Mutation of Haemophilus influenzae transforming DNA in vitro with near-ultraviolet radiation: action spectrum

Mutations were produced in purified transforming DNA from Haemophilus influenzae by near-UV radiation and were assayed as mutants among cells transformed with irradiated DNA. The maximum efficiency of mutation induction was at around 334 nm, and the efficiency dropped off steeply at lower and higher wavelengths. The difference between the action spectrum for mutation and that for the oxygen-independent inactivation of transforming DNA, which had a shoulder at 365 nm, indicates that there are different lesions involved in the inactivating and mutagenic effects of near-UV. The presence of histidine during irradiation enhanced the mutagenic effect at 334 and 365 nm, although it protected against inactivation at 365 nm. The effective near-UV wavelengths for in vitro mutation are to some extent the same as the effective wavelengths for mutation in vivo reported previously. These findings indicate that mutations are produced in vivo by near-UV with DNA as the primary target molecule rather than by a secondary non-photochemical reaction between DNA and some other cell component.     

Effect of γ Irradiation on the Fatty Acid Composition of Soybean and Soybean Oil

Food irradiation is a form of food processing to extend the shelf life and reduce spoilage of food. We examined the effects of γ radiation on the fatty acid composition, lipid peroxidation level, and antioxidative activity of soybean and soybean oil which both contain a large amount of unsaturated fatty acids. Irradiation at 10 to 80 kGy under aerobic conditions did not markedly change the fatty acid composition of soybean. While 10-kGy irradiation did not markedly affect the fatty acid composition of soybean oil under either aerobic or anaerobic conditions, 40-kGy irradiation considerably altered the fatty acid composition of soybean oil under aerobic conditions, but not under anaerobic conditions. Moreover, 40-kGy irradiation produced a significant amount of trans fatty acids under aerobic conditions, but not under anaerobic conditions. Irradiating soybean oil induced lipid peroxidation and reduced the radical scavenging activity under aerobic conditions, but had no effect under anaerobic conditions. These results indicate that the fatty acid composition of soybean was not markedly affected by radiation at 10 kGy, and that anaerobic conditions reduced the degradation of soybean oil that occurred with high doses of γ radiation.     

Effect of γ irradiation on the fatty acid composition of soybean and soybean oil

Food irradiation is a form of food processing to extend the shelf life and reduce spoilage of food. We examined the effects of γ radiation on the fatty acid composition, lipid peroxidation level, and antioxidative activity of soybean and soybean oil which both contain a large amount of unsaturated fatty acids. Irradiation at 10 to 80 kGy under aerobic conditions did not markedly change the fatty acid composition of soybean. While 10-kGy irradiation did not markedly affect the fatty acid composition of soybean oil under either aerobic or anaerobic conditions, 40-kGy irradiation considerably altered the fatty acid composition of soybean oil under aerobic conditions, but not under anaerobic conditions. Moreover, 40-kGy irradiation produced a significant amount of trans fatty acids under aerobic conditions, but not under anaerobic conditions. Irradiating soybean oil induced lipid peroxidation and reduced the radical scavenging activity under aerobic conditions, but had no effect under anaerobic conditions. These results indicate that the fatty acid composition of soybean was not markedly affected by radiation at 10 kGy, and that anaerobic conditions reduced the degradation of soybean oil that occurred with high doses of γ radiation.     

Loss of photoreversibility for UV mutation in E. coli using 405 nm or near-UV challenge

E. coli mutagenized with germicidal ultraviolet light (UV) were incubated to allow for development of mutation-fixation processes. Fixation was estimated from the effects on mutation frequency of photoreactivation challenge during the first 60 min post-UV. Two different light sources were used for photoreactivation, one providing effective light primarily at 405 nm and another providing a broad range of near-UV around 365 nm. Kinetics for the loss of photoreversibility (LOP) were determined. The times for completion of LOP in wild-type cells indicated one fixation process for back mutation and another for de novo or converted suppressor mutation regardless of the light source. Using 405-nm light for photoreactivation, the LOP kinetics for back mutation and de novo suppressor mutation in uvrA cells were similar. Hence, classical observations were confirmed here. Immediately post-UV all mutation frequencies were more sensitive to near-UV than 405-nm light. Experiments with rel cells supported the idea that growth delay and inhibition of induced lexA-coordinated responses may be responsible for this early, pronounced sensitivity to photoreactivation by near UV. For back mutation and de novo suppressor mutation, the sensitivity to 405-nm light was initially small and actually increased for 10-15 min. Possibly genome conformation changes are induced by UV and this affects the efficiency of photoenzymatic monomerization of 405-nm light during the first 10-15 min after irradiation.     

