Spatial structure increases the benefits of antibiotic production in Streptomyces.

Bacteria in the soil compete for limited resources. One of the ways they might do this is by producing antibiotics, but the metabolic costs of antibiotics and their low concentrations have caused uncertainty about the ecological role of these products for the bacteria that produce them. Here, we examine the benefits of streptomycin production by the filamentous bacterium Streptomyces griseus. We first provide evidence that streptomycin production enables S. griseus to kill and invade the susceptible species, S. coelicolor, but not a streptomycin-resistant mutant of this species. Next, we show that the benefits of streptomycin production are density dependent, because production scales positively with cell number, and frequency dependent, with a threshold of invasion of S. griseus at around 1%. Finally, using serial transfer experiments where spatial structure is either maintained or destroyed, we show that spatial structure reduces the threshold frequency of invasion by more than 100-fold, indicating that antibiotic production can permit invasion from extreme rarity. Our results show that streptomycin is both an offensive and defensive weapon that facilitates invasion into occupied habitats and also protects against invasion by competitors. They also indicate that the benefits of antibiotic production rely on ecological interactions occurring at small local scales.

Detection of antibiotics synthetized in microfluidic picolitre-droplets by various actinobacteria.

The natural bacterial diversity is regarded as a treasure trove for natural products. However, accessing complex cell mixtures derived from environmental samples in standardized high-throughput screenings is challenging. Here, we present a droplet-based microfluidic platform for ultrahigh-throughput screenings able to directly harness the diversity of entire microbial communities. This platform combines extensive cultivation protocols in aqueous droplets starting from single cells or spores with modular detection methods for produced antimicrobial compounds. After long-term incubation for bacterial cell propagation and metabolite production, we implemented a setup for mass spectrometric analysis relying on direct electrospray ionization and injection of single droplets. Even in the presence of dense biomass we show robust detection of streptomycin on the single droplet level. Furthermore, we developed an ultrahigh-throughput screening based on a functional whole-cell assay by picoinjecting reporter cells into droplets. Depending on the survival of reporter cells, droplets were selected for the isolation of producing bacteria, which we demonstrated for a microbial soil community. The established ultrahigh-throughput screening for producers of antibiotics in miniaturized bioreactors in which diverse cell mixtures can be screened on the single cell level is a promising approach to find novel antimicrobial scaffolds.

Regulation of antibiotic production in Actinobacteria: new perspectives from the post-genomic era.

Covering: 2000 to 2018 The antimicrobial activity of many of their natural products has brought prominence to the Streptomycetaceae, a family of Gram-positive bacteria that inhabit both soil and aquatic sediments. In the natural environment, antimicrobial compounds are likely to limit the growth of competitors, thereby offering a selective advantage to the producer, in particular when nutrients become limited and the developmental programme leading to spores commences. The study of the control of this secondary metabolism continues to offer insights into its integration with a complex lifecycle that takes multiple cues from the environment and primary metabolism. Such information can then be harnessed to devise laboratory screening conditions to discover compounds with new or improved clinical value. Here we provide an update of the review we published in NPR in 2011. Besides providing the essential background, we focus on recent developments in our understanding of the underlying regulatory networks, ecological triggers of natural product biosynthesis, contributions from comparative genomics and approaches to awaken the biosynthesis of otherwise silent or cryptic natural products. In addition, we highlight recent discoveries on the control of antibiotic production in other Actinobacteria, which have gained considerable attention since the start of the genomics revolution. New technologies that have the potential to produce a step change in our understanding of the regulation of secondary metabolism are also described.

Protein acetylation involved in streptomycin biosynthesis in Streptomyces griseus.

