Pharmacological Characterization of the Novel and Selective α7 Nicotinic Acetylcholine Receptor-Positive Allosteric Modulator BNC375.

Treatments for cognitive deficits associated with central nervous system (CNS) disorders such as Alzheimer disease and schizophrenia remain significant unmet medical needs that incur substantial pressure on the health care system. The α7 nicotinic acetylcholine receptor (nAChR) has garnered substantial attention as a target for cognitive deficits based on receptor localization, robust preclinical effects, genetics implicating its involvement in cognitive disorders, and encouraging, albeit mixed, clinical data with α7 nAChR orthosteric agonists. Importantly, previous orthosteric agonists at this receptor suffered from off-target activity, receptor desensitization, and an inverted U-shaped dose-effect curve in preclinical assays that limit their clinical utility. To overcome the challenges with orthosteric agonists, we have identified a novel selective α7 positive allosteric modulator (PAM), BNC375. This compound is selective over related receptors and potentiates acetylcholine-evoked α7 currents with only marginal effect on the receptor desensitization kinetics. In addition, BNC375 enhances long-term potentiation of electrically evoked synaptic responses in rat hippocampal slices and in vivo. Systemic administration of BNC375 reverses scopolamine-induced cognitive deficits in rat novel object recognition and rhesus monkey object retrieval detour (ORD) task over a wide range of exposures, showing no evidence of an inverted U-shaped dose-effect curve. The compound also improves performance in the ORD task in aged African green monkeys. Moreover, ex vivo 13C-NMR analysis indicates that BNC375 treatment can enhance neurotransmitter release in rat medial prefrontal cortex. These findings suggest that α7 nAChR PAMs have multiple advantages over orthosteric α7 nAChR agonists for the treatment of cognitive dysfunction associated with CNS diseases. SIGNIFICANCE STATEMENT: BNC375 is a novel and selective α7 nicotinic acetylcholine receptor (nAChR) positive allosteric modulator (PAM) that potentiates acetylcholine-evoked α7 currents in in vitro assays with little to no effect on the desensitization kinetics. In vivo, BNC375 demonstrated robust procognitive effects in multiple preclinical models across a wide exposure range. These results suggest that α7 nAChR PAMs have therapeutic potential in central nervous system diseases with cognitive impairments.

A perspective on the contribution of animal models to the pharmacological treatment of posttraumatic stress disorder.

OBJECTIVES: Posttraumatic stress disorder (PTSD) is a prevalent, chronic, disabling disorder that may develop following exposure to a traumatic event. This review summarizes currently used animal models of PTSD and their potential role in the development of better therapeutics. Heterogeneity is one of the main characteristics of PTSD with the consequence that many pharmacological approaches are used to relieve symptoms of PTSD. To address the translational properties of the animal models, we discuss the types of stressors used, the rodent correlates of human PTSD (DSM-5) symptoms, and the efficacy of approved, recommended and off-label drugs used to treat PTSD in 'PTSD-animals'. CONCLUSIONS: Currently available animal models reproduce most PTSD symptoms and are validated by existing therapeutics. However, novel therapeutics are needed for this disorder as not one drug alleviates all symptoms and many have side effects that lead to non-compliance among PTSD patients. The true translational power of animal models of PTSD will only be demonstrated when new therapeutics acting through novel mechanisms become available for clinical practice.

BNC210, a negative allosteric modulator of the alpha 7 nicotinic acetylcholine receptor, demonstrates anxiolytic- and antidepressant-like effects in rodents.

