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PubMed1983

Genetic manipulation of antibiotic-producing microorganisms.

The application of directed selection techniques and genetic engineering methods for manipulation of antibiotic-producing microorganisms is generating a new era in industrial microbiology. Modern methods, based on advances in the knowledge of the biosynthetic pathways and regulatory mechanisms involved in the induction and repression of genes involved in antibiotic synthesis, provide a means of increasing antibiotic activity. Hence, recombinant DNA and protoplast fusion methods are used to alter the genetics of antibiotic producers in a semirandom fashion for the development of novel hybrid antibiotics. Directed mutation and selection, protoplast fusion, and both semirandom and specific recombinant DNA methods are examples of alternative procedures for manipulating the biosynthetic pathways of microorganisms for strain improvement and for new hybrid antibiotic synthesis.

Anti-Bacterial Agents↗
PubMed2004

Cold shock response and adaptation at near-freezing temperature in microorganisms.

Microorganisms that naturally encounter sharp temperature shifts must develop strategies for responding and adapting to these shifts. Escherichia coli, which are adapted to living at both warm temperatures inside animals and cooler ambient temperatures, respond to low temperatures (10 degrees to 15 degrees C) by adjusting membrane lipid composition and increasing the production of proteins that act as "RNA chaperones" required for transcription and translation and proteins that facilitate ribosomal assembly. In contrast, yeast, which are adapted to cooler temperatures, show a relatively minor cold shock response after temperature shifts from 30 degrees to 10 degrees C but respond with a dramatic increase in the synthesis of trehalose and a heat shock protein when exposed to freezing or near-freezing temperatures. This emphasizes the fact that different groups of microorganisms exhibit distinct types of cold shock responses.

Adaptation, Physiological↗
PubMed2006

Identification of acetate-assimilating microorganisms under methanogenic conditions in anoxic rice field soil by comparative stable isotope probing of RNA.

Acetate is the most abundant intermediate of organic matter degradation in anoxic rice field soil and is converted to CH(4) and/or CO(2). Aceticlastic methanogens are the primary microorganisms dissimilating acetate in the absence of sulfate and reducible ferric iron. In contrast, very little is known about bacteria capable of assimilating acetate under methanogenic conditions. Here, we identified active acetate-assimilating microorganisms by using a combined approach of frequent label application at a low concentration and comparative RNA-stable isotope probing with (13)C-labeled and unlabeled acetate. Rice field soil was incubated anaerobically at 25 degrees C for 12 days, during which (13)C-labeled acetate was added at a concentration of 500 muM every 3 days. (13)C-labeled CH(4) and CO(2) were produced from the beginning of the incubation and accounted for about 60% of the supplied acetate (13)C. RNA was extracted from the cells in each sample taken and separated by isopycnic centrifugation according to molecular weight. Bacterial and archaeal populations in each density fraction were screened by reverse transcription-PCR-mediated terminal restriction fragment polymorphism analysis. No differences in the bacterial populations were observed throughout the density fractions of the unlabeled treatment. However, in the heavy fractions of the (13)C treatment, terminal restriction fragments (T-RFs) of 161 bp and 129 bp in length predominated. These T-RFs were identified by cloning and sequencing of 16S rRNA as from a Geobacter sp. and an Anaeromyxobacter sp., respectively. Apparently these bacteria, which are known as dissimilatory iron reducers, were able to assimilate acetate under methanogenic conditions, i.e., when CO(2) was the predominant electron acceptor. We hypothesize that ferric iron minerals with low bioavailability might have served as electron acceptors for Geobacter spp. and Anaeromyxobacter spp. under these conditions.

Acetates↗
PubMed1976

Sensitivity of environmental microorganisms to antimicrobial agents.