Protein acetylation, the reversible addition of an acetyl group to lysine residues, is a protein post-translational modification ubiquitous in living cells. Although the involvement of protein acetylation in the regulation of primary metabolism has been revealed, the function of protein acetylation is largely unknown in secondary metabolism. Here, we characterized protein acetylation in Streptomyces griseus, a streptomycin producer. Protein acetylation was induced in the stationary and sporulation phases in liquid and solid cultures, respectively, in S. griseus. By comprehensive acetylome analysis, we identified 134 acetylated proteins with 162 specific acetylated sites. Acetylation was found in proteins related to primary metabolism and translation, as in other bacteria. However, StrM, a deoxysugar epimerase involved in streptomycin biosynthesis, was identified as a highly acetylated protein by 2-DE-based proteomic analysis. The Lys70 residue, which is critical for the enzymatic activity of StrM, was the major acetylation site. Thus, acetylation of Lys70 was presumed to abolish enzymatic activity of StrM. In accordance with this notion, an S. griseus mutant producing the acetylation-mimic K70Q StrM hardly produced streptomycin, though the K70Q mutation apparently decreased the stability of StrM. A putative lysine acetyltransferase (KAT) SGR1683 in S. griseus, as well as the Escherichia coli KAT YfiQ, acetylated Lys70 of StrM in vitro. Furthermore, absolute quantification analysis estimated that 13% of StrM molecules were acetylated in mycelium grown in solid culture for 3days. These results indicate that StrM acetylation is of biological significance. We propose that StrM acetylation functions as a limiter of streptomycin biosynthesis in S. griseus. BIOLOGICAL SIGNIFICANCE: Protein acetylation has been extensively studied not only in eukaryotes, but also in prokaryotes. The acetylome has been analyzed in more than 14 bacterial species. Here, by comprehensive acetylome analysis, we showed that acetylation was found in proteins related to primary metabolism and translation in Streptomyces griseus, similarly to other bacteria. However, five proteins involved in secondary metabolism were also identified as acetylated proteins; these proteins are enzymes in the biosynthesis of streptomycin (StrB1 and StrS), grixazone (GriF), a nonribosomal peptide (NRPS1-2), and a siderophore (AlcC). Additionally, StrM in streptomycin biosynthesis was identified as a highly acetylated protein by 2-DE-based proteomic analysis; approximately 13% of StrM molecules were acetylated. The acetylation occurs at Lys70 to abolish the enzymatic activity of StrM, suggesting that StrM acetylation functions as a limiter of streptomycin biosynthesis in S. griseus. This is the first detailed analysis of protein acetylation of an enzyme involved in secondary metabolism.

An ABC transporter involved in the control of streptomycin production in Streptomyces griseus.

We screened for a gene that inhibits streptomycin production in Streptomyces griseus when it is introduced on a high-copy-number plasmid pIJ702, and obtained a plasmid pKM545. The introduction of pKM545 abolished streptomycin production on all media tested including YMP-sugar and Nutrient broth. S1 protection analysis demonstrated that the introduction of this plasmid downregulated the transcriptional activity of the promoter preceding strR, the pathway-specific transcriptional regulator for streptomycin biosynthesis. The 2.8-kb BamHI fragment cloned onto pKM545 contained two coding sequences SGR_5442 and 5443. These coding sequences and the two downstream ones (SGR_5444 and 5445) constituted a possible operon structure designated to be rspABCD (regulation of streptomycin production). RspB and RspC exhibited a marked similarity with an ATP-binding domain and a membrane-associating domain of an ABC-2 type transporter, respectively, suggesting that the Rsp proteins comprise a membrane exporter. The gene cluster consisting of the rsp operon and the upstream divergent small coding sequence (SGR_5441) was widely distributed to Streptomyces genome. An rspB mutant of S. griseus produced 3-fold streptomycin of the parental strain in YMP liquid medium. The evidence implies that the Rsp translocator is involved in the export of a substance that specifies the expression level of streptomycin biosynthesis genes in S. griseus.

Historical and hygienic aspects on roles of quality requirements for antibiotic products in Japan: Part 5 - Introduction of technology and knowledge on streptomycin production from the United States of Americat.