Action spectrum for lethality of near-UV light on Haemophilus influenzae and lack of mutation.

Mutation and inactivation of H. influenzae have been measured following irradiation at various near-UV wavelengths. Inactivation takes place most readily at 334 nm (but is unaffected by absence of excision or postreplication repair), and decreases markedly at longer wavelengths. No induced mutations to resistance to novobiocin or streptomycin or to ability to utilize protoporphyrin instead of hemin were detected at any of the wavelengths used. There were also no detectable induced mutations in an excision-defective strain after 334-nm irradiation. These results are in contrast to the in vitro mutation of purified transforming DNA we previously observed.     

Effect of oxygen on Bacteroides fragilis survival after far-ultraviolet irradiation.

Bacteroides fragilis cells were more sensitive to far-ultraviolet radiation under aerobic conditions than under anaerobic conditions. The percentage of pyrimidine dimers assayed after irradiation under both conditions was similar.     

哮喘患儿口咽部菌群分析及3株优势菌的拮抗作用

了解哮喘患儿呼吸道菌群多样性及其组成特征, 同时研究所分离的3株优势菌对流感嗜血杆菌的抑制作用, 探究哮喘与呼吸道菌群之间的关系。

采集沈阳市儿童医院呼吸内科2019年3月至2019年12月收治的21例4~12岁急性发作期哮喘患儿咽拭标本, 并同时采集23例同龄健康儿童的咽拭标本作为对照, 对呼吸道菌群进行分离培养、纯化和16S rRNA鉴定。采用牛津杯法检测健康儿童口咽部分离的3株优势菌对流感嗜血杆菌的拮抗作用。

哮喘和健康儿童呼吸道培养出的需氧菌(t=2.143, P=0.038)和厌氧菌(t=3.270, P=0.002)的密度差异有统计学意义。哮喘患儿咽部需氧菌以肺炎链球菌和流感嗜血杆菌为主, 厌氧菌以韦荣球菌为主。健康儿童咽部需氧菌以缓症链球菌和口腔链球菌为主, 厌氧菌以干酪乳杆菌为主。健康儿童口咽部的3株优势菌对哮喘儿童口咽部流感嗜血杆菌的生长具有显著抑制作用。

与健康儿童相比, 哮喘患儿口咽部菌群发生紊乱, 且哮喘患儿口咽部需氧菌、厌氧菌密度显著增加。健康儿童口咽部的某些优势菌可能对哮喘致病菌的定植有一定的拮抗作用。

     

Enhancement on biodegradation and anaerobic digestion efficiency of activated sludge using a dual irradiation process

A dual irradiation process involving aerobic thermophilic irradiation pretreatment (ATIP) and intermittent irradiation anaerobic digestion was developed to improve the digestion of waste-activated sludge. First, the effect of ATIP on further anaerobic digestion of activated sludge in batch mode was investigated. When exposed to ATIP for 24 h, the digestion reactor gave the highest methane yield, removed the most dissolved organic carbon (DOC) and showed the most effective reduction of VS compared to other irradiation times. This process was further enhanced by using an anaerobic fluidised-bed reactor packed with carbon felt in semi-continuous mode for digesting the pretreated activated sludge under intermittent irradiation conditions. Dual irradiation for 24 h followed by 60 min of anaerobic irradiation processing per day turned out to be optimal. This resulted in 65.3% of VS reduction, 83.9% of DOC removal ratio and 538 ml/g-VS of methane yield.     

