Effects of acuC on the growth development and spinosad biosynthesis of Saccharopolyspora spinosa.

BACKGROUND: Acetoin utilization protein (acuC) is a type I histone deacetylase which is highly conserved in bacteria. The acuC gene is related to the acetylation/deacetylation posttranslational modification (PTM) system in S. spinosa. Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. However, the specific functions and influences of acuC protein in S. spinosa are yet to be characterized. RESULTS: The knockout strain and overexpression strain were constructed separately with the shuttle vector pOJ260. The production of spinosyns A and D from S. spinosa-acuC were 105.02 mg/L and 20.63 mg/L, which were 1.82-fold and 1.63-fold higher than those of the wild-type strain (57.76 mg/L and 12.64 mg/L), respectively. The production of spinosyns A and D from S. spinosa-ΔacuC were 32.78 mg/L and 10.89 mg/L, respectively. The qRT-PCR results of three selected genes (bldD, ssgA and whiA) confirmed that the overexpression of acuC affected the capacities of mycelial differentiation and sporulation. Comparative proteomics analysis was performed on these strains to investigate the underlying mechanism leading to the enhancement of spinosad yield. CONCLUSIONS: This study first systematically analysed the effects of overexpression acuC on the growth of S. spinosa and the production of spinosad. The results identify the differentially expressed proteins and provide evidences to understand the acetylation metabolic mechanisms which can lead to the increase of secondary metabolites.

GlnR and PhoP regulate ß-glucosidases involved in cellulose digestion in response to nitrogen and phosphate availability.

The limited catalytic efficiency of cellulose-degrading enzymes restricts cellulose digestion. We investigated the transcriptional regulation of genes encoding key cellulose degrading enzymes, namely ß-glucosidases, in the industrial actinobacterium Saccharopolyspora erythraea. We observed that the expression of most ß-glucosidase-encoding genes was controlled by the availability of nitrogen and phosphate via their respective global regulators, namely GlnR and PhoP. Electrophoretic mobility shift assay demonstrated that GlnR and PhoP bound directly to the promoters of ß-glucosidase-encoding genes. Deletion of glnR resulted in lower transcript levels and activity of ß-glucosidases, leading to decreased bacterial growth on cellulose. Overexpression of glnR and phoP or nitrogen/phosphate starvation increased the transcript levels and total activity of ß-glucosidases. Moreover, GlnR/PhoP-mediated cellobiose utilization was also observed in Streptomyces coelicolor A3(2). These findings provide insights into the regulatory roles played by GlnR and PhoP in coordinating nitrogen/phosphate metabolism and carbohydrate utilization, and indicate potential strategies for cellulose fermentation in the production of bio-based chemicals by actinobacteria.

Engineering of an Lrp family regulator SACE_Lrp improves erythromycin production in Saccharopolyspora erythraea.

Leucine-responsive regulatory proteins (Lrps) are a group of transcriptional regulators that regulate diverse cellular processes in bacteria and archaea. However, the regulatory role of Lrps in antibiotic biosynthesis remains poorly understood. In this study, we show that SACE_5388, an Lrp family regulator named as SACE_Lrp, is an efficient regulator for transporting and catabolizing branched-chain amino acids (BCAAs), playing an important role in regulating erythromycin production in Saccharopolyspora erythraea. SACE_Lrp directly controlled the expression of the divergently transcribed SACE_5387-5386 operon putatively encoding a BCAA ABC transporter by interacting with the intergenic region between SACE_Lrp and SACE_5387 (SACE_Lrp-5387-int), and indirectly controlled the expression of ilvE putatively encoding an aminotransferase catabolizing BCAAs. BCAA catabolism is one source of the precursors for erythromycin biosynthesis. Lysine and arginine promoted the dissociation of SACE_Lrp from SACE_Lrp -5387-int, whereas histidine increased their binding. Gene disruption of SACE_Lrp (ΔSACE_Lrp) in S. erythraea A226 resulted in a 25% increase in erythromycin production, while overexpression of SACE_5387-5386 in A226 enhanced erythromycin production by 36%. Deletion of SACE_Lrp (WBΔSACE_Lrp) in the industrial strain S. erythraea WB enhanced erythromycin production by 19%, and overexpression of SACE_5387-5386 in WBΔSACE_Lrp (WBΔSACE_Lrp/5387-5386) increased erythromycin production by 41% compared to WB. Additionally, supplement of 10mM valine to WBΔSACE_Lrp/5387-5386 culture further increased total erythromycin production up to 48%. In a 5-L fermenter, the erythromycin accumulation in the engineered strain WBΔSACE_Lrp/5387-5386 with 10mM extra valine in the industrial culture media reached 5001mg/L, a 41% increase over 3503mg/L of WB. These insights into the molecular regulation of antibiotic biosynthesis by SACE_Lrp in S. erythraea are instrumental in increasing industrial production of secondary metabolites.

