The behaviour of NAD+ and NADH inAcinetobacter calcoaceticus duringn-alkane assimilation

The behaviour of the nicotinamide adenine dinucleotides NAD+ and NADH in Acinetobacter calcoaceticus during n-alkane assimilation was studied, acetate and succinate being used as reference carbon sources. The intracellular concentration of the two nucleotides was found to increase during the exponential growth phase, reaching its maximum in the phase of decreasing growth rates. In the exponential phase, the NAD+/NADH quotients were less than 1 and showed only unimportant variations. In the phase of decreasing growth rates, the concentration of NADH showed a distinct decrease, reaching its minimum in the stationary phase. Parallel to this, the concentration of NAD+ showed a continuous increase until the stationary phase was reached. This resulted in an increase, during the phase of decreasing growth rates, of the NAD+/NADH quotients to values greater than 1, similarly as recorded in the stationary phase. There were no fundamental differences in this behaviour between the individual carbon sources.     

Effects of growth rate and oxygen tension on glucose dehydrogenase activity inAcinetobacter calcoaceticus LMD 79.41

The regulation of the synthesis of the quinoprotein glucose dehydrogenase (EC 1.1.99.17) has been studied inAcinetobacter calcoaceticus LMD 79.41, an organism able to oxidize glucose to gluconic acid, but unable to grow on both compounds. Glucose dehydrogenase was synthesized constitutively in both batch and carbon-limited chemostat cultures on a variety of substrates. In acetate-limited chemostat cultures glucose dehydrogenase levels and the glucose-oxidizing capacity of whole cells were dependent on the growth rate. They strongly increased at low growth rates at which the maintenance requirement of the cells had a pronounced effect on biomass yield. Cultures grown on a mixture of acetate and glucose in carbon and energy-limited chemostat cultures oxidized glucose quantitatively to gluconic acid. However, during oxygen-limited growth on this mixture glucose was not oxidized and only very low levels of glucose dehydrogenase were detected in cell-free extracts. After introduction of excess oxygen, however, cultures or washed cell suspensions almost instantaneously gained the capacity to oxidize glucose at a high rate, by an as yet unknown mechanism.     

Physiological regulation of competence induction for natural transformation in Acinetobacter calcoaceticus

Acinetobacter calcoaceticus induced competence for natural transformation maximally after dilution of a stationary culture into fresh medium. Competence was gradually lost during prolonged exponential growth and after entrance into the stationary state. Growth cessation and nutrient upshift were involved in the induction of competence. The level of competence of a chemostat culture of A. calcoaceticus was dependent on the nature of the growth limitation. Under potassium limitation a transformation frequency of ±1x10^-4 was obtained. This frequency was independent of the specific growth rate. In phosphate-, nitrogen-, and carbon-limited chemostat cultures, in contrast, the transformation frequency depended on the specific growth rate; the transformation frequency equalled±10^-4 at dilution rates close to µ_max of 0.6h^-1 and decreased to ±10^-7 at a dilution rate of 0.1 h^-1. We conclude that (1) DNA uptake for natural transformation in A. calcoaceticus does not serve a nutrient function and (2) competence induction is regulated via a promoter(s) that resembles the fis promoter from Escherichia coli.     

Quinoprotein D-glucose dehydrogenases inAcinetobacter calcoaceticus LMD 79. 41: Purification and characterization of the membrane-bound enzyme distinct from the soluble enzyme

