Decomposition of nucleic acids and some of their degradation products by microorganisms

A study was made of the decomposition of nucleic acids, uric acid and urea by different groups of soil microorganisms including bacilli, non-coryneform rods, corynebacteria (arthrobacters and non-arthrobacters), streptomycetes, fungi and yeasts. Hydrolysis of nucleic acids was found to be a common phenomenon. The decomposition of uric acid was readily carried out by arthrobacters and streptomycetes. Most bacilli, however, lacked this capacity. Few soil microorganisms degraded urea. Additional investigations were made of the breakdown of nucleic acids and their degradation products by coryneform bacteria from soil, cheese, sea-fish, sewage, the phyllosphere and poultry litter. Generally, coryneforms from soil could utilize allantoin as the sole source of nitrogen, carbon and energy in contrast to most of those from cheese which could not. Breakdown of uric acid and allantoin by washed cells of a coryneform strain from soil resulted in formation of ammonia; breakdown of these compounds by washed cells of a strain from cheese resulted in accumulation of urea.     

Identification of dimethyl disulfide-forming bacteria isolated from activated sludge

Twenty-four strains with high dimethyl disulfide (DMDS)-forming ability were isolated from activated sludge and identified to the genus level. These bacteria were classified into four groups (A, B, C, and D) by the API ZYM System (API System S.A., Montalieu, France). Group A (three strains) was identified as genus Lactobacillus by the API 20B System, by the method of Cowan and Steel, and by production of lactic acid as confirmed by gas-liquid chromatography. Group B (eight strains) was identified as genus Corynebacterium by API 20B and the Cowan and Steel method. Group C (one strain) was suggested to belong to genus Corynebacterium by the API 20B System. Group D (12 strains) was identified as genus Pseudomonas or Alcaligenes by the API 20B System, as genus Alcaligenes by the Cowan and Steel method, and as Achromobacter group Vd by the API 20NE System. However, on the basis of guanine-plus-cytosine contents in DNA and form of flagella, these strains were identified as genus Pseudomonas. Formation of DMDS from DL-methionine and S-methyl-L-cysteine was tested. DMDS-forming bacteria isolated from activated sludge formed DMDS from both precursors. In genus Pseudomonas, P. aeruginosa could not form DMDS from either precursor, but P. acidovorans, P. alcaligenes, P. pseudoalcaligenes, and P. testosteroni formed DMDS. In genus Alcaligenes, A. denitrificans subsp. xylosoxydans, A. denitrificans subsp. denitrificans, A. faecalis, and A. odorans formed DMDS from both precursors. Achromobacter group Vd formed DMDS from S-methyl-L-cysteine, but could not from DL-methionine.     

Nucleic Acid Homologies Among Oxidase-Negative Moraxella Species

The deoxyribonucleic acid (DNA) base composition and DNA homologies of more than 40 strains of oxidase-negative Moraxella species were determined. These bacteria have also been identified as belonging to the Mima-Herellea-Acinetobacter group and the Bacterium anitratum group, as well as to several other genera including Achromobacter and Alcaligenes. The DNA base content of these strains ranged from 40 to 46% guanine plus cytosine. DNA-DNA competition experiments distinguished five groups whose members were determined by showing 50% or more homology to one of the reference strains: B. anitratum type B5W, Achromobacter haemolyticus var. haemolyticus, Alcaligenes haemolysans, Achromobacter metalcaligenes, and Moraxella lwoffi. A sixth group comprised those strains showing less than 50% homology to any of the reference strains. Negligible homology was found between strains of oxidase-negative and oxidase-positive Moraxella species in DNA-DNA competition experiments. However, evidence of a distant relationship between the two groups was obtained in competition experiments by using ribosomal ribonucleic acid.     

