Studies of diurnal variations of nitrate reductase activity in barley leaves using various assay methods

The in vitro and various modifications of the in vivo assay for nitrate reductase have been compared in order to elucidate their usefulness in studies of diurnal variations of enzyme activity in barley leaves ( Hordeum vulgare L. cv. Herta). Generally, activity was low in the morning and increased rapidly during the first hours of the photoperiod. In the in vivo assay the leaf tissue was vacuum-infiltrated, whereafter either N₂ was bubbled through the assay buffer (anaerobic assay), or no N₂ was used (aerobic assay). Activity was 2–25 times higher in the anaerobic than in the aerobic assay. Anaerobiosis enhanced activity most during the dark period when the nitrate reductase level was low. Aerobic in vivo activity usually showed a more rapid decrease towards the end of the light period than did anaerobic activity. Addition of glucose and/or nitrate to the in vivo assay buffer usually stimulated activity more in the aerobic than in the anaerobic assay. In the morning, at the end of the dark period, these additives stimulated activity by 20–400% depending on growth and assay conditions. Later in the day stimulation was usually less, and even a slight inhibition was observed when only nitrate (0.1 M ) was added. The effect of these additives on the activity patterns determined was to dampen the oscillations. The additives were therefore not advantageous when testing diurnal variations. However, when the plants were grown under relatively poor light conditions it was necessary to add nitrate and glucose to the aerobic in vivo assay buffer since activity was otherwise too low to be measured. The in vitro assay gave about 5 times higher activity than the anaerobic in vivo assay. During the last part of the dark period in vivo activity (without glucose and KNO₃ in the assay buffer) decreased while in vitro activity remained constant.     

Collagenolytic activity of bacteria

Actively growing aerobic and anaerobic bacteria were screened by a plate assay, with reconstituted guinea pig collagen as a substrate, for their ability to produce a collagenolytic factor. Collagenolytic activity was not demonstrated among the aerobic organisms tested, with the exception of one strain of Staphylococcus aureus (only when grown under anaerobic conditions). Collagenolytic activity, however, was detected in cultures of Clostridium tetani and Bacteroides species other than B. melaninogenicus. Collagenolytic activity of these organisms could be confirmed by measuring the amount of hydroxyproline liberated from the collagen gel during growth. Although collagenase production by Pseudomonas aeruginosa has been suggested in previous reports, our results were negative. An extracellular fraction of P. aeruginosa was able to hydrolyze a synthetic hexapeptide Cbz-glycyl-l-prolyl-glycyl-glycyl-l-prolyl-l- alanine, but was without detectable effect on reconstituted collagen.     

Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.     

Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora.

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.     

Superoxide dismutase in ruminal bacteria.

Of 13 species of anaerobic ruminal bacteria examined, 11 were found to contain measurable levels of superoxide dismutase activity. Four of five other strict anaerobic species studied for comparison were found to contain superoxide dismutase activity.     

Superoxide dismutase in ruminal bacteria

Of 13 species of anaerobic ruminal bacteria examined, 11 were found to contain measurable levels of superoxide dismutase activity. Four of five other strict anaerobic species studied for comparison were found to contain superoxide dismutase activity.     

In vitro activity of linezolid and eperezolid, two novel oxazolidinone antimicrobial agents, against anaerobic bacteria

Linezolid (formerly U-100766) and eperezolid (formerly U-100592) are novel oxazolidinone antimicrobial agents that are active against multi-drug-resistant staphylococci, streptococci, enterococci, corynebacteria, and mycobacteria. Preliminary studies also demonstrated that the compounds inhibited some test strains of anaerobic bacteria. Therefore, we extended the in vitro evaluation of these agents to include a total of 54 different anaerobic species. Minimal inhibitory concentration (MIC) values were determined using a standard agar dilution method for 143 anaerobic bacterial isolates. Eperezolid and linezolid demonstrated potent activity against the anaerobic Gram-positive organisms with most MIC values in the range of 0.25-4 microg/mL. Viridans streptococci demonstrated MICs of 1-2 microg/mL; Peptostreptococcus species and Propionibacterium species were inhibited by 相似文献   

Transformation of glutamate to delta-aminolevulinic acid by soluble extracts of Chlorobium vibrioforme.