The sensitivity of different microorganisms, considered as typical representatives of the microflora of soil and water, was established to evaluate the influence of the nonmedical use of antimicrobial agents on bacterial ecology. Only seven strains, six chemoorganotrophs and one chemolithotroph, could be considered as relatively sensitive to the 21 antimicrobial compounds tested. The other 29 microorganisms may be regarded as resistant to most antimicrobial agents. Streptomyces were sensitive to high concentrations of active substances. Broad-spectrum antibiotics showed an effect on environmental bacteria similar to that on human pathogens. Cephalothin stimulated the growth of a Chlorella sp. From these experiments, it appears that spilled antimicrobial agents have little chance of causing an alteration in the microbial ecology.

Anti-Bacterial Agents↗
PubMed1977

Rapid identification and quantitation of small numbers of microorganisms by a chemiluminescent immunoreaction.

A method (patent pending) for rapidly identifying and quantitating small numbers of microorganisms was developed based on the specific immunoreaction of microorganisms with homologous antibodies linked by conjugation to peroxidase. The high sensitivity of the method is due to the use of a chemiluminescent reaction for the determination of the enzyme. The reaction was performed on Alcar supports with low nonspecific adsorption. The very low noise achieved permitted the detection of as few as 30 to 300 bacterial cells.

Bacteria↗
PubMed1979

Mechanism of lethal action of 2,450-MHz radiation on microorganisms.

Various bacteria, actinomycetes, fungi, and bacteriophages were exposed to microwaves of 2,450 +/- 20 MHz in the presence and in the absence of water. It was found that microorganisms were inactivated only when in the presence of water and that dry or lyophilized organisms were not affected even by extended exposures. The data presented here prove that microorganisms are killed by "thermal effect" only and that, most likely, there is no "nonthermal effect"; cell constituents other than water do not absorb sufficient energy to kill microbial cells.

Bacteria↗
PubMed1979

14C-most-probable-number method for enumeration of active heterotrophic microorganisms in natural waters.

A most-probable-number method using 14C-labeled substrates is described for the enumeration of aquatic populations of heterotrophic microorganisms. Natural populations of microorganisms are inoculated into dilution replicates prepared from the natural water from which the organisms originated. The natural water is supplemented with a 14C-labeled compound added so as to approximate a true environmental concentration. 14CO2 evolved by individual replicates is trapped in NaOH and counted by liquid scintillation techniques for use in scoring replicates as positive or negative. Positives (14CO2 evolution) are easily distinguished from negatives (no 14CO2 evolution). The results from a variety of environments using the 14CO2 procedure agreed well with previously described methods, in most instances. The 14C-most-probable-number method described here reduces handling procedures over previously described most-probable-number procedures using 14C-labeled substrates. It also appears to have advantages over other enumeration methods in its attempt to approximate natural conditions more closely.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1980

Production of nitrous oxide by ammonia-oxidizing chemoautotrophic microorganisms in soil.

Gas chromatographic studies showed that nitrous oxide was produced in each instance when sterilized (autoclaved) soil was incubated after treatment with ammonium sulfate and inoculation with pure cultures of ammonia-oxidizing chemoautotrophic microorganisms (strains of Nitrosomonas, Nitrosospira, and Nitrosolobus). Production of N(2)O in ammonium-treated sterilized soil inoculated with Nitrosomonas europaea increased with the concentration of ammonium and the moisture content of the soil and was completely inhibited by both nitrapyrin and acetylene. Similar effects of nitrapyrin, acetylene, ammonium concentration, and soil moisture content were observed in studies of factors affecting N(2)O production in nonsterile soil treated with ammonium sulfate. These observations support the conclusion that, at least under some conditions, most of the N(2)O evolved from soils treated with ammonium or ammonium-producing fertilizers is generated by chemoautotrophic nitrifying microorganisms during oxidation of ammonium to nitrite.

Journal Article↗
PubMed1982

Rapid enumeration of microorganisms in foods by the direct epifluorescent filter technique.