Acinetobacter calcoaceticus is known to contain soluble and membrane-bound quinoprotein D-glucose dehydrogenases while other oxidative bacteria such asPseudomonas orGluconobacter contain only membrane-bound enzyme. The two different forms were believed to be the same enzyme or interconvertible. Present results show that the two different forms of glucose dehydrogenase are distinct from each other in their enzymatic and immunological properties as well as in their molecular size.The soluble and membrane-bound glucose dehydrogenases were separated after French press-disruption by repeated ultracentrifugation, and then purified to nearly homogeneous state. The soluble enzyme was a polypeptide of 55 Kdaltons, while the membrane-bound enzyme was a polypeptide of 83 Kdaltons which is mainly monomeric in detergent solution. Both enzymes showed different enzymatic properties including substrate specificity, optimum pH, kinetics for glucose, and reactivity for ubiquinone-homologues. Furthermore, the two enzymes could be distinguished immunochemically: the membrane-bound enzyme is cross-reactive with an antibody raised against membrane-bound enzyme purified fromPseudomonas but not with antibody elicited against the soluble enzyme, while the soluble enzyme is not cross-reactive with the antibody of membrane-bound enzyme.Data also suggest that the membrane-bound enzyme functions by linking to the respiratory chain via ubiquinone though the function of the soluble enzyme remains unclear.     

Mapping of the tryptophan genes of Acinetobacter calcoaceticus by transformation

Auxotrophs of Acinetobacter calcoaceticus blocked in each reaction of the synthetic pathway from chorismic acid to tryptophan were obtained after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. One novel class was found to be blocked in both anthranilate and p-aminobenzoate synthesis; these mutants (trpG) require p-aminobenzoate or folate as well as tryptophan (or anthranilate) for growth. The loci of six other auxotrophic classes requiring only tryptophan were defined by growth, accumulation, and enzymatic analysis where appropriate. The trp mutations map in three chromosomal locations. One group contains trpC and trpD (indoleglycerol phosphate synthetase and phosphoribosyl transferase) in addition to trpG mutations; this group is closely linked to a locus conferring a glutamate requirement. Another cluster contains trpA and trpB, coding for the two tryptophan synthetase (EC 4.2.1.20) subunits, along with trpF (phosphoribosylanthranilate isomerase); this group is weakly linked to a his marker. The trpE gene, coding for the large subunit of anthranilate synthetase, is unlinked to any of the above. This chromosomal distribution of the trp genes has not been observed in other organisms.     

Inorganic phosphate transport inAcinetobacter lwoffi

The transport of inorganic phosphate has been studied inAcinetobacter lwoffi JW11. During growth on excess phosphate, only one transport system was present, with an apparent K_m of 1.4 M. When cells were starved for phosphate, a second uptake system with an apparent K_m of 110 nM was also synthesized. The two transport systems could be distinguished by differing sensitivities to the phosphate analogs arsenate and 2-aminoethylphosphonate. Both systems were inhibited by carbonylcyanidem-chlorophenylhydrazone, and to a lesser extent by Na azide. The high-affinity transport system was inactivated by osmotic shock treatment and by spheroplast formation. Preliminary evidence for a phosphate-binding protein in the osmotic shock fluid is presented. The isolation of a mutant constitutive for the high-affinity transport system is described.     

Conditions for natural transformation of Ralstonia solanacearum.

The development of competence allowing natural transformation of Ralstonia solanacearum was found to occur during exponential growth and not in response to any excreted factors. Linear DNAs were effectively integrated by recombination requiring a minimum of 50 bp of homologous DNA. Therefore, DNA from other genera and species were ineffective.     

Plasmid transformation of naturally competent Acinetobacter calcoaceticus in non-sterile soil extract and groundwater

