Magnesium Starvation of Aerobacter aerogenes IV. Cytochemical Changes

A number of cytochemical changes were revealed by microscopic observations of Aerobacter aerogenes populations starving for Mg(++). During the first few hours, while the synthesis of deoxyribonucleic acid (DNA) was paralleled by an increase in viable bacteria, the cells became progressively smaller. Subsequently, the number of viable cells in the culture remained constant in spite of continuing DNA synthesis, and the cells progressively elongated into filamentous forms. During this time, a second population of very small bacteria could be identified. These cells, whose number increased progressively, were inert with respect to (i) growth or reproduction when returned to a complete medium and (ii) biosynthetic activity as judged by autoradiographic estimation of uracil-H(3) incorporation into nucleic acids. When observed by electron microscopy, many thin sections from bacteria that had been starved of Mg(++) for 20 hr appeared to be almost devoid of ribosomal particles. Thionine staining indicated that the inert cells contain DNA. Furthermore, the rate of DNA synthesis in the culture corresponded to the rate of accumulation of inert cells, suggesting that their presence can account for the difference between total DNA and viable count.     

Magnesium Starvation of Aerobacter aerogenes III. Protein Metabolism

The metabolism of the ribosomal and soluble protein components of Aerobacter aerogenes was examined during its incubation in a Mg(++)-deficient medium. Bacteria were exposed to leucine-H(3) during the exponential growth period preceding Mg(++) starvation, and extracts were prepared after intervals of starvation and were centrifuged through gradients of sucrose to separate ribosomal from soluble proteins. Ribosomal proteins synthesized during the preceding exponential growth were slowly lost from the ribosomes; after 8 hr of starvation, few, if any, sedimented with ribosomes. Losses of total protein, together with the known rate of ribosome decay during Mg(++) starvation, suggested that these ribosomal proteins are ultimately degraded to acid-soluble products and account for all protein lost by the starving cells. These conclusions were supported by studies of Mg(++) starvation in a uracil-requiring strain of A. aerogenes: during uracil starvation a smaller fraction of the proteins synthesized were ribosomal, and the fraction of protein which subsequently decayed during Mg(++) starvation was correspondingly less. During recovery from Mg(++) starvation, proteins, lost from disintegrated ribosomes, were not detectably reutilized into new particles even before their degradation to acid-soluble products was complete. Synthesis of soluble proteins continued for more than 24 hr of starvation at a rate per milliliter close to 45% of the instantaneous rate per milliliter of the exponentially growing bacteria at the time Mg(++) was removed. This value agreed with that found previously for synthetic rates of deoxyribonucleic acid, transfer ribonucleic acid, and ribosomal ribonucleic acid during starvation relative to rates during exponential growth.     

Magnesium starvation of Aerobacter aerogenes. I. Changes in nucleic acid composition

Aerobacter aerogenes incubated in a medium containing all factors necessary for exponential growth except Mg⁺⁺ continued to synthesize nucleic acids and proteins for more than 70 hr, provided the major carbon source was in excess at all times. After 24 hr of Mg⁺⁺ starvation, deoxyribonucleic acid content in the culture had increased 10-fold. In contrast, the viable-cell count increased only about threefold during the first few hours and then remained approximately constant for the subsequent 70 hr. After specified intervals of Mg⁺⁺ starvation, extracts of the bacteria, or ribonucleic acid (RNA) purified from them, was centrifuged through gradients of sucrose to separate transfer RNA from ribosomal components. After correcting for losses, we obtained the following results. (i) There was a progressive rise in the content of transfer RNA competent to accept amino acids and during starvation it remained completely stable. (ii) In contrast, the contents of normally sedimenting ribosomal RNA and ribosomal subunits (30 and 50S) remained approximately constant for more than 24 hr. This did not result from stability of ribosomes made prior to starvation together with an inhibition of synthesis of new particles. Rather, ribosomes were continually breaking down and being replaced by an equivalent number of new ones. (iii) The breakdown of ribosomes appeared to be sequentially ordered; polysomes yielded 70S monomers, which then gave 30 and 50S particles, and these disintegrated to smaller units and finally to acid-soluble products. (iv) Furthermore, the particles derived from breakdown do not appear to exchange with subparticles on the path of assembly. Thus, ribosome decay was age-dependent and ribosomal RNA molecules had a minimal life expectancy of 90 min; however, some survived much longer.     

