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New polyenic antibiotics active against gram-positive and gram-negative bacteria. VIII. Construction of synthetic medium for production of mono-chloro-congeners of enacyloxins.

New antibiotics enacyloxins (ENXs) are a family of non-lactonic polyene antibiotics produced by Frateuria sp. W-315. For the production of antibiotics, we had to employ two-step fermentations, the first is the production of spent medium of Neurospora crassa and the second is the production of antibiotics by Frateuria. To simplify the production of antibiotics, systematic analyses have been done on the spent medium, and factors which affect the production of antibiotics characterized. From the above results, we constructed a new medium for antibiotic production. Moreover, we could get a new antibiotic named enacyloxin IIIa (1), C33H48O11NCl (m/z 669). 1 was deduced to be one of the congeners of enacyloxins because it was similar to ENX IIa or ENX IVa both in biological and physico-chemical properties. Chlorine of 1 could be replaced by bromine, biosynthetically, and the resultant bromine-containing antibiotic also showed an antibacterial activity comparable to 1.

Acetobacteraceae↗

Direct fermentation of 2-keto-L-gulonic acid in recombinant Gluconobacter oxydans.

We isolated Gluconobacter oxydans T-100 that had an activity to produce 2-KLGA from D-sorbitol; however, the yield of 2-KLGA was quite insufficient. Therefore, enzymes involved in the biosynthesis of L-sorbosone and 2-KLGA, L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH), respectively, were purified from G. oxydans T-100. A genomic library of G. oxydans T-100 was screened to clone both genes for SDH and SNDH based on their amino acid sequences. SNDH and SDH were encoded in sequential open reading frames with 1497 and 1596 nucleotides, respectively, which were verified by the expression in Escherichia coli. The amino acid sequence of SDH and SNDH showed close similarity with E. coli choline dehydrogenase (CDH) and betaine-aldehyde dehydrogenase (BADH), respectively, which cooperatively play a key role for conferring osmotic tolerance. Because the yield of 2-KLGA by G. oxydans introduced with the genes for SDH and SNDH were insufficient, replacement of the promoter with that of Escherichia coli tufB1 in combination with chemical mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine resulted in improvement of the production level.

Acetobacteraceae↗

Sequence analysis of the Gluconobacter oxydans RecA protein and construction of a recA-deficient mutant.

The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotacticum, Escherichia coli, and Pseudomonas aeruginosa, respectively. The amino acid residues essential for function of the recombinase, protease, and ATPase in E. coli recA protein are conserved in G. oxydans. Of 24 amino acid residues believed to be the ATP binding domain of E. coli RecA, 17 are found to be identical in G. oxydans RecA. Interestingly, nucleotide sequence alignment between the SOS box of G. orphans recA gene and those from different microorganisms revealed that all the DNA sequences examined have dyad symmetry that can form a stem-loop structure. A G. oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette. Such replacement of the wild-type recA with a kanamycin resistance gene in the chromosome was further verified by Southern hybridization. Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation than the wild-type strain, suggesting that the recA gene of G. oxydans ATCC9324 plays a role in repairing DNA damage caused by UV irradiation. Moreover, the mutant strain is much more plasmid transformable than its parent strain, illustrating that G. oxydans LCC96 could be used as a host to take up the recombinant plasmid for gene manipulation.

Acetobacteraceae↗

[Alleviation of type I restriction in Escherichia coli K12 in the presence of the arsR gene from pKW301 of Acidiphilium multivorum AIU 301].

A study was made of the antirestriction activity of Acidiphilium multivorum AIU 301 ArsR, a repressor of the ars operon which confers resistance to arsenite and arsenate and is on pKW301. In Escherichia coli, arsR cloned under the control of Plac in a multi-copy vector alleviated restriction of nonmodified lambda DNA by a factor of 120, six times more efficiently than its analogs of conjugal plasmids R64 (incI1) and R773 (incFI). Amino acid sequence analysis showed that the three ArsR proteins have a homologous region of 38 residues, including the antirestriction motif, in their N domains, whereas the motif is in the C domain in the Ard proteins. The other regions are nonhomologous, and pKW301 ArsR is 33 residues shorter than R64 and R773 ArsRs. The total charge is -4 in pKW301 ArsR and +2 in R64 and R733 ArsRs. A total negative charge was assumed to contribute to the antirestriction activity.

Acetobacteraceae↗

Reductive biodegradation of 1,2-dichloroethane by methanogenic granular sludge: perspectives for in situ remediation.

Granular methanogenic sludge was able to dechlorinate 1,2-dichloroethane (1,2-DCA) to ethene in UASB reactors. Ethanol served as the sole carbon and energy source. The average dechlorination rate measured on the basis of ethene production varied between 1.7 and 2.1 micromol 1,2-DCA/(h.gVSS) (46.7 and 57.4 mg/L.d). In order to elucidate the microbial origin of this bioconversion, enrichment cultures of the methanogenic sludge were prepared with different carbon and electron sources: pyruvate, lactate, H2/CO2, ethanol and formate. Dithiothreitol (DTT) was the strong reductant in order to increase the negative redox potential in the media. A homo-acetogenic gram-positive strain could be isolated in the presence of formate. 16S rRNA of the isolated strain showed that the bacterium was closely related (99.7%) to Acetobacterium wieringae. The strain also grew on pyruvate, lactate, H2/CO2 and ethanol, although dechlorination rates of 1,2-DCA were at least 5 times higher when formate was the (only) electron source. Average conversion rates reached 3 micromol/(h.g(dry cells)) and appeared t relate to cometabolic biocatalysis on the corrinoid centers of the homo-acetogenic strain. Some perspectives of anaerobic in situ bioremediation of groundwater polluted with chloroethanes are presented.

