Detection of low numbers of Bacillus anthracis spores in three soils using five commercial DNA extraction methods with and without an enrichment step.

AIMS: To (i) compare the limits of detection of Bacillus anthracis spores in three soils (one Florida, one Texas, and one a commercial Garden product) by PCR using DNA extracted with five commercial extraction kits and (ii) examine if removing organic acids or adding an enrichment step utilizing a growth medium will improve the detection limits. METHODS AND RESULTS: Bacillus anthracis spores were added to soil aliquots and used immediately with a DNA extraction kit or pretreated to remove organics or incubated overnight in a selective growth medium before the DNA extraction was performed. Using hybridization and PCR assays for capC, pag and lef genes, 10(5) -10(6) B. anthracis spores were detected in untreated Florida soil, 10(4) -10(7) spores in untreated Texas soil and 10(6) -10(7) in Garden soil. Pretreatment did not reliably improve detection. DNA from untreated and pretreated soils was suitable for hybridization but not always for PCR. When 10(1) -10(2) spores were added to the soils and allowed to amplify in a growth medium selective for B. anthracis, DNA extracted using four methods reliably produced PCR acceptable DNA positive for the B. anthracis genes. CONCLUSIONS: The quality of DNA extracted with commercial kits appears to be influenced by the soil type and pretreatment. Yet, with an enrichment step added, four of five extraction methods produced PCR suitable DNA and detected ≤10(2) spores. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment step could enhance the detection of B. anthracis spores in soils and small samples contaminated with soil.

Procurement of spore-free Bacillus anthracis for molecular typing outside of BSL3 environment.

AIMS: To (i) develop a protocol that would eliminate or greatly reduce sporulation within Bacillus anthracis vegetative cells, and (ii) harvest an adequate number of cells and sufficient DNA suitable for molecular methods including Riboprint analysis and pulse field gel electrophoresis (PFGE). METHODS AND RESULTS: Seven strains of B. anthracis (Ames, French B2, Heluky, Kruger, Pasteur, Sterne, and Vollum) were grown at 37, 42 and 45 degrees C under normal air, enhanced CO(2), microaerophilic, and anaerobic conditions on solid media and subcultured in two broths with and without supplements. The bacterial cells were centrifuged and washed. Slides made from the cell pellets were stained with Malachite Green and observed for the presence of spores. Cell preparations were subjected to 80 degrees C for 30 min and processed for and analysed by either Riboprinte or PFGE. Multiple pellets of each strain were processed, stained, placed onto solid culture media, incubated for 7 days and observed for growth. The cell preparations yielded clear and reproducible results with both molecular methods. None of the cell preparations yielded growth on the culture media. CONCLUSIONS: This method eliminated viable spores in cell preparations of B. anthracis, yet still allowed the growth of vegetative cells to provide sufficient DNA suitable for analysis by Riboprinter and PFGE. SIGNIFICANCE AND IMPACT OF THE STUDY: This method will provide safe cell preparations, prevent instrument contamination, and may be useful for other aerobic and anaerobic spore-formers.

The inactivation and removal of airborne Bacillus atrophaeus endospores from air circulation systems using UVC and HEPA filters.

AIMS: To (i) evaluate the UV radiation in the 'C' band/high efficient particulate air (UVC/HEPA) instrument's potential to inactivate spores of Bacillus atrophaeus and selected Bacillus species and (ii) test whether a titanium dioxide coating inside the cylindrical HEPA filter improves the system's efficacy. METHODS AND RESULTS: Known amounts of dried spore preparations of B. atrophaeus, Bacillus cereus, Bacillus megaterium, Bacillus stearothermophilus and Bacillus thuringiensis were exposed to the UVC lamp within a cylindrical HEPA filter for different time lengths (30 min to 48 h) and with different air flow speeds (0-235 l s(-1)). The log(10) reduction (range 5-16 logs) of colony forming units for spores exposed to the UVC compared with the unexposed spores was significant (P < 0.0001). The addition of a titanium dioxide (TiO(2)) veneer to the interior surface of the HEPA filter significantly increased the inactivation of spores (P < 0.0001). CONCLUSIONS: The UVC/HEPA unit could inactivate spores of B. atrophaeus, B. cereus, B. megaterium, B. stearothermophilus and B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The UVC/HEPA unit represents an effective method of decontaminating circulating air within an air-duct system as found in a building.

Transcriptional regulation of the nitrate reductase gene in Chlorella vulgaris: identification of regulatory elements controlling expression.