The Action of 2, 4-dichlorophenoxyacetic Acid on Bacterium lactis aerogenes (Aerobacter aerogenes)

2, 4-Dichlorophenoxyacetic acid (2, 4-D) had little effect onthe growth of Bacterium lactis aerogenes at concentrations lowerthan 1,000 mg per 1. As the concentration was raised the lagincreased and the growth-rate decreased gradually. Serial subcultureat 5,000 mg per 1 caused an unusual pattern of behaviour. Firstthe lag at 5,000 mg per 1 fell gradually to a low level overabout 25 subcultures; then, although the lag did not increase,growth became gradually slower over a further 91 subcultures.During the first subculture in 5,000 mg per 1 of 2, 4-D, RNA/masswas low throughout, but DNA/mass and protein/mass were neverlower than normal; polysaccharide/mass passed through a maximumand cell size was unaffected. Possible mechanisms for the actionof the drug are discussed in view of the findings that (i) after1,000 generations of growth in drug the fermentative propertiesand the utilization of glucose by the cells were unimpairedand (ii) the concentration of magnesium ions had little effecton the action of 2, 4-D (unlike EDTA).     

Aerobacter aerogenes PRL-R3 urease. Purification and properties.

A method for the preparation of a 150-fold purified and homogenous A. aerogenes urease is reported. The enzyme exhibited two pH optima at pH 7.0 and 7.5 in triethanolamine and phosphate buffer, respectively. The affinity of the enzyme toward its substrate increased with the increase of pH. No effect of the pH was observed on the measured temperature coefficient (Q10). There was no discontinuity in the Arrhenius plots at pH 5.4 and 7.5 but an upward discontinuity at pH 6.15 and 8.7 with transition temperature at 30 degrees C. Also, the calculated activation energies are greatly affected by the pH of the enzyme reaction mixture.     

Production of N-Succinyl-l-diaminopimelic Acid by Auxotrophic Mutants of Aerobacter aerogenes and Serratia marcescens

α,ε-Diaminopimelic acid (DAP)-requiring mutants isolated from Aerobacter aerogenes ATCC 8308 and Serratia marcescens ATCC 19180 were found to accumulate N-succinyl-l-diaminopimelic acid (SDAP) which was an intermediate in the biosynthesis of lysine in Escherichia coli. SDAP was isolated from the culture broth and identified by the behavior in paper chromatography, melting point, elementary analysis, infrared spectrum, and optical rotation.

The culture conditions for SDAP production by A. aerogenes KY 7049 (DAP^?) and S. marcescens KY 8921 (DAP^?/Lys^?) were investigated. A. aerogenes KY 7049 has an absolute requirement for DAP together with a relative requirement for l-lysine. High levels of DAP (2000~4000 μg/ml) were proved to be favorable for SDAP accumulation, while if lysine along with DAP was added to the fermentation medium, optimal level of DAP for SDAP production was relatively low (about 200 μg/ml at 200 μg/ml of lysine). A variety of compounds which may conceivably affect the course of a fermentation process, i.e., carbon source, inorganic nitrogen source, amino acids, vitamines, precursors, were screened at optimal levels of lysine and DAP. Thus, the amount of SDAP accumulation reached a level of 19.9 mg/ml with the medium containing 10% glucose and 2000 μg/ml of DAP. S. marcescens KY 8921 requires either DAP or lysine for growth. Optimal level of DAP and lysine for SDAP accumulation was 50~100μg/ml.