Cytotoxic and Genotoxic Consequences of Heat Stress Are Dependent on the Presence of Oxygen in Saccharomyces cerevisiae

Lethal heat stress generates oxidative stress in Saccharomyces cerevisiae, and anaerobic cells are several orders of magnitude more resistant than aerobic cells to a 50 degrees C heat shock. Here we characterize the oxidative effects of this heat stress. The thermoprotective effect in anaerobic cells was not due to expression of HSP104 or any other heat shock gene, raising the possibility that the toxicity of lethal heat shock is due mainly to oxidative stress. Aerobic but not anaerobic heat stress caused elevated frequencies of forward mutations and interchromosomal DNA recombination. Oxidative DNA repair glycosylase-deficient strains under aerobic conditions showed a powerful induction of forward mutation frequencies compared to wild-type cells, which was completely abolished under anaerobiosis. We also investigated potential causes for this oxygen-dependent heat shock-induced genetic instability. Levels of sulfhydryl groups, dominated mainly by the high levels of the antioxidant glutathione (reduced form) and levels of vitamin E, decreased after aerobic heat stress but not after anaerobic heat stress. Aerobic heat stress also led to an increase in mitochondrial membrane disruption of several hundredfold, which was 100-fold reduced under anaerobic conditions.     

Inhibition of respiration in yeast by light

Irradiation of starved cultures of Saccharomyces cerevisiae with blue light under aerobic conditions inhibited the capacity of the yeast cells to respire added substrates (e.g., ethanol) and stimulated endogenous respiration. Spectroscopic examination of the cells showed that the irradiation destroyed both cytochrome a and a₃ components of cytochrome oxidase and a part of the cytochrome b. Irradiation under anaerobic conditions had no effect on the respiratory capacity or the cytochrome content of the cells. Under aerobic conditions cytochrome a₃ was protected against photodestruction when complexed with cyanide and cytochrome a was protected when complexed with azide.     

The occurrence of denitrification in extremely halophilic bacteria

Abstract A total of 97 aerobic and facultatively anaerobic bacteria, and 3 Candida albicans , were tested for their ability to inhibit the growth of Haemophilus influenzae . Only strains of Pseudomonas aeruginosa showed any inhibitory effect and all 5 strains tested clearly inhibited the growth of all 10 strains of H. influenzae . The inhibition of H. influenzae . by Ps. aeruginosa may play a role in the establishment of chronic Ps. aeruginosa infections in the respiratory tracts of patients with bronchiectasis and cystic fibrosis (CF).     

Photoinactivation of acetylcholinesterase by erythrosin B and related compounds

Acetylcholinesterase was rapidly inactivated when exposed to light in the presence of xanthene dyes. Photosensitizing efficiency paralleled the dye triplet state quantum yields, increasing in the order fluorescein less than eosin B less than eosin Y less than erythrosin B less than rose bengal. The observed first-order rate constants of photoinactivation increased hyperbolically with dye concentration. Evidence for the formation of a dye-enzyme complex prior to inactivation was obtained from spectrophotometric and protein fluorescence quenching methods. The latter technique allowed estimates of the dye-enzyme dissociation constants for rose bengal (20 microM) and erythrosin B (30 microM). After photoinactivation, a portion of the dye became covalently bound to the enzyme. The photoinactivation reaction occurs in both aerobic (air saturated) and anaerobic (argon saturated) solution, with the rates of photoinactivation being about three to five times greater under the latter conditions. The aerobic reaction exhibits a large deuterium isotope enhancement effect and is largely (but not completely) quenched by 10(-2) M azide. The anaerobic reaction is unaffected by azide and exhibits only a small deuterium isotope effect. These results indicate that the photoinactivation reaction proceeds mainly by a type II (singlet oxygen mediated) pathway under aerobic conditions and by a type I (radical) pathway under anaerobic conditions. The enzyme was protected from inactivation by edrophonium, a competitive inhibitor, but not by d-tubocurarine, a peripheral-site ligand, indicating that destruction of a crucial residue at or near the catalytic site is an important component of the inactivation process. Extensive destruction of tryptophan undoubtedly occurs, at least under aerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacterial DNA supercoiling and [ATP]/[ADP]. Changes associated with a transition to anaerobic growth.