Nitrate reductase (NR), the rate-limiting and primary control point of the nitrate assimilation pathway, is regulated at transcriptional and post-transcriptional levels. To better understand how NR is regulated at the transcriptional level in Chlorella vulgaris, studies were performed to identify the factors regulating NR expression. Sequence analysis of the NR promoter identified a number of potential sites that were investigated by mobility shift assays. Of the protein-binding sites found, several., such as USF and E2F are likely involved in the basal NR gene transcription. An indirect repeat sequence with similarity to the sequence recognized by the common plant regulatory factor was identified and shown to bind a Chlorella protein in vitro. Mobility shift assays of a consensus GATA element indicated that proteins able to specifically bind this element are constitutive, regardless of the nitrogen source. Mutational analysis revealed that the GATA core is required for protein binding in vitro. Additionally, a NIT2 zinc-finger domain/glutathione S-transferase fusion protein was found to bind in a sequence-specific manner to this site. In Neurospora crassa and Aspergillus nidulans, consensus GATA elements are bound by the NIT2 protein, which has a major role in the expression of nitrogen-metabolizing genes. The ability of the GATA element to function as a nitrogen response element (NRE) was further examined by in vivo foot-printing. The protection of guanines in the GATA core and surrounding region was observed only in cells grown in the presence of nitrate. These data confirm that a single GATA element has a role in regulating the expression of NR in C. vulgaris.

1H and 13C NMR studies of a truncated heme domain from Chlorella vulgaris nitrate reductase: signal assignment of the heme moiety.

A water soluble truncated heme domain (a tetramer of MW = 45 kDa) of the tetrameric nitrate reductase complex from the green alga Chlorella vulgaris has been overexpressed and purified. This truncated heme domain with four identical subunits has a high redox potential (midpoint potential E1/2 = +16 mV) as compared with other heme-containing flavoproteins. We have undertaken a determination of the detailed configuration of the heme moiety in order to understand the unique electrochemical property of the heme moiety of this enzyme. We report here the study of the heme prosthetic group of the truncated heme domain by the use of 2D 1H and 13C NMR techniques. A complete signal assignment of the heme has been achieved. Our observations suggest that the heme configuration is similar to that of the crystal structure of the membrane-bound bovine liver cytochrome b5.

Cloning and characterization of the nitrate reductase-encoding gene from Chlorella vulgaris: structure and identification of transcription start points and initiator sequences.

The reduction of nitrate to nitrite catalyzed by nitrate reductase (NR) is considered to be the rate-limiting and regulated step of nitrate assimilation, a major metabolic pathway occurring in a wide range of organisms which in turn supply the nutritional nitrogen requirements for other forms of life. Chlorella vulgaris NR mRNA levels are very responsive to changes in nitrogen source. In the presence of ammonia as the sole nitrogen source, under repressed conditions, NR mRNA is undetectable. Under inducing conditions, the removal of ammonia and addition of nitrate, rapid NR mRNA synthesis occurs. We are studying the elements involved in regulating the expression of this important gene. Two overlapping genomic clones (NRS1 and NR5') were isolated from a cosmid library. The two clones were sequenced and their sequences were aligned with that of a full-length NR cDNA. The gene is approximately 8 kb long and consists of 19 exons and 18 introns. Unlike NR isolated from other species, the exons which code for the functional domains of C. vulgaris are separated by introns. Two transcription start points (tsp) were identified and each is surrounded by potential initiator sequences. No TATA, CAAT or GC-rich promoter elements were located. A time course of NR induction revealed that while transcription initiation from one tsp remains at a constant level from the point of induction through steady state, the level of initiation from another tsp is high upon induction, but decreases as steady state is attained.

Possible role for mRNA stability in the ammonium-controlled regulation of nitrate reductase expression.

Ammonium, or a metabolite of ammonium, represses the expression of nitrate reductase (NR) in Chlorella vulgaris. The removal of ammonium and addition of nitrate (induction) resulted in a rapid (20 min) peaked synthesis of NR mRNA. Nitrate reductase protein and activity increased at a much lower rate, reaching their maxima by 8 h. Ammonium added to nitrate-grown cells resulted in a dramatic decrease in NR mRNA from a steady-state level to undetectable levels within 15 min of ammonium addition. Nitrate reductase activity and protein levels decreased to 20% and 40% of initial levels respectively over 8 h. The half-life for NR mRNA under these conditions was estimated to be less than 5 min, compared with 120 min for NR protein. Such rapid decreases in NR mRNA suggested a degradation and/or cessation in NR mRNA transcription. No apparent difference in NR mRNA-specific RNAase activity of crude cell extracts (NR-induced or repressed) was observed. However, a significant difference in the susceptibility to degradation of NR mRNA from long-term nitrate-grown cells compared with the NR mRNA isolated from short-term induced cells (20 min in nitrate) was observed. NR mRNA isolated from long-term-nitrate-grown cells was completely degraded by RNAases in cell extracts under conditions in which the NR mRNA isolated from short-term induced cells was resistant to degradation. These results suggest that mRNA stability may be an important factor in the metabolic regulation of assimilatory nitrate reductase in Chlorella.