Immunoreactive proteins of Saccharopolyspora rectivirgula for farmer's lung serodiagnosis.

PURPOSE: Saccharopolyspora rectivirgula is the principal cause of farmer's lung disease (FLD). Serodiagnosis is based on immunoprecipitation techniques or enzyme immunoassays with homemade crude antigens and is not standardized. We aimed to produce specific recombinant antigens for the development of a standardized ELISA. EXPERIMENTAL DESIGN: We recruited 41 patients and 43 healthy exposed controls from five university hospital pneumology departments in France and Switzerland. S. rectivirgula proteins were extracted, separated by 2D electrophoresis, and subjected to Western blotting, with sera from FLD patients or controls. FLD-specific proteins were identified by MS and were produced as recombinant antigens. The diagnostic performance of ELISA tests using the recombinant antigens was assessed with all the sera from FLD patients and controls. RESULTS: We identified 25 FLD-specific proteins, some of which play important roles in transport, nutrition, or virulence. We produced 17 of these proteins as recombinant antigens and assessed their suitability for inclusion in the ELISA test. A combination of three of these proteins (SR1FA, SR17, and SR22) proved remarkably effective at discriminating between patients and controls, with a sensitivity of 83% and a specificity of 77%. CONCLUSIONS AND CLINICAL RELEVANCE: The recombinant antigens produced in this study constitute a major step toward the improvement of diagnostic performance and the standardization of FLD serodiagnosis.

Saccharopolyspora ghardaiensis sp. nov., an extremely halophilic actinomycete isolated from Algerian Saharan soil.

A novel halophilic actinomycete, strain designated H53(T), was isolated from a Saharan soil sample collected from Chaâbet Ntissa, Béni-isguen, Ghardaïa (South of Algeria) and was characterized taxonomically by means of polyphasic approach. Optimal growth was found to occur at 30-35 °C, pH 6-7 and in the presence of 15-25% (w/v) NaCl. The strain was observed to produce abundant aerial mycelium, which formed long chains of rod-shaped spores at maturity, and well developed and fragmented substrate mycelium. The cell wall was determined to contain meso-diaminopimelic acid; the diagnostic whole-cell sugars were arabinose and galactose. The predominant menaquinones were found to be MK-9(H4) and MK-9(H6). The predominant cellular fatty acids were determined to be iso- and anteiso-C17:0, iso-C15:0, and cis9 iso-C17:1. The diagnostic phospholipid detected was phosphatidylcholine. The morphological and chemotaxonomic characteristics of the strain were consistent with those of members of the genus Saccharopolyspora. Phylogenetic analyses on the basis of the 16S ribosomal RNA (rRNA) gene sequence showed that this strain formed a distinct phyletic line within the radiation of the genus Saccharopolyspora. The 16S rRNA sequence similarities between strain H53(T) and other members of the genus Saccharopolyspora ranged from 92.1 to 94.3%. The DNA G+C content of strain H53(T) was 72.6%. The genotypic and phenotypic data showed that the strain H53(T) represents a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora ghardaiensis sp. nov. is proposed, with the type strain H53(T) (=DSM 45606(T)=CCUG 63370(T)=CECT 8304(T)).