Shifting Escherichia coli from aerobic to anaerobic growth caused changes in the ratio of [ATP]/[ADP] and in negative supercoiling of chromosomal and plasmid DNA. Shortly after lowering oxygen tension, both [ATP]/[ADP] and supercoiling transiently decreased. Under conditions of exponential anaerobic growth, both were higher than under aerobic conditions. These correlations may reflect an effect of [ATP]/[ADP] on DNA gyrase, since in vitro [ATP]/[ADP] influences the level of plasmid supercoiling attained when gyrase is either introducing or removing supercoils. When the supercoiling activity of gyrase was perturbed by a mutation in gyrB, a shift to anaerobic conditions resulted in plasmid supercoil relaxation similar to that seen with wild-type. However, the low level of supercoiling in the mutant persisted during a time when supercoiling in wild-type recovered and then exceeded aerobic levels. Thus, changes in oxygen tension can alter DNA supercoiling through an effect on gyrase, and correlations exist between changes in supercoiling and changes in the intracellular ratio of [ATP]/[ADP].     

The ArcA regulon and oxidative stress resistance in Haemophilus influenzae

Haemophilus influenzae transits between niches within the human host that are predicted to differ in oxygen levels. The ArcAB two-component signal transduction system controls gene expression in response to respiratory conditions of growth and has been implicated in bacterial pathogenesis, yet the mechanism is not understood. We undertook a genome-scale study to identify genes of the H. influenzae ArcA regulon. Deletion of arcA resulted in increased anaerobic expression of genes of the respiratory chain and of H. influenzae's partial tricarboxylic acid cycle, and decreased anaerobic expression levels of genes of polyamine metabolism, and iron sequestration. Deletion of arcA also conferred a susceptibility to transient exposure to hydrogen peroxide that was greater following anaerobic growth than after aerobic growth. Array data revealed that the dps gene, not previously assigned to the ArcA modulon in bacteria, exhibited decreased expression in the arcA mutant. Deletion of dps resulted in hydrogen peroxide sensitivity and complementation restored resistance, providing insight into the previously uncharacterized mechanism of arcA-mediated H(2)O(2) resistance. The results indicate a role for H. influenzae arcA and dps in pre-emptive defence against transitions from growth in low oxygen environments to aerobic exposure to hydrogen peroxide, an antibacterial oxidant produced by phagocytes during infection.     

Effect of antimicrobial therapy on bowel flora and bacterial infection in irradiated mice

Mice exposed to 10 Gy cobalt-60 radiation were given intramuscular antimicrobial therapy of gentamicin, or metronidazole, or a combination of the two. Mortality in the mice treated with metronidazole alone or in combination with gentamicin occurred earlier than in the controls (P less than 0.001). Microorganisms were recovered from the blood, spleen, and liver of the metronidazole-treated mice earlier than from other groups. The predominant organisms recovered from these animals were Enterobacteriaceae. Quantitative cultures of the ileal flora showed a decrease in the number of aerobic, facultative anaerobic and strict anaerobic bacteria after irradiation, and a subsequent increase only in the number of strict aerobic bacteria. As compared to untreated mice, a rapid decrease (by 8.8 logs) in the number of anaerobic flora occurred in the mice treated with metronidazole 5 days after irradiation. This was followed by a rapid increase in the number of aerobic organisms which coincided with the earlier mortality in this group. These data suggest that antimicrobial agents that decrease the number of the strict anaerobic component of the gut flora enhance systemic infection by aerobic or facultative anaerobic bacteria, and this facilitates mortality after irradiation.     

