Bacterial ATP Research Articles

PHOTOSYNTHETIC AND HETEROTROPHIC IMPACTS OF NUTRIENT LOADING TO BLACKWATER STREAMS

Blackwater stream systems are the most abundant type of freshwater lotic system on the Coastal Plain of the eastern United States. Many of these ecosystems drain watersheds that receive large anthropogenic nutrient inputs, whereas some blackwater systems remain relatively pristine. A series of nutrient addition experiments was carried out over a four‐year period to assess the roles of nitrogen and phosphorus loading on the phytoplankton, bacterioplankton, and respiration of two third‐order and two fifth‐order blackwater streams in southeastern North Carolina. Stream water was distributed into 4‐L containers, amended with various nutrient addition treatments, and incubated in gently agitated outdoor pools over a six‐day period. Chlorophyll a production, direct bacterial counts, ATP, and biochemical oxygen demand (BOD) were measured as response variables. Significant phytoplankton production over control occurred in most experiments involving nitrogen additions, regardless of whether it was in the form of ammonium, nitrate, or urea. Concentrations of nitrate or a urea–nitrate combination of 14.3 μM (0.2 mg N/L) or higher increased chlorophyll a production and significantly stimulated BOD. Organic or inorganic phosphorus additions did not stimulate phytoplankton production. Combined organic–inorganic phosphorus additions of 16.1–32.3 μM P (0.5–1.0 mg P/L) significantly stimulated bacterial abundance, ATP, and BOD on most occasions. Nitrogen inputs stimulate blackwater stream phytoplankton growth, which in turn dies and decomposes in deeper, higher order streams, becoming sources of BOD and lowering dissolved oxygen (DO). Phosphorus inputs directly stimulate bacterial growth, increasing BOD, and lowering stream DO. Thus, changes in nutrient loading stimulate two different biological pathways (photosynthetic and heterotrophic activity), which in turn reduce dissolved oxygen, potentially causing hypoxic conditions stressful to resident aquatic life. The low slope, slow summer flows, and naturally low summer DO conditions make Coastal Plain blackwater streams particularly susceptible to additional DO losses through BOD formation.

The action of bismuth against Helicobacter pylori mimics but is not caused by intracellular iron deprivation.

Helicobacter pylori is highly susceptible to bismuth, a heavy metal with antimicrobial activity linked to its effect on bacterial iron uptake. Three strains of H. pylori were analyzed for indicators of iron limitation following exposure to the MIC of colloidal bismuth subcitrate (MIC(CBS)). Similar morphologic and outer membrane changes were observed following growth in iron-limiting medium and at the MIC(CBS) that inhibited the growth of all three strains. These changes, which were also observed for iron-limited bacteria, were alleviated by the addition of iron to the cultures. H. pylori ATP levels, reduced in iron-limiting medium, were below the limits of detection in two of the three strains following exposure to bismuth. The addition of iron partially restored bacterial ATP levels in these two strains, although not to normal concentrations. In contrast, exposure of the same strains to the MIC(CBS) failed to deplete intracellular levels of iron, which were significantly reduced by culturing in iron-limiting medium. Thus, the antimicrobial effect of bismuth and of iron limitation on H. pylori may be similar. However, the respective mechanisms of intracellular action would appear to be mediated by different pathways within the cell.

Winding and unwinding ATP synthase

![][1] ATP synthase turns one way to make ATP and the other to break it down. Borsch Like a revolving door, bacterial ATP synthase turns two ways, according to Manuel Diez, Michael Borsch (Universitat Stuttgart, Germany), and colleagues. One direction makes ATP, whereas the other

Evaluation of a rapid bacterial ATP assay for screening BAL samples from ICU patients submitted for quantitative bacterial cultures