TLR2 regulates neutrophil recruitment and cytokine production with minor contributions from TLR9 during hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is an interstitial lung disease that develops following repeated exposure to environmental antigens. The disease results in alveolitis, granuloma formation and may progress to a fibrotic chronic form, which is associated with significant morbidity and mortality. The severity of the disease correlates with a neutrophil rich influx and an IL-17 response. We used the Saccharopolysporarectivirgula (SR) model of HP to determine whether Toll-like receptors (TLR) 2 and 9 cooperate in neutrophil recruitment and IL-17-associated cytokine production during the development of HP. Stimulation of bone marrow derived macrophages (BMDMs) from C57BL/6, MyD88(-/-) and TLR2/9(-/-) mice with SR demonstrate that SR is a strong inducer of neutrophil chemokines and growth factors. The cytokines induced by SR were MyD88-dependent and, of those, most were partially or completely dependent on TLRs 2 and 9. Following in vivo exposure to SR, CXCL2 production and neutrophil recruitment were reduced in TLR2(-/-) and TLR2/9(-/-) mice suggesting that the response was largely dependent on TLR2; however the reduction was greatest in the TLR2/9(-/-) double knockout mice indicating TLR9 may also contribute to the response. There was a reduction in the levels of pro-inflammatory cytokines TNFα and IL-6 as well as CCL3 and CCL4 in the BALF from TLR2/9(-/-) mice compared to WT and single knockout (SKO) mice exposed one time to SR. The decrease in neutrophil recruitment and TNFα production in the TLR2/9(-/-) mice was maintained throughout 3 weeks of SR exposures in comparison to WT and SKO mice. Both TLRs 2 and 9 contributed to the Th17 response; there was a decrease in Th17 cells and IL-17 mRNA in the TLR2/9(-/-) mice in comparison to the WT and SKO mice. Despite the effects on neutrophil recruitment and the IL-17 response, TLR2/9(-/-) mice developed granuloma formation similarly to WT and SKO mice suggesting that there are additional mediators and pattern recognition receptors involved in the disease.

Saccharopolyspora dendranthemae sp. nov. a halotolerant endophytic actinomycete isolated from a coastal salt marsh plant in Jiangsu, China.

A halotolerant actinomycete strain, designated strain KLBMP 1305(T), was isolated from a salt marsh plant Dendranthema indicum (Linn.) Des Moul collected from the coastal region of Nantong, Jiangsu Province, in east China and was studied in detail for its taxonomic position. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain KLBMP 1305(T) is a member of the genus Saccharopolyspora. The 16S rRNA gene sequence similarity indicated that strain KLBMP 1305(T) was most closely related to 'Saccharopolyspora pathumthaniensis' S582(T) (99.31 %), 'Saccharopolyspora endophytica' YIM 61095(T) (99.17 %) and Saccharopolyspora tripterygii YIM 65359(T) (99.15 %); similarity to other type strains of the genus Saccharopolyspora was <97.2 %. The organism had chemical and morphological features consistent with its classification in the genus Saccharopolyspora such as meso-diaminopimelic acid as the diagnostic diamino acid in the cell wall peptidoglycan and arabinose and galactose as the diagnostic sugars. The predominant menaquinone was MK-9(H4). The polar lipids detected were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unknown glycolipid and an unknown lipid. The major fatty acids were iso-C16:0, iso-C15:0, anteiso-C15:0, anteiso-C17:0 and sum in feature 8 (18:1ω7c/18:1ω6c). The G+C content of the genomic DNA of the type strain was 68.7 mol%. DNA-DNA relatedness data, together with phenotypic differences, clearly distinguished the isolate from its closest relatives. On the basis of these phenotypic and genotypic data, the isolate represents a novel species, for which the name Saccharopolyspora dendranthemae sp. nov. is proposed. The type strain is KLBMP 1305(T) (=KCTC 19889(T) = NBRC 108675(T)).

An assessment of seed quality on erythromycin production by recombinant Saccharopolyspora erythraea strain.

An assessment of seed quality on erythromycin production by recombinant strain Saccharopolyspora erythraea ZL1004 was investigated in 15 l fermenter. Adding 10 g/l corn steep liquor and 30 g/l soybean flour in seed medium were beneficial to improve cell growth, and the maximal biomass reached 36% at 40 h. Enzyme activity in cell showed that the maximal protease and minimum amylase were appeared in this stage. Compared with the control in 50 l fermenter, the cell metabolism with inoculation of the optimized seed cultivation was obviously quicker, and physiological response such as oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) were also improved. The maximal erythromycin A production was 9160 U/ml at 215 h, which was increased by 21.63% with respect to the control. It was the first report to integrate cell growth characteristics and physiological response method to assess the seed quality for erythromycin production.

Saccharopolyspora gloriosae sp. nov., an endophytic actinomycete isolated from the stem of Gloriosa superba L.