The natural transformation of Acinetobacter calcoaceticus BD413 (trp E27) was characterized with respect to features that might be important for a possible gene transfer by extracellular DNA in natural environments. Transformation of competent cells with chromosomal DNA (marker trp ⁺) occurred in aqueous solutions of single divalent cations. Uptake of DNA into the DNase I-resistant state but not the binding of DNA to cells was strongly stimulated by divalent cations. An increase of transformation of nearly 3 orders of magnitude was obtained as a response to the presence of 0.25 mM Ca²⁺. With CaCl₂ solutions the transformation frequencies approached the highest values obtained under standard broth conditions, followed by MnCl₂ and MgCl₂. It is concluded that transformation requires divalent cations. DNA competition experiments showed that A. calcoaceticus does not discriminate between homologous and heterologous DNA. Furthermore, circular plasmid DNA competed with chromosomal DNA fragments and vice versa. The equally efficient transformation with plasmid pKT210 isolated from A. calcoaceticus or Escherichia coli indicated absence of DNA restriction in transformation. High efficiency plasmid transformation was obtained in samples of non-sterile natural groundwater and in non-sterile extracts of fresh and air-dried soil. Heat-treatment (10 min, 80°C) of the non-sterile liquid samples increased transformation only in the dried soil extract, probably by inactivation of DNases. The results presented suggest that competent cells of A. calcoaceticus can take up free high molecular weight DNA including plasmids of any source in natural environments such as soil, sediment or groundwater.     

Natural transformation and availability of transforming DNA to Acinetobacter calcoaceticus in soil microcosms.

A small microcosm, based on optimized in vitro transformation conditions, was used to study the ecological factors affecting the transformation of Acinetobacter calcoaceticus BD413 in soil. The transforming DNA used was A. calcoaceticus homologous chromosomal DNA with an inserted gene cassette containing a kanamycin resistance gene, nptII. The effects of soil type (silt loam or loamy sand), bacterial cell density, time of residence of A. calcoaceticus or of DNA in soil before transformation, transformation period, and nutrient input were investigated. There were clear inhibitory effects of the soil matrix on transformation and DNA availability. A. calcoaceticus cells reached stationary phase and lost the ability to be transformed shortly after introduction into sterile soil. The use of an initially small number of A. calcoaceticus cells and nutrients, resulting in bacterial growth, enhanced transformation frequencies within a limited period. The availability of introduced DNA for transformation of A. calcoaceticus cells disappeared within a few hours in soil. Differences in transformation frequencies between soils were found; A. calcoaceticus cells were transformed at a higher rate and for a longer period in a silt loam than in a loamy sand. Physical separation of DNA and A. calcoaceticus cells had a negative effect on transformation. Transformation was also detected in nonsterile soil microcosms, albeit only in the presence of added nutrients and at a reduced frequency. These results suggest that chromosomal DNA released into soil rapidly becomes unavailable for transformation of A. calcoaceticus. In addition, strain BD413 quickly loses the ability to receive, stabilize, and/or express exogenous DNA after introduction into soil.     

Genetic analysis of supraoperonic clustering by use of natural transformation in Acinetobacter calcoaceticus.

DNA within Escherichia coli colonies carrying cloned Acinetobacter calcoaceticus genes transforms mutant A. calocaceticus cells with high efficiency. Therefore, E. coli colonies containing such cloned genes can be identified by replica plating onto a lawn of A. calcoaceticus mutant cells. Transformation of A. calcoaceticus also facilitates gap repair and thus allows recovery of specified chromosomal segments in recombinant plasmids. These procedures were used to demonstrate the clustering of A. calcoaceticus genes required for utilization of p-hydroxybenzoate. Chromosomal linkage of the bacterial genes, contained in different operons separated by about 10 kbp of DNA, may have been selected on the basis of their physiological interdependence.     

微生物转化浮萍资源生产蛋白酶的条件

利用微生物转化富含蛋白质的浮萍生产蛋白酶,为浮萍的高值化利用开辟一条新途径。在摇瓶转化的基础上,探索在5L发酵罐上沙雷氏菌(Serratiasp.)SYBC H转化浮萍生产蛋白酶的最佳转化条件。通过对温度、通风量、搅拌转速、pH等参数的研究,发现在5L发酵罐上,沙雷氏菌SYBC H转化浮萍生产蛋白酶的最佳条件为通风量6vvm,搅拌转速400r/min,发酵温度30℃。转化过程中,采用全自动控制pH值(9.0),蛋白酶的产量比不控制pH的对照组提高了23.3%。进一步研究发现,利用微生物沙雷氏菌SYBC H转化浮萍生产的蛋白酶具有耐有机溶剂的特性,在某些亲水性有机溶剂中保留90%以上的活性,是一种稀少珍贵的极端酶。     

Distance between alleles as a determinant of linkage in natural transformation of Acinetobacter calcoaceticus.

