An altered Pseudomonas diversity is recovered from soil by using nutrient-poor Pseudomonas-selective soil extract media.

We designed five Pseudomonas-selective soil extract NAA media containing the selective properties of trimethoprim and sodium lauroyl sarcosine and 0 to 100% of the amount of Casamino Acids used in the classical Pseudomonas-selective Gould's S1 medium. All of the isolates were confirmed to be Pseudomonas by a Pseudomonas-specific OprF antibody and a Pseudomonas-specific PCR targeting 16S ribosomal DNA. The Pseudomonas isolates were characterized by classical physiological tests, repetitive extragenic palindromic-PCR, Fourier transform infrared spectroscopy, and carbon source utilization patterns. Several of these analyses showed that the amount of Casamino Acids significantly influenced the diversity of the recovered Pseudomonas isolates. Furthermore, the data suggested that specific Pseudomonas subpopulations were represented on the nutrient-poor media. The NAA 1:100 medium, containing ca. 15 mg of organic carbon per liter, consistently gave significantly higher Pseudomonas CFU counts than Gould's S1 when tested on four Danish soils. NAA 1:100 may, therefore, be a better medium than Gould's S1 for enumeration and isolation of Pseudomonas from the low-nutrient soil environment.     

Uptake and Incorporation of Amino Acids and Peptides by Bacteroides amylophilus

Bacteroides amylophilus H-18 demonstrated a higher growth yield, a slightly higher growth rate, and a diminished lag period when Tryptose was added to the basal medium. This uptake of labeled amino acids was concentration-dependent, as the contribution of exogenous amino acid to the cell protein increased from 15.4 to 24.1% when the concentration of Casamino Acids in the medium was increased from 1.4 to 2.8 mg/ml. There was considerable redistribution of (14)C-label to other amino acids. Tryptic peptides of casein competed effectively with the amino acids for uptake. The (14)C-label from a protein was incorporated into B. amylophilus H-18 cells presumably after breakdown of the protein by the B. amylophilus H-18 protease.     

Casamino acids enhance growth of Bacteroides melaninogenicus.

Casamino Acids enhance the growth of Bacteroides melaninogenicus when added to various concentrations of Trypticase. Absence of a peptide, not amino acids, is responsible for the inability of Casamino Acids to support growth.     

Malachite green agar,a new selective medium for Fusarium spp.

Malachite Green Agar 2.5 ppm (MGA 2.5) is a potent selective medium for isolation and enumeration of Fusarium spp. It has been tested with pure and mixed cultures as well as in naturally contaminated samples. The recoveries of Fusarium species in MGA 2.5 were the same as the recoveries obtained in Nash and Snyder medium. However, this medium is a more selective culture medium for Fusarium spp. than Nash and Snyder medium, because it does not allow the development of colonies belonging to other fungal genera. MGA 2.5 is simple to prepare and less hazardous than other Fusarium selective media containing pentachloronitrobenzene (PCNB). This revised version was published online in June 2006 with corrections to the Cover Date.     

Selective medium for isolation of Fusarium species and dematiaceous hyphomycetes from cereals

A selective medium containing 2 micrograms of dichloran per ml, 200 micrograms of chloramphenicol per ml, and 1.5% bacteriological peptone was developed for the isolation of Fusarium species and dematiaceous hyphomycetes from cereals. The medium, designated DCPA, was shown to select against species of Aspergillus, Penicillium, Cladosporium, and mucoraceous fungi. DCPA was evaluated for use as an enumeration medium and compared satisfactorily with dichloran-rose bengal-chloramphenicol agar when both media were tested with a range of cereal samples. Fusarium species and dematiaceous hyphomycetes produced well-formed colonies with good conidial production on DCPA, permitting rapid identification of such isolates on this medium.     

Selective medium for isolation of Fusarium species and dematiaceous hyphomycetes from cereals.