In order to investigate the roles of quality requirements for antibiotics products in Japan, from historical and hygienic aspects, we examined how technology and knowledge in the production and quality control of streptomycin were introduced from the United States of America. In this study, through detailed investigations and analyses, it was confirmed that the introduction of technology and knowledge on streptomycin was strongly supported by Brigadier General CRAWFORD SAms, the chief of the Public Health and Welfare Section (PHW) of the Supreme Commander for Allied Powers/General Headquarters, via the Ministry of Welfare in Japan. Dr. SELMAN WAKSMAN, the discoverer of streptomycin, along with scientists of Merck & Co., also helped Japanese industries extensively, via PHW, by providing the original streptomycin-producing strains and transferring expertise in streptomycin production. With the technology and knowledge being introduced from the USA, domestic production of streptomycin preparations increased very rapidly. As noted in our previous report, domestic production reached amounts enough to satisfy national demand within three years. Japanese people have a racial tendency to be highly susceptible to tuberculosis known as an incurable national disease. Thanks to streptomycin therapy, the tuberculosis mortality rate (per 100,000 population) had fallen dramatically within only five years from 187.2 in 1947 to 82.2 in 1952.

N-methylphenylalanyl-dehydrobutyrine diketopiperazine, an A-factor mimic that restores antibiotic biosynthesis and morphogenesis in Streptomyces globisporus 1912-B2 and Streptomyces griseus 1439.

The cell-free extracts of a landomycin E-producing strain, Streptomyces globisporus 1912-2, were shown to contain a low-molecular-weight compound that, like A-factor, restored the landomycin E and streptomycin biosynthesis and sporulation of the defective mutants S. globisporus 1912-B2 and S. griseus 1439, respectively. The compound was purified by thin layer chromatography and HPLC. It had an absorption maximum at λmax=245 nm and a molecular mass of m/z 244. On the basis of NMR spectroscopy ((1)H, (13)C, HSQC, HMBC, COSY and NOE) the chemical structure of the compound was elucidated as 6-benzyl-3-eth-(Z)-ylidene-1-methyl-piperazine-2,6-dione ((L)-N-methylphenylalanyl-dehydrobutyrine diketopiperazine (MDD)). The sequences of arpA genes in S. globisporus 1912-2 and S. griseus NBRC 13350 are highly conserved. An explanation for the observed biological activity of MDD was proposed.

Sensitivity of photosynthesis to UV radiation in several Cosmarium strains (Zygnematophyceae, Streptophyta) is related to their geographical distribution.

Photoinhibitory effects of ultraviolet radiation (UVR) on four Cosmarium strains were studied with respect to their geographical distribution pattern. This study dealt with two strains of a cosmopolitan taxon (C. punctulatum var. subpunctulatum) collected from high-mountain tropical and lowland polar regions, one typical tropical species (C. beatum) and one typical polar representative (C. crenatum var. boldtianum). Physiological characteristics of the strains during and after various UVR spectral combinations at two temperature gradients were determined by the measurement of chlorophyll fluorescence, oxygen evolution rates and using an inhibitor of chloroplast-encoded protein synthesis (streptomycin). All of the Cosmarium strains investigated exhibited consistent geographical distribution patterns in accordance with the UVR prevailing at their sampling sites, despite a long-term cultivation under constant laboratory conditions. It appeared that moderate ultraviolet-B radiation (UVBR) treatment did not exert large damages to photosystem II in all of the Cosmarium strains, compared to ultraviolet-A radiation (UVAR) treatment at 21 °C. Interestingly, an ameliorating effect of UVBR at 21 °C was observed in C. beatum as concluded from higher rates of recovery of maximum quantum yield after moderate UVBR treatment, compared to that after UVAR application. This study also reveals that the mucilage of desmids has a limited role in the protection against UVR as demonstrated by the measurements of absorption in the UVR range, in contrast to previous assumptions. Increased UVBR (i.e. high UVBR : PAR ratio) severely decreases oxygen evolution in all of the Cosmarium strains, pointing to possible consequences for peat bogs which are native habitats of desmids, as they are particularly poor in oxygen.

[Contribution of microbiologists of Kirov City to development of penicillin and streptomycin production processes (70 years since development of technology for submerged production of first domestic antibiotics)].

The publication is concerned with development of the technological processes for submered production of the first domestic antibiotics 70 years age. The literature data on the contribution of the microbiologists of the Kirov City and mainly the workers of the Red Army Research Institute of Epidemiology and Hygiene (nowadays Central Research Institute No. 48 of the Ministry of Defense of the Russian Federation, Kirov), to development of the manufacture processes for production of penicillin and streptomycin are reviewed.