Formation of the tetrapyrrole pigment precursor delta-aminolevulinic acid (ALA) from glutamate was detected and partially characterized in extracts of the strictly anaerobic green photosynthetic bacterial species Chlorobium vibrioforme by using assay methods derived from those developed for algae and cyanobacteria. ALA formation in Chlorobium extracts was saturated at 10 mM glutamate and required NADPH and ATP at optimal concentrations of 0.3 and 3 mM, respectively. Preincubation of the enzyme extract with RNase A destroyed the ALA-forming activity completely. Activity in the RNase-treated extract was restored by supplementation with Chlorobium RNA after addition of RNasin to block further RNase action. RNA from the cyanobacterium Synechocystis sp. strain PCC 6803 and Escherichia coli tRNAGlu also restored activity. Activity was inhibited 50% by 0.2 microM hemin. ALA formation was completely abolished by the addition of 5 microM 3-amino-2,3-dihydrobenzoic acid (gabaculine). These results indicate that Chlorobium extracts share with those of plants, eucaryotic algae, cyanobacteria, prochlorophytes, and methanogens the capacity for RNA-dependent ALA formation from glutamate.     

PCR-based diagnostics for anaerobic infections

Conventional methods to identify anaerobic bacteria have often relied on unique clinical findings, isolation of organisms, and laboratory identification by morphology and biochemical tests (phenotypic tests). Although these methods are still fundamental, there is an increasing move toward molecular diagnostics of anaerobes. In this review, some of the molecular approaches to anaerobic diagnostics based on the polymerase chain reaction (PCR) are discussed. This includes several technological advances in PCR-based methods for the detection, identification, and quantitation of anaerobes including real-time PCR which has been successfully used to provide rapid, quantitative data on anaerobic species on clinical samples. Since its introduction in the mid-1980s, PCR has provided many molecular diagnostic tools, some of which are discussed within this review. With the advances in micro-array technology and real-time PCR methods, the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual anaerobic species but also on whole communities.     

Desulfoferrodoxin of Clostridium acetobutylicum functions as a superoxide reductase

Desulfoferrodoxin (cac2450) of Clostridium acetobutylicum was purified after overexpression in E. coli. In an in vitro assay the enzyme exhibited superoxide reductase activity with rubredoxin (cac2778) of C. acetobutylicum as the proximal electron donor. Rubredoxin was reduced by ferredoxin:NADP(+) reductase from spinach and NADPH. The superoxide anions, generated from dissolved oxygen using Xanthine and Xanthine oxidase, were reduced to hydrogen peroxide. Thus, we assume that desulfoferrodoxin is the key factor in the superoxide reductase dependent part of an alternative pathway for detoxification of reactive oxygen species in this obligate anaerobic bacterium.     

A rapid assay of the sialidase activity in species of the Bacteroides fragilis group by using peanut lectin hemagglutination

In this study, a novel, simple and rapid hemagglutination assay by using a peanut lectin to detect a neuraminidase activity in strains of the Bacteroides fragilis group was developed. One hundred and fourteen species of the B. fragilis group isolated from children with and without diarrhea and 15 reference strains were evaluated. Neuraminidase production was determined by using the method above described and its inhibition was observed by using galactose. The neuraminidase production was observed in 54 (84.37%) diarrhea and in 43 (86%) non-diarrhea strains. HA titers were ranged from 2 to 32. This neuraminidase assays based on PNA hemagglutination is highly sensitive, reproducible and could be used as a tool to detect the sialidase activity in anaerobic bacteria, particularly, in species of the B. fragilis group.     