A selective medium containing 2 micrograms of dichloran per ml, 200 micrograms of chloramphenicol per ml, and 1.5% bacteriological peptone was developed for the isolation of Fusarium species and dematiaceous hyphomycetes from cereals. The medium, designated DCPA, was shown to select against species of Aspergillus, Penicillium, Cladosporium, and mucoraceous fungi. DCPA was evaluated for use as an enumeration medium and compared satisfactorily with dichloran-rose bengal-chloramphenicol agar when both media were tested with a range of cereal samples. Fusarium species and dematiaceous hyphomycetes produced well-formed colonies with good conidial production on DCPA, permitting rapid identification of such isolates on this medium.     

Differential Reduction of Tellurite by Growing Colonies of Normal Yeast and Respiration-Deficient Mutants.

Nagai, Susumu (National Women's University, Nara, Japan). Differential reduction of tellurite by growing colonies of normal yeast and respiration-deficient mutants. J. Bacteriol. 90:220-222. 1965.-A differential reduction of sodium tellurite was observed between normal and respiration-deficient mutant colonies of several species of Saccharomyces. Normal colonies turned black in contrast to mutant colonies which remained nearly white when grown on an agar medium containing 30 to 40 mg per liter of tellurite. Schopfer's medium enriched with yeast extract and a mixture of vitamins was most suitable to develop such black-and-white contrast. The difference was far less obvious when the asparagine of this medium was replaced by other nitrogen sources such as glutamate, peptone, or Casamino Acids. Addition of ammonium sulfate to the medium weakened and sometimes completely reversed the contrast. The usefulness of tellurite medium for diagnostic color differentiation of respiration deficiency was considered.     

Bias Caused by Using Different Isolation Media for Assessing the Genetic Diversity of a Natural Microbial Population

Abstract The influence of isolation medium on the biodiversity of Burkholderia cepacia strains recovered from the rhizosphere of Zea mays was evaluated by comparing the genetic diversity of isolates obtained by plating serial dilutions of root macerates on the two selective media TB-T and PCAT. From each medium, 50 randomly chosen colonies were isolated. On the basis of the restriction patterns of DNA coding for 16S rRNA (16S rDNA) amplified by means of PCR (ARDRA), all strains isolated from TB-T medium were assigned to the B. cepacia species, whereas among PCAT isolates only 74% were assigned to the B. cepacia species. Genetic diversity among the PCAT and TB-T isolates was evaluated by the random amplified polymorphic DNA (RAPD) technique. The analysis of molecular variance (AMOVA) method was applied to determine the variance component for RAPD patterns. Most of the genetic diversity (90.59%) was found within the two groups of isolates, but an appreciable amount (9.41%) still separated the two groups (P < 0.001). Mean genetic distances among PCAT isolates (10.39) and TB-T isolates (9.36) were significantly different (P < 0.0001). The results indicate that the two different isolation media select for B. cepacia populations with a different degree of genetic diversity. Moreover, a higher degree of genetic diversity was observed among strains isolated from PCAT medium than among those isolated from TB-T medium. Received: 29 April 1999; Accepted: 27 January 2000; Online Publication: 28 August 2000     

Established and abandoned tea (Camillia sinensis L.) rhizosphere: dominant bacteria and their antagonism

Some parts of the Indian Himalayan region are covered by established and abandoned tea bushes. Rhizospheric soils of these plants were studied for bacterial dominance and antagonism. Representatives of Bacillus and Pseudomonas genera were found to dominate the rhizosphere of established and abandoned tea bushes, respectively. Amongst the isolated species Bacillus subtilis and Bacillus mycoides appeared to be closely associated with roots of established tea bushes while the rhizosphere of abandoned tea bushes was dominated by Pseudomonas putida. Four isolates of both B. subtilis and P. putida were selected on the basis of maximum antibacterial activity. The bacteriocin-like activity of B. subtilis and P putida strains was detected to be active over a range of temperature 0-50 degrees C and was sensitive to proteolytic enzymes. Incubation of indicator strains with different concentrations of bacteriocin-like substances confirmed their bactericidal activity. Various species of Bacillus and Pseudomonas behaved antagonistically amongst themselves due to the production of bacteriocins under in vitro conditions.     