Evidence that the glutamine-stimulated loss of nitrate reductase protein from the yeast Candida nitratophila is not the result of inducer exclusion.

Synthesis of nitrate reductase protein and increases in nitrate reductase activity occurred in cultures of the yeast Candida nitratophila when they were incubated in medium containing ammonium nitrate. Similar treatment with glutamine plus nitrate resulted in little increase in nitrate reductase activity, in cultures grown previously with reduced nitrogen compounds, and decreases in enzyme activity, in cultures adapted to nitrate. Labelling studies conducted in vivo revealed a rapid cessation of de novo nitrate reductase synthesis when glutamine was supplied to nitrate-adapted cultures in the presence of nitrate. Intracellular glutamine concentrations increased rapidly under these conditions and these cultures exhibited high glutamine: glutamate ratios. As nitrate was taken up in the presence of glutamine in these experiments, it is concluded that the glutamine-stimulated inhibition of nitrate reductase synthesis is a consequence of repression and rapid turnover of nitrate reductase mRNA and not inducer (nitrate) exclusion.

Further characterization of the assimilatory nitrate reductase from the yeast Candida nitratophila.

Nitrate reductase from the yeast Candida nitratophila was found to contain one molecule of cytochrome b557 and one atom of molybdenum per subunit. FAD/haem-dependent diaphorase activity (haem domain) was associated with a 40 kDa tryptic fragment of the subunit. The 50 amino-terminal residues of this fragment were determined, and the sequence did not show significant similarity to deduced sequences of other nitrate reductases previously published. Increasing ionic strength in vitro had a stimulatory effect on enzymic activity via stimulation of the molybdenum-dependent terminal nitrate-reducing activity. Stimulation of activity by exogenous protein (bovine serum albumin or casein) also appeared to be an ionic effect. Stimulation of catalytic activity by phosphate was a separate effect.

Expression and characterization of the heme-binding domain of Chlorella nitrate reductase.

A recombinant protein corresponding to the putative heme-binding domain of assimilatory NADH:nitrate reductase from Chlorella vulgaris has been expressed and purified from transformed Escherichia coli BL21 cells. The recombinant protein, exhibited a subunit molecular mass of approximately 10 kDa with a N-terminal sequence beginning with the residues PAGA in agreement with that predicted by cDNA analysis. The UV-visible spectrum of the protein confirmed the incorporation of heme with maxima at 413 nm and 423, 528, and 557 nm for the oxidized and reduced forms, respectively. Circular dichroism spectra indicated the environment of the heme chromophore was very similar to that of the native enzyme. Potentiometric titrations of the recombinant heme domain yielded a midpoint potential of +16 mV (n = 1, pH 7), substantially higher than the values of -160 mV obtained for the native enzyme and -28 mV obtained for a previously expressed recombinant heme domain that contained part of the Mo-pterin domain. These results indicate that portions of the amino acid sequence that are involved in the formation of the Mo-pterin domain of Chlorella nitrate reductase influence the redox potential of the heme prosthetic group.

Expression of a cDNA clone encoding the haem-binding domain of Chlorella nitrate reductase.

A partial cDNA clone coding for the haem-binding domain of NADH:nitrate reductase (EC 1.6.6.1) (NR) from the unicellular green alga Chlorella vulgaris has been isolated, sequenced and expressed. A 1.2 kb cDNA (pCVNR1) was isolated from a lambda gt11 expression library produced from polyadenylated RNA extracted from nitrate-grown Chlorella cells. pCVNR1 hybridized to a 3.5 kb mRNA transcript that was nitrate-inducible and absent from ammonium-grown cells. The entire sequence of pCVNR1 was obtained and found to have a single uninterrupted reading frame. The derived amino acid sequence of 318 amino acids has a 45-50% similarity to higher-plant NRs, including Arabidopsis thaliana, spinach (Spinacia oleracea) and tobacco (Nicotiana tabacum). A comparison with the putative domain structure of higher-plant nitrate reductases suggested that this sequence contains the complete haem-binding domain, approximately one-third of the Mo-pterin domain and no FAD-binding domain. A 32% sequence similarity is evident when comparing the Chlorella NR haem domain with that of calf cytochrome b5. Expression of pCVNR1 in a pET vector synthesized a 35 kDa protein that was antigenic to anti-(Chlorella NR) antibody. The spectral properties of this protein (reduced and oxidized) in the 400-600 nm region are identical with those of native Chlorella NR and indicate that haem is associated with the protein.