Filtration of "stomachered" food suspensions through nylon filters (pore size, 5 microns) removed most of the food debris without affecting the recovery of microorganisms. Two to ten milliliters of these prefiltered suspensions could be filtered in the direct epifluorescent filter technique (DEFT). The technique takes less than 30 min to complete and has a lower sensitivity of less than 60,000 microorganisms per g for all products examined. Vegetative bacterial cells, spores, fungal hyphae, and yeasts could be distinguished with the technique. For fresh meat and fish, the DEFT count of prefiltered suspensions agreed well with the plate count of unfiltered suspensions over the range of 10(4) to 10(10)/g (correlation coefficient of 0.91). For frozen meat and fish and frozen vegetables, the two counting methods had correlation coefficients of 0.87 and 0.66, respectively. The poor correlation for frozen vegetables was due to the inclusion in the DEFT count of nonviable bacteria killed by the blanching process used to inactivate enzymes. Good agreement was obtained between the prefiltered DEFT count and unfiltered plate count for cooked meats, cream doughnut, and whole peppers. Possible reasons for the poor agreement between the DEFT count and plate count for certain products are discussed.

Bacteria↗
PubMed1982

Absence of surface-associated microorganisms in adult oysters (Crassostrea gigas).

Healthy, actively feeding intertidal oysters were removed from an estuarine environment (Pipeclay Lagoon, Tasmania). The epithelial surfaces of various organs of the mantle cavity and alimentary tract were explored by scanning and transmission electron microscopy. All epithelial tissues examined were ciliated, and nearly all were partly covered with secreted mucus. However, microorganisms were seen rarely in the adhesive mucus and never attached to the epithelium. Electron microscopy also failed to demonstrate a surface microflora in emersed oysters which had been incubated at 5 to 25 degrees C for 6 or 24 h. The absence of an internal surface microflora did not vary on a seasonal basis. In laboratory experiments, oysters were allowed to filter feed from seawater containing diverse types of marine bacteria at concentrations of 10(3) to 10(7)/mL. However, no surface microflora could be found within actively feeding oysters or in emersed animals incubated at 20 degrees C for 6 or 24 h. In contrast, surface-associated microorganisms were detected readily by scanning electron microscopy on the external shell of healthy oysters and on various internal tissues in spoiled oysters. It is suggested that the major mechanisms restricting microbial growth within oysters are ciliary movement and mucus secretion.

Animals↗
PubMed1984

Phyllosphere of cotton as a habitat for diazotrophic microorganisms.

Positive nitrogenase activities ranging from 0.18 to 0.78 nmol of C(2)H(4) cm h were detected on the leaf surfaces of different varieties of cotton (Gossypium hirsutum L. and G. herbaceum L.) plants. Beijerinckia sp. was observed to be the predominant nitrogen-fixing microorganism in the phyllosphere of these varieties. A higher level of phyllosphere nitrogen-fixing activity was recorded in the variety Varalaxmi despite a low C/N ratio in the leaf leachates. Leaf surfaces of the above variety possessed the largest number of hairy outgrowths (trichomes) which entrapped a majority of microbes. Immersion of plant roots in nutrient medium containing P(i) led to the accumulation of label in the trichome-borne microorganisms, thereby indicating a possible transfer of nutrients from leaf to microbes via trichomes. Extrapolation of acetylene reduction values suggested that 1.6 to 3.2 kg of N ha might be contributed by diazotrophs in the phyllosphere of the variety Varalaxmi during the entire growth period.

Journal Article↗
PubMed1985

Incorporation of phenol carbon at trace concentrations by phenol-mineralizing microorganisms in fresh water.

