Glyn Hobbs

Dispersed growth of Streptomyces in liquid culture

SummaryThe study of the physiology of the filamentous bacterium Streptomyces is inhibited by its formation of mycelial pellets in liquid cultures. It is demonstrated that dispersed growth may be achieved by the addition of polymers to the culture medium. Uncharged polymers, such as polyethylene glycol, are relatively ineffective but polyanions such as agar, Carbopol and Junlon produce dispersed cultures when included in a defined growth medium at low concentrations. Junlon-containing media enable optical density measurements to be used to follow batch growth of Streptomyces. Improvements in both biomass yield and product yield of the pigmented antibiotic actinorhodin were found to result from the incorporation of Junlon into minimal medium.

PIGMENTED ANTIBIOTIC PRODUCTION BY STREPTOMYCES COELICOLOR A3(2) : KINETICS AND THE INFLUENCE OF NUTRIENTS

SUMMARY: The production of the pigments actinorhodin and undecylprodigiosin by Streptomyces coelicolor A3(2) was examined in a chemically defined medium which permits dispersed growth of the organism. The physiological controls on the production of the two pigments were markedly disparate. Actinorhodin production occurred mainly in the stationary phase of batch cultures grown with glucose and sodium nitrate as the principal carbon and nitrogen sources. In the same batch cultures, undecylprodigiosin accumulated during the exponential growth phase. The production of both pigments was sensitive to the levels of ammonium and phosphate in the medium. Actinorhodin production was exquisitely sensitive to ammonium concentration, and was completely inhibited by as little as 1 mM-ammonium chloride, whereas more than 50 mM-ammonium chloride was required to prevent undecylprodigiosin production. A similar, but less extreme effect was seen with phosphate: actinorhodin production was completely inhibited by 24 mM-phosphate, whereas undecylprodigiosin was still formed at this high phosphate concentration. The effects of ammonium inhibition of pigmented antibiotic production were relieved by reducing the concentration of phosphate in the medium, but changing the ammonium concentration had no effect on phosphate inhibition. Thus the regulation of pigment production by these two nutrients is interrelated, with phosphate control being epistatic to that of ammonium. The results implicate a phosphorylated intermediate as a major regulator of secondary metabolite synthesis by S. coelicolor.

Application of Toxocara canis excretory–secretory antigens and IgG subclass antibodies (IgG1-4) in serodiagnostic assays of human toxocariasis

A major problem in the serodiagnosis of human toxocariasis in tropical countries is cross-reaction with antibodies to other helminthic diseases and a lack of sensitivity. The majority of tests currently available use total IgG and, in this study, the use of peroxidase-conjugated anti-human IgG subclass antibodies (IgG1-4) was compared with total IgG for the diagnosis of human toxocariasis by using Toxocara excretory-secretory (TES) antigens in an enzyme-linked immunosorbent assay (ELISA) format. All four IgG subclass antibodies gave approximately 10-fold increases in optical density (OD) values for 50 toxocariasis patients compared to 29 healthy normals; this was significantly greater than the approximate doubling of OD values seen in the total IgG-ELISA format. IgG2 gave by far the greatest sensitivity (values: IgG, 50%; IgG1, 60%; IgG2, 98%; IgG3, 78%; IgG4, 64%). Significant cross-reactivity using all IgG subclasses in the TES ELISA was seen with 141 serum samples from patients with 10 other helminthic infections. However, IgG3 gave the best specificity (values: IgG, 73%; IgG1, 76%; IgG2, 71%; IgG3, 81%; IgG4, 71%). Thus, of the IgG subclass antibodies, IgG2 appeared best and employing this subclass can improve the serodiagnosis of human toxocariasis since it recognises carbohydrate epitopes of TES antigens.

Differentiation of Streptomyces coelicolor A3(2) under nitrate-limited conditions

The life cycle of Streptomyces coelicolor during development on solid medium has been studied from a physiological perspective. A biphasic growth pattern was demonstrated, evidenced by a continuous transition from an initial exponential growth period into a slower phase of biomass accretion. The switch between the two phases coincided with the exhaustion of nitrate from the medium. The depletion of nitrate from the medium coincided with the initiation of aerial mycelium formation within the cultures and the development of hydrophobic surface properties. During secondary growth, cultures remained metabolically active, continuing to accumulate DNA, despite a cessation in the levels of RMA and cell protein accretion. In addition, the accumulation of glycogen and lipid contributed to the observed accretion of biomass in this phase. The depletion of nitrate also marked an increase in the production of α-ketoglutarate by the culture and a coincident decrease in medium pH. Latter stages of the secondary growth phase saw the development of spores within the culture, this in turn was associated with a decrease in cellular glycogen. This supported previous observations that glycogen degradation and spore maturation were intimately associated.