Spectroscopic, thermodynamic and kinetic properties of Candida nitratophila nitrate reductase.

Visible spectra of oxidized and reduced Candida nitratophila assimilatory NAD(P)H:nitrate reductase yielded absorbance maxima of 413 nm and 423 nm, and 525 nm and 555 nm respectively, characteristic of a b5-type cytochrome. E.p.r. spectra of the partially reduced enzyme revealed a single Mo(V) species (g1 = 1.9957, g2 = 1.9664 and g3 = 1.9658) exhibiting superhyperfine coupling to a single proton [A(1H)av. = 1.4 mT]. Oxidation-reduction midpoint potentials (E'0) (25 degrees C, pH 7) for the haem and Mo-pterin prosthetic groups were determined by visible and e.p.r. potentiometric titrations and yielded values of E'0 = -174 mV (n = 1) for the haem and E'0 = -3 mV and E'0 = -27 mV for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples respectively. Comparison of initial rates of the NADH-oxidizing and nitrate-reducing partial activities at various ionic strengths indicated electron transfer from reduced haem to Mo was rate-limiting during turnover. These results suggest a close similarity between Candida nitratophila and Chlorella vulgaris nitrate reductases.

Ammonium assimilation by the nitrate-utilizing yeast, Candida nitratophila.

Ammonium-nitrogen was assimilated rapidly by nitrogen-replete cultures of the nitrate-utilizing yeast, Candida nitratophila as long as a suitable source of carbon was available. These cultures contained high activities of an NADPH-dependent glutamate dehydrogenase with a relatively high affinity for ammonium (Km = 0.27 mM) and high glutamine synthetase activity. Both enzyme activities were apparently derepressed when glutamine-grown cultures were starved of nitrogen or transferred to nitrate medium. Nitrogen-deficient cultures also contained NADH-dependent glutamate synthase activity that was inhibited by azaserine in vitro. Ammonium assimilation in vivo, was inhibited by methionine sulphoximine whilst addition of azaserine resulted in an accumulation of intracellular glutamine and an inhibition of glutamate production. Our results suggest that, in C. nitratophila, there is a potential for ammonium assimilation via both the glutamate dehydrogenase pathway and the glutamine synthetase/glutamate synthase pathway with the latter pathway predominating in nitrogen-deficient cells.

Evidence for the transcriptional control of nitrate reductase in Candida nitratophila from in vitro translation studies.

In vivo labelling and in vitro translation studies were used to study the regulation of the synthesis of nitrate reductase in the yeast Candida nitratophila. These studies showed that synthesis of the enzyme subunit took place when ammonium-grown cells were nitrogen-starved and this was stimulated by subsequent addition of nitrate. Ammonium-grown cultures did not contain mRNA that could be translated into the nitrate reductase subunit in an in vitro system. Nitrate reductase mRNA could be extracted from nitrogen-starved and nitrate cultures. Synthesis of the enzyme is apparently controlled at the level of transcription in this yeast.

Regulation of synthesis of citrate synthase in regreening Euglena gracilis.

Exposure of dark-grown Euglena to white or red light, but not blue light, produced a twofold increase in the specific activity of citrate synthase. A 400-fold purification of mitochondrial citrate synthase (subunit Mr = 44000) was achieved from cells of Euglena gracilis by affinity chromatography on ATP-activated agarose. Antisera, raised against the homogeneously pure enzyme, were used to demonstrate that the increase in citrate synthase activity on exposure of dark-grown cells to light resulted from an increase in citrate synthase protein. Anti-(citrate synthase) was used to detect precursor citrate synthase resulting from the translation of total polyadenylated RNA from Euglena in a cell-free rabbit reticulocyte lysate system. Citrate synthase mRNA was found to be present in cells at all stages of regreening. However, extraction and translation of polyadenylated RNA from free polysomes isolated from darkgrown and regreening cells demonstrated that appreciable translation of citrate synthase mRNA was only occurring in regreening cells.