Amino acid precursor supply in the biosynthesis of the RNA polymerase inhibitor streptolydigin by Streptomyces lydicus

Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the Streptomyces lydicus streptolydigin gene cluster slgE1-slgE2-slgE3 are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to slgE3, housekeeping NADPH- and ferredoxin-dependent glutamate synthase genes have been identified in S. lydicus. The expression of slgE3 is increased up to 9-fold at the onset of streptolydigin biosynthesis and later decreases to ～2-fold over the basal level. In contrast, the expression of housekeeping glutamate synthases decreases when streptolydigin begins to be synthesized. SlgE1 and SlgE2 are the two subunits of a glutamate mutase that would convert glutamate into 3-methylaspartate. Deletion of slgE1-slgE2 led to the production of two compounds containing a lateral side chain derived from glutamate instead of 3-methylaspartate. Expression of this glutamate mutase also reaches a peak increase of up to 5.5-fold coinciding with the onset of antibiotic production. Overexpression of either slgE3 or slgE1-slgE2 in S. lydicus led to an increase in the yield of streptolydigin.     

Localization of mutation leading to resistance of E. coli RNA polymerase to the antibiotic streptolydigin in the gene rpoB coding for the beta-subunit of the enzyme

For the first time a mutation of streptolydigin resistance was localized. It was discovered to be a double substitution, namely Gly544----Asp, Phe545----Ser, in the region where most rif-r mutations are located. One may suppose that this region takes part in the formation of both elongation NTP binding site, blocked by streptolydigin, and RNA chain binding and translocation site that is blocked by rifampicin.     

Wood Chip-Polyacrylamide Medium for Biocontrol Bacteria Decreases Verticillium dahliae Infection on Potato

The lack of consistent success of biological control of soilborne plant pathogens may be due to the introduction of the organism into a foreign environment. We hypothesized that wood chip-polyacrylamide (PAM) cores surrounding host plant roots could alter the soil environment to favour growth of introduced biocontrol microorganisms, thereby reducing Verticillium dahliae infection of potato ( Solanum tuberosum L.) in a greenhouse. A 7cm diameter ×15cm deep hole (core) was drilled in the center of a 20 ×30cm deep pot (1.9 kg) containing soil infested with V. dahliae inoculum. Cores were then filled with wood chip-PAM-biocontrol organism mixtures. Soils that had Streptomyces lydicus inoculated into wood chip-PAM cores had lower levels of V. dahliae symptoms ( V vis ) and V. dahliae isolations ( V iso ) than all other treatments in three soils. V vis and V iso on plants growing in soils amended with S. lydicus or Pseudomona corrugata inoculated into the soil itself (without wood chip-PAM cores) did not differ from soils that were unamended with these biocontrol organisms. V. dahliae biomass was lower in wood chip-PAM cores inoculated with S. lydicus than control or wood chip-PAM cores without biocontrol bacteria. Soils with wood chip-PAM cores inoculated with S. lydicus or P. corrugata generally had higher microbial biomass/ V. dahliae biomass (MB/VB) ratios than control soils, or soils with S. lydicus or P. corrugata inoculated into the soil. Wood chipPAM cores alone and wood chip-PAM cores inoculated with S. lydicus had higher MB/VB ratios than wood chip-PAM cores inoculated with P. corrugata . V vis and V iso were curvilinearly correlated with the MB/VB ratios in negative relationships, respectively (r 2 = 0.68, r 2 = 0.68). As the MB/VB ratio increased, V vis and V iso decreased. Although field studies and economic evaluations are necessary, amending soil with wood chips-PAM and a biocontrol bacterium may be a valuable method to increase the effectiveness of biocontrol organisms.     

<Emphasis Type="Italic">fabC</Emphasis> of <Emphasis Type="Italic">Streptomyces lydicus</Emphasis> involvement in the biosynthesis of streptolydigin

Streptolydigin, a secondary metabolite produced by Streptomyces lydicus, is a potent inhibitor of bacterial RNA polymerases. It has been suggested that streptolydigin biosynthesis is associated with polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS). Thus, there is great interest in understanding the role of fatty acid biosynthesis in the biosynthesis of streptolydigin. In this paper, we cloned a type II fatty acid synthase (FAS II) gene cluster of fabDHCF from the genome of S. lydicus and constructed the SlyfabCF-disrupted mutant. Sequence analysis showed that SlyfabDHCF is 3.7 kb in length and encodes four separated proteins with conserved motifs and active residues, as shown in the FAS II of other bacteria. The SlyfabCF disruption inhibited streptolydigin biosynthesis and retarded mycelial growth, which were likely caused by the inhibition of fatty acid synthesis. Streptolydigin was not detected in the culture of the mutant strain by liquid chromatography–mass spectrometry. Meanwhile, the streptolol moiety of streptolydigin accumulated in cultures. As encoded by fabCF, acyl carrier protein (ACP) and β-ketoacyl-ACP synthase II are required for streptolydigin biosynthesis and likely involved in the step between PKS and NRPS. Our results provide the first genetic and metabolic evidence that SlyfabCF is shared by fatty acid synthesis and antibiotic streptolydigin synthesis.     