This work describes the characterization of BNC210 (6-[(2,3-dihydro-1H-inden-2-yl)amino]-1-ethyl-3-(4-morpholinylcarbonyl)-1,8-naphthyridin-4(1H)-one), a selective, small molecule, negative allosteric modulator (NAM) of α7 nicotinic acetylcholine receptors (α7 nAChR). With the aim to discover a non-sedating, anxiolytic compound, BNC210 was identified during phenotypic screening of a focused medicinal chemistry library using the mouse Light Dark (LD) box to evaluate anxiolytic-like activity and the mouse Open Field (OF) (dark) test to detect sedative and/or motor effects. BNC210 exhibited anxiolytic-like activity with no measurable sedative or motor effects. Electrophysiology showed that BNC210 did not induce α7 nAChR currents by itself but inhibited EC80 agonist-evoked currents in recombinant GH4C1 cell lines stably expressing the rat or human α7 nAChR. BNC210 was not active when tested on cell lines expressing other members of the cys-loop ligand-gated ion channel family. Screening over 400 other targets did not reveal any activity for BNC210 confirming its selectivity for α7 nAChR. Oral administration of BNC210 to male mice and rats in several tests of behavior related to anxiety- and stress- related disorders, demonstrated significant reduction of these behaviors over a broad therapeutic range up to 500 times the minimum effective dose. Further testing for potential adverse effects in suitable rat and mouse tests showed that BNC210 did not produce sedation, memory and motor impairment or physical dependence, symptoms associated with current anxiolytic therapeutics. These data suggest that allosteric inhibition of α7 nAChR function may represent a differentiated approach to treating anxiety- and stress- related disorders with an improved safety profile compared to current treatments.

A bioassay for the detection of perchlorate in the ppb range.

A bioassay for the determination of ppb (µg·L(-1)) concentrations of perchlorate has been developed and is described herein. The assay uses the enzyme perchlorate reductase (PR) from the perchlorate-reducing organism Dechloromonas agitata in purified and partially purified forms to detect perchlorate. The redox active dye phenazine methosulfate (PMS) is shown to efficiently shuttle electrons to PR from NADH. Perchlorate can be determined indirectly by monitoring NADH oxidization by PR. To lower the detection limit, we have shown that perchlorate can be concentrated on a solid-phase extraction (SPE) column that is pretreated with the cation decyltrimethylammonium bromide (DTAB). Perchlorate is eluted from these columns with a solution of 2 M NaCl and 200 mM morpholine propane sulfonic acid (MOPS, pH 12.5). By washing these columns with 15 mL of 2.5 mM DTAB and 15% acetone, contaminating ions, such as chlorate and nitrate, are removed without affecting the bioassay. Because of the effect of complex matrices on the SPE columns, the method of standard additions is used to analyze tap water and groundwater samples. The efficacy of the developed bioassay was demonstrated by analyzing samples from 2-17000 ppb in deionized lab water, tap water, and contaminated groundwater.

Cholinergic modulation of disorder-relevant human defensive behaviour in generalised anxiety disorder.

Drugs that are clinically effective against anxiety disorders modulate the innate defensive behaviour of rodents, suggesting these illnesses reflect altered functioning in brain systems that process threat. This hypothesis is supported in humans by the discovery that the intensity of threat-avoidance behaviour is altered by the benzodiazepine anxiolytic lorazepam. However, these studies used healthy human participants, raising questions as to their validity in anxiety disorder patients, as well as their generalisability beyond GABAergic benzodiazepine drugs. BNC210 is a novel negative allosteric modulator of the alpha 7 nicotinic acetylcholine receptor and we recently used functional Magnetic Resonance Imaging to show it reduced amygdala responses to fearful faces in generalised anxiety disorder patients. Here we report the effect of BNC210 on the intensity of threat-avoidance behaviour in 21 female GAD patients from the same cohort. We used the Joystick Operated Runway Task as our behavioural measure, which is a computerised human translation of the Mouse Defense Test Battery, and the Spielberger state anxiety inventory as our measure of state affect. Using a repeated-measures, within-subjects design we assessed the effect of BNC210 at two dose levels versus placebo (300 mg and 2000 mg) upon two types of threat-avoidance behaviour (Flight Intensity and Risk Assessment Intensity). We also tested the effects of 1.5 mg of the benzodiazepine lorazepam as an active control. BNC210 significantly reduced Flight Intensity relative to placebo and the low dose of BNC210 also significantly reduced self-reported state anxiety. Risk Assessment Intensity was not significantly affected. Results show both human defensive behaviour and state anxiety are influenced by cholinergic neurotransmission and there provide converging evidence that this system has potential as a novel target for anxiolytic pharmacotherapy.

Cholinergic Modulation of Disorder-Relevant Neural Circuits in Generalized Anxiety Disorder.