Effect of oxygen partial pressure on nitrogen fixation and acetylene reduction in a sandy loam soil amended with glucose

Summary Small samples of soil amended with 2% (w/w) of glucose were preincubated either aerobically or anaerobically and then assayed (N₂ ¹⁵ and C₂H₂-C₂H₄) either aerobically or anaerobically for different time periods. One-hour C₂H₂-C₂H₄ assays showed greatest activity when anaerobic assay followed anaerobic preincubation. During the anaerobic preincubation a lag of 12–24 h occurred before rapid increase in one-hour assay activity was observed. When aerobic assay followed aerobic preincubation a longer lag was observed and lower activities were obtained. When anaerobic assay followed aerobic preincubation (orvice versa) negligible activities were observed in short assays, and longer assays showed increasing activity related to changes in atmosphere and/or microbial population in the closed system. Preincubation of soil on a diffusion gradient at a series of different partial pressures of oxygen confirmed the above pattern and showed that as preincubation pO₂ increased, the anaerobic assay activity rapidly decreased. As preincubation pO₂ decreased from 0.2 atm the aerobic assay activity decreased but less rapidly. The activities observed were related to the sizes of the Azotobacter and Clostridium populations. There was no evidence of aerobic or anaerobic C₂H₂ reduction in any cultures of ‘oligonitrophiles’ isolated. Incorporation of N₂ ¹⁵ was related to C₂H₂ reduction activity in the soil system studied. However, observed C₂H₄/N₂ molar ratios ranged from 10 to 22 and appeared to be highest in samples which were preincubated anaerobically. Issued as Macdonald College Journal Series No.618 and as Canadian IBP contribution No.84.     

The place of molecular genetic methods in the diagnostics of human pathogenic anaerobic bacteria. A minireview

Anaerobic infections are common and can cause diseases associated with severe morbidity, but are easily overlooked in clinical settings. Both the relatively small number of infections due to exogenous anaerobes and the much larger number of infections involving anaerobic species that are originally members of the normal flora, may lead to a life-threatening situation unless appropriate treatment is instituted. Special laboratory procedures are needed for the isolation, identification and susceptibility testing of this diverse group of bacteria. Since many anaerobes grow more slowly than the facultative or aerobic bacteria, and particularly since clinical specimens yielding anaerobic bacteria commonly contain several organisms and often very complex mixtures of aerobic and anaerobic bacteria, considerable time may elapse before the laboratory is able to provide a final report. Species definition based on phenotypic features is often time-consuming and is not always easy to carry out. Molecular genetic methods may help in the everyday clinical microbiological practice in laboratories dealing with the diagnostics of anaerobic infections. Methods have been introduced for species diagnostics, such as 16S rRNA PCR-RFLP profile determination, which can help to distinguish species of Bacteroides, Prevotella, Actinomyces, etc. that are otherwise difficult to differentiate. The use of DNA-DNA hybridization and the sequencing of special regions of the 16S rRNA have revealed fundamental taxonomic changes among anaerobic bacteria. Some anaerobic bacteria are extremely slow growing or not cultivatable at all. To detect them in special infections involving flora changes due to oral malignancy or periodontitis, for instance, a PCR-based hybridization technique is used. Molecular methods have demonstrated the spread of specific resistance genes among the most important anaerobic bacteria, the members of the Bacteroides genus. Their detection and investigation of the IS elements involved in their expression may facilitate following of the spread of antibiotic resistance among anaerobic bacteria involved in infections and in the normal flora members. Molecular methods (a search for toxin genes and ribotyping) may promote a better understanding of the pathogenic features of some anaerobic infections, such as the nosocomial diarrhoea caused by C. difficile and its spread in the hospital environment and the community. The investigation of toxin production at a molecular level helps in the detection of new toxin types. This mini-review surveys some of the results obtained by our group and others using molecular genetic methods in anaerobic diagnostics.     

Evaluation for anaerobic culture system: Anoxomat Mart II]