Eradication of Polymyxa betae by Thermal and Anaerobic Conditions and in the Presence of Compost Leachate

The abiotic conditions required for eradication of Polymyxa betae, the vector of Beet necrotic yellow vein virus in sugar beet, were investigated. Survival of resting spores of P. betae was determined under aerobic (30 min, 4 days and 21 days) and anaerobic (4 days) conditions under several temperature regimes in a water suspension and in leachate extracted from an aerobic compost heap. In water under aerobic conditions the lethal temperature was 60, 55 and 40°C for exposure times of 30 min, 4 days and 21 days, respectively. The effect of compost leachate and/or anaerobic conditions on survival of P. betae depended on temperature. After incubation for 4 days at 20°C, no significant effects of anaerobic conditions or leachate on the survival of P. betae were found. However, at 40°C for 4 days under anaerobic conditions, survival of P. betae was significantly lower than survival under aerobic conditions in water as well as in leachate. In leachate taken from an aerobic compost heap, aerobically incubated at 40°C for 4 days, survival of P. betae was significantly lower than survival in water at the same temperature. As anaerobic spots are prevalent in aerobic compost heaps, especially during the thermophilic phase, actual inactivation temperatures under composting conditions are likely to be lower than the temperatures we found for eradication in water under aerobic conditions.     

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.     

Control of porphyrin biosynthesis in Rhodopseudomonas spheroides and Propionibacterium shermanii. A direct 13C nuclear-magnetic-resonance spectroscopy study.

The facultative anaerobes Rhodopseudomonas spheroides and Propionibacterium shermanii were grown under anaerobic and aerobic conditions. The effect of light was studied with the photosynthetic R. spheroides, and the adaptation of both species to dark anaerobic life was monitored by direct observation of 5-amino[5-13C]laevulinic acid metabolism by using 13C nuclear-magnetic-resonance spectroscopy.     

Requirement for terminal cytochromes in generation of the aerobic signal for the arc regulatory system in Escherichia coli: study utilizing deletions and lac fusions of cyo and cyd.

Escherichia coli has two terminal oxidases for its respiratory chain: cytochrome o (low O2 affinity) and cytochrome d (high O2 affinity). Expression of the cyo operon, encoding cytochrome o, is decreased by anaerobic growth, whereas expression of the cyd operon, encoding cytochrome d, is increased by anaerobic growth. We show by the use of lac gene fusion that the expressions of cyo and cyd are under the control of the two-component arc system. In a cyo+ cyd+ background, expression of phi(cyo-lac) is higher when the organism is grown aerobically than when it is grown anaerobically. A mutation in either the sensor gene arcB or the pleiotropic regulator gene arcA almost abolishes the anaerobic repression. In the same background, expression of phi(cyd-lac) is higher under anaerobic growth conditions than under aerobic growth conditions. A mutation in arcA or arcB lowers both the aerobic and anaerobic expressions, suggesting that ArcA plays an activating role instead of the typical repressing role. Under aerobic growth conditions, double deletions of cyo and cyd lower phi(cyo-lac) expression but enhance phi(cyd-lac) expression. The double deletions also prevent elevated aerobic induction of the lct operon (encoding L-lactate dehydrogenase), another target operon of the arc system. In contrast, these deletions do not circumvent aerobic repression of the nar operon (encoding the anaerobic respiratory enzyme nitrate reductase) under the control of the pleiotropic fnr gene product. It thus appears that ArcB senses the presence of O2 by level of an electron transport component in reduced form or that of an nonautoxidizable compound linked to the process by a redox reaction, whereas Fnr senses O2 by a different mechanism.     

Characterization of a nalidixic-acid-resistant mutant of Escherichia coli as a strict aerobe

An Escherichia coli mutant, C18, which plates at an efficiency of 5.0 x 10(-4) under anaerobic condition, was isolated among spontaneous nalidixic-acid-resistant mutants. This strict aerobic mutation was mapped by P1 cotransduction with a gyrA linked transposon Tn10 and found to be at the gyrA gene. A low degree of superhelicity of pBR322 DNA isolated from C18 was demonstrated by agarose gel electrophoresis with various concentrations of ethidium bromide. The superhelical density of pBR322 isolated from C18 was 80% of the value of pBR322 isolated from wild-type bacteria cultured under aerobic condition, and 50% cultured under anaerobic condition. These results lead us to conclude that a certain mutation of the gyrA gene causes a decrease in DNA superhelicity and prevents anaerobic growth.