Isolation of Frankia Strains from Alder Actinorhizal Root Nodules

A simple procedure, based on the rapid filtration and washing of Frankia vesicle clusters, was devised for the isolation of Frankia strains from alder actinorhizal root nodules. Of 46 Alnus incana subsp. rugosa nodules prepared, 42 yielded isolates. A simple medium containing mineral salts, Casamino Acids, and sodium pyruvate proved to be the most effective for isolation. In general, colonies appeared 6 to 20 days after inoculation. On the basis of hyphal morphology, two distinct types of Frankia strains were characterized. Randomly selected isolates were tested for infectivity, and all formed root nodules on A. glutinosa. Because of its simplicity and efficiency, the procedure is an improved method for the study of Frankia diversity in alder root nodules.     

Phomopsin A production by Phomopsis leptostromiformis in liquid media.

Phomopsis leptostromiformis WA1515 produced 75 to 150 mg of phomopsin A per liter in stationary cultures in a Czapek-Dox medium supplemented with 5 to 10 g of yeast extract per liter. pH and temperature optima were approximately 6.0 and 25 degrees C, respectively. A commercial tryptic digest of casein was a satisfactory alternative to the yeast extract, but poor growth and very little phomopsin were obtained when the yeast was replaced by vitamin-free Casamino Acids or a mixture of 18 amino acids. Approximately 95% of the phomopsin A produced was found in the cutlure liquid. No phomopsin was detected in shaken cultures. No phomopsin B was found in any culture. Methods are described for recovery and estimation of phomopsin A from culture liquids.     

Phomopsin A production by Phomopsis leptostromiformis in liquid media.

Phomopsis leptostromiformis WA1515 produced 75 to 150 mg of phomopsin A per liter in stationary cultures in a Czapek-Dox medium supplemented with 5 to 10 g of yeast extract per liter. pH and temperature optima were approximately 6.0 and 25 degrees C, respectively. A commercial tryptic digest of casein was a satisfactory alternative to the yeast extract, but poor growth and very little phomopsin were obtained when the yeast was replaced by vitamin-free Casamino Acids or a mixture of 18 amino acids. Approximately 95% of the phomopsin A produced was found in the cutlure liquid. No phomopsin was detected in shaken cultures. No phomopsin B was found in any culture. Methods are described for recovery and estimation of phomopsin A from culture liquids.     

Comparison of aerobic heterotrophic taxa isolated from four root domains of mature sugar beet (Beta vulgaris)

Abstract: The distribution of bacteria in the rhizosphere, rhizoplane, interior root tissues (core) and lower root (all tissues) of mature sugar beet roots ( Beta vulgaris ) was compared. Of 556 isolates, 102 species from 40 genera were identified by fatty acid methyl ester gas-chromatographic (FAME-GC) analysis. The ten most common genera ( Bacillus , 14%; Arthrobacter , 12%; Pseudomonas , 11%; Aureobacterium , 9%; Micrococcus , 6%; Xanthomonas , 5%; Alcaligenes , 4%; Flavobacterium , 3%; Agrobacterium , 3%; Microbacterium , 3%) accounted for 70% of isolates, and were found in each of three root domains (rhizosphere, rhizoplane and interior root tissues) on the two principal sampling occasions. Gram-positive strains were more abundant in the rhizosphere than the rhizoplane. Compared to the rhizoplane, rhizosphere bacterial communities were represented by a less diverse, more hierarchical distribution of species where twice as many isolates formed late developing colonies on isolation plates. Between October and January, the bacteria isolated from root interior tissues acquired a distinct change in taxonomic pattern, with decreased diversity and increased hierarchy. A bacterial continuum of similar taxa was observed which extended from the rhizosphere to interior root tissues.     

A note on a selective agar medium for the enumeration of Flavobacterium species in water

A selective nutrient agar medium containing kanamycin at 50 μg/ml was developed for the isolation and enumeration of yellow-pigmented colonies from the River Sowe, Coventry. Such organisms were shown to be members of the heterogeneous genus Flavobacterium . Typically, yellow pigmented colonies constituted less than 10% of the colonies on nutrient agar alone but up to 70% on nutrient agar plus kanamycin. This medium is a useful addition to the range of media available for the isolation and further ecological study of particular species of this important group of micro-organisms.     