Cotransformation frequencies of 16, 39, 51, and 60% were observed when donor alleles were separated by distances of 9.2, 7.4, 6.3, and 5.1 kb, respectively, in donor Acinetobacter calcoaceticus DNA. A different and unexpected pattern was observed when the distance between recipient alleles was reduced from 9.2 to 5.1 kb. Ligation of unlinked chromosomal DNA fragments allowed them to be linked genetically through natural transformation.     

Physiological Conditions Conducive to High Cyanophycin Content in Biomass of Acinetobacter calcoaceticus Strain ADP1

The effects of the inorganic medium components, the initial pH, the incubation temperature, the oxygen supply, the carbon-to-nitrogen ratio, and chloramphenicol on the synthesis of cyanophycin (CGP) by Acinetobacter calcoaceticus strain ADP1 were studied in a mineral salts medium containing sodium glutamate and ammonium sulfate as carbon and nitrogen sources, respectively. Variation of all these factors resulted in maximum CGP contents of only about 3.5% (wt/wt) of the cell dry matter (CDM), and phosphate depletion triggered CGP accumulation most substantially. However, addition of arginine to the medium as the sole carbon source for growth promoted CGP accumulation most strikingly. This effect was systematically studied, and an optimized phosphate-limited medium containing 75 mM arginine and 10 mM ammonium sulfate yielded a CGP content of 41.4% (wt/wt) of the CDM at 30°C. The CGP content of the cells was further increased to 46.0% (wt/wt) of the CDM by adding 2.5 μg of chloramphenicol per ml of medium in the accumulation phase. These contents are by far the highest CGP contents of bacterial cells ever reported. CGP was easily isolated from the cells by using an acid extraction method, and this CGP contained about equimolar amounts of aspartic acid and arginine and no detectable lysine; the molecular masses ranged from 21 to 29 kDa, and the average molecular mass was about 25 kDa. Transmission electron micrographs of thin sections of cells revealed large CGP granules that frequently had an irregular shape with protuberances at the surface and often severely deformed the cells. A cphI::ΩKm mutant of strain ADP1 with a disrupted putative cyanophycinase gene accumulated significantly less CGP than the wild type accumulated, although the cells expressed cyanophycin synthetase at about the same high level. It is possible that the intact CphI protein is involved in the release of CGP primer molecules from initially synthesized CGP. The resulting lower concentration of primer molecules could explain the observed low rate of accumulation at similar specific activities.     

Influence of environmental parameters on polyphosphate accumulation inAcinetobacter sp.

The regulation of and the optimum conditions for polyphosphate accumulation inAcinetobacter sp. were determined.Acinetobacter strain 210A accumulated polyphosphate in the presence of an intra- or extracellular energy source. The accumulation of polyphosphate during endogenous respiration was stimulated by streptomycin and inhibited by KCN. The highest amount of polyphosphate was found in cells in which energy supply was not limited, namely at low growth rates under sulphur limitation, and in the stationary phase of growth when either the nitrogen or the sulphur source was depleted. The phosphorus accumulation was not affected by the pH between 6.5 and 9. There was a pronounced effect of the temperature on phosphorus accumulation but is varied from strain to strain.Acinetobacter strain 210A accumulated more phosphate at low temperatures, strain B8 showed an optimum accumulation at 27.5° C, while strain P accumulated phosphorus independently of the temperature. The optimum temperature for growth ofAcinetobacter strains tested ranged from 25 to 33° C, and the optimum pH was between 6 and 9.     