Evaluation of broiler litter with reference to the microbial composition as assessed by using 16S rRNA and functional gene markers

Very little is known about the microbial composition of animal bedding wastes, including poultry litter, and what is known has been deduced from standard culture methods, by which some fastidious organisms that exist in the environment may not be detected. We evaluated the bacterial composition of poultry litter by using a combination of culture and molecular detection. Total aerobic bacteria in poultry litter were detected by culture at 10(9) CFU/g of material. Enteric bacteria such as Enterococcus spp. and coliforms composed 0.1 and 0.01%, respectively, of the total aerobic cultivatable bacteria in poultry litter; no Salmonella strains were detected by culture. In order to characterize the most abundant bacterial groups, we sequenced 16S ribosomal DNA (rDNA) genes amplified by PCR with microbial community DNA isolated from poultry litter as the template. From the 16S rDNA library, 31 genera were identified. Twelve families or groups were identified with lactobacilli and Salinococcus spp. forming the most abundant groups. In fact, 82% of the total sequences were identified as gram-positive bacteria with 62% of total belonging to low G+C gram-positive groups. In addition to detection of 16S rDNA sequences associated with the expected fecal bacteria present in manure, we detected many bacterial sequences for organisms, such as Globicatella sulfidofaciens, Corynebacterium ammoniagenes, Corynebacterium urealyticum, Clostridium aminovalericum, Arthrobacter sp., and Denitrobacter permanens, that may be involved in the degradation of wood and cycling of nitrogen and sulfur. Several sequences were identified in the library for bacteria associated with disease in humans and poultry such as clostridia, staphylococci, and Bordetella spp. However, specific PCR targeting other human and veterinary pathogens did not detect the presence of Salmonella, pathogenic Escherichia coli, Campylobacter spp., Yersinia spp., Listeria spp., or toxigenic staphylococci. PCR and DNA hybridization revealed the presence of class 1 integrons with gene cassettes that specify resistance to aminoglycosides and chloramphenicol. Only from understanding the microbial community of animal wastes such as poultry litter can we manage animal disease and limit the impact of animal waste on the environment and human and animal health.     

THE DIFFERENTIATION OF PSEUDOMONAS FROM OTHER GRAM-NEGATIVE BACTERIA ON THE BASIS OF ARGININE METABOLISM

SUMMARY: Of 391 Gram-negative bacteria isolated from chicken meat spoiled at a low temperature and classified by the commonly used methods, 156 were considered to be Pseudomonas and 188 Achromobacter , and 47 others belonged to the coli-aerogenes group or remained unclassified. A test for the production of alkaline conditions in an arginine medium incubated under a vaseline seal gave positive results for 155 of the Pseudomonas isolates, and negative results for 1 Pseudomonas and all the 188 Achromobacter strains. When named strains from culture collections were tested under these conditions, 63 Pseudomonas strains produced alkalinity while two plant pathogenic Pseudomonas species and two non-pigmented strains did not. These last two, which produced no acid from glucose, could not be regarded as typical Pseudomonas. All the Achromobacter strains gave negative results, as did four Alcaligenes , but one species, Alcaligenes bookeri , produced slightly alkaline conditions. One strain of Chromobacterium and three of Vibrio were also positive. These could be distinguished from Pseudomonas by their metabolism of glucose.     

The Microbiology of Built Up Poultry Litter

The numbers of viable bacteria in built up poultry litter were found to be 10¹⁰-10¹¹/g fresh weight and appeared to be little affected by factors such as age, temperature, moisture content and pH. Counts for unused litter and poultry droppings were lower. In built up litter of high alkalinity coryneform bacteria were predominant; micrococci occurred sporadically and small numbers of nocardias, streptomycetes, aerobic spore formers and streptococci were encountered. A variety of Gram negative bacteria also occurred, the numbers of which appeared to be controlled by alkalinity; they were less abundant in litters where the pH and buffering capacity were high. Strongly alkaline conditions also tended to lower the fungal counts but had no effect on the count of enterococci.     

