Genetic transfers inBrevibacterium sp.

A positive genetic transfer by protoplast fusion was obtained in auxotrophic mutants Brevibacterium sp. M27 his and Brevibacterium sp. M27 arg. Transformation and protoplast fusion with liposomes (as genetic transfers in intact cells and their protoplasts by both the chromosomal and plasmid DNA) did not lead to transfer of the markers followed.     

Induction of Yellow Pigmentation in Serratia marcescens

The appearance of yellow pigmentation in nonpigmented strains of Serratia sp. has been demonstrated to be due to the production of a muconic acid, 2-hydroxy-5-carboxymethylmuconic acid semialdehyde. The 3,4-dihydroxyphenylacetate 2,3-dioxygenase responsible for the synthesis of this muconic acid was induced in all strains tested. Another muconic acid, the β-cis-cis-carboxymuconic acid, could also be synthesized from 3,4-dihydroxybenzoate, but this product was not colored. Mutants that were unable to grow on tyrosine and produced yellow pigment were isolated from nonpigmented strains. These mutants had properties similar to those of the yellow-pigmented strains. The ability to produce pigment may be more widespread among Serratia marcescens strains than is currently known.     

Fed-batch culture for L-lysine production via on-line state estimation and control

Computer application for fed-batch culture of Brevibacterium flavum for L-lysine production has been developed. The organisms are auxotrophic mutants for L-homoserine and are resistant to S-(2-aminoethyl)-L-cysteine. Adaptive control is applied for substrate addition. The sugar concentration is estimated using online respiratory measurement. During the period of fed-batch culture, the total sugar concentration is maintained at a given value. The cultivation strategy results in high productivity and high conversion yield.     

Correlation between pigmentation and antifouling compounds produced by Pseudoalteromonas tunicata

Pseudoalteromonas tunicata is a marine bacterium with the ability to prevent biofouling by the production of at least four target-specific compounds. In addition to these antifouling compounds, P. tunicata produces at least two pigments. These include a yellow and a purple pigment which, when combined, give the bacterium a dark green appearance. Transposon mutagenesis was used in this study to investigate the correlation between pigment production and the expression of specific antifouling phenotypes in P. tunicata. Four different categories of pigmentation mutants were isolated including yellow, dark-purple, light-purple and white mutants. The mutants were tested for their ability to inhibit the settlement of invertebrate larvae, algal spore germination, fungal growth and bacterial growth. The results showed that the yellow-pigmented mutants retained full antifouling activity, whereas the purple and white mutant strains had lost some, or all, of their ability to inhibit target organisms. This demonstrates that the loss of antifouling capabilities correlates with the loss of yellow pigment and not purple pigment. Sequencing and analysis of the genes disrupted by the transposons in these mutants identified a number of potential biosynthetic enzymes and transport systems involved in the synthesis and regulation of pigmentation and fouling inhibitors in this organism.     

Mutation analysis of the feedback inhibition site of aspartokinase III of Escherichia coli K-12 and its use in L-threonine production.

Aspartokinase III (AKIII), one of three isozymes of Escherichia coli K-12, is inhibited allosterically by L-lysine. This enzyme is encoded by the lysC gene and has 449 amino acid residues. We analyzed the feedback inhibition site of AKIII by generating various lysC mutants in a plasmid vector. These mutants conferred resistance to L-lysine and/or an L-lysine analogue on their host. The inhibitory effects of L-lysine on and heat tolerance of 14 mutant enzymes were examined and DNA sequencing showed that the types of mutants were 12. Two hot spots, amino acid residue positions 318-325 and 345-352, were detected in the C-terminal region of AKIII and these enzyme regions may be important in L-lysine-mediated feedback inhibition of AKIII. Feedback resistant lysC relieved on L-threonine hyper-producing strain, B-3996, from reduced L-threonine productivity by addition of L-lysine, and furthermore increased L-threonine productivity even when no addition of L-lysine. It suggested that the bottleneck of L-threonine production of B-3996 was AK and feedback resistant lysC was effective because of the strict inhibition by cytoplasmic L-lysine.     