A note on a selective agar medium for the enumeration of Flavobacterium species in water

A selective nutrient agar medium containing kanamycin at 50 micrograms/ml was developed for the isolation and enumeration of yellow-pigmented colonies from the River Sowe, Coventry. Such organisms were shown to be members of the heterogeneous genus Flavobacterium. Typically, yellow pigmented colonies constituted less than 10% of the colonies on nutrient agar alone but up to 70% on nutrient agar plus kanamycin. This medium is a useful addition to the range of media available for the isolation and further ecological study of particular species of this important group of micro-organisms.     

Purification, properties, and regulation of glutamic dehydrogenase of Bacillus licheniformis

Cell-free extracts of Bacillus licheniformis and B. cereus were found to contain high specific activities of nicotinamide adenine dinucleotide phosphate (NADP)-dependent-l-glutamate dehydrogenase [EC 1.4.1.4; l-glutamate: NADP oxidoreductase (deaminating)]. Maximum specific activities were found in extracts of cells during the late exponential phase of growth when ammonium ion served as the sole source of nitrogen. Extremely low specific activities were detected throughout the growth cycle when l-glutamate or Casamino Acids served as the source of carbon and nitrogen. The enzyme was purified 55-fold from crude extracts of B. licheniformis, and apparent kinetic constants were determined. Sigmoidal saturation kinetics were not observed, and various adenylates had no effect on the enzyme. Repression of enzyme synthesis during growth on l-glutamate or Casamino Acids was partially overcome by additions of glucose or pyruvate, and this apparent derepression was totally abolished by inhibitors of ribonucleic acid and protein synthesis. Similarly, additions of l-glutamate or Casamino Acids to cells growing on glucose-ammonium ion resulted in strong repression of enzyme synthesis. It is suggested that the enzyme serves an anabolic role in metabolism. Nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase activity was not detected in five species of Bacillus, irrespective of nutritional conditions or of the physiological age of cells.     

Quantitative procedure for enumeration of bifidobacteria.

A membrane filter technique has been developed for the enumeration of bifidobacteria in natural aquatic environments. The technique is quantitative, selective, and differential. The medium (YN-6) contains: yeast extract, 2.0 g; agar, 1.5 g; polypeptone peptone, 1.0 g; vitamin-free Casamino Acids, 0.8 g; sodium chloride, 0.32 g; and L-cysteine hydrochloride, 0.003 g; in 100 ml of deionized water. The medium is adjusted to pH 7.0 before autoclaving. Nalidixic acid (80 micrograms/ml), neomycin sulfate (2.5 micrograms/ml), and bromcresol green (300 micrograms/ml) are included as selective and differential agents. After incubation for 48 h at 37 degrees C in an anaerobic environment, Gram-stained smears from green, glistening, smooth entire colonies are examined microscopically for typical bifidobacterial morphology. No significant difference in recoveries was observed when YN-6 was compared with reinforced clostridial agar, using bifidobacteria freshly isolated from feces and raw sewage. Using this technique with aquatic and fecal samples, less than 9% false-positive and 8% false-negative isolates were observed. These results indicated that the medium was able to satisfactorily recover organisms from a variety of situations.     

Selective utilization of exogenous amino acids by Dehalococcoides ethenogenes strain 195 and its effects on growth and dechlorination activity

Bacteria of the genus Dehalococcoides are important members of bioremediation communities because of their ability to detoxify chloroethenes to the benign end product ethene. Genome-enabled studies conducted with Dehalococcoides ethenogenes 195 have revealed that two ATP-binding cassette (ABC)-type amino acid transporters are expressed during its exponential growth stages. In light of previous findings that Casamino Acids enhanced its dechlorination activity, we hypothesized that strain 195 is capable of importing amino acids from its environment to facilitate dechlorination and growth. To test this hypothesis, we applied isotopomer-based dilution analysis with (13)C-labeled acetate to differentiate the amino acids that were taken up by strain 195 from those synthesized de novo and to determine the physiological changes caused by the significantly incorporated amino acids. Our results showed that glutamate/glutamine and aspartate/asparagine were almost exclusively synthesized by strain 195, even when provided in excess in the medium. In contrast, phenylalanine, isoleucine, leucine, and methionine were identified as the four most highly incorporated amino acids, at levels >30% of respective proteinogenic amino acids. When either phenylalanine or all four highly incorporated amino acids were added to the defined mineral medium, the growth rates, dechlorination activities, and yields of strain 195 were enhanced to levels similar to those observed with supplementation with 20 amino acids. However, genes for the putative ABC-type amino acids transporters and phenylalanine biosynthesis exhibited insignificant regulation in response to the imported amino acids. This study also demonstrates that using isotopomer-based metabolite analysis can be an efficient strategy for optimizing nutritional conditions for slow-growing microorganisms.     