Metabolic analysis reveals the amino acid responses of Streptomyces lydicus to pitching ratios during improving streptolydigin production

Pitching ratio has been reported to impact not only on the primary metabolism, but also the secondary metabolism. Comparative metabolomics was used to explore the metabolic responses of Streptomyces lydicus E9 to pitching ratios (1, 10, and 30 %, v/v). We identified more than 120 metabolites involved in glycolysis, tricarboxylic acid cycle, and amino acid and secondary metabolism, of which there are significant differences in the quantified 32 metabolites under different pitching ratios by gas chromatography coupled to time-of-flight mass spectrometry. The intracellular levels of most amino acids (e.g., valine, alanine, and isoleucine) declined with the increases of pitching ratios. Especially, the relative abundances of glutamate and proline were not only decreased with the increases of pitching rations, but also had much low level at stages II and III, which might be related to the significant enhancement in streptolydigin of S. lydicus E9 under 30 % high pitching ratio. Moreover, principal component analysis revealed that eight metabolites, including glucopyranoside, maltose, cAMP, glycine, proline, lysine, isoleucine, and valine, were considered as potential biomarkers to distinguish the influences of pitching ratios on streptolydigin production. Further investigations demonstrated that the additions of exogenous glutamate and proline (100 mg?L^?1) enhanced significantly the accumulation of streptolydigin, indicating that glutamate was the synthetic precursor of streptolydigin, while proline in S. lydicus E9 was converted into glutamate and consequently improved streptolydigin biosynthesis. Therefore, these findings provide new insights into the amino acid responses of S. lydicus E9 to pitching ratios and provide potential strategies to improve streptolydigin production.     

Novel plant-microbe rhizosphere interaction involving Streptomyces lydicus WYEC108 and the pea plant (Pisum sativum)

A previously undescribed plant-microbe interaction between a root-colonizing Streptomyces species, S. lydicus WYEC108, and the legume Pisum sativum is described. The interaction is potentially of great importance to the health and growth in nature of this nodulating legume. The root-colonizing soil actinomycete S. lydicus WYEC108 influences pea root nodulation by increasing root nodulation frequency, possibly at the level of infection by Rhizobium spp. S. lydicus also colonizes and then sporulates within the surface cell layers of the nodules. Colonization leads to an increase in the average size of the nodules that form and improves the vigor of bacteroids within the nodules by enhancing nodular assimilation of iron and possibly other soil nutrients. Bacteroid accumulation of the carbon storage polymer, poly-beta-hydroxybutyrate, is reduced in colonized nodules. Root nodules of peas taken from agricultural fields in the Palouse hills of northern Idaho were also found to be colonized by actinomycete hyphae. We hypothesize that root and nodule colonization is one of several mechanisms by which Streptomyces acts as a naturally occurring plant growth-promoting bacterium in pea and possibly other leguminous plants.     

RNA polymerase is required for DNA initiation in vitro

Summary We have previously reported in vitro complementation assays for chromosome initiation that enable dnaA and dnaC mutant extracts to synthesize DNA. To examine the role of RNA polymerase in chromosome initiation, inhibitors of the enzyme and anti-RNA polymerase antibody were used. Though rifampicin failed to efficiently inhibit ribonucleoside triphosphate polymerization under the assay conditions, both streptolydigin and anti-RNA plymerase antibody abolished ribonucleic acid synthesis completely. Antibody effectively inhibited chromosome initiation in the dnoA mutant based reaction but streptolydigin did not. Neither streptolydigin nor antibody affected the dnaC-dependent assay. It was concluded that RNA polymerase is required for initiation but not necessarily to polymerize a polyribonucleotide. A scheme for the sequence of initiation events is presented.     