Estimation of solvent-tolerance of bacteria by the solvent parameter log P

The solvent-tolerance of Gram-negative and Gram-positive bacteria were determined by the solvent parameter, log P. The solvent-tolerance of bacteria tested were in the following order, Pseudomonas>Escherichia, Serratia, Klebsiella>Proteus, Achromobacter, Acinetobacter>Alteromonas, Aeromonas, Flavobacterium>Alcaligenes, Bacillus>Agrobacterium, Micrococcus, Staphylococcus>Streptococcus>Leuconostoc, Lactobacillus>Corynebacterium, Chromobacterium, Brevibacterium, Rhodococcus.     

A Study of 5920 Strains of Psychrotrophic Bacteria Isolated from Chickens

The psychrotrotrophic flora of chicken carcases in 3 different industrial processing plants has been studied. The micro-organisms, 5920 strains, were placed in 4 categories (arranged in decreasing order of the number of isolates): (1), Pseudomonas and Achromobacter (Acineto-bacter) ; (2) Flavobacterium, Corynebacterium and yeasts; (3), Aeromonas and Entero-bacteriaceae; (4), lactic acid bacteria, Micrococcaceae and Bacillus . Different types of strains belonging to these groups are described and the importance of each is discussed with reference to the observations of other authors. The proteolytic action of 1717 of the 5920 isolates was studied.     

Classification of coryneform bacteria associated with human urinary tract infection (group D2) as Corynebacterium urealyticum sp. nov.

Urealytic strains of coryneform bacteria that are designated Corynebacterium group D2 and are isolated from human urine are a cause of urinary tract infections. Cell wall and lipid analyses confirmed that these organisms are members of the genus Corynebacterium but can be separated from other species in the genus on the basis of DNA base composition and DNA-DNA hybridization values. Biochemically, strains in this taxon can be distinguished from other Corynebacterium spp. by their failure to produce acid from carbohydrates, by their failure to reduce nitrates, and by their ability to hydrolyze urea. We regard these bacteria as a new species of the genus Corynebacterium and propose the name Corynebacterium urealyticum. The type strain is strain NCTC 12011 (= ATCC 43042).     

Studies on the biodegradation of nonionic surfactants applied in the polyester fiber industry. I. Activated sludge bacteria degrading the surfactants.

The paper presents characteristics of 76 strains of bacteria capable of utilizing nonionic surfactants Cirrasol FP, Cirrasol SF 200 and Cirrasol TCS as the source of carbon. The strains were isolated from two activated sludges adapted to the purification of wastes containing the above compounds at concentration 150--200 mg/l. The isolated strains belonged to the genera: Achromobacter, Alcaligenes, Arthrobacter, Flavobacterium, Mycobacterium, Nocardia, Pseudomonas and Xanthomonas. With load 0.11 mg surfactant/mg d.w./day bacteria belonging to Alcaligenes were dominating. With load 0.18--0.31 mg surfactant/mg d.w./day microorganisms were dominated by Pseudomonas. The highest intensity of degradation of the studied surfactant was shown by species: Alcaligenes viscolactis, Nocardia blackwellii and Pseudomonas rathonis.     

Long-term activation of semicarbazide-sensitive amine oxidase lowers circulating levels of uric acid in diabetic conditions

