A rapid and effective method of extracting fully intact RNA from thermophilic geobacilli that is suitable for gene expression analysis

Sharkey, Freddie H.; Banat, Ibrahim M.; Marchant, Roger

doi:10.1007/s00792-003-0363-2

Cited by 14 publications

(8 citation statements)

References 11 publications

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“…In this study the RT/PCR assay was only used to detect and quantify toxin mRNA levels in C. botulinum under laboratory conditions. However, the template format for this RT/PCR assay has also been successfully applied to the monitoring of alkane monooxygenase (alkB) gene expression in extremophilic Geobacillus thermoleovorans species isolated from crude soil samples [Sharkey et al, 2004a]. Whilst this was not a directly related study, the work clearly illustrates the robustness of the assay and potential it may hold for environmental and food analysis purposes.…”

Section: Resultsmentioning

confidence: 94%

Quantitative Effects of Carbohydrates and Aromatic Amino Acids on <i>Clostridium botulinum </i>Toxin Gene Expression Using a Rapid Competitive RT/PCR Assay

2005

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A rapid competitive RT/PCR assay was developed to determine the effects of nutrients on Clostridium botulinum type E toxin gene expression. The type E strain (EVH) was grown in a nutrient-rich broth containing 1% glucose (base medium). Toxin gene expression was quantified at both mid and late exponential phases of growth. It was found that toxin encoding mRNA levels were highly growth phase dependent with elevated levels found in late exponential phase compared to mid exponential phase. Changing the carbohydrate source had a smaller effect on toxin encoding mRNA levels but as earlier results have suggested, toxin encoding mRNA levels show a strong correlation with type E growth rate. The results have important implications for the food industry whereby risk of type E botulism could be correlated to the nutrient composition of the contaminated food or assessed from C. botulinum growth rates in challenged foodstuffs.

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Section: Resultsmentioning

confidence: 94%

Quantitative Effects of Carbohydrates and Aromatic Amino Acids on <i>Clostridium botulinum </i>Toxin Gene Expression Using a Rapid Competitive RT/PCR Assay

2005

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“…RNA was extracted from pure cultures of T70 and soil samples using a TNS–PAS‐based technique (Sharkey et al ., 2004a). The RNA extraction buffer containing TNS and PAS has been shown to be superior in terms of isolating mRNA to be used as a template in cDNA synthesis for RT‐PCR (Bowler et al ., 1999).…”

Section: Resultsmentioning

confidence: 99%

“…We have investigated the effect of temperature on degradation of hexadecane, as a representative alkane, in soil microcosms and then supplemented this study with an investigation of expression of the alkane mono‐oxygenase gene ( alkB ) in thermophilic geobacilli, in pure culture and in soil environments, using reverse transcription PCR (RT‐PCR). Despite the availability of various assays for detection and localization of gene expression, RT‐PCR is generally considered the most sensitive method for detecting low copy numbers of mRNA (Sharkey et al ., 2004a, b). Because of the short half‐life of bacterial mRNAs (1–3 min) (Belasco & Higgins, 1988; Arraiano, 1993), RT‐PCR provides the opportunity of obtaining an immediate indication of the expression status.…”

Section: Introductionmentioning

confidence: 99%

The degradation of n-hexadecane in soil by thermophilic geobacilli

Marchant¹,

Sharkey²,

Banat³

et al. 2006

FEMS Microbiology Ecology

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Soil microcosms have been used to demonstrate the ability of indigenous soil thermophiles to degrade effectively a representative alkane (n-hexadecane). A fragment of the alkane mono-oxygenase gene (alkB) was amplified from thermophilic Geobacillus thermoleovorans strain T70 by PCR using degenerate primers. The amplicon demonstrated 96% sequence similarity with the alkB gene from Rhodococcus erythropolis. Critical controls ensured that the positive PCR signal detected was not a result of mesophilic soil organisms. A reverse transcription PCR (RT-PCR) assay was developed to determine if expression of the gene was inducible in the presence of an alkane or constitutively expressed in soil. In the presence of n-hexadecane, expression of the alkane mono-oxygenase gene was induced in pure cultures and soil samples and was dependent on temperature. No positive RT-PCR signal was detected at mesophilic growth temperatures either in pure cultures or in soil microcosms, whereas at 55 degrees C positive RT-PCR signals were obtained for both pure cultures of T70 and soil samples. Many different amplicons of the alkB gene fragment were obtained from the soil used in the microcosms. Thirty cloned fragments yielded 27 different sequences showing 85-96% sequence similarity with the alkB sequence of T70. To establish that the amplified alkB gene sequences from soil were derived from thermophilic geobacilli, additional strains were isolated on a selective medium containing n-hexadecane as sole carbon source. The 16S rRNA gene sequences were determined to identify the 50 isolates obtained (G. thermoleovorans, 27; G. caldoxylosilyticus, 17; G. pallidus, 2; G. toebiii, 1; Geobacillus sp., 3) representing 18 different strains and alkB gene sequences determined and deposited with the European Bioinformatics Institute.