BACKGROUND: Generalized anxiety disorder is associated with hyperactivity in the amygdala-prefrontal networks, and normalization of this aberrant function is thought to be critical for successful treatment. Preclinical evidence implicates cholinergic neurotransmission in the function of these systems and suggests that cholinergic modulation may have anxiolytic effects. However, the effects of cholinergic modulators on the function of anxiety-related networks in humans have not been investigated. METHODS: We administered a novel α7 nicotinic acetylcholine receptor-negative allosteric modulator, BNC210, to 24 individuals (3 male subjects) with generalized anxiety disorder and assessed its effects on neural responses to fearful face stimuli. RESULTS: BNC210 reduced amygdala reactivity to fearful faces relative to placebo and similarly to lorazepam and also reduced connectivity between the amygdala and the anterior cingulate cortex, a network involved in regulating anxious responses to aversive stimuli. CONCLUSIONS: These results demonstrate for the first time that the function of disorder-relevant neural circuits in generalized anxiety disorder can be beneficially altered through modulation of cholinergic neurotransmission and suggest potential for this system as a novel target for anxiolytic pharmacotherapy.

Discovery of BNC375, a Potent, Selective, and Orally Available Type I Positive Allosteric Modulator of α7 nAChRs.

Positive allosteric modulators (PAMs) of α7 nAChRs can have different properties with respect to their effects on channel kinetics. Type I PAMs amplify peak channel response to acetylcholine but do not appear to influence channel desensitization kinetics, whereas Type II PAMs both increase channel response and delay receptor desensitization. Both Type I and Type II PAMs are reported in literature, but there are limited reports describing their structure-kinetic profile relationships. Here, we report a novel class of compounds with either Type I or Type II behavior that can be tuned by the relative stereochemistry around the central cyclopropyl ring: for example, (R,R)-13 (BNC375) and its analogues with RR stereochemistry around the central cyclopropyl ring are Type I PAMs, whereas compounds in the same series with SS stereochemistry (e.g., (S,S)-13) are Type II PAMs as measured using patch-clamp electrophysiology. Further fine control over the kinetics has been achieved by changing the substitutions on the aniline ring: generally the substitution of aniline with strong electron withdrawing groups reduces the Type II character of these compounds. Our structure-activity optimization efforts have led to the discovery of BNC375, a small molecule with good CNS-drug like properties and clinical candidate potential.

The prediction of self-care behaviors in end-stage renal disease patients using Leventhal's Self-Regulatory Model.

OBJECTIVE: To assess the utility of Leventhal's Self-Regulatory Model (SRM) to predict self-care behavior with regard to dietary, medication, and fluid regimes in end-stage renal disease (ESRD) patients. METHODS: In a prospective study, ESRD patients treated via hospital-based haemodialysis (N=73) were screened for cognitive deficits and completed questionnaires that enquired about illness perceptions, coping strategies, knowledge of kidney disease, and psychological distress at Time 1. Physiological proxy measures of self-care behaviors regarding diet (serum potassium levels), fluid intake (mean and standard deviation of interdialytic weight gain), and medication (serum phosphate levels) regimes were collected 3 weeks later at Time 2. RESULTS: Illness representations (emotional and timeline perceptions) predicted self-care behaviors with regard to diet and medication. Emotion-focused coping strategies predicted higher levels of variation in adherence to fluid restrictions. Younger males were less likely to adhere to the fluid restrictions. CONCLUSIONS: The SRM has predictive utility. Psychological interventions should focus on alleviating disease-specific distress and challenging erroneous timeline perceptions in order to increase adherence to dietary and medication regimes in ESRD patients. A more specific measure of coping for ESRD is required to clarify the role of coping strategies in this population. Younger, male patients should be targeted for extra support with fluid restrictions.

Galantamine is not a positive allosteric modulator of human α4ß2 or α7 nicotinic acetylcholine receptors.