Insights into the roles of exogenous glutamate and proline in improving streptolydigin production of Streptomyces lydicus with metabolomic analysis

The addition of precursors was one strategy to improve antibiotic production. The exogenous proline and glutamate, as precursors of streptolydigin, could significantly improve the streptolydigin production, but their underlying molecular mechanisms remain unknown. Herein, metabolomic analysis was carried out to explore the metabolic responses of Streptomyces lydicus to the additions of proline and glutamine. The significant differences in the quantified 53 metabolites after adding the exogenous proline and glutamate were enunciated by gas chromatography coupled to time-of-flight mass spectrometry. Among them, the levels of some fatty acids (e.g., dodecanoic acid, octadecanoic acid, hexadecanoic acid) were significantly decreased after adding glutamate and proline, indicating that the inhibition of fatty acid synthesis might be benefit for the accumulation of streptolydigin. Particularly, the dramatic changes of the identified metabolites, which are involved in glycolysis, the tricarboxylic acid cycle, and the amino acid and fatty acid metabolism, revealed that the additions of glutamate and proline possibly caused the metabolic cross-talk in S. lydicus. Additionally, the level of intracellular glutamate dramatically enhanced at 12 h after adding proline, showing that exogenous proline may be firstly convert into glutamate and consequently result in crease of the streptolydigin production. The high levels of streptolydigin at 12 and 24 h after adding glutamate unveiled that part glutamate were rapidly used to synthesize the streptolydigin. Furthermore, there is the significant difference in metabolomic characteristics of S. lydicus after adding glutamate and proline, uncovering that multiple regulatory pathways are involved in responses to the additions of exogenous glutamate and proline. Taken together, exogenous glutamate and proline not only directly provided the precursors of streptolydigin biosynthesis, but also might alter the metabolic homeostasis of S. lydicus E9 during improving the production of streptolydigin.     

Streptomyces lydicus A_(01) affects soil microbial diversity,improving growth and resilience in tomato

The actinomycete Streptomyces lydicus A_(01) promotes tomato seedling growth; however, the underlying mechanism is unclear. In this study, we investigated whether changes in soil microbial diversity, following Streptomyces lydicus A_(01) treatment, were responsible for the increased tomato seedling growth. Eukaryotic 18 S ribosomal DNA(rDNA) sequencing showed that S. lydicus A_(01)-treated and untreated soil shared 193 operational taxonomic units(OTUs), whereas bacterial 16 S rDNA sequencing identified 1,219 shared OTUs between the treated and untreated soil. Of the 42 dominant eukaryotic OTUs, eight were significantly increased and six were significantly decreased after A_(01) treatment. Of the 25 dominant bacterial OTUs, 12 were significantly increased and eight were significantly decreased after A_(01) treatment.Most of the eukaryotes and bacteria that increased in abundance exhibited growth promoting characteristics,which were mainly predicted to be associated with mineralization of nitrogen and phosphorus, phosphate solubilization, nutrient accumulation, and secretion of auxin, whereas some were related to plant protection,such as the degradation of toxic and hazardous substances. Soil composition tests showed that S. lydicus A_(01) treatment enhanced the utilization of nitrogen, phosphorus, and potassium in tomato seedlings. Thus, microbial fertilizers based on S. lydicus A_(01) may improve plant growth, without the detriment effects of chemical fertilizers.     

Use of randomly amplified polymorphic DNA as a means of developing genus- and strain-specific Streptomyces DNA probes

We have analyzed 20 randomly amplified polymorphic DNA (RAPD) primers against 36 Streptomyces strains, including 17 taxonomically undefined strains, 25 nonstreptomycete actinomycetes, and 12 outgroups consisting of gram-positive and -negative species. Most of the primers were useful in identifying unique DNA polymorphisms of all strains tested. We have used RAPD techniques to develop a genus-specific probe, one not necessarily targeting the ribosomal gene, for Streptomyces, and a strain-specific probe for the biological control agent Streptomyces lydicus WYEC108. In the course of these investigations, small-scale DNA isolations were also developed for efficiently isolating actinomycete DNA. Various modifications of isolation procedures for soil DNA were compared, and the reliability and specificity of the RAPD methodology were tested by specifically detecting the S. lydicus WYEC108 in DNA isolated from soil.     

Isolation and Characterization of Rifampin-Resistant and Streptolydigin-Resistant Mutants of Bacillus subtilis with Altered Sporulation Properties

Mutants of Bacillus subtilis with altered deoxyribonucleic-dependent ribonucleic acid polymerase activity have been isolated and characterized. These mutants, selected as strains resistant to rifampin or streptolydigin, demonstrate drug-resistant in vitro ribonucleic acid synthesis. Sporeforming ability and support of phage infection are altered in many of the mutants. Mutations to rifampin and streptolydigin resistance have been located on the B. subtilis chromosome and ordered relative to the markers cysA14 and str.     

