Habitat, applications and genomics of the aerobic, thermophilic genus Geobacillus.

Thermophilic bacteria belonging to Bacillus genetic group 5 have been reclassified as being members of Geobacillus gen. nov., with G. stearothermophilus as the type strain. Geobacillus species, literally meaning earth or soil Bacillus, are widely distributed and readily isolated from natural and man-made thermophilic biotopes. Work within our group has however shown that an abundance of genetically distinct Geobacillus isolates can be obtained from temperate Irish soils. As with many thermophiles there is considerable interest in potential industrial application of these bacteria and their gene products. This review describes two novel applications for Geobacillus isolates, firstly in the metabolism of the herbicide glyphosate and secondly in the metabolism of quorum-sensing signal molecules from Gram-negative bacteria. Finally the current state of the art is described for Bacillus genomics, with details given of three independent genome-sequencing projects of Geobacillus isolates.

Low-temperature brewing using yeast immobilized on dried figs.

Dried figs, following exhaustive extraction of their residual sugars with water, were used for immobilization of Saccharomyces cerevisiae AXAZ-1. The immobilized biocatalyst was used in repeated batch fermentations of glucose at 30 degrees C, where significant reduction of the fermentation time was observed, falling from 65 h in the first batch to 7 h after the sixth batch. Repeated fermentations of wort at room and low temperatures resulted in fermentation times that fell from 26 to 20 h and from 27 to 24 days at 18 and 3 degrees C, respectively. Ethanol and beer productivities were high, showing suitability of the biocatalyst for low-temperature brewing. Diacetyl concentrations were low (0.3-0.5 mg/L), and polyphenols were lower than in commercial products and decreased as the fermentation temperature was decreased (126-50 mg/L). Ethyl acetate concentrations increased from 53 to 88 mg/L as the temperature was decreased, while the concentration of amyl alcohols at 3 degrees C (58 mg/L) was lower than half of that at 18 degrees C (125 mg/L). The beers produced at the end of the main fermentation had a fine clarity and a special fruity figlike aroma and taste, distinct from commercial products and more intense than beers produced by cells immobilized on other food-grade supports (gluten pellets or delignified cellulosic materials). GC-MS analysis did not show significant differences in the qualitative composition of the aroma compounds of the beers produced by immobilized and free cells.

Organophosphonate metabolism by a moderately halophilic bacterial isolate.

A Gram-negative halophile isolated from soil beneath a road gritting salt pile grew optimally at 10% (w/v) NaCl and was shown most likely to be Chromohalobacter marismortui or Pseudomonas beijerinckii on the basis of 16S rRNA analysis. The strain utilised phosphonoacetate, 2-aminoethyl-, 3-aminopropyl-, 4-aminobutyl-, methyl- and ethyl-phosphonates as phosphorus sources for growth. Differences were observed in the growth rate on different phosphonates and the range of phosphonates utilised at elevated NaCl concentrations, possibly as a result of differentially-induced transport mechanisms. An assay of cell-free extracts of 2-aminoethylphosphonate (2AEP) grown cells showed no detectable 2AEP:pyruvate aminotransferase or phosphonoacetaldehyde hydrolase activity.

The utilization of 4-aminobutylphosphonate as sole nitrogen source by a strain of Kluyveromyces fragilis.

A strain of the yeast Kluyveromyces fragilis was screened for its ability to utilize a range of synthetic and natural organophosphonate compounds as the sole source of phosphorus, nitrogen or carbon. Only 4-aminobutylphosphonate was utilized as sole nitrogen source with protein yields increasing proportionally with substrate concentrations up to 10 mM. No 4-aminobutylphosphonate metabolizing enzyme activity was detectable in cell-free extracts prepared from K. fragilis pregrown on 2.5 mM 4-aminobutylphosphonate. None of the organophosphonates tested served as a source of carbon or phosphorus for K. fragilis.

Initial in vitro characterisation of phosphonopyruvate hydrolase, a novel phosphate starvation-independent, carbon-phosphorus bond cleavage enzyme in Burkholderia cepacia Pal6.

A novel, inducible carbon-phosphorus bond cleavage enzyme, phosphonopyruvate hydrolase, was detected in cell-free extracts of Burkholderia cepacia Pal6, an environmental isolate capable of mineralising L-phosphonoalanine as carbon, nitrogen and phosphorus source. The activity was induced only in the presence of phosphonoalanine, did not require phosphate starvation for induction and was uniquely specific for phosphonopyruvate, producing equimolar quantities of pyruvate and inorganic phosphate. The native enzyme had a molecular mass of some 232 kDa and showed activation by metal ions in the order Co2+ > Ni2+ > Mg2+ > Zn2+ > Fe2+ > Cu2+. Temperature and pH optima in crude cell extracts were 50 degrees C and 7.5, respectively, and activity was inhibited by EDTA, phosphite, sulfite, mercaptoethanol and sodium azide. Phosphonopyruvate hydrolase is the third bacterial C-P bond cleavage enzyme reported to date that proceeds via a hydrolytic mechanism.

Phosphate starvation-independent 2-aminoethylphosphonic acid biodegradation in a newly isolated strain of Pseudomonas putida, NG2.

A strain of Pseudomonas putida that utilized the biogenic organophosphonate 2-aminoethylphosphonic acid as sole carbon and energy, nitrogen and phosphorus source contained 2-aminoethylphosphonic acid: pyruvate aminotransferase and phosphonoacetaldehyde hydrolase (phosphonatase) activities which were inducible by the presence of 2-aminoethylphosphonic acid in the culture medium, regardless of the phosphate status of the cells. Neither of these activities were induced in their phosphate-free or phosphate-replete medium in the absence of 2-aminoethylphosphonic acid. Alkaline phosphatase activity was induced in phosphate limited medium, however, indicating a phosphate-starvation inducible response. In Enterobacter aerogenes IFO 12010, 2-aminoethylphosphonate: pyruvate aminotransferase and phosphonatase activities were induced only when cells were both phosphate limited and supplied with 2-aminoethylphosphonic acid as sole phosphorus source for growth. Neither enzyme activity was induced in phosphate-replete medium, or in medium where both 2-aminoethylphosphonic acid and inorganic phosphate were supplied as sources of phosphorus. The results point to the presence of a substrate inducible 2-aminoethylphosphonic acid biodegradation pathway in the isolated strain of Pseudomonas putida. Uniquely, therefore, the pathway is not under pho regulon control in this strain.

In vitro cleavage of the carbon-phosphorus bond of phosphonopyruvate by cell extracts of an environmental Burkholderia cepacia isolate.

Cell-free extracts of Burkholderia cepacia strain Pal6 catalysed the degradation of 3-phosphonopyruvate to pyruvate and inorganic phosphate; the products were detected in equimolar quantities. The stable in vitro activity responsible was distinct from both phosphonoactealdehyde hydrolase and phosphonoacetate hydrolase and from phosphoenolpyruvate phosphomutase and appears to represent a novel mode of carbon-phosphorus bond cleavage.