BACKGROUND AND PURPOSE: The alkaloid galantamine was originally isolated from the green snowdrop Galanthus woronowii and is currently marketed as a drug for treatment of mild to moderate dementia in patients with Alzheimer's disease. In addition to a well-documented proficiency to inhibit acetylcholinesterase, galantamine has been reported to increase neuronal nicotinic ACh (nACh) receptor function by acting as a positive allosteric modulator. Yet there remains controversy regarding these findings in the literature. To resolve this conundrum, we evaluated galantamine actions at α4ß2 and α7, which represent the nACh receptors most commonly associated with mammalian cognitive domains. EXPERIMENTAL APPROACH: α4ß2 [in (α4)3 (ß2)2 and (α4)2 (ß2)3 stoichiometries] and α7 nACh receptors were expressed in Xenopus laevis oocytes and subjected to two-electrode voltage-clamp electrophysiological experiments. Galantamine (10 nM to 100 µM) was evaluated for direct agonist effects and for positive modulation by co-application with sub-maximally efficacious concentrations of ACh. In addition, similar experiments were performed with α7 nACh receptors stably expressed in HEK293 cells using patch-clamp electrophysiology. KEY RESULTS: In concentrations ranging from 10 nM to 1 µM, galantamine did not display direct agonism nor positive modulatory effects at any receptor combination tested. At concentrations from 10 µM and above, galantamine inhibited the activity with a mechanism of action consistent with open-channel pore blockade at all receptor types. CONCLUSION AND IMPLICATIONS: Based on our data, we conclude that galantamine is not a positive allosteric modulator of α7 or α4ß2 receptors, which represent the majority of nACh receptors in mammalian brain.

Metabolic patterns associated with the clinical response to galantamine therapy: a fludeoxyglucose f 18 positron emission tomographic study.

BACKGROUND: Regional brain correlates of treatment with cholinesterase inhibitors in those with Alzheimer disease are unknown. OBJECTIVE: To map brain metabolism associated with the treatment response to galantamine with fludeoxyglucose F 18 positron emission tomography in patients with Alzheimer disease. DESIGN: This is a hypothesis-driven, prospective, open-label study of 19 patients with mild to moderate Alzheimer disease examined before and after treatment with the cholinesterase inhibitor galantamine. Clinical examinations included the cognitive portion of the Alzheimer Disease Assessment Scale, the Mini-Mental State Examination, and the Neuropsychiatric Inventory. Imaging was performed using fludeoxyglucose F 18 positron emission tomography. The positron emission tomographic data, registered to a probabilistic anatomical atlas, were subjected to a voxel-based analysis of 3 subgroups: total patient analysis, cognitive analysis, and behavioral analysis. Subvolume thresholding corrected random lobar noise to produce 3-dimensional significance maps. RESULTS: The total group analysis showed an increase in left caudate metabolism with no significant change in clinical outcomes for the total group with treatment. Subgroup analysis of cognitive and behavioral responders demonstrated a significant activation of a striatal-thalamofrontal network with galantamine treatment that was not present in patients whose condition worsened or was unchanged by therapy. In cognitive subgroups, change in left anterior cingulate metabolism significantly correlated with change in the cognitive portion of the Alzheimer Disease Assessment Scale (r = 0.70, P = .02); in behavioral subgroups, right cingulate metabolic change significantly correlated with improvement in depression and right ventral putamen metabolic change with improvement in apathy (r = 0.63, P<.05 for both). CONCLUSION: Cognitive and behavioral responders to galantamine therapy show clinically related improvements in prefrontal network metabolism along with thalamic activation.

Reduced accumulation of specific microRNAs in colorectal neoplasia.

Short non-coding RNAs are known to regulate cellular processes including development, heterochromatin formation, and genomic stability in eukaryotes. Given the impact of these processes on cellular identity, a study was undertaken to investigate possible changes in microRNA (miRNA) levels during tumorigenesis. A total of 28 different miRNA sequences was identified in a colonic adenocarcinoma and normal mucosa, including 3 novel sequences and a further 7 that had previously been cloned only from mice. Human homologues of murine miRNA sequences, miR-143 and miR-145, consistently display reduced steady-state levels of the mature miRNA at the adenomatous and cancer stages of colorectal neoplasia.

Physiological characterisation of recombinant Aspergillus nidulans strains with different creA genotypes expressing A. oryzae alpha-amylase.