The fate of phenol carbon at phenol concentrations ranging from 1 ng/ml to 1 microgram/ml was determined in freshwater samples. Approximately 20% of the parent phenol was incorporated into trichloroacetic acid-precipitable material by the microorganisms capable of mineralizing phenol. There was no apparent lag period before phenol incorporation commenced, and incorporation was complete within 2 h at all concentrations tested. A direct relationship was found between the initial phenol concentrations and both phenol mineralization and incorporation rates, indicating that cometabolism of phenol does not occur at concentrations that are environmentally significant. At all concentrations, approximately 80% of the initial phenol concentration was mineralized. This percentage plus the percentage of phenol incorporated at the various concentrations equaled approximately 100%. Therefore, the parent phenol does not remain in fresh water; it is either incorporated into cellular biomass or mineralized. However, the incorporated phenol carbon is subject to bioaccumulation in nature. There was no apparent lag period before thymidine was incorporated into biomass, and incorporation was complete within 4 to 8 h at all of the phenol concentrations tested. Thymidine incorporation was independent of phenol concentration at all levels tested. This is probably due to the small amount of thymidine incorporated by the phenol-mineralizing microorganisms in comparison with the amount of thymidine incorporated by the total microbial population.

Bacteria↗
PubMed1985

UV inactivation of pathogenic and indicator microorganisms.

Survival was measured as a function of the dose of germicidal UV light for the bacteria Escherichia coli, Salmonella typhi, Shigella sonnei, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis spores, the enteric viruses poliovirus type 1 and simian rotavirus SA11, the cysts of the protozoan Acanthamoeba castellanii, as well as for total coliforms and standard plate count microorganisms from secondary effluent. The doses of UV light necessary for a 99.9% inactivation of the cultured vegetative bacteria, total coliforms, and standard plate count microorganisms were comparable. However, the viruses, the bacterial spores, and the amoebic cysts required about 3 to 4 times, 9 times, and 15 times, respectively, the dose required for E. coli. These ratios covered a narrower relative dose range than that previously reported for chlorine disinfection of E. coli, viruses, spores, and cysts.

Amoeba↗
PubMed1985

Effects of chemical speciation on the mineralization of organic compounds by microorganisms.

The mineralization of 1.0 to 100 ng each of four complexing compounds-oxalate, citrate, nitrilotriacetate (NTA), and EDTA-per ml was tested in media prepared in accordance with equilibrium calculations by a computer program so that the H, Ca, Mg, Fe, or Al complex (chemical species) was predominant. Sewage microorganisms mineralized calcium citrate more rapidly than iron, aluminum, or hydrogen citrate, and magnesium citrate was degraded slowest. Aluminum, hydrogen, and iron oxalates were mineralized more rapidly than calcium oxalate, and magnesium oxalate was decomposed slowest. Sewage microorganisms mineralized calcium NTA but not aluminum, magnesium, hydrogen, or iron NTA or any of the EDTA complexes. Pseudomonas sp. mineralized calcium and iron citrates but had no activity on hydrogen, aluminum, or magnesium citrate. Pseudomonas pseudoalcaligenes mineralized calcium, iron, hydrogen, and aluminum citrates but had little activity on magnesium citrate. Pseudomonas alcaligenes used calcium, iron, hydrogen, and aluminum oxalates readily, but it used magnesium oxalate at a slower rate. Listeria sp. destroyed calcium NTA but had no effect on hydrogen, iron, or magnesium NTA. Increasing the Ca concentration in the medium enhanced the breakdown of NTA by Listeria sp. The different activities of the bacterial isolates were not a result of the toxicity of the complexes or the lack of availability of a nutrient element. NTA mineralization was not enhanced by the addition of Ca to Beebe Lake water, but it was enhanced when Ca and an NTA-degrading inoculum were added to water from an oligotrophic lake. The data show that chemical speciation influences the mineralization of organic compounds by naturally occurring microbial communities and by individual bacterial populations.

Journal Article↗
PubMed1985

Reactions of fish to microorganisms in wastewater.