Correlation between carbon flux through the pentose phosphate pathway and production of the antibiotic methylenomycin in Streptomyces coelicolor A3(2)

Radiorespirometry was employed to study carbon metabolism during the growth of Streptomyces coelicolor A3(2) in a minimal medium which permitted the production of methylenomycin as the sole detectable secondary metabolite. A switch in the pattern of carbon metabolism from the Embden-Myerhof-Parnas pathway to the pentose phosphate pathway occurred during the period of slower growth in batch culture which immediately preceded entry into the stationary phase. This coincided with the period of methylenomycin production. It is proposed that the biosynthesis of methylenomycin is supported by the generation of NADPH during the latter part of growth.

Loop-Mediated Isothermal Amplification Test for Detection of Neisseria gonorrhoeae in Urine Samples and Tolerance of the Assay to the Presence of Urea

ABSTRACT A loop-mediated isothermal amplification (LAMP) assay for open reading frame 1 (ORF1) of the glutamine synthetase gene of Neisseria gonorrhoeae was able to tolerate urea concentrations of ≤1.8 M, compared with a PCR assay that was functional at concentrations of <100 mM. The LAMP assay was as sensitive as the PCR assay while being faster and simpler to perform.

A new approach to isolating siderophore‐producing actinobacteria

Aims: This study was conducted to investigate the application of 2,2′‐dipyridyl as a new approach to isolating siderophore‐producing actinobacteria.

Trichomonas vaginalis: Clinical relevance, pathogenicity and diagnosis.

Abstract Trichomonas vaginalis is the etiological agent of trichomoniasis, the most prevalent non-viral sexually transmitted disease worldwide. Trichomoniasis is a widespread, global health concern and occurring at an increasing rate. Infections of the female genital tract can cause a range of symptoms, including vaginitis and cervicitis, while infections in males are generally asymptomatic. The relatively mild symptoms, and lack of evidence for any serious sequelae, have historically led to this disease being under diagnosed, and under researched. However, growing evidence that T. vaginalis infection is associated with other disease states with high morbidity in both men and women has increased the efforts to diagnose and treat patients harboring this parasite. The pathology of trichomoniasis results from damage to the host epithelia, caused by a variety of processes during infection and recent work has highlighted the complex interactions between the parasite and host, commensal microbiome and accompanying symbionts. The commercial release of a number of nucleic acid amplification tests (NAATs) has added to the available diagnostic options. Immunoassay based Point of Care testing is currently available, and a recent initial evaluation of a NAAT Point of Care system has given promising results, which would enable testing and treatment in a single visit.

The importance of amino acids as carbon sources for Micromonospora echinospora (ATCC 15837)

Aims: This study set out to investigate the effect of amino acids on the uptake of glucose by Micromonospora eichinospora (ATCC 15837).

Response of Micromonospora echinospora (NCIMB 12744) spores to heat treatment with evidence of a heat activation phenomenon

P.A. HOSKISSON, G. HOBBS and G.P. SHARPLES.2000. The effects of heat treatment on spores of the actinomycete Micromonospora echinospora were investigated. The percentage of culturable spores in untreated spore stocks was found to be approximately 20%. A 60 °C treatment of spores in phosphate buffer for 10 min led to an approximately five‐fold increase in the number of culturable units. This indicated that a large proportion of the spores were constitutively dormant. Within 10 min and in the absence of an external energy‐yielding substrate, the heat treatment was found to stimulate spore respiration suggesting that endogenous storage compounds were being utilized. Heating spores at 70 °C shortened the time period required for activation; holding times greater than 10 min, however, resulted in a reduction of culturable cells. Classic thermal death characteristics were seen at temperatures of 80 °C and above with d‐values of 21·43, 2·67,0·45 and 0·09 min being recorded at 70, 80, 90 and 100 °C, respectively. Spores of this organism, while being weakly heat resistant in comparison with bacterial endospores, are significantly more resistant than vegetative cells.