The physiology of three strains of Aspergillus nidulans was examined--a creA deletion strain, a wild type creA genotype and a strain containing extra copies of the creA gene, all producing Aspergillus oryzae alpha-amylase. The strains were cultured in batch and continuous cultivations and the biomass formation and alpha-amylase production was characterised. Overexpression of the creA gene resulted in a lower maximum specific growth rate and a slightly higher repression of the alpha-amylase production during conditions with high glucose concentration. No expression of creA also resulted in a decreased maximum specific growth rate, but also in drastic changes in morphology. Furthermore, the expression of alpha-amylase was completely derepressed and creA thus seems to be the only regulatory protein responsible for glucose repression of alpha-amylase expression. The effect of different carbon sources on the alpha-amylase production in the creA deletion strain was investigated and it was found that starch was the best inducer. The degree of induction by starch increased almost linearly with the concentration of starch in starch/glucose mixtures. High-density batch cultivation was performed with the creA deletion strain and a final titre of 6.0 g l(-1) of alpha-amylase was reached after 162 h of cultivation.

Anaerobic nitrate-dependent iron(II) bio-oxidation by a novel lithoautotrophic betaproteobacterium, strain 2002.

Microbial nitrate-dependent Fe(II) oxidation is known to contribute to iron biogeochemical cycling; however, the microorganisms responsible are virtually unknown. In an effort to elucidate this microbial metabolic process in the context of an environmental system, a 14-cm sediment core was collected from a freshwater lake and geochemically characterized concurrently with the enumeration of the nitrate-dependent Fe(II)-oxidizing microbial community and subsequent isolation of a nitrate-dependent Fe(II)-oxidizing microorganism. Throughout the sediment core, ambient concentrations of Fe(II) and nitrate were observed to coexist. Concomitant most probable number enumeration revealed the presence of an abundant nitrate-dependent Fe(II)-oxidizing microbial community (2.4 x 10(3) to 1.5 x 10(4) cells g(-1) wet sediment) from which a novel anaerobic, lithoautotrophic, Fe(II)-oxidizing bacterium, strain 2002, was isolated. Analysis of the complete 16S rRNA gene sequence revealed that strain 2002 was a member of the beta subclass of the proteobacteria with 94.8% similarity to Chromobacterium violaceum, a bacterium not previously recognized for the ability to oxidize nitrate-dependent Fe(II). Under nongrowth conditions, both strain 2002 and C. violaceum incompletely reduced nitrate to nitrite with Fe(II) as the electron donor, while under growth conditions nitrate was reduced to gaseous end products (N2 and N2O). Lithoautotrophic metabolism under nitrate-dependent Fe(II)-oxidizing conditions was verified by the requirement of CO2 for growth as well as the assimilation of 14C-labeled CO2 into biomass. The isolation of strain 2002 represents the first example of an anaerobic, mesophilic, neutrophilic Fe(II)-oxidizing lithoautotroph isolated from freshwater samples. Our studies further demonstrate the abundance of nitrate-dependent Fe(II) oxidizers in freshwater lake sediments and provide further evidence for the potential of microbially mediated Fe(II) oxidation in anoxic environments.

Anaerobic degradation of benzene, toluene, ethylbenzene, and xylene compounds by Dechloromonas strain RCB.

Dechloromonas strain RCB has been shown to be capable of anaerobic degradation of benzene coupled to nitrate reduction. As a continuation of these studies, the metabolic versatility and hydrocarbon biodegradative capability of this organism were investigated. The results of these revealed that in addition to nitrate, strain RCB could alternatively degrade benzene both aerobically and anaerobically with perchlorate or chlorate [(per)chlorate] as a suitable electron acceptor. Furthermore, with nitrate as the electron acceptor, strain RCB could also utilize toluene, ethylbenzene, and all three isomers of xylene (ortho-, meta-, and para-) as electron donors. While toluene and ethylbenzene were completely mineralized to CO2, strain RCB did not completely mineralize para-xylene but rather transformed it to some as-yet-unidentified metabolite. Interestingly, with nitrate as the electron acceptor, strain RCB degraded benzene and toluene concurrently when the hydrocarbons were added as a mixture and almost 92 microM total hydrocarbons were oxidized within 15 days. The results of these studies emphasize the unique metabolic versatility of this organism, highlighting its potential applicability to bioremediative technologies.

Universal immunoprobe for (per)chlorate-reducing bacteria.