Effect of Preirradiation Ribonucleic Acid Synthesis Inhibition on Resistance to Ultraviolet Light with Resistant and Sensitive Strains of Escherichia coli B/r

The ultraviolet resistance of a streptolydigin-susceptible strain of Escherichia coli B/r hcr(-) increased during preirradiation treatment with streptolydigin (an inhibitor of deoxyribonucleic acid-dependent ribonucleic acid polymerase) for 20 min and then remained constant. During preirradiation treatment with chloramphenicol (an inhibitor of protein synthesis), resistance to ultraviolet light increased for 1 to 2 h, and reached a maximal level significantly above that attained in streptolydigin-containing medium. These results suggest that there are two mechanisms that function in Hcr(-) cells during chloramphenicol treatment which contribute to the concomitant ultraviolet resistance enhancement. One is ribonucleic acid dependent and is inhibited by streptolydigin. This ribonucleic acid-dependent mechanism appears to be absent in wild-type and RecA E. coli B/r strains.     

Investigation of proteomic responses of Streptomyces lydicus to pitching ratios for improving streptolydigin production

Streptomyces lydicus has been reported to produce antibiotic streptolydigin. Pitching ratios play crucial roles in primary and secondary metabolism of Streptomyces bacteria. The higher pitching ratio (30%, v/v) significantly enhanced the levels of streptolydigin products in S. lydicus. Proteome analysis revealed that betaglucosidase and UTP-glucose-1-phosphate uridylyltransferase were up-regulated to accelerate the starch hydrolyzation at the high pitching ratios. Enhancement in the levels of UDPN-acetylmuramoylalanyl-D-glutamate-2, 6-diaminopimelate ligase and glycine cleavage system aminomethyltransferase were involved in the conversion of amino acids into secondary metabolites. Additionally, the expression levels of PfkA2, PfkA3, Zwf2, SucD, GalE1, GatB, TktA1 and ThcA, associated with glycolysis, pentose phosphate pathway, TCA cycle and amino acid metabolism, were dramatically elevated at high pitching ratios, which play important roles in the enhanced streptolydigin production in S. lydicus E9. Interestingly, the levels of proteins (glutamine synthetase I, glutamate synthase subunit beta and glutamine synthetase) were down-regulated with the increases of pitching ratios and fermentation progress, revealing that pitching ratio altered the glutamine synthetase levels and consequently regulated the streptolydigin production of S. lydicus E9. The up-regulation of proteins (eg, aldehyde dehydrogenase and alkyl hydroperoxide reductase) was involved in the redox-based regulation network triggered by an imbalance of the intracellular cell redox homeostasis and by crosstalk with secondary metabolism at the higher pitching ratio. These results settle new insights into physiological facts of S. lydicus E9 in responses to pitching ratios and will eventually improve the antibiotic production schemes in industry.     

NOVEL Escherichia coli dnaB mutant: direct involvement of the dnaB252 gene product in the synthesis of an origin-ribonucleic acid species during initiaion of a round of deoxyribonucleic acid replication.

The initiation process of deoxyribonucleic acid (DNA) replication in Escherichia coli has been studied using the thermoreversible dna initiation mutant E. coli HfrHl65/120/6 dna-252. This dna mutation was incorrectly classed as a dnaA mutation. Biochemical and genetic evidence suggests that the dna-252 mutant is a novel dnaB mutant, possessing phenotypic properties which distinguish it from other dnaB mutants. Sensitivity of reinitiation in the dna-252 mutant to specific inhibitors of protein, ribonucleic acid (RNA), and DNA synthesis was studied. Reinitiation is shown to be sensitive to rifampin and streptolydigin but not to cholramphenicol. Thus, the dna-252 gene product appears to be required during the initiation process for a step occurring either before or during synthesis of an RNA species (origin-RNA). Using reversible inhibition of RNA synthesis by streptolydigin of a streptolydigin-sensitive derivative of the dna-252 mutant, the dna-252 gene product is shown to be directly involved in the synthesis of an orgin-RNA species. These results are included in a schematic model presented in the accompanying paper of the temporal sequence of events occurring during the initiation process.     

Identification of some streptomycetes producing oxytetracycline

A study was made of Streptomyces rimosus and mutant strains to compare the phenotype of high and low oxytetracycline producers. Strains were identified using a probabilistic identification matrix for the genus Streptomyces. Mutant strains separated into two groups: high-titre strains and blocked mutants. The former identified with the S. rimosus cluster whereas the latter were not identified. Two further oxytetracycline producers identified with the Streptomyces lydicus cluster.