Growth,differentiation and secondary metabolites of colour mutants ofTrichoderma viride

The parental strainTrichoderma viride and 3 colour mutants (milk white, yellow and brown) blocked at various stages of colony pigmentation derived from it were characterized. The parental strain and the mutants exhibited different growth rates. The identical type of induced fluorescence was observed in all the strains. Hyphae and septa lighted first, whereas reproduction structures did not; after treatment with fluorescein-isothiocyanate and Blankophor RKH the growing hyphal apices were accentuated. In the mutants conidiation was induced at 1-, 2- and 3-d intervals, similarly to the parent strain. Pigmentation of conidiation rings depended on their type and age. The yellow and brown mutants excreted chromatographically different pigments, extractable with ethylacetate, into the medium. Two anthraquinone pigments,viz. 1,3,6,8-tetrahydroxyanthraquinone (1) and 1-acetyl-2,4,5,7-tetrahydroxyanthraquinone (2) were isolated from the brown mutant (Betinaet al. 1986).     

The capacity of orotic acid production in pyrimidinedeficient mutants ofBrevibacterium ammoniagenes

Eight uracil-dependent mutants ofBrevibacterium ammoniagenes CCEB 364 and three mutants ofCorynebacterium sp. 9366 were checked for the production of precursors of nucleic acids. Four of the strains liberated into the medium a substantial amount of orotic acid. The production of orotic acid by a mutant ofBrevibacterium ammoniagenes (1043) was examined on mineral media containing varying amounts of glucose in the presence of uracil. The optimum concentration of glucose for the production of orotic acid was found to be 5–8%. On media to which natural substrates were added the orotic acid production increased substantially. The maximum production (6.5 g orotic acid/liter) was reached in a medium containing 0.5% yeast extract and 5% glucose; addition of uracil to this medium had no effect on the production. The maximum rate of production occurred between 24 and 72 h of fermentation. After this period the concentration of orotic acid in the medium decreases.     

Frequency of antibiotic and heavy metal resistance, pigmentation, and plasmids in bacteria of the marine air-water interface.

A field investigation of marine coastal waters revealed that the frequency of pigmented bacteria and the occurrence of bacterial antibiotic resistance were higher at the air-water interface than in the bulk water. The differences in the frequency of pigmented colonies at the surface and in the bulk-water samples could not be explained by the degree of cell surface hydrophobicity or by bacterial adhesion to air-water interfaces. Pigmented strains exhibited a higher degree of multiple drug resistance than did nonpigmented strains. However, the frequency of multiple drug resistance in nonpigmented strains was also substantial. An average of 91% of all strains were resistant to more than one antibiotic, and 21% of the bacteria isolated were resistant to five of the eight antibiotics tested. High numbers of plasmid-carrying strains were found among selected surface isolates, but the presence of detectable plasmids could not be correlated with either pigmentation or multiple drug resistance. Furthermore, selected surface isolates were significantly more resistant to mercury than were bulk-water bacteria. The higher frequency of pigmented, antibiotic-resistant, and mercury resistant strains at the air-water interface than in the bulk water are discussed in terms of various forms of selective pressure and genetic exchange at the surface.     

Frequency of antibiotic and heavy metal resistance, pigmentation, and plasmids in bacteria of the marine air-water interface

A field investigation of marine coastal waters revealed that the frequency of pigmented bacteria and the occurrence of bacterial antibiotic resistance were higher at the air-water interface than in the bulk water. The differences in the frequency of pigmented colonies at the surface and in the bulk-water samples could not be explained by the degree of cell surface hydrophobicity or by bacterial adhesion to air-water interfaces. Pigmented strains exhibited a higher degree of multiple drug resistance than did nonpigmented strains. However, the frequency of multiple drug resistance in nonpigmented strains was also substantial. An average of 91% of all strains were resistant to more than one antibiotic, and 21% of the bacteria isolated were resistant to five of the eight antibiotics tested. High numbers of plasmid-carrying strains were found among selected surface isolates, but the presence of detectable plasmids could not be correlated with either pigmentation or multiple drug resistance. Furthermore, selected surface isolates were significantly more resistant to mercury than were bulk-water bacteria. The higher frequency of pigmented, antibiotic-resistant, and mercury resistant strains at the air-water interface than in the bulk water are discussed in terms of various forms of selective pressure and genetic exchange at the surface.     