Anoxomat Mart II (Mart Microbiology BV, Lichtenvooorde, Netherlands, Central Scientific Commerce Inc., Tokyo, Japan) is an anaerobic jar apparatus which uses a vacuum pump in combination with catalyst as gas replacement procedure to remove all traces of oxygen. As we had a chance to use Anoxomat Mart II, we compared it with other two anaerobic culture methods; namely AnaeroPack anaero (Mitsubishi Gas Chemical Co., Tokyo, Japan) which employs anaerobic jar method, and Concept400 (RUSKINN TECHNOLOGY LTD, England; Central Scientific Commerce INc., Tokyo, Japan) which uses anaerobic chamber method. We used 10 different species of anaerobic bacteria obtained from ATCC. One strain each of 10 species was cultured and examined for measurement of the sensitibity of an anaerobic indicator, th number of bacteria after 48 hour culture, the diameter of colonies, and MIC value. As a result, the time to reach the anaerobic condition was around 30 minutes by the Mart II against around 60 minutes by the AnaeroPack anaero. There was no difference concerning the number of bacteria after 48 hour culture among three methods. But anaerobic bacteria cultured by Mart II tended to make bigger colonies compared to other two methods in the 5 strains out of 9, except for one strain in which the diameter of colonies could not be measured. On the other hand, the comparison of MIC value showed good correlation in 11 antibiotics out of 12 among three methods. The MIC value of 11 antibiotics fitted within 1-fold difference, and 2-fold difference was observed in only one antibiotic. Mart II is so small that it does cheep consumables. From these reasons, we concluded that Mart II can be one of the useful anerobic culture methods.     

木麻黄和桤木离体根瘤和立地土壤的固氮酶与N2O还原酶活性的研究

为了解非豆科固氮树种的固氮酶和N_2O还原酶(Nos)活性,采用乙炔还原法和乙炔抑制技术对细枝木麻黄(Casuarina cunninghamiana)和江南桤木(Alnus trabeculosa)离体根瘤及立地土壤的两种酶活性进行了研究。结果表明,离体根瘤只在厌氧条件下有固氮酶活性,在好氧条件下有Nos活性。根瘤区根际土和非根瘤区根际土的固氮酶活性在好氧条件大于厌氧条件,Nos活性只表现在厌氧条件下。在好氧条件下,根瘤区根际土和非根瘤区根际土的固氮酶活性无显著差异;根瘤区根际土的Nos活性显著大于非根瘤区根际土。除离体根瘤在好氧条件下不表现固氮酶活性外,细枝木麻黄和桤木的离体根瘤、根瘤区根际土和非根瘤区根际土的固氮酶活性均都大于Nos活性。好氧条件下根瘤区根际土的固氮酶活性与非根瘤区根际土的呈极显著正相关,而厌氧条件下根瘤的固氮酶活性与好氧条件下根瘤区根际土和非根瘤区根际土固氮酶活性、好氧条件下根瘤的Nos活性与厌氧条件下根瘤区根际土和非根瘤区根际土Nos活性均呈极显著负相关。这为研究弗兰克氏菌结瘤植物共生固氮体系对N2O汇强度的影响和调控奠定基础。     

Detection of collagenase activity in oral bacteria

Collagenolytic activity of 12 species of oral bacteria was assessed using two methods of detection. Except for two species, all bacterial strains tested were capable of degrading at least one general protein substrate. Results of collagenolytic activity in a growth assay indicate that Bacteroides gingivalis is the only bacterium capable of degrading collagen when the substrate is sterilized using ethylene oxide. However, if the substrate is sterilized by autoclaving, in the presence or absence of the growth medium, other bacterial species could be shown to be collagenolytic. Collagenolytic activity was also demonstrated when whole or broken cells were used in a [14C]collagen assay. Results from this assay and from inhibition studies indicate that collagenolytic activity can either be the result of the combined activities of both a specific collagenase and nonspecific proteases (B. gingivalis) or nonspecific proteases only (other strains in this study), although in the latter case, the time taken to hydrolyze collagen can be 10 times longer than with a specific collagenase.     

Production and consumption of nitric oxide by denitrifying bacteria under anaerobic and aerobic conditions

Abstract: Pseudomonas aeruginosa, P. stutzeri and Azospirillum brasilense showed highest NO production rates and NO consumption rate constants when anaerobically grown cells were tested under anaerobic conditions. Aerobic assay conditions resulted in 20–75-fold lower NO production rates. NO consumption rate constants, however, decreased by less than a factor of four. NO consumption activity was observed even in aerobically grown P. aeruginosa , provided the assay was done under anaerobic conditions. Obviously, NO consumption was less O₂-sensitive than NO production so that compensation between production and consumption occurred at lower NO mixing ratios under aerobic than under anaerobic conditions.     