A Gram-stain-positive, aerobic actinomycete, strain YIM 60513(T), was isolated from the stem of Gloriosa superba L. collected from tropical rainforest at Xishuangbanna, Yunnan Province, south-west China. 16S rRNA gene sequence analysis indicated that strain YIM 60513(T) belonged to the genus Saccharopolyspora and was closely related to Saccharopolyspora gregorii NCIB 12823(T) (99.1 % similarity) and Saccharopolyspora cebuensis SPE 10-1(T) (97.3 % similarity). Data for the predominant quinone [MK-9(H(4))], major fatty acids (iso-C(16 : 0), anteiso-C(17 : 0) and C(17 : 1) cis9) and G+C content of the genomic DNA (71.6 mol%) were similar to those for members of the genus Saccharopolyspora. The level of DNA-DNA relatedness between strain YIM 60513(T) and S. gregorii NCIB 12823(T) was 43 %. The combination of phylogenetic analysis, phenotypic differences, chemotaxonomic characteristics and DNA-DNA hybridization data supported the view that strain YIM 60513(T) should be distinguished from S. gregorii NCIB 12823(T) and S. cebuensis SPE 10-1(T). Strain YIM 60513(T) therefore represents a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora gloriosae sp. nov. is proposed. The type strain is YIM 60513(T) (=KCTC 19243(T) =CCTCC AA 207006(T)).

[Conditions for protoplast preparation of spinosyn-producing strain and the physiological properties of protoplast-regenerated strains].

To improve spinosyn-producing strain and enhance spinosyns yield, we studied the effects of glycin concentration and the operational time, temperature and lysozyme concentration on protoplast preparation of Saccharopolyspora spinosa SP06081. We also studied different regeneration media and osmotic stabilizing agents. In addition, we compared the change of morphology and spinosyns yield of the regenerated strains. The results showed that the Saccharopolyspora spinosa SP06081 protoplast yield was the highest under these conditions: the collected mycelium from SP06081 grown in Tryptic Soy Broth (TSB) medium with 0.2% glycin for 48 h was treated by 0.1 mg/mL lysozyme at 28 degrees C for 20 min, then plated on the R2YE medium with sucrose as osmotic stabilizer, the number of regeneration protoplast was up to 10(8)/mL. The protoplast-regenerated strains exhibited changes in morphology and antibiotic production, 29.3% protoplast-regenerated strains was characterized by loose mycelium and abundant broken branches as did their parent. Among them, 58.2% strains presented the trend to positive variation in spinosad yield, with the highest spinosad yield of up to 582.0 mg/L, 85.6% higher than that of their parent. There is significant correlation between the morphological differentiation and antibiotic yield of the protoplast-regenerated strains from spinosyn-producing strain.

Saccharopolyspora jiangxiensis sp. nov., isolated from grass-field soil.

An actinomycete isolated from soil from a field of grass was analysed using a polyphasic taxonomic approach. The organism, designated strain W12(T), was found to have a range of chemical and morphological properties consistent with its classification in the genus Saccharopolyspora. An almost-complete 16S rRNA gene sequence determined for the strain was aligned with corresponding sequences for representatives of the genus Saccharopolyspora, using two tree-making algorithms. The novel organism formed a monophyletic clade with the type strain of Saccharopolyspora antimicrobica, but the DNA-DNA relatedness value was low. Strain W12(T) also had a phenotypic profile that readily distinguished it from recognized representatives of the genus Saccharopolyspora. It is evident from the combined genotypic and phenotypic data that the new organism should be classified as a novel species of the genus Saccharopolyspora. The name proposed for this new taxon is Saccharopolyspora jiangxiensis sp. nov., with strain W12(T) (=CGMCC 4.3529(T)=JCM 14613(T)) as the type strain.

Saccharopolyspora rosea sp. nov., isolated from a patient with bronchial carcinoma.

The taxonomic status of a bacterium isolated from a patient with bronchial carcinoma was established using a polyphasic taxonomic approach. The strain had morphological and chemotaxonomic characteristics consistent with those of members of the genus Saccharopolyspora. The generic assignment was confirmed by comparative analysis of the 16S rRNA gene sequence, which showed that the strain constituted a distinct phyletic line, displaying 93.5-96.9 % sequence similarity with respect to members of the genus Saccharopolyspora. The isolate was distinguished from the type strains of recognized members of the genus Saccharopolyspora by means of various biochemical tests. The genotypic and phenotypic data obtained demonstrated that strain IMMIB L-1070(T) represents a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora rosea sp. nov. is proposed. The type strain is IMMIB L-1070(T) (=DSM 45226(T)=CCUG 56401(T)).

Saccharopolyspora endophytica sp. nov., an endophytic actinomycete isolated from the root of Maytenus austroyunnanensis.