Ammonia-Hyperproducing Bacteria from New Zealand Ruminants

Pasture-grazed dairy cows, deer, and sheep were tested for the presence of ammonia-hyperproducing (HAP) bacteria in roll tubes containing a medium in which tryptone and Casamino Acids were the sole nitrogen and energy sources. Colonies able to grow on this medium represented 5.2, 1.3, and 11.6% of the total bacterial counts of dairy cows, deer, and sheep, respectively. A total of 14 morphologically distinct colonies were purified and studied further. Restriction fragment length polymorphisms of 16S rRNA genes indicated that all isolates differed from the previously described HAP bacteria, Clostridium aminophilum, Clostridium sticklandii, and Peptostreptococcus anaerobius. Carbon source utilization experiments showed that five isolates (C2, D1, D4, D5, and S1) were unable to use any, or very few, of the carbon sources tested. Biochemical tests and phylogenetic analyses of 16S ribosomal DNA sequences indicated that all isolates were monensin sensitive; that D1 and S1 belonged to the genus Peptostreptococcus, that D4 and D5 belonged to the family Bacteroidaceae, where D4 was similar to Fusobacterium necrophorum; and that C2 was most similar to an unidentified species from the genus Eubacterium. Growth on liquid medium containing tryptone and Casamino Acids as the sole nitrogen and energy source showed that D1, D4, and S1 grew rapidly (specific growth rates of 0.40, 0.35, and 0.29 h⁻¹, respectively), while C2 and D5 were slow growers (0.25 and 0.10 h⁻¹, respectively). Ammonia production rates were highest in D1 and D4, which produced 945.5 and 748.3 nmol/min per mg of protein, respectively. Tests of individual nitrogen sources indicated that D1 and D4 grew best on tryptone, S1 grew equally well on Casamino Acids or tryptone, and C2 and D5 grew poorly on all nitrogen sources. The intact proteins casein and gelatin did not support significant growth of any of the isolates. These isolates extend the diversity of known HAP rumen bacteria and indicate the presence of significant HAP bacterial populations in pasture-grazed New Zealand ruminants.     

Characterization of bacterial strains capable of desulphurisation in soil and sediment samples from Antarctica

The presence of sulphur in fossil fuels and the natural environment justifies the study of sulphur-utilising bacterial species and genes involved in the biodesulphurisation process. Technology has been developed based on the natural ability of microorganisms to remove sulphur from polycyclic aromatic hydrocarbon chains. This biotechnology aims to minimise the emission of sulphur oxides into the atmosphere during combustion and prevent the formation of acid rain. In this study, the isolation and characterization of desulphurising microorganisms in rhizosphere and bulk soil samples from Antarctica that were either contaminated with oil or uncontaminated was described. The growth of selected isolates and their capacity to utilise sulphur based on the formation of the terminal product of desulphurisation via the 4S pathway, 2-hydroxybiphenyl, was analysed. DNA was extracted from the isolates and BOX-PCR and DNA sequencing were performed to obtain a genomic diversity profile of cultivable desulphurising bacterial species. Fifty isolates were obtained showing the ability of utilising dibenzothiophene as a substrate and sulphur source for maintenance and growth when plated on selective media. However, only seven genetically diverse isolates tested positive for sulphur removal using the Gibbs assay. DNA sequencing revealed that these isolates were related to the genera Acinetobacter and Pseudomonas.