A novel application of a rapid diagnostic technique for the detection of significant bacterial pathogens (≥10 4 cfu/mL) in bronchoalveolar lavage (BAL) samples from critically ill ventilated patients is described. This rapid diagnostic assay (UTIscreen™, Coral Biotechnology, San Diego, CA) utilizes a luciferin-luciferase reaction to detect bacterial adenosine triphosphate (ATP) and is currently commercially available for screening bacteriuria in urine specimens. One hundred and twenty-eight BAL samples were examined microscopically with Gram's stain and tested in parallel using the bacterial ATP assay and standard quantitative culture. The sensitivity (Sn), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) for the detection of bacteria ≥10 4 cfu/ml in BAL specimens for the bacterial ATP assay was 87%, 59%, 39%, and 94%, and for the Gram's stain was 73%, 65%, 39%, and 89%, respectively. The diagnostic utility was improved by combining the results of Gram's stain/bacterial ATP assay results with Sn, Sp, PPV, and NPV of 97%, 38%, 32%, and 97% respectively. A combined negative rapid test consisting of Gram's stain/bacterial ATP assay rules out significant bacteria in BAL samples with a high degree of certainty. Future studies are needed to clinically validate these observations.

An ATP-based method for monitoring the microbiological drinking water quality in a distribution network

The titration of adenosine triphosphate (ATP) by bioluminescence permits rapid evaluation of the quantity of viable micro-organisms present in a water sample. During two sampling campaigns, Société Anonyme de Gestion des Eaux de Paris (SAGEP) tested a new extraction and titration system of bacterial ATP in the Paris drinking water distribution network. As far as the entire set of results of analyses of water in the network is concerned there is a linear relationship between log [ATP] and log(HPC-R2A/ml). Furthermore, as regards the drinking water originating from treatment of surface waters, some of the results obtained indicate a slight change as regards the Paris network in the microbiological quality. This is certainly linked to the distance travelled from the production location as well as to a reservoir effect observed on a site. Conversely, no change is apparent with regard to waters of underground origin. Lastly, despite changes in temperature and chlorine residual, no significant influence has been observed, essentially because of the very low density of culturable bacteria.

Laser Impact on Bacterial ATP: Insights into the Mechanism of Laser-Bacteria Interactions

The mechanisms of laser action on bacteria are not adequately understood. Here, an attempt has been made to study the fluctuation in ATP (adenosine triphosphate) concentration following laser irradiation from a pulsed Nd:YAG laser on a marine biofilm-forming bacterium Pseudoalteromonas carrageenovora. A stationary phase bacterial suspension (density 107-8 mlm 1) was exposed to pulsed laser irradiations at a fluence of 0.1 J cmm 2 (pulse width 5 ns, repetition rate 10 Hz) for different durations, ranging from 2 s to 15 min. The total viable count (TVC) and ATP concentration of the irradiated samples were determined immediately after the laser irradiation. While the maximum reduction in the TVC observed with respect to the control was 59% immediately after 15 min irradiation, the ATP concentration showed a reduction of about 86% for the same duration. The ATP concentration showed an abrupt reduction from 3 min of laser irradiation and continued to reduce significantly with increasing duration of irradiation. Thus, 3 min irradiation at a fluence of 0.1 J cmm 2 is considered as an approximate threshold for ATP production in this bacterium. As the decreased level of ATP production continued, bacterial mortality resulted. The reduction in ATP production could be due to damage caused by the laser irradiations on bacterial metabolic processes such as cellular respiration.

Observing Membrane Protein Diffusion at Subnanometer Resolution

Single sodium-driven rotors from a bacterial ATP synthase were embedded into a lipid membrane and observed in buffer solution at subnanometer resolution using atomic force microscopy (AFM). Time-lapse AFM topographs show the movement of single proteins within the membrane. Subsequent analysis of their individual trajectories, in consideration of the environment surrounding the moving protein, allow principal modes of the protein motion to be distinguished. Within one trajectory, individual proteins can undergo movements assigned to free as well as to obstacled diffusion. The diffusion constants of these two modes of motion are considerably different. Without the structural information about the membrane environment restricting the moving proteins, it would not be possible to reveal insight into these mechanisms. The high-resolution AFM topographs suggest that, in future studies, such data revealed under various physiological conditions will provide novel insights into molecular mechanisms that drive membrane protein assembly and supply excellent boundary conditions to model protein–protein arrangements.

Cationic liposomes enhanced firefly bioluminescent assay of bacterial ATP in the presence of an ATP extractant.