Regulation of alpha-aminoadipate reductase from Penicillium chrysogenum in relation to the flux from alpha-aminoadipate into penicillin biosynthesis.

The activity and regulation of alpha-aminoadipate reductase in three Penicillium chrysogenum strains (Q176, D6/1014/A, and P2), producing different amounts of penicillin, were studied. The enzyme exhibited decreasing affinity for alpha-aminoadipate with increasing capacity of the respective strain to produce penicillin. The enzyme from all three strains was inhibited by L-lysine, and the enzyme from the lowest producer, Q176, was least sensitive. Between pH 7.5 and 6.5, inhibition of alpha-aminoadipate reductase by L-lysine was pH dependent, being more pronounced at lower pH. The highest producer strain, P2, displayed the lowest alpha-aminoadipate reductase activity at pH 7.0. In Q176, the addition of 0.5-1 mM of exogenous lysine stimulated penicillin formation, whereas the same concentration was ineffective or inhibitory with strains D6/1014/A and P2. The addition of higher (up to 5 mM) lysine concentrations inhibited penicillin production in all three strains. In mutants of P. chrysogenum D6/1014/A, selected for resistance to 20 mM alpha-aminoadipate, highest penicillin production was observed in those strains whose alpha-aminoadipate reductase was most strongly inhibited by L-lysine. The results support the conclusion that the in vivo activity of alpha-aminoadipate reductase from superior penicillin producer strains of P. chrysogenum is more strongly inhibited by lysine, and that this is related to their ability to accumulate increased amounts of alpha-aminoadipate, and hence penicillin.     

Plasmid-borne determinants of pigmentation and thiamine prototrophy in Erwinia herbicola.

Strains of Erwinia herbicola lost yellow pigmentation and thiamine prototrophy at high frequency when grown at elevated temperature (38 degrees C) or in the presence of sodium dodecyl sulfate. All pigmentless, thiamine-auxotrophic variants had lost a large plasmid (ca. 350 megadaltons). Conversely, all pigmented, thiamine-prototrophic strains contained the large plasmid. The evidence presented indicates that pigmentation and thiamine prototrophy are specified or controlled by genes carried on the 350-megadalton plasmid.     

Formation and reversion of protoplasts in mutant strains ofBrevibacterium sp. M27

In mutant strains ofBrevibacterium sp. M27 requiring amino acids and resistant to antibiotics four methods were verified and a method inducing a high formation of protoplasts and their reversion was developed. Formation of protoplasts is 60–99% and their reversion 17–76%. In induced mutants a correlation between protoplast formation and growth rate could be demonstrated.     

Induction of mutants resistant to antibiotics inBrevibacterium flavum

The optimum conditions for the induction of mutants resistant to antibiotics in Brevibacterium flavum ATCC 14067 were determined. UV irradiation at the energy fluence of 6.5 kJ/m2 and N-methyl-N'-nitro-N-nitrosoguanidine (1 mg/mL) at pH 6.0 were used for the induction of mutants. Mutant strains resistant to rifampicin, oleandomycin, streptomycin and erythromycin were prepared.     

Lysine excretion by S-(2-aminoethyl)L-cysteine resistant mutants of Bacillus subtilis

S-(2-Aminoethyl)L-cysteine (AEC) at 2 X 10(-1) mM concentration completely inhibited the growth of Bacillus subtilis. This inhibitory effect was readily reversed by 2 X 10(-2) mM L-lysine. Besides L-lysine, L-aspartic acid was only effective of all the natural amino acids tested in reversing the AEC-mediated growth inhibition. AEC resistant mutants of B. subtilis were isolated and found to excrete L-lysine in high yields.     