Recent studies in our lab have demonstrated the ubiquity and diversity of microorganisms which couple growth to the reduction of chlorate or perchlorate [(per)chlorate] under anaerobic conditions. We identified two taxonomic groups, the Dechloromonas and the Dechlorosoma groups, which represent the dominant (per)chlorate-reducing bacteria (ClRB) in the environment. As part of these studies we demonstrated that chlorite dismutation is a central step in the reductive pathway of (per)chlorate that is common to all ClRB and which is mediated by the enzyme chlorite dismutase (CD). Initial studies on CD suggested that this enzyme is highly conserved among the ClRB, regardless of their phylogenetic affiliation. As such, this enzyme makes an ideal target for a probe specific for these organisms. Polyclonal antibodies were commercially raised against the purified CD from the ClRB Dechloromonas agitata strain CKB. The obtained antiserum was deproteinated by ammonium sulfate precipitation, and the antigen binding activity was assessed using dot blot analysis of a serial dilution of the antiserum. The titers obtained with purified CD indicated that the antiserum had a high affinity for the CD enzyme, and activity was observed in dilutions as low as 10(-6) of the original antiserum. The antiserum was active against both cell lysates and whole cells of D. agitata, but only if the cells were grown anaerobically with (per)chlorate. No response was obtained with aerobically grown cultures. In addition to D. agitata, dot blot analysis employed with both whole-cell suspensions and cell lysates of several diverse ClRB representing the alpha, beta, and gamma subclasses of Proteobacteria tested positive regardless of phylogenetic affiliation. Interestingly, the dot blot response obtained for each of the ClRB cell lysates was different, suggesting that there may be some differences in the antigenic sites of the CD protein produced in these organisms. In general, no reactions were observed with cells or cell lysates of the organisms closely related to the ClRB which could not grow by (per)chlorate reduction. These studies have resulted in the development of a highly specific and sensitive immunoprobe based on the commonality of the CD enzyme in ClRB which can be used to assess dissimilatory (per)chlorate-reducing populations in environmental samples regardless of their phylogenetic affiliations.

Hypersexuality after right pallidotomy for Parkinson's disease.

The authors describe hypersexuality following atypical right pallidotomy for intractable Parkinson's Disease (PD). This patient and literature review suggest important roles for the pallidum in sexual behavior and dopamine in sexual arousal.

An alternative method for predicting attrition from an alcohol treatment programme.

AIMS: To test the predictive validity of a vignette methodology based on a Signal Detection model by examining treatment attrition within an alcohol clinic. METHODS: Participants were asked to categorize vignettes that described individuals drinking alcohol as problem or nonproblem alcohol use at the beginning of a 4-week intensive course of treatment. These participants were divided retrospectively into two groups: those who completed treatment and those who dropped-out of treatment. A matched post-treatment long-term abstainer group was also tested. RESULTS: Signal Detection analyses demonstrated that response bias scores predicted who would drop out of treatment (P = 0.01). CONCLUSIONS: The vignette methodology provided useful levels of prediction in an applied clinical setting. It was argued that verbal reports from problem alcohol users may be more usefully conceptualized in terms of sensitivity and response bias than in terms of memory or 'truth'.

Diversity and ubiquity of bacteria capable of utilizing humic substances as electron donors for anaerobic respiration.

Previous studies have demonstrated that reduced humic substances (HS) can be reoxidized by anaerobic bacteria such as Geobacter, Geothrix, and Wolinella species with a suitable electron acceptor; however, little is known of the importance of this metabolism in the environment. Recently we investigated this metabolism in a diversity of environments including marine and aquatic sediments, forest soils, and drainage ditch soils. Most-probable-number enumeration studies were performed using 2,6-anthrahydroquinone disulfonate (AHDS), an analog for reduced HS, as the electron donor with nitrate as the electron acceptor. Anaerobic organisms capable of utilizing reduced HS as an electron donor were found in all environments tested and ranged from a low of 2.31 x 10(1) in aquifer sediments to a high of 9.33 x 10(6) in lake sediments. As part of this study we isolated six novel organisms capable of anaerobic AHDS oxidation. All of the isolates coupled the oxidation of AHDS to the reduction of nitrate with acetate (0.1 mM) as the carbon source. In the absence of cells, no AHDS oxidation was apparent, and in the absence of AHDS, no cell density increase was observed. Generally, nitrate was reduced to N(2). Analysis of the AHDS and its oxidized form, 2,6-anthraquinone disulfonate (AQDS), in the medium during growth revealed that the anthraquinone was not being biodegraded as a carbon source and was simply being oxidized as an energy source. Determination of the AHDS oxidized and nitrate reduced accounted for 109% of the theoretical electron transfer. In addition to AHDS, all of these isolates could also couple the oxidation of reduced humic substances to the reduction of nitrate. No HS oxidation occurred in the absence of cells and in the absence of a suitable electron acceptor, demonstrating that these organisms were capable of utilizing natural HS as an energy source and that AHDS serves as a suitable analog for studying this metabolism. Alternative electron donors included simple volatile fatty acids such as propionate, butyrate, and valerate as well as simple organic acids such as lactate and pyruvate. Analysis of the complete sequences of the 16S rRNA genes revealed that the isolates were not closely related to each other and were phylogenetically diverse, with members in the alpha, beta, gamma, and delta subdivisions of the PROTEOBACTERIA: Most of the isolates were closely related to known genera not previously recognized for their ability to couple growth to HS oxidation, while one of the isolates represented a new genus in the delta subclass of the PROTEOBACTERIA: The results presented here demonstrate that microbial oxidation of HS is a ubiquitous metabolism in the environment. This study represents the first description of HS-oxidizing isolates and demonstrates that microorganisms capable of HS oxidation are phylogenetically diverse.