Conditions affecting transformation of a group H streptococcus

Schlissel, Harvey J. (The University of Kansas, Lawrence), and C. P. Sword. Conditions affecting transformation of a group H streptococcus. J. Bacteriol. 92:1357-1363. 1966.-A defined transforming medium (DTM) containing buffer and 5 to 10 mug per ml of deoxyribonucleic acid was developed to study the physical and chemical requirements for optimal transformation in streptococcal strain SBE. Optimal transformation in DTM occurred at pH 7.5 and 7.0 in 0.07 m sodium phosphate buffer and 0.05 m tris(hydroxymethyl)aminomethane buffer, respectively. In the presence of either a monovalent or a divalent cation, transformation was stimulated maximally by Mn(+2) (10(-3)m) and K(+) (0.05 m). Other cations tested (Na(+), Mg(+2), Ca(+2)) were less stimulatory. A mixture of K(+) and Mn(+2) stimulated transformation to a level higher than either cation alone. Kinetic studies showed that the stimulating effect of cations was greatest during the early part of the transformation reaction and decreased with time. Transformation was inhibited by Cu(+2) (10(-5)m) and Mn(+2) (10(-2)m). Ethylenediaminetetraacetic acid (EDTA) inhibited transformation at 10(-5)m. The inhibition by EDTA could be overcome by Mn(+2) during the early part of the transformation reaction.     

Mechanisms for biopolymer accumulation in immobilized Acinetobacter calcoaceticus system

The gram-positive bacteria, Acinetobacter calcoaceticus, is capable of accumulating biopolymer in the carrier matrix of an immobilized cell system. Several possible mechanisms for the biopolymer accumulation are evaluated. It appears that direct solid surface polymer adsorption and polymer diffusion limitation within the pore space are minor factors in biopolymer accumulation. Calculations demonstrate that the cell bound polymer to dry cell weight ratio is much higher for immobilized cells than for free cells. The higher cell-bound polymer to dry-cell-weight ratio for immobilized cells as well as the accumulation of the immobilized cells in the Celite matrix are believed to be the main factors for biopolymer accumulation in the Carrier matrix. Further studies reveal that the cell-bound polymer to dry-cell-weight ratio is strongly affected by shear forces. At zero shear stress, such as would be present in the carrier matrix, cell bound polymer to dry cell weight ratio can be as high as 1.6. As the shear stress increases, this ratio decreases. When shear stress increases above 5 dyn/cm(2), a level equivalent to the shear experienced by free cells in a stirred tank fermentation, cell-bound polymer decreases to less than 20% of dry cell weight. A macroscopic model is developed to describe the effect of shear stress on the cell-bound polymer to dry-cell-weight ratio.     

Pyrimidine metabolism in Acinetobacter calcoaceticus

The metabolism of thymine, thymidine, uracil, and uridine has been investigated in five different strains of Acinetobacter calcoaceticus. Attempts to isolate thymine and thymidine auxotrophic mutants were not successful. Consistent with this finding was the observation that uptake of radioactive thymine or thymidine could not be demonstrated. Search for enzymes capable of transforming thymine via thymidine to thymidine-5'-monophosphate in crude extracts was performed, and the following enzymes were absent judging from enzyme assays: thymidine phosphorylase (EC 2.4.2.4), trans-N-deoxyribosylase (EC 2.4.2.6), and thymidine kinase (EC 2.7.1.21). The enzymes responsible for the phosphorylation of thymidine-5'-monophosphate to thymidine-5'-triphosphate were present in crude extracts. Radioactive uracil was readily incorporated into both ribonucleic acid and deoxyribonucleic acid, the ratio being 6:1, and radioactivity was found only in pyrimidine bases. No uptake of uridine could be demonstrated. Uridine-5'-monophosphate pyrophosphorylase (EC 2.4.2.9) activity was detected in crude extracts, suggesting that uracil is converted directly to uridine-5'-monophosphate which is then phosphorylated to uridine-5'-triphosphate or transformed to other ribo- and deoxypyrimidine nucleotides.