Cationic liposomes composed of two components, diethylaminoethyl-carbamoyl cholesterol and phosphatidylcholine, were applied to an enhancer for a firefly bioluminescent (BL) assay of bacterial ATP in the presence of an ATP extractant. Trichloroacetic acid (TCA), which inhibits the activity of luciferase, was used as an ATP extractant. Cationic liposomes enhanced the BL intensity as long as luciferase was active. The detection limits for cell numbers of Escherichia coli extracts in the presence of cationic liposomes and in water alone were 199 and 897 colony forming units ml(-1), respectively. The sensitivity for bacterial ATP in the presence of cationic liposomes was improved by a factor of 2.5 times compared to that in the presence of diethylaminoethyl-dextran.

Estimation of bacterial concentrations in commercial foods by measuring ATP changes during incubation.

Recently we have developed new kinetic methods of bacterial cell estimation in food products by measurement of the increase in bacterial adenosine-triphosphate (ATP) content during incubation using a conventional firefly luminometer [Fujikawa and Morozumi: Jpn. J. Food Hyg. (2002)]. The methods were the end point method and the delay method. We, in this study, evaluated these methods for bacterial concentration estimation in commercial foods. The methods were successfully applied to food products inoculated with Escherichia coli or Staphylococcus aureus. The methods were then used to estimate bacterial concentrations in commercial foods. For estimation, the kinetic parameter values were determined first from the data of bacterial strains studied previously. The bacterial concentrations estimated with these parameter values were slightly smaller than the measured concentrations for most of the food samples. The parameter values obtained with the least-squares method gave better estimations in both methods. The delay method predicted the bacterial concentrations in food products better than the end point method. This study showed that with appropriate parameter values, these methods could be useful tools for bacterial concentration estimation of commercial foods.

The structure of Escherichia coli BtuF and binding to its cognate ATP binding cassette transporter.

Bacterial binding protein-dependent ATP binding cassette (ABC) transporters facilitate uptake of essential nutrients. The crystal structure of Escherichia coli BtuF, the protein that binds vitamin B12 and delivers it to the periplasmic surface of the ABC transporter BtuCD, reveals a bi-lobed fold resembling that of the ferrichrome binding protein FhuD. B12 is bound in the "base-on" conformation in a deep cleft formed at the interface between the two lobes of BtuF. A stable complex between BtuF and BtuCD (with the stoichiometry BtuC2D2F) is demonstrated to form in vitro and was modeled using the individual crystal structures. Two surface glutamates from BtuF may interact with arginine residues on the periplasmic surface of the BtuCD transporter. These glutamate and arginine residues are conserved among binding proteins and ABC transporters mediating iron and B12 uptake, suggesting that they may have a role in docking and the transmission of conformational changes.

Development of a bioluminescent ATP assay to quantify mammalian and bacterial cell number from a mixed population

Modification of biomaterial surfaces is one approach aimed at improving cellular interactions and the subsequent integration of medical devices into the body. However, by optimising surfaces to enhance mammalian cell adhesion there is the potential risk that adhesion of bacteria will also be increased. Bacterial colonisation of biomaterials can be problematic as infection often results in morbidity and the consequent removal of the failing device from the body. Currently, quantifying cellular adhesion from a mixed population of bacterial and mammalian cells can only be determined by slow and laborious methods such as microscopy. ATP is a key molecule in the metabolism of both mammalian and bacterial cells and can be used to quantify cell numbers. In this study, we have modified a bioluminescence-based ATP assay to enable the differential measurement of both mammalian and bacterial cell ATP levels within the same culture. Mixed populations of Staphylococcus epidermidis and 3T3 fibroblasts were assessed both in suspension and adhered to a surface. ATP levels from cultures in suspension were selectively extracted and measured from both cell types, revealing a linear trend that would enable the differentiation of cell numbers from a mixed population. The application of the assay to adhered mixed cultures also allowed differences in ATP levels from both cell types to be distinguished. The data presented reveals that this assay would be useful for the rapid screening of cellular adhesion to modified surfaces although, its use in detecting subtle differences in ATP levels may be limited due to natural interactions between the two cell types.