Environmental factors that control microbial perchlorate reduction.

As part of a study to elucidate the environmental parameters that control microbial perchlorate respiration, we investigated the reduction of perchlorate by the dissimilatory perchlorate reducer Dechlorosoma suillum under a diverse set of environmental conditions. Our results demonstrated that perchlorate reduction by D. suillum only occurred under anaerobic conditions in the presence of perchlorate and was dependent on the presence of molybdenum. Perchlorate reduction was dependent on the presence of the enzyme chlorite dismutase, which was induced during metabolism of perchlorate. Anaerobic conditions alone were not enough to induce expression of this enzyme. Dissolved oxygen concentrations less than 2 mg liter(-1) were enough to inhibit perchlorate reduction by D. suillum. Similarly to oxygen, nitrate also regulated chlorite dismutase expression and repressed perchlorate reduction by D. suillum. Perchlorate-grown cultures of D. suillum preferentially reduced nitrate in media with equimolar amounts of perchlorate and nitrate. In contrast, an extended (40 h) lag phase was observed if a similar nitrate-perchlorate medium was inoculated with a nitrate-grown culture. Perchlorate reduction commenced only when nitrate was completely removed in either of these experiments. In contrast to D. suillum, nitrate had no inhibitory effects on perchlorate reduction by the perchlorate reducer Dechloromonas agitata strain CKB. Nitrate was reduced to nitrite concomitant with perchlorate reduction to chloride. These studies demonstrate that microbial respiration of perchlorate is significantly affected by environmental conditions and perchlorate reduction is directly dependent on bioavailable molybdenum and the presence or absence of competing electron acceptors. A microbial treatment strategy can achieve and maintain perchlorate concentrations below the recommended regulatory level, but only in environments in which the variables described above can be controlled.

Sequencing and transcriptional analysis of the chlorite dismutase gene of Dechloromonas agitata and its use as a metabolic probe.

The dismutation of chlorite into chloride and O(2) represents a central step in the reductive pathway of perchlorate that is common to all dissimilatory perchlorate-reducing bacteria and is mediated by a single enzyme, chlorite dismutase. The chlorite dismutase gene cld was isolated and sequenced from the perchlorate-reducing bacterium Dechloromonas agitata strain CKB. Sequence analysis identified an open reading frame of 834 bp that would encode a mature protein with an N-terminal sequence identical to that of the previously purified D. agitata chlorite dismutase enzyme. The predicted translation product of the D. agitata cld gene is a protein of 277 amino acids (aa), including a leader peptide of 26 aa. Primer extension analysis identified a single transcription start site directly downstream of an AT-rich region that could represent the -10 promoter region of the D. agitata cld gene. Northern blot analysis indicated that the cld gene was transcriptionally up-regulated when D. agitata cells were grown in perchlorate-reducing versus aerobic conditions. Slot blot hybridizations with a D. agitata cld probe demonstrated the conservation of the cld gene among perchlorate-reducing bacteria. This study represents the first description of a functional gene associated with microbial perchlorate reduction.