC 7002 resulted in roseoflavin-sensitive recombinant strains which in turn could be employed to select roseoflavin-resistant, riboflavin-overproducing strains as a chassis for further improvement.     

Structure–function relationships of two paralogous single-stranded DNA-binding proteins from Streptomyces coelicolor: implication of SsbB in chromosome segregation during sporulation

The linear chromosome of Streptomyces coelicolor contains two paralogous ssb genes, ssbA and ssbB. Following mutational analysis, we concluded that ssbA is essential, whereas ssbB plays a key role in chromosome segregation during sporulation. In the ssbB mutant, ∼30% of spores lacked DNA. The two ssb genes were expressed differently; in minimal medium, gene expression was prolonged for both genes and significantly upregulated for ssbB. The ssbA gene is transcribed as part of a polycistronic mRNA from two initiation sites, 163 bp and 75 bp upstream of the rpsF translational start codon. The ssbB gene is transcribed as a monocistronic mRNA, from an unusual promoter region, 73 bp upstream of the AUG codon. Distinctive DNA-binding affinities of single-stranded DNA-binding proteins monitored by tryptophan fluorescent quenching and electrophoretic mobility shift were observed. The crystal structure of SsbB at 1.7 Å resolution revealed a common OB-fold, lack of the clamp-like structure conserved in SsbA and previously unpublished S-S bridges between the A/B and C/D subunits. This is the first report of the determination of paralogous single-stranded DNA-binding protein structures from the same organism. Phylogenetic analysis revealed frequent duplication of ssb genes in Actinobacteria, whereas their strong retention suggests that they are involved in important cellular functions.