Taxonomic studies on filamentous bacteria from sewage belonging to the Flavobacterium-Cytophaga complex

Ten strains of gram-negative nonmotile pigmented filamentous bacteria were isolated from activated sludge. All strains were negative in nitrate reduction, hydrolysis of gelatin, indole production and acid production from glucose (except one strain) but positive in aminopeptidase, oxidase and catalase and oxidative in the O/F-test. Two groups could be differentiated by pigmentation and acid production from saccharose (group P, pink) or maltose (group Y, yellow). Group P (6 strains) had a G+C-content in the range from 41.9 to 43.7 mol%. The menaquinone isolated was predominantly MK-7. The pattern of branched and hydroxylated fatty acids was similar to the strains in phenovar 4–5 of Oyaizu and Komagata (1981). Group Y (4 strains) had a G+C-content of 33.7–34.6 mol%; menaquinones determined were predominantly MK-6. Fatty acid patterns were similar to those of the strains of the phenovar 6–1 of Oyaizu and Komagata (1981).     

Fatty acids and carbohydrate-containing lipids in four Micrococcaceae strains

Fatty acid composition and lipidic carbohydrate to lipidic phosphorus molar ratio of yellow pigmented micrococci are compared to red pigmented ones and may be summarized by three indexes. These bacteria show wide differences in their fatty acid composition: three strains possess saturated branched chain fatty acids and one has unsaturated straight chain ones. A significant increase in 'anteiso/iso indexes' is observed between pink (M. roseus) and yellow colored bacteria (M. lysodeikticus, S. lutea). There is no significant difference (P greater than 0.05) between the 'unsaturation indexes' of the red pigmented parental D. radiodurans strain and its colorless mutant. Radioresistant strains exhibit a higher 'carbohydrate/phosphorus index' than other strains. There seems to be a relationship between a high carbohydrate-containing lipid content and a high resistance to physical and chemical agents, in particular to radiations. These differences observed in the lipid composition have implications in taxonomy and in establishing an evolutionary scheme.     

Pigmented variants of Rhizobium

Summary Non-infective pigmented variants of Rhizobium have been isolated from several cultures after treatment with antibiotics, heat and U.V. irradiation. These variants tolerated much higher doses of heat, U.V. and certain antibiotics than the wild type strains from which they originated. Some of the pigmented variants produced reverse mutants on subculture which had lost their pigment but recovered their infectivity. These reverse mutants also lost their extreme U.V.-resistance. However, many pigmented mutants were very stable and reverted only after U.V. irradiation.This investigation shows that pigmentation in Rhizobium and loss of ability to form nodules on the roots of leguminous plants are genetically linked and may be pleiotropic effects. So far, more than 10 pigmented variants have been isolated, none of them was infective, while reverse mutants which had lost their pigment always gave rise to nodules.     

UVC-mutagenesis in acetogens: resistance to methanol,ethanol, acetone,or n-butanol in recombinants with tailored genomes as the step in engineering of commercial biocatalysts for continuous CO2/H2 blend fermentations

Time- and cost-efficient six-step UVC-mutagenesis was developed and validated to generate acetogen mutants with preliminary reduced genomes to prevent product inhibition in the to-be-engineered commercial biocatalysts. Genome reduction was performed via elimination of pta, ack, spo0A, spo0J and some pro-phage genes. UVC-mutants such as Clostridium sp. MT1784RG, Clostridium sp. MT653RG, Clostridium sp. MT896RG, and Clostridium sp. MT1962RG (all 4 share 97 % DNA homology with Clostridium ljungdahlii ATCC 55383) were selected based on resistance to methanol (3 M), ethanol (3.6 M), acetone (2.5 M), or n-butanol (0.688 M), respectively. As a part of the biocatalyst engineering algorithm, genome reduction step was associated with integration of attTn7 recognition sequence to the chromosomes of each of the above strains to prepare the defined integration sites for future integration of multi-copy synthetic operons encoding biosynthesis of methanol, ethanol, acetone or n-butanol. Reduced genome mutants had cell duplication times decreased compared to the same for the respective parental strains. All groups of mutants had decreased share of palmitic (C16:0) and increased share of oleic (C18:1) acids along with detection of isopropylstearate (C20) compared to the parental strains. Mutants resistant to acetone and n-butanol also had monounsaturated fatty acid (C20:1) not found in parental strains. Cyclopropane fatty acid (C21) was identified only in n-butanol resistant mutants.