Uric acid is involved in nitrogenous waste in animals, together with ammonia and urea. Uric acid has also antioxidant properties and is a surrogate marker of metabolic syndrome. We observed that the elevated plasma uric acid of high-fat fed mice was normalized by benzylamine treatment. Indeed, benzylamine is the reference substrate of semicarbazide-sensitive amine oxidase (SSAO), an enzyme highly expressed in fat depots and vessels, which generates ammonia when catalysing oxidative deamination. Ammonia interferes with uric acid metabolism/solubility. Our aim was therefore to investigate whether the lowering action of benzylamine on uric acid was related to an improvement of diabetic complications, or was connected with SSAO-dependent ammonia production. First, we observed that benzylamine administration lowered plasma uric acid in diabetic db/db mice while it did not modify uric acid levels in normoglycemic and lean mice. In parallel, benzylamine improved the glycemic control in diabetic but not in normoglycemic mice, while plasma urea remained unaltered. Then, uric acid plasma levels were measured in mice invalidated for AOC3 gene, encoding for SSAO. These mice were unable to oxidize benzylamine but were not diabetic and exhibited unaltered plasma uric levels. Therefore, activated or abolished ammonia production by SSAO was without influence on uric acid in the context of normoglycemia. Our observations confirm that plasma uric acid increases with diabetes and can be normalized when glucose tolerance is improved. They also show that uric acid, a multifunctional metabolite at the crossroads of nitrogen waste and of antioxidant defences, can be influenced by SSAO, in a manner apparently related to changes in glucose homeostasis.     

Studies on the Utilization of Hydrocarbons by Microorganisms

Taxonomical studies on ten strains of hydrocarbon-utilizing bacteria reported in previous paper, which produced various kinds of amino acid, were carried out. They were Achromo-bacter cycloclastes, Achromobacter delmarvae, Bacillus species, Corynebacterium species, Micrococcus species. Many of them were not identical with the species which are described in Bergey’s Manual of 7th Edition.     

Excretion of nitrogen compounds in sweat during a sauna]

The aim of the study was to determine a loss of nitrogen compounds with sweat in sauna and to estimate their plasma concentration. Sweat was collided during 30 min stay in sauna. Blood was taken before and immediately after the sauna. Concentrations of ammonia, urea, creatinine and uric acid were determined in the both fluids. It has been found, that the concentration of ammonia in sweat exceeds, that in plasma by 77 times. Ammonia plasma concentration following sauna increased by about 60%. Sweat urea concentration exceeded that in plasma by 3.5 times. Plasma urea concentration was significantly reduced after sauna. Sweat creatinine concentration was about two times higher than that in plasma. No uric acid was detected in sweat. Sweating did not affect plasma creatinine and uric acid concentrations. Results indicate that considerable amount of nitrogen is lost with sweat during sauna.     

Diversity of culturable heterotrophic aerobic bacteria in pristine stream bed sediments

More than 900 culturable, heterotrophic aerobic isolates were obtained from the sediments of a forested, pristine stream and analyzed using three classical microbiological tests: API 20E, amplified ribosomal DNA restriction analysis (ARDRA), and fatty acid analysis. Gram-negative bacteria comprised most of the heterotrophic aerobic isolates (66.7%), similar to other oligotrophic environments. The isolates were assigned to the genus level as Pseudomonas, Flavobacterium, Micrococcus, Bacillus, Chromobacterium, Acinetobacter, Alcaligenes, Aeromonas, Methylobacterium, Enterobacter, Corynebacterium, and Sporolactobacillus. Genotypic analysis by ARDRA facilitated the comparison among strains within Pseudomonas, Bacillus, and Enterobacter groups. Temperature and predation may influence the survival of bacteria during seasons, as shown previously by others. Our results showed that the number of heterotrophic aerobic bacteria, especially Enterobacter, Alcaligenes, and Aeromonas, and Gram-positive bacteria, decreased in winter compared to summer conditions.     

Nonfermentative gram-negative bacteria encountered in clinical specimens

The characteristics of 1192 gram-negative nonfermentative bacteria isolated from human clinical specimens and 32 reference strains representing six genera (Pseudomonas, Alcaligenes, Achromobacter, Acinetobacter, Moraxella, Flavobacterium) encountered in clinical bacteriology are presented. Salient features for their identification in the routine diagnostic laboratory are summarized.     