Effect of thermal-alkaline pretreatment on the anaerobic digestion of streptomycin bacterial residues for methane production.

The anaerobic digestion of streptomycin bacterial residues, solutions with hazardous waste treatments and bioenergy recovery, was tested in laboratory-scale digesters at 35°C at various organic loading rates (OLRs). The methane production and biomass digestion were efficient at OLRs below 2.33 gVS L(-1) d(-1) but were deteriorated as OLR increased because of the increased total ammonia nitrogen (TAN) concentration from cell protein decay. The thermal-alkaline pretreatment with 0.10 NaOH/TS at 70°C for 2 h significantly improved the digestion performance. With the thermal-alkaline pretreatment, the volumetric reactor productivity and specific methane yield of the pretreated streptomycin bacterial residue increased by 22.08-27.08% compared with those of the unpretreated streptomycin bacterial residue at an OLR of 2.33 gVS L(-1) d(-1). The volatile solid removal was 64.09%, with less accumulation of TAN and total volatile fatty acid.

[Influence of perfluorodecalin on growth of actinomycetes and intensification of Streptomycin and daunorubicin production by the genus Streptomyces kind bacteria in submerged culture].

Addition of perfluorodecalin with gas-transporting function to the liquid medium during submerged cultivation of actinomycetes of the genus Streptomyces resulted in higher intensity and level of the biomass synthesis and increased production of streptomycin and daunorubicin. Addition of perfluorodecalin to the medium provided a 2.0-2.3-fold surpass of the maximum antibiotic production (achieved by the 120th-144th hours of the culture growth) vs. the antibiotic accumulation peaks in the control.

Diversity analysis of streptomycetes and associated phosphotranspherase genes in soil.

An attempt was made to verify the observation that Streptomyces griseus was prevalent in soil based on isolation work. A genus-specific PCR was developed for Streptomyces based on the housekeeping gene atpD and used to investigate species diversity within selected soils. The presence of S. griseus was investigated to determine coexistence of resistance-only streptomycin phosphotransferase (strA) in the same soil as streptomycin producers. Two additional PCR-based assays were developed; one specific for strA in association with production, the other for more diverse strA and other related phosphotranferases. Both the S. griseus atpD and strA genes were below the PCR detection limit in all soils examined. A number of more diverse phosphotransferase genes were amplified, a minority of which may be associated with streptomycin production. We conclude that neither streptomycin producers nor S. griseus are prevalent in the fresh or chitin and starch-amended soils examined (less than 0.1% of soil actinobacteria). One of the soil sites had received plantomycin (active ingredient: streptomycin) and diversity studies suggested that this altered the streptomycete populations present in the soil.

Effect of pamamycin-607 on secondary metabolite production by Streptomyces spp.

The effect of the aerial mycelium-inducing compound, pamamycin-607, on antibiotic production by several Streptomyces spp. was examined. Exposure to 6.6 µM pamamycin-607 stimulated by 2.7 fold the puromycin production by Streptomyces alboniger NBRC 12738, in which pamamycin-607 had first been isolated, and restored aerial mycelium formation. Pamamycin-607 also stimulated the respective production of streptomycin by S. griseus NBRC 12875 and that of cinerubins A and B by S. tauricus JCM 4837 by approximately 1.5, 1.7 and 1.9 fold. The antibiotic produced by Streptomyces sp. 91-a was identified as virginiamycin M(1), and its synthesis was enhanced 2.6 fold by pamamycin-607. These results demonstrate that pamamycin-607 not only restored or stimulated aerial mycelium formation, but also stimulated secondary metabolite production.

Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism.