Evolving an enzyme into different metabolic roles: structures of fungal and bacterial ATP sulfurylase

Evolving an enzyme into different metabolic roles: structures of fungal and bacterial ATP sulfurylase

Purinergic (P2X7) Receptor Activation of Microglia Induces Cell Death via an Interleukin-1-Independent Mechanism

Activation of purinergic P2X7 receptors, principally by extracellular ATP, promotes the processing and release of the cytokine interleukin-1β (IL-1β) and induces cell death in activated microglia and macrophages. The objective of this study was to determine if IL-1β release contributes directly to this cell death in microglia. Exposure of microglia to bacterial lipopolysaccharide (LPS) and ATP induced release of IL-1β and IL-1α, as well as cell death. Neither cell death nor IL-1 release was observed in microglia lacking the P2X7 receptor. Microglia from mice lacking the IL-1β gene demonstrated a profile of death identical to that of wild-type microglia in response to LPS and ATP. Thus, IL-1β is not required for P2X7 receptor-stimulated microglial death.

New estimation methods of bacterial concentration by measuring ATP changes during incubation.

New estimation methods of bacterial cell concentration in samples by measurement of the increase in bacterial adenosine-triphosphate (ATP) content during incubation using a conventional firefly luminometer were established. When an Escherichia coli cell suspension was incubated in nutrient broth, the increase in the ATP content of the suspension during the incubation period followed a sigmoidal curve. The increase ratio of the ATP content of the suspension at a given period of incubation (5 hours in this study) to the initial ATP content was greater at higher initial cell concentrations. With this relationship, the initial cell concentration of a test suspension could be predicted from the measured ratio; this was called the end point method. On the other hand, the lag period in the ATP increase curve was longer at lower initial cell concentrations. A highly linear relationship was observed between the lag period and the logarithm of the initial cell concentrations. Based on this relationship, a delay method was developed for prediction. The two relationships were also observed for bacterial suspensions of Klebsiella sp., Staphylococcus aureus, Bacillus subtilis, and Pseudomonas sp. These results suggested that the two methods have the potential to estimate the bacterial cell concentration of a sample suspension.

Modes of active inorganic carbon uptake in the cyanobacterium, Synechococcus sp. PCC7942.

Cyanobacteria (blue-green algae) have evolved a remarkable environmental adaptation for survival at limiting CO2 concentrations. The adaptation is known as a CO2 concentrating mechanism, and functions to actively transport and accumulate inorganic carbon (Ci; HCO3- and CO2) within the cell. Thereafter, this Ci pool is utilised to provide elevated CO2 concentrations around the primary CO2 fixing enzyme, Rubisco, which is encapsulated in a unique micro-compartment known as the carboxysome. Recently, significant progress has been gained in understanding the different types of Ci transport in cyanobacteria. This semi-review centres on the model cyanobacterium, Synechococcus sp. PCC7942, which possesses at least four distinct modes of Ci uptake when grown under Ci limitation, each possessing a high degree of functional redundancy. The four modes so far identified are: (i) BCT1, an inducible, high affinity HCO3- transporter of the bacterial ATP binding cassette transporter family, encoded by cmpABCD; (ii) a constitutive, Na+-dependent HCO3- transport system that can be allosterically activated (possibly by phosphorylation) in as little as 10 min; (iii) and (iv) two CO2 uptake systems, one constitutive and the other inducible, based on specialised forms of thylakoid-based, type 1, NAD(P)H dehydrogenase complexes (NDH-1). Here, we forward a speculative model that proposes that two unique proteins, ChpX and ChpY, possess CO2 hydration activity in the light, and when coupled to photosynthetic electron transport through the two specialised NDH-1 complexes, result in net hydration of CO2 to HCO3- as a crucial component of the CO2 uptake process.

The central plug in the reconstituted undecameric c cylinder of a bacterial ATP synthase consists of phospholipids

The isolated rotor cylinder of the ATP synthase from Ilyobacter tartaricus was reconstituted into two-dimensional crystalline arrays. Atomic force microscopy imaging indicated a central cavity on one side of the rotor and a central plug protruding from the other side. Upon incubation with phospholipase C, the plug disappeared, but the appearance of the surrounding c subunit oligomer was not affected. This indicates that the plug consists of phospholipids. As the detergent-purified c cylinder is completely devoid of phospholipids, these are incorporated into the central hole from one side of the cylinder during the reconstitution procedure.

