Hiromi Maruyama

The participation of ribonuclease in the degradation of Escherichia coli ribosomal ribonucleic acid as revealed by oligonucleotides accumulation in the phosphorus-deficient stage

Abstract 1. 1. Escherichia coli ribosomal RNA degradation during the phosphorus-deficient stage 1,2 was investigated by analysis of its degradation products. 2. 2. After exhaustion of the phosphorus in the medium, 32 P in the 50 % ethanolsoluble fraction of the bacterial cell increased greatly in parallel with the degradation of ribosomal RNA, while trichloroacetic acid-soluble 32 P did not. 3. 3. Soon after phosphorus exhaustion, most of the ethanol-soluble 32 P consisted of characteristic oligonucleotides. Later, inorganic phosphate increased gradually parallel with a decrease in oligonucleotides in ethanol extracts. 4. 4. The differences in the base ratios, the terminal structures and other properties of these oligonucleotides suggest that the breakdown of ribosomal RNA was caused mainly by ribosomal latent ribonuclease (EC 2.7.7.17) 3 . These oligonucleotides, in their turn, were found to be finally converted to inorganic phosphate and nucleosides by the cooperation of alkaline phosphomonoesterase (EC 3.1.3.1) 2,4 and cyclic phosphodiesterase 5,6 .

Ribosome degradation and the degradation products in starved Escherichia coli. I. Comparison of the degradation rate and of the nucleotide pool between Escherichia coli B and Q-13 strains in phosphate deficiency.

Abstract The differences in ribosomal degradation and the fate of degradation products between Escherichia coli wild-type (B) and a ribonuclease less mutant (Q-13) during phosphate starvation were compared. The degradation rate of 32P-labeled ribosomes was found to be of the same order at the early stage but to differ at the later stage of deficiency between Escherichia coli B strain and Q-13 strain. The degradation in Q-13 nearly stopped after 6-h starvation, while it continued for more than 10 h in B strain, resulting in more than 80% degradation of 32P-prelabeled ribosomes. In the acid-soluble 32P pool of Q-13 strain, there was an increase in the nucleoside monophosphates but no increase and a continuous decrease in nucleoside di-and triphosphates, respectively. Q-13 contained no nucleoside 2′,3′-cyclic phosphate or nucleoside 3′-phosphate and little oligonucleotide, all of which had been detected in B strain during this stage. From these facts it was suggested that ribosomal RNA degradation in Escherichia coli Q-13 is mainly caused by ribonuclease II in vivo.

Reutilization of degradation products of ribosomal ribonucleic acid in Escherichia coli strain B during the phosphorus-deficient stage.

Abstract A closer investigation was carried out into the acid-soluble nucleotides of Escherichia coli B during the phosphorus-deficient stage in order to clarify the mechanism of reutilization of the degradation products of ribosomal RNA by the ribonuclease system. Inorganic phosphate and nucleosides were found to be final products arising from the hydrolysis of Nuc-2′,3′-P to Nuc-3′-P, and the conversion of these to precursors of DNA or mRNA via Nuc-5′-P is described. 1. 1. After exhaustion of the phosphorus in the medium, the amounts of polyphosphorylated nucleotides, P1 and NAD were first shown to decrease greatly and then all, except NAD, increased gradually in proportion to the degradation of rRNA. 2. 2. In the early stages of phosphorus deficiency, the amount of monophosphorylated nucleotides was very high but this decreased with time. The peaks corresponding to the four authentic Nuc-5′-P compounds on Dowex-1 chromatograms at this stage were found to represent mainly Nuc-5′-P and Nuc-3′-P which seemed to be intermediates in the degradation of rRNA. The amounts of Nuc-5′-P were initially low but increased gradually with time. 3. 3. On the whole, the ratio of deoxyribonucleotide to ribonucleotide in acidsoluble extracts of phosphorus-deficient cells is higher than that of normal cells, especially in the di- and triphosphorylated nucleotide regions. However, the absolute amount of deoxyribonucleotides present was not significantly different from normal, indicating that there was only a relative decrease in these ribonucleotides. 4. 4. Nucleosides in trichloroacetic acid extracts from phosphorus-deficient cells increased and these were composed mainly of uridine and inosine.

Ribosome degradation and the degradation products in starved Escherichia coli: II. Changes in base sequence of ribosomal RNA during degradation induced by phosphate and magnesium starvation

Abstract Changes in the base sequence of ribosomal RNA isolated from ribosomes in phosphate- or Mg 2+ -deficient Escherichia coli were investigated. The frequency of purine tracts differed to various extents from that of rRNA obtained from normal cells. The purine tract distribution was determined by ribonuclease digestion followed by separation of the products by DEAE-cellulose chromatography. This difference from normal rRNA sequence was greater in the phosphate-deficient ribosomes from the wild strain than it was in those of Mg 2+ -deficient wild stain or the phosphate deficient ribonuclease I-less mutant strain, Q-13. The size of the RNAs in intact ribosomes in these deficient states was normal as judged by sedimentation analysis and the total base composition of rRNA was slightly different from that in normal cells. The implication of the results is discussed proposing two possible hypothesis.

Ribosome degradation and the degradation products in starved Escherichia coli: III. Ribosomal RNA degradation during the complete deprivation of nutrients

Abstract Escherichia coli B, which had been precultured in a normal medium containing 32P, harvested during the exponential growth phase, washed, resuspended in a buffer solution without any nutrient sources and then incubated at 37°, was studied with special regard to the degradation of 32P-labeled ribosomes. 1. 1. During the incubation, after complete deprivation of nutrient, the cell suspensions showed a remarkable decrease in turbidity, viable cell count and ribosome content. These decreases were more marked at alkaline pH and greater with Tris than with saline. 2. 2. The rate of degradation of 32