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“…mRNA differential display. Isolation of RNA from Rhodococcus rhodochrous PY11 was performed according to a previously published method (19). Reverse transcription (RT)-PCRs were performed as described in reference 20 with modifications.…”

Section: Methodsmentioning

confidence: 99%

A 2-Hydroxypyridine Catabolism Pathway in Rhodococcus rhodochrous Strain PY11

Vaitekūnas

Gasparavičiūtė

Rutkienė

et al. 2016

Appl Environ Microbiol

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Rhodococcus rhodochrous PY11 (DSM 101666) is able to use 2-hydroxypyridine as a sole source of carbon and energy. By investigating a gene cluster (hpo) from this bacterium, we were able to reconstruct the catabolic pathway of 2-hydroxypyridine degradation. Here, we report that in Rhodococcus rhodochrous PY11, the initial hydroxylation of 2-hydroxypyridine is catalyzed by a four-component dioxygenase (HpoBCDF). A product of the dioxygenase reaction (3,6-dihydroxy-1,2,3,6-tetrahydropyridin-2-one) is further oxidized by HpoE to 2,3,6-trihydroxypyridine, which spontaneously forms a blue pigment. In addition, we show that the subsequent 2,3,6-trihydroxypyridine ring opening is catalyzed by the hypothetical cyclase HpoH. The final products of 2-hydroxypyridine degradation in Rhodococcus rhodochrous PY11 are ammonium ion and ␣-ketoglutarate. Pyridine and its derivatives are ubiquitous in nature. The pyridine ring is found in alkaloids (e.g., nicotine, actinidine), coenzymes [NAD(P)H, pyridoxal], and man-made solvents, pesticides, and herbicides (e.g., paraquat). Hydroxypyridines are common intermediate metabolites produced during microbial biodegradation of various N-heterocycles (pyridine, nicotine, picoline, 2,6-dipicolinic acid) (1-3).It has previously been reported that Arthrobacter crystallopoietes, Arthrobacter pyridinolis, and Arthrobacter viridescens (4), Achromobacter sp. strain G2 (5), and Nocardia sp. strain PNO (6) use 2-hydroxypyridine (2HP) as a sole carbon and energy source. Through more than 50 years of investigation of pyridine ring metabolism, many intermediates have been identified and metabolic pathways have been proposed. However, the genes and enzymes responsible for 2HP biodegradation have seldom been reported.In Achromobacter sp. G2, 2HP is metabolized via the maleamate pathway (5) (Fig. 1). No enzymes responsible for the initial hydroxylation step of 2HP leading to the formation of 2,5-dihydroxypyridine (2,5DHP) have been reported to date. Nevertheless, the degradation of 2,5DHP, an intermediate of nicotinic acid metabolism, has been fully investigated by Jiménez et al. (7), and all genes encoding the enzymes involved in the maleamate pathway have been identified and characterized (7).Arthrobacter crystallopoietes, A. pyridinolis, and A. viridescens (4) and Arthrobacter sp. strain PY22 (8) produce a blue pigment (nicotine blue) in the medium when grown on 2HP. The nicotine blue has been shown to be a 4,5,4=,5=-tetrahydroxy-3,3=-diazadiphenoquinone-(2,2=) (9) that is an autoxidation product of 2,3,6-trihydroxypyridine (THP). THP can be synthesized via hydroxylation of 2,5DHP, 2,3-dihydroxypyridine (2,3DHP), or 2,6-dihydroxypyridine (2,6DHP); however, only the 2,6DHP 3-hydroxylase, which is involved in the biodegradation of nicotine by Arthrobacter nicotinovorans, has been identified to date (10).We have previously reported that HpyB monooxygenase from Arthrobacter sp. PY22 is sufficient for the conversion of 2HP to THP (8). Since no reaction intermediates have been detected, a consecutive two-st...

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A rapid and effective method of extracting fully intact RNA from thermophilic geobacilli that is suitable for gene expression analysis

Cited by 14 publications

References 11 publications

Quantitative Effects of Carbohydrates and Aromatic Amino Acids on <i>Clostridium botulinum </i>Toxin Gene Expression Using a Rapid Competitive RT/PCR Assay

Quantitative Effects of Carbohydrates and Aromatic Amino Acids on <i>Clostridium botulinum </i>Toxin Gene Expression Using a Rapid Competitive RT/PCR Assay

The degradation of n-hexadecane in soil by thermophilic geobacilli

A 2-Hydroxypyridine Catabolism Pathway in Rhodococcus rhodochrous Strain PY11

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