Antibiotic susceptibility of bacterial strains isolated from patients with community-acquired urinary tract infections

Isolates from urine samples obtained during 1999 were indentified and their susceptibility to antimicrobial agents studied along with any production of extended-spectrum β-lactamases (ESBL) by Escherichia coli and Klebsiella pneumoniae. A total of 13 774 samples were analysed using an automatic system for the detection of bacterial ATP (Coral, USA). Of these samples, 49% were reported to be positive and uncontaminated; bacteria most frequently isolated were E. coli (47%), Proteus mirabilis (7%), Enterococcus faecalis (6%) and K. pneumoniae (5%). The susceptibility studies showed 37% E. coli strains resistant to amoxycillin+clavulanate 33% to cotrimoxazole and 22% to ciprofloxacin . Seven strains of E. coli produced ESBL. Thirteen per cent of strains were resistant to cefuroxime but only (1%) to fosfomycin. Resistance to nitrofurantoin in K. pneumoniae was 38% . P. mirabilis showed 52% resistance to cotrimoxazole and 13% Staphylococcus aureus, were methicillin-resistant. E. faecalis did not show any special resistance to normal medication. Fosfomycin continued to show high activity against Gram-negative bacilli. However, enterococci, some species of staphylococci and yeasts were difficult to treat empirically. ESBL were detected in the isolates of E. coli and there were some methicillin-resistant strains of S. aureus.

Inhibition of phosphofructokinases by copper(II)

The biochemical inhibition by Cu 2+ on eight phylogenetically and biochemically different phosphofructokinases (PFKs) was investigated. The enzymes screened included representatives from thermophilic and mesophilic bacteria, a hyperthermophilic archaeon and a eukaryote, covering all three phosphoryl donor subtypes (ATP, ADP and pyrophosphate). The sensitivities of the enzymes to Cu 2+ varied greatly, with the archaeal ADP-PFK being the least and the eukaryote ATP-PFK being the most sensitive. The bacterial ATP- and pyrophosphate-dependent PFKs showed intermediate sensitivity with the exception of the Spirochaeta thermophila enzyme (pyrophosphate-dependent) which was relatively resistant.

Inhibition of phosphofructokinases by copper(II).

The biochemical inhibition by Cu2+ on eight phylogenetically and biochemically different phosphofructokinases (PFKs) was investigated. The enzymes screened included representatives from thermophilic and mesophilic bacteria, a hyperthermophilic archaeon and a eukaryote, covering all three phosphoryl donor subtypes (ATP, ADP and pyrophosphate). The sensitivities of the enzymes to Cu2+ varied greatly, with the archaeal ADP-PFK being the least and the eukaryote ATP-PFK being the most sensitive. The bacterial ATP- and pyrophosphate-dependent PFKs showed intermediate sensitivity with the exception of the Spirochaeta thermophila enzyme (pyrophosphate-dependent) which was relatively resistant.

Salivary biomass assessed by bioluminescence ATP assay related to (bacterial and somatic) cell counts

The present work aimed (1) to evaluate ATP content in saliva by the bioluminescent luciferin–luciferase method, (2) to evaluate the relationships between ATP content, bacterial count and epithelial cell numbers in saliva, (3) to study the effect of two different antiseptics (peroxidase system producing hypothiocyanite and chlorhexidine) on the salivary biomass. In 45 young adults, the salivary ATP content ranged from 8 to 1515 nm. Salivary ATP content was significantly and directly correlated to bacterial count and epithelial cell numbers (Spearman–Rank correlation, P≤0·001). Regression analysis allowed the inference of a mean epithelial cell and bacterial ATP content of 152·7 fg and 8·3 fg per cell, respectively. The salivary ATP content decreased significantly to 38·8±12·3 per cent (mean±SEM, N=6) of its initial value after a 30-min incubation in the presence of a peroxidase system producing hypothiocyanite (OSCN−). Chlorhexidine (CHX) reduced salivary ATP content to 52·0±16·7 per cent. OSCN− did not affect the transformed logarithm of bacterial count but CHX reduced it from 7·02±0·26 to 0·52±0·33. No effect of OSCN− was seen on the ratio of epithelial cell viability while CHX reduced it from 46·7±5·1 to 3·9±1·1 per cent. It is concluded that the combination of the evaluations of the ATP content and cell numbers in saliva can provide reliable data about the effects of oral antiseptics on salivary biomass. Copyright © 2000 John Wiley & Sons, Ltd.