A Gram-positive, aerobic, non-motile, non-acid-alcohol-fast strain, designated YIM 61095(T), was isolated from the root of Maytenus austroyunnanensis collected from a tropical rainforest of Xishuangbanna in Yunnan Province, south-west China. Strain YIM 61095(T) exhibited chemotaxonomic and morphological characteristics that were consistent with members of the genus Saccharopolyspora. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 61095(T) was a member of the genus Saccharopolyspora and was most closely related to Saccharopolyspora flava AS 4.1520(T) (97.7% sequence similarity). The major fatty acids were iso-C(15:0), iso-C(16:0), iso-C(17:0) and anteiso-C(17:0). The predominant quinone detected was MK-9(H(4)). The DNA G+C content was 66.2 mol%. The phenotypic characteristics and DNA-DNA hybridization relatedness data indicated that strain YIM 61095(T) should be distinguished from Saccharopolyspora flava AS 4.1520(T). On the basis of the evidence presented in this study, strain YIM 61095(T) represents a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora endophytica sp. nov. is proposed. The type strain is YIM 61095(T) (=KCTC 19397(T)=CCTCC AA 208003(T)).

Saccharopolyspora shandongensis sp. nov., isolated from wheat-field soil.

A polyphasic study was undertaken to establish the taxonomic status of a wheat-field soil isolate. The organism, strain 88T, was found to have a range of chemical and morphological properties consistent with its classification in the genus Saccharopolyspora. Phylogenetic analysis based on an almost-complete 16S rRNA gene sequence of the strain showed that it formed a monophyletic clade with the type strain of Saccharopolyspora spinosa. However, the two strains shared low DNA-DNA relatedness. Strain 88T also had a profile of phenotypic properties that readily distinguished it from representatives of the species of Saccharopolyspora with validly published names. It is evident from the combination of genotypic and phenotypic data that the organism should be classified within a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora shandongensis sp. nov. is proposed. The type strain is strain 88T (=CGMCC 4.3530T =JCM 14614T).

Saccharopolyspora antimicrobica sp. nov., an actinomycete from soil.

Three Gram-positive, aerobic, non-motile, non-acid-alcohol-fast strains, designated I05-00051, I05-00074T and I03-00808, were isolated from different soil samples in Beijing and Sichuan, China. Phylogenetic analysis based on 16S rRNA gene sequences and DNA-DNA hybridization experiments revealed that these three isolates represented the same genospecies. These three strains showed <97.0 % 16S rRNA gene sequence similarity with the type strains of recognized species of the genus Saccharopolyspora, with the exception of Saccharopolyspora hirsuta subsp. hirsuta DSM 43463T (98.1 % gene sequence similarity) and Saccharopolyspora spinosa DSM 44228T (98.0 % similarity). Chemotaxonomic data, including meso-diaminopimelic acid as the diagnostic diamino acid, arabinose and galactose as predominant sugars, iso-C15 : 0, iso-C16 : 0, iso-C17 : 0 and anteiso-C17 : 0 as major fatty acids, MK-9(H4) as predominant menaquinone and polar lipids dominated by diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol, supported the affiliation of these three organisms to the genus Saccharopolyspora. The genomic DNA G+C contents of the three isolates were 68.2-69.9 mol%. The results of DNA-DNA hybridization experiments among these three isolates and S. hirsuta subsp. hirsuta DSM 43463T and S. spinosa DSM 44228T, in combination with chemotaxonomic and physiological data, demonstrated that the three new isolates represent a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora antimicrobica sp. nov. is proposed. The type strain is I05-00074T (=CCM 7463T=KCTC 19303T).

Intelligent modelling of bioprocesses: a comparison of structured and unstructured approaches.

This contribution moves in the direction of answering some general questions about the most effective and useful ways of modelling bioprocesses. We investigate the characteristics of models that are good at extrapolating. We trained three fully predictive models with different representational structures (differential equations, differential equations with inheritance of rates and a network of reactions) on Saccharopolyspora erythraea shake flask fermentation data using genetic programming. The models were then tested on unseen data outside the range of the training data and the resulting performances were compared. It was found that constrained models with mathematical forms analogous to internal mass balancing and stoichiometric relations were superior to flexible unconstrained models, even though no a priori knowledge of this fermentation was used.

[Exposure to Saccharopolyspora rectivirgula among cattle breeders in the province of Reggio Emilia and the risk of extrinsic allergic alveolitis (farmer's lung)]. / Esposizione a Saccharopolyspora rectivirgula degli allevatori di bestiame bovino della provincia di Reggio Emilia e rischio di alveolite allergica estrinseca (Farmer's Lung).