Acetobacteraceae↗

[Microbial biodiversity in the Lake Baikal water].

The investigation of the microbial community of Lake Baikal by the methods of general and molecular microbiology showed that culturable bacterial strains were represented by various known genera. The lake water contains a great number of bacterial morphotypes, as revealed by electron microscopy, and a great diversity of nonculturable microorganisms belonging to different phylogenetic groups, as revealed by 16S rRNA gene fragment sequencing. The inference is made that the microbial community of Lake Baikal contains not only the known species but also new, possibly endemic to the lake, bacterial species.

Acetobacteraceae↗

Production of cephalexin by Gluconobacter oxydans CCRC 10383.

Intact cells of Gluconobacter oxydans CCRC 10383 produced cephalexin from 7-amino-3-deacetoxy cephalosporanic acid (7-ADCA) and D-alpha-phenylglycine methylester HC1 (PGM). Factors affecting the production of cephalexin by G. oxydans CCRC 10383 were studied. The optimum pH and temperature for the synthetic reaction of cephalexin were 6.0 and 42 degrees C, respectively. A higher concentration of PGM than 7-ADCA was required to obtain a good yield of cephalexin.

Acetobacter↗

Genetics of metal resistance in acidophilic prokaryotes of acidic mine environments.

Acidophilic bacteria inhabiting acidic mine regions cause natural leaching of sulphidic ores. They are now exploited in industrial operations for leaching of metals and beneficiation of low-grade and recalcitrant ores. Recent trends emphasize application of thermoacidophiles and genetic engineering of ore-leaching bacteria for greater success in this area. This requires an in-depth understanding on the molecular genetics of these bacteria and construction of cloning vectors for them. Metal resistance is considered as the most suitable phenotypic trait for cloning vectors of bio-mining chemolithoautotrophic (viz. Acidithiobacillus ferrooxidans) and heterotrophic (Acidiphilium and Acidocella species) bacteria of mine environments. These bacteria take part in ore-leaching either directly or indirectly, exhibit low to high level of resistance/tolerance to various metals under different conditions. Majority of these bacteria contain one or more plasmids--the genetic elements that usually carry metal resistant genes. But none of the At. ferrooxidans plasmids has been definitely proved to harbour metal-resistant genes which have mostly been found in the chromosome of this bacterium. Plasmids of acidophilic heterotrophs of the genera Acidiphilium and Acidocella, on the other hand, carry metal resistant genes. While genes bestowing arsenic resistance in Acidiphilium multivorum are similar to those analyzed from other sources, the metal (Cd and Zn)-resistance conferring cloned plasmid DNA fragments from Acidiphilium symbioticum KM2 and Acidocella GS19h strains were found to have no sequence similarity with the reported Cd- and Zn-resistant genes. Such observations indicate some novel aspects of metal resistance in acidophilic bacteria.

Acetobacteraceae↗

Manufacture of a beverage from cheese whey using a "tea fungus" fermentation.

Kombucha is a sour beverage reported to have potential health effects prepared from the fermentation of black tea and sugar with a "tea fungus", a symbiotic culture of acetic acid bacteria and yeasts. Although black tea is the preferred substrate for Kombucha fermentation, other beverages have also been tested as substrates with fair results. Cheese whey is a by-product with a good amount of fermentable lactose that has been used before in the production of beverages, so the objective of this study was to test three types of whey (fresh sweet, fresh acid and reconstituted sweet) in the elaboration of a fermented beverage using a kombucha culture as inoculum. The isolation and identification of bacteria and yeasts from the fermented tea and wheys was done along with the study of the rates of change in sugar consumption, acid production and pH decrease. Several species of acetic acid bacteria (Acetobacter aceti subsp. aceti, Gluconobacter oxydans subsp. industrius, subsp. oxydans and Gluconoacetobacter xylinus) were isolated from the different kombuchas along with the yeasts Saccharomyces cerevisiae, Kluyveromyces marxianus, and Brettanomyces bruxelensis. The main metabolic products in the fermented wheys included ethanol, lactic and acetic acids. A good growth was obtained in both sweet wheys in which a pH of 3.3 and a total acid content (mainly lactic and acetic acids) of 0.07 mol/l was reached after 96 h. The sweet whey fermented beverages contained a relatively low lactose concentration (< 12 g/l). The final ethanol content was low (5 g/l) in all the fermented wheys. The whey products were strongly sour and salty non sparkling beverages.

Acetobacteraceae↗

[Sensor for rapid measurement of blood glucose].

Bacterial biosensor consisting of Gluconobacter oxydans cells immobilized on the surface of oxygen electrode was used for measuring glucose concentration in human blood serum. The results were compared with those of standard clinical method (color reaction with ortho-toluidine). The proposed biosensor permits highly accurate measurements (mean quadratic deviation no more than 2% of the mean arithmetic), the coefficient of correlation with the results of standard method being 0.970.

Acetobacteraceae↗