To construct a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis, the genome of the industrial microorganism Streptomyces avermitilis was systematically deleted to remove nonessential genes. A region of more than 1.4 Mb was deleted stepwise from the 9.02-Mb S. avermitilis linear chromosome to generate a series of defined deletion mutants, corresponding to 83.12-81.46% of the wild-type chromosome, that did not produce any of the major endogenous secondary metabolites found in the parent strain. The suitability of the mutants as hosts for efficient production of foreign metabolites was shown by heterologous expression of three different exogenous biosynthetic gene clusters encoding the biosynthesis of streptomycin (from S. griseus Institute for Fermentation, Osaka [IFO] 13350), cephamycin C (from S. clavuligerus American type culture collection (ATCC) 27064), and pladienolide (from S. platensis Mer-11107). Both streptomycin and cephamycin C were efficiently produced by individual transformants at levels higher than those of the native-producing species. Although pladienolide was not produced by a deletion mutant transformed with the corresponding intact biosynthetic gene cluster, production of the macrolide was enabled by introduction of an extra copy of the regulatory gene pldR expressed under control of an alternative promoter. Another mutant optimized for terpenoid production efficiently produced the plant terpenoid intermediate, amorpha-4,11-diene, by introduction of a synthetic gene optimized for Streptomyces codon usage. These findings highlight the strength and flexibility of engineered S. avermitilis as a model host for heterologous gene expression, resulting in the production of exogenous natural and unnatural metabolites.

Engineering of a genome-reduced host: practical application of synthetic biology in the overproduction of desired secondary metabolites.

Synthetic biology aims to design and build new biological systems with desirable properties, providing the foundation for the biosynthesis of secondary metabolites. The most prominent representation of synthetic biology has been used in microbial engineering by recombinant DNA technology. However, there are advantages of using a deleted host, and therefore an increasing number of biotechnology studies follow similar strategies to dissect cellular networks and construct genome-reduced microbes. This review will give an overview of the strategies used for constructing and engineering reduced-genome factories by synthetic biology to improve production of secondary metabolites.

Activation of secondary metabolite-biosynthetic gene clusters by generating rsmG mutations in Streptomyces griseus.

Unlike other Streptomyces spp., the streptomycin producer Streptomyces griseus IFO13189 shows emergence of a small fraction of rsmG and rpsL mutants among spontaneous low- or high-level streptomycin-resistant mutants. rsmG, but not rpsL, mutants showed greater ability (two- to threefold) to produce streptomycin, accompanied by enhanced transcription of metK and strR, together with streptomycin biosynthetic genes, such as strB1, strD and strF, thus underlying the observed increase in streptomycin production in the rsmG mutants. Moreover, rsmG mutation was effective for activating the 'silent' or poorly expressed secondary metabolite-biosynthetic genes present in S. griseus.

DNA microarray analysis of global gene regulation by A-factor in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is a microbial hormone that triggers morphological differentiation and secondary metabolism in Streptomyces griseus. The effects of A-factor on global gene expression were determined by DNA microarray analysis of transcriptomes obtained with the A-factor-deficient mutant DeltaafsA. A-factor was added at a concentration of 25 ng ml(-1) to mutant DeltaafsA at the middle of the exponential growth phase, and RNA samples were prepared from the cells grown after A-factor addition for a further 5, 15 and 30 min, and 1, 2, 4, 8 and 12 h. The effects of A-factor on transcription of all protein-coding genes of S. griseus were evaluated by comparison of the transcriptomes with those obtained from cells grown in the absence of A-factor. Analysis of variance among the transcriptomes revealed that 477 genes, which were dispersed throughout the chromosome, were differentially expressed during the 12 h after addition of A-factor, when evaluated by specific criteria. Quality threshold clustering analysis with regard to putative polycistronic transcriptional units and levels of upregulation predicted that 152 genes belonging to 74 transcriptional units were probable A-factor-inducible genes. Competitive electrophoretic mobility shift assays using DNA fragments including putative promoter regions of these 74 transcriptional units suggested that AdpA bound 37 regions to activate 72 genes in total. Many of these A-factor-inducible genes encoded proteins of unknown function, suggesting that the A-factor regulatory cascade of S. griseus affects gene expression at a specific time point more profoundly than expected.

Genome sequence of the streptomycin-producing microorganism Streptomyces griseus IFO 13350.