Decomposition of forest litters

Summary Studies have been made on the production of ammonia and nitrate nitrogen, rate of decomposition along with comparative changes in microbial population during decomposition of deciduous and coniferous litters. At the same time the effect of addition of antibiotics on the microbial population and rate of decomposition of coniferous litter was investigated.Liberation of nitrogen in the form of ammonia occurred somewhat more rapidly in decomposing deciduous litter than in that of the coniferous litter. Towards the end of the experiment the nitrification process commenced in the deciduous litter but was absent in the coniferous litter.The deciduous litter decomposed more rapidly than the coniferous litter.The bacteria and actinomycetes were consistently more numerous in the deciduous litter than in the coniferous litter; in the case of fungi the reverse occured. An antifungal antibiotic (cycloheximide) had little or no effect on numbers of fungi in the decomposing coniferous litter while antibacterial antibiotics (streptomycin and chloramphenicol) reduced slightly the numbers of bacteria and actinomycetes. Numbers of bacteria and actinomycetes were increased greatly in cultures receiving the antifungal antibiotic and fungal growth was apparently stimulated where antibacterial antibiotics were added. In spite of the observed shift in the microbial population brought about by the addition of antibiotics the rate of decomposition of the coniferous litter appeared to be unaffected. This would suggest that the type of microbia l population is not as important as the type of litter or some other factor in regard to decomposition rate.Joint contribution from the Divisions of Bacteriology (No. 469) and Chemistry (No. 412), Science Service.     

Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria

Polyclonal antibodies were used to identify heme or copper nitrite reductases in the following groups: 23 taxonomically diverse denitrifiers from culture collections, 100 numerically dominant denitrifiers from geographically diverse environments, and 51 denitrifiers from a culture collection not selected for denitrification. Antisera were raised against heme nitrite reductases from Pseudomonas aeruginosa and Pseudomonas stutzeri and against copper nitrite reductase from Achromobacter cycloclastes. Nitrite reductases were identified by Western immunoblot. Diethyldithiocarbamate, which specifically inhibits copper nitrite reductases, was used to confirm the immunological characterization and determine which type was present in strains nonreactive with any antiserum. For groups in which the type of nitrite reductase has not been previously described, we found that Alcaligenes eutrophus, Bacillus azotoformans, Bradyrhizobium japonicum, Corynebacterium nephridii, and Rhizobium spp. contained copper nitrite reductase, while Aquaspirillum itersonii, Flavobacterium spp., and Pseudomonas fluorescens contained heme nitrite reductase. Heme nitrite reductases dominated, regardless of soil type or geographic origin. They occurred in 64 and 92%, respectively, of denitrifiers in the numerically dominant and nonselected collections. The two nitrite reductase types were mutually exclusive in individual bacteria, but both appeared in different strains from the Alcaligenes and Pseudomonas genera. The heme type predominated in Pseudomonas strains. The heme-type nitrite reductase appeared more conserved if judged by similarities in molecular weights and immunological reactions. The Cu type was found in more taxonomically unrelated strains and varied in molecular weight and antiserum recognition.     

Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria.

Polyclonal antibodies were used to identify heme or copper nitrite reductases in the following groups: 23 taxonomically diverse denitrifiers from culture collections, 100 numerically dominant denitrifiers from geographically diverse environments, and 51 denitrifiers from a culture collection not selected for denitrification. Antisera were raised against heme nitrite reductases from Pseudomonas aeruginosa and Pseudomonas stutzeri and against copper nitrite reductase from Achromobacter cycloclastes. Nitrite reductases were identified by Western immunoblot. Diethyldithiocarbamate, which specifically inhibits copper nitrite reductases, was used to confirm the immunological characterization and determine which type was present in strains nonreactive with any antiserum. For groups in which the type of nitrite reductase has not been previously described, we found that Alcaligenes eutrophus, Bacillus azotoformans, Bradyrhizobium japonicum, Corynebacterium nephridii, and Rhizobium spp. contained copper nitrite reductase, while Aquaspirillum itersonii, Flavobacterium spp., and Pseudomonas fluorescens contained heme nitrite reductase. Heme nitrite reductases dominated, regardless of soil type or geographic origin. They occurred in 64 and 92%, respectively, of denitrifiers in the numerically dominant and nonselected collections. The two nitrite reductase types were mutually exclusive in individual bacteria, but both appeared in different strains from the Alcaligenes and Pseudomonas genera. The heme type predominated in Pseudomonas strains. The heme-type nitrite reductase appeared more conserved if judged by similarities in molecular weights and immunological reactions. The Cu type was found in more taxonomically unrelated strains and varied in molecular weight and antiserum recognition.     