INTRODUCTION: Nearly 2.350 dairy farms (and 137.000 milk cows) are located in the province of Reggio Emilia, Italy, to produce the famous Parmigiano-Reggiano" cheese. Feeding is hay-based both in the cold season and (together with grazing) in the warm season. This requires a large production of hay and frequent handling by the farmers. Hay is packed in large cylindrical bales, "round bales" (nearly 2.41 m3), or, rarely, in traditional small prisms-shaped bales (about 0.15 m3), only used on small farms. We estimated there were 6.000-9.000 the workers exposed to hay dust. The risks for the farmer's health due to the hay dust exposure are well known; in particular Farmer's Lung disease (FL) is rather frequent in this Region (1.5%-3.0% among people exposed). We studied hay and air pollution by Saccharopolyspora Rectivirgula (SR) in relation to these two different hay-packing techniques (hay dried in the open air) both in flat and in hilly areas. METHODS: On 56 cattle-farms, hay and air samples were collected and analyzed using a six-stage Andersen sampler and a sedimentation chamber (SC) for hay samples with plastic Petri dishes containing culture medium. RESULTS: Round bales were richer in SR spores than the small prism-shaped bales (n = 37, mean = 6.20 logn ufc/m3 in SC, ds: 3.87 vs n = 15, mean = 2.40 logn ufc/m3 in SC; ds: 4.16) and they seem to produce higher air pollution (n = 30, mean = 5.30 logn ufc/m3; ds: 3.71 vs n = 15, mean = 2.32 logn ufc/m3; ds: 2.99). In hilly areas the pollution produced by round bales (in hay and air) was higher than in flat areas. On the contrary hay from small bales produced in hilly areas was poorest in SR spores. CONCLUSIONS: An heavy exposure to actinomycetes spores, therefore, comes from "round bales" hay handling, especially when the bales are produced in mountain areas. New drying systems, probably, can reduce this risk and raise hay quality.

Decreasing the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338 leads to increased resistance to breakage and increased antibiotic production.

Mutation and selection for increased resistance to cell-wall synthesis inhibitors led to alterations in the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338. Mutants with decreased branching frequency exhibited increased hyphal strength (estimated by in vitro micromanipulation). As the hyphal strength was increased, this led to a greater proportion of hyphal particles in liquid culture with a hyphal fragment diameter of greater than 88 microm. This, in turn, coincided with proportionately increased antibiotic production.

Viability, strength, and fragmentation of Saccharopolyspora erythraea in submerged fermentation.

Two fermentations of the commercially important erythromycin-producing filamentous bacterium Saccharopolyspora erythraea were conducted in defined media. One was glucose-limited and the other nitrate-limited. The viability of the hyphae was determined using the fluorescent stain BacLight (Molecular Probes, Eugene, OR). Also, the force required to strain hyphae to breakage was determined using micromanipulation and a sensitive force transducer. In both fermentations, fragmentation coincided with the appearance of regions in the mycelia with permeabilised membranes (considered nonviable). Under glucose-limitation, hyphal breaking force rose to 1,050 +/- 130 nN at the end of the growth phase and fell to an undetectable value as a result of glucose exhaustion. Under nitrate-limitation, hyphal breaking force fell from 900 +/- 160 nN during the growth phase to 550 +/- 40 nN in the stationary phase. In both cases image analysis showed that the dimensions of mycelia were of the same order, suggesting that the major factor influencing fragmentation was the appearance of nonviable regions (assumed to be weak). The location in which nonviable regions first appear within hyphae could not be determined because of their appearance coinciding with fragmentation.

Rapid and sensitive quantitation of antibiotics in fermentations by electrospray mass spectrometry.

Electrospray ionisation mass spectrometry (ESI-MS) has been used for the determination and quantitation of a broad range of 24 antibiotics, from groups including aminoglycosides, beta-lactams, tetracyclines, antifungals and glycopeptides. Spectra have been acquired for all 24 antibiotics derived from pure samples dissolved in acetonitrile/water, along with samples extracted from complex fermentation liquor. Quantitation was carried out by the detection of the protonated molecules, using time-scheduled single-ion monitoring (SIM). ESI-MS was used to detect and quantify to 5-microM levels. A one-step extraction of antibiotics with an organic solvent (methanol) was used for this rapid and simple procedure. Specificity is not matched by other methods and antibiotic analogues (e.g. the five forms of erythromycin) can be determined within minutes.