We determined the complete genome sequence of Streptomyces griseus IFO 13350, a soil bacterium producing an antituberculosis agent, streptomycin, which is the first aminoglycoside antibiotic, discovered more than 60 years ago. The linear chromosome consists of 8,545,929 base pairs (bp), with an average G+C content of 72.2%, predicting 7,138 open reading frames, six rRNA operons (16S-23S-5S), and 66 tRNA genes. It contains extremely long terminal inverted repeats (TIRs) of 132,910 bp each. The telomere's nucleotide sequence and secondary structure, consisting of several palindromes with a loop sequence of 5'-GGA-3', are different from those of typical telomeres conserved among other Streptomyces species. In accordance with the difference, the chromosome has pseudogenes for a conserved terminal protein (Tpg) and a telomere-associated protein (Tap), and a novel pair of Tpg and Tap proteins is instead encoded by the TIRs. Comparisons with the genomes of two related species, Streptomyces coelicolor A3(2) and Streptomyces avermitilis, clarified not only the characteristics of the S. griseus genome but also the existence of 24 Streptomyces-specific proteins. The S. griseus genome contains 34 gene clusters or genes for the biosynthesis of known or unknown secondary metabolites. Transcriptome analysis using a DNA microarray showed that at least four of these clusters, in addition to the streptomycin biosynthesis gene cluster, were activated directly or indirectly by AdpA, which is a central transcriptional activator for secondary metabolism and morphogenesis in the A-factor (a gamma-butyrolactone signaling molecule) regulatory cascade in S. griseus.

Conditionally positive effect of the TetR-family transcriptional regulator AtrA on streptomycin production by Streptomyces griseus.

AtrA, a transcriptional activator for actII-ORF4, encoding the pathway-specific transcriptional activator of the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor A3(2), has been shown to bind the region upstream from the promoter of strR, encoding the pathway-specific transcriptional activator of the streptomycin biosynthetic gene cluster in Streptomyces griseus [Uguru et al. (2005) Mol Microbiol 58, 131-150]. The atrA orthologue (atrA-g) in S. griseus was constitutively transcribed throughout growth from a promoter located about 250 nt upstream of the translational start codon, as determined by S1 nuclease mapping. DNase I footprinting showed that histidine-tagged AtrA-g bound an inverted repeat located upstream of strR at positions -117 to -142 relative to the transcriptional start point of strR as +1. This AtrA-g-binding site was between two AdpA-binding sites at approximately nucleotide positions -270 and -50. AdpA is a central transcriptional activator in the A-factor regulatory cascade and essential for the transcription of strR. AtrA-g and AdpA simultaneously bound the respective binding sites. In contrast to AdpA, AtrA-g was non-essential for strR transcription; an atrA-g-disrupted strain produced streptomycin on routine agar media to the same extent as the wild-type strain. However, the atrA-g-disrupted strain tended to produce a smaller amount of streptomycin than the wild-type strain under some conditions, for example, on Bennett agar containing 1 % maltose and on a minimal medium. Therefore, AtrA-g had a conditionally positive effect on streptomycin production, as a tuner, probably by enhancing the AdpA-dependent transcriptional activation of strR in a still unknown manner.

S-Adenosylmethionine induces BldH and activates secondary metabolism by involving the TTA-codon control of bldH expression in Streptomyces lividans.

In the present study, a mechanism for S-adenosylmethionine (SAM) to promote secondary metabolism was characterized in terms of bldH sl) expression in Streptomyces lividans. A previous study demonstrated that SAM, on application at 2 microM, induces the transcription of the strR promoter (strRp), which originates from Streptomyces griseus, in S. lividans. An inactivation study verified that bldH sl is essential to strRp transcription in S. lividans and it was demonstrated that the effects of SAM on the induction of strRp activity, on the transcription of redZ and actII-orf4, and on antibiotic production were compromised when the unique leucine TTA-codon of bldH sl was changed to TTG. Western blot analysis revealed that SAM supplementation enhances the expression of bldH sl when the TTA-codon was intact but not when the TTG replacement was provided. This study validates the involvement of BldH sl in the potentiating effect of SAM on the antibiotic production and substantiates that SAM controls the expression of bldH sl through the TTA-codon control in translating bldH mRNA. It is argued here that the intracellular SAM-level modulates the maturation of bldA, which encodes the UUA-codon tRNA and controls secondary metabolism in S. lividans.