Do mammals,birds, reptiles and fish have similar nitrogen conserving systems?

Comparative physiological studies are a powerful tool for revealing common animal adaptations. Amino acid catabolism produces ammonia which is detoxified through the synthesis of urea (mammals, some fish), uric acid (birds), or urea and uric acid (reptiles). In mammalian herbivores and omnivores, urea nitrogen is salvaged by a series of steps involving urea transfer into the intestine, microbial mediated urea hydrolysis with synthesis of amino acids utilizing the liberated ammonia and transfer of the amino acids back to the host. A similar series of steps occur in omnivorous/granivorous and herbivorous birds, although in this case urine, containing uric acid, is refluxed directly into the intestine where microbes degrade the uric acid and utilize the liberated ammonia for amino acid synthesis. These amino acids are transferred back to the host. In reptiles and ureotelic fish not all of these steps have been experimentally confirmed. Reptiles like birds, reflux urine into the intestine where it is exposed to the microflora. However, the capacity of these microbes to breakdown the uric acid and urea and utilize ammonia for amino acid synthesis has not been documented. Ureotelic fish transfer urea into the intestine where urease (presumably of bacterial origin) hydrolyzes the urea. However, the amino acid synthesizing capacity of the intestinal microflora has not been studied. The series of steps, as outlined, would define the prevailing nitrogen conservation system for herbivores and omnivores at least. However, it would appear that some animals, in particular the fruit-eating bat and perhaps the fruit-eating bird, may have evolved alternative, as yet uncharacterized, adaptations to a very limited nitrogen intake.     

Nitrogen metabolism in broiler chickens consuming the bacterial strain Alcaligenes eutrophus

The facultative autotrophic H₂-oxidising bacterium Alcaligenes eutrophus strain H 16 (mutant lacking PHB = poly-β-hydroxybutyric acid) is a potential source of single-cell protein.As compared to soya bean protein, A. eutrophus is more adequate in nearly all amino acids (expressed as g/16 g non-nucleic acid-N) essential for the growth of poultry. The relatively high values for methionine and lysine (2.5 g and 7.5 g/16 g non-nucleic acid-N, respectively) have to be emphasized.The present study was designed to investigate the N-utilisation of a PHB-free mutant of A. eutrophus grown under autotrophic conditions when given at increasing levels in broiler diets. The influence of bacterial-N on the distribution of excreted nitrogen in uric acid, urea and ammonia was examined in metabolic studies. For determination of apparent N-digestibility, faecal and urinary nitrogen were separated chemically.The dietary protein was replaced by the bacterial dry matter (on the basis of its content of non-nucleic acid-N) at levels of 15, 30, 45 and 61.7% at the expense of soya bean meal. At a level of 45% or more of the protein of the diet replaced by the bacterial strain, values for apparent N-digestibility and N-retention decreased slightly. The excreted uric acid-N (g per g of N ingested) was not influenced by the bacterial N intake. The excretion of ammonia-N increased with increasing dietary levels of bacterial-N while the output of urea-N was not clearly influenced.From the N-balance data it is concluded that replacement of soya bean meal by the bacterial matter (autotrophically grown A. eutrophus) may be recommended up to 30% of the dietary protein on the basis of non-nucleic acid-N, and should not exceed a level of 45%.