Fish were inoculated with various microorganisms present in wastewater. A threshold concentration was determined over which these microorganisms were recovered from the muscles. The threshold concentrations were different for bacteria, bacteriophages, and polio 1 LSc virus. The threshold values were lower when fish were inoculated than when they were immersed in water containing these organisms. Depuration experiments were efficient when the fish did not contain high concentrations of bacteria in their muscles. As the threshold concentrations are an expression of the capability of the immune system of the fish, these values can be useful for the design and management of fishponds in which treated wastewater is used.

Animals↗
PubMed1986

Glyphosate-degrading microorganisms from industrial activated sludge.

A plating medium was developed to isolate N-phosphonomethylglycine (glyphosate)-degrading microorganisms, with glyphosate as the sole phosphorus source. Two industrial biosystems treating glyphosate wastes contained elevated microbial counts on the medium. One purified isolate metabolized glyphosate to aminomethylphosphonic acid, mineralizing this accumulating intermediate during log growth. This microorganism has been identified as a Flavobacterium species.

Journal Article↗
PubMed1986

Rapid assay for screening and characterizing microorganisms for the ability to degrade polychlorinated biphenyls.

We designed a rapid assay that assesses the polychlorinated biphenyl (PCB)-degradative competence and congener specificity of aerobic microorganisms, identifies strains capable of degrading highly chlorinated biphenyls, and distinguishes among those that degrade PCBs by alternative pathways. Prior attempts to assay PCB-degradative competence by measuring disappearance of Aroclors (commercial PCB mixtures) have frequently produced false-positive findings because of volatilization, adsorption, or absorption losses. Furthermore, these assays have generally left the chemical nature of the competence obscure because of incomplete gas chromatographic resolution and uncertain identification of Aroclor peaks. We avoided these problems by using defined mixtures of PCB congeners and by adopting incubation and extraction methods that prevent physical loss of PCBs. Our assay mixtures include PCB congeners ranging from dichloro- to hexachlorobiphenyls and representing various structural classes, e.g., congeners chlorinated on a single ring (2,3-dichlorobiphenyl), blocked at 2,3 sites (2,5,2'5'-tetrachlorobiphenyl), blocked at 3,4 sites (4,4'-dichlorobiphenyl), and lacking adjacent unchlorinated sites (2,4,5,2',4',5'-hexachlorobiphenyl). The PCB-degrative ability of microorganisms is assessed by packed-column gas chromatographic analysis of these defined congener mixtures following 24-h incubation with resting cells. When tested with 25 environmental isolates, this assay revealed a broad range of PCB-degradative competence, highlighted differences in congener specificity and in the extent of degradation of individual congeners, predicted degradative competence on commercial PCBs, and (iv) identified strains with superior PCB-degradative ability.

Alcaligenes↗
PubMed1986

Pattern recognition analysis of in vivo enzyme-substrate fluorescence velocities in microorganism detection and identification.

A spectrometric technique is presented that combines most of the important criteria necessary for efficient detection and identification of microorganisms. These criteria include simplicity of experimental design, various degrees of sensitivity and selectivity, convenience, and total reaction times of less than 15 min. The study takes advantage of the inherent extracellular enzymes present in living as opposed to dead, non-enzyme-producing organisms. Sequentially these are harnessed in in vivo reactions with a substrate containing a select organic functional group that is known to be cleaved or hydrolyzed by a certain enzyme. The substrate is tailored so that one of the products can be induced to fluoresce, and by using a conventional spectrofluorimeter the rate at which the fluorescence appears can be recorded. By subjecting the same bacterial sample to a number of different enzyme substrates, a pattern of fluorescence response rates emerges from a 7 by 7 microorganism-substrate matrix. Detection limits ranged from 3.6 X 10(2) to 3.5 X 10(8) cells per ml for the Bacillus globigii-indoxyl acetate and Escherichia coli-diacetylfluorescein pairs, respectively. The specificity and versatility of the method for bacterial determination is demonstrated in probing different bacterial enzymes through their spectrally active metabolic products.

Bacillus↗