Species and Environmental Variations in the Effect of Inorganic Phosphate on Sucrose-Phosphate Synthase Activity : Reliability of Assays Based Upon UDP Formation

The effect of inorganic phosphate (Pi) on sucrose-phosphate synthase (SPS) activity was determined for the enzyme from five plant species (Nicotiana tabacum L., Spinacia oleracea L., Triticum aestivum L., Zea mays L., Glycine max L.) using two assay methods. The assay method based on determination of uridine diphosphate glucose- (UDPG) and fructose-6-phosphate-dependent sucrose formation was linear up to 15 minutes for all species tested. When assayed in this way, the effect of Pi at levels of 5 or 10 millimolar in the assay was variable, ranging from 0 to 35% inhibition of SPS activity. The assay method based on substrate dependent UDP formation was linear for some, but not for all of the species tested. Deviations from linearity were caused by loss of UDP from the assay medium. In some species, the extent of UDP loss was influenced by the level of Pi in the assay medium and, for at least one species (tobacco), it was influenced by the environment in which the plants were grown. The results indicated that (a) the role of Pi as an effector of SPS may vary depending on the species, and (b) the UDP assay method should be used with caution for assays of crude or desalted extracts, particularly when evaluating the effect of Pi on SPS activity.     

Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen

Traditional methods for enumerating and identifying microbial populations within the rumen can be time consuming and cumbersome. Methods that involve culturing and microscopy can also be inconclusive, particularly when studying anaerobic rumen fungi. A real-time PCR SYBR Green assay, using PCR primers to target total rumen fungi and the cellulolytic bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes, is described, including design and validation. The DNA and crude protein contents with respect to the fungal biomass of both polycentric and monocentric fungal isolates were investigated across the fungal growth stages to aid in standard curve generation. The primer sets used were found to be target specific with no detectable cross-reactivity. Subsequently, the real-time PCR assay was employed in a study to detect these populations within cattle rumen. The anaerobic fungal target was observed to increase 3.6-fold from 0 to 12 h after feeding. The results also indicated a 5.4-fold increase in F. succinogenes target between 0 and 12 h after feeding, whereas R. flavefaciens was observed to maintain more or less consistent levels. This is the first report of a real-time PCR assay to estimate the rumen anaerobic fungal population.     

Survey of wastewater indicators and human pathogen genomes in biosolids produced by class a and class B stabilization treatments

Accurate modeling of the infectious aerosol risk associated with the land application of biosolids requires an in-depth knowledge of the magnitudes and changes in pathogen concentrations for a variety of class A and class B stabilization methods. The following survey used quantitative PCR (qPCR) and culture assays to detect environmentally resistant bacterial and viral pathogens and biosolid indicator organisms for 36 biosolid grab samples. Biosolids were collected from 14 U.S. states and included 16 class B mesophilic anaerobic digestion (MAD) samples and 20 class A biosolid samples from temperature-phased anaerobic digestion (TPAD), MAD plus composting (COM), and MAD plus heat pelletization processes. The indicator concentrations of fecal coliforms and male-specific coliphages as well as pathogen genome concentrations for human adenovirus species, Legionella pneumophila, Staphylococcus aureus, and Clostridium difficile were significantly lower in the class A samples, and a multivariate analysis of variance ranked the stabilization processes from the lowest pathogen/indicator load to the highest as (i) class A COM, (ii) class A TPAD, and (iii) class B MAD. Human adenovirus genomes were found in 88% of the class B samples and 70 to 100% of the class A samples. L. pneumophila, S. aureus, and C. difficile genomes were detected at the qPCR assay detection limits in 19 to 50% of the class B and class A anaerobic digestion samples, while L. pneumophila was detected in 50% of the class A compost samples. When considering all the stabilization methods, both the fecal coliform and the male-specific coliphage concentrations show a significant linear correlation with the pathogen genome concentrations. This survey provides the necessary pathogen concentrations to add to biosolid aerosol risk and pathogen exposure analyses and clarifies the effectiveness of class A stabilization methods with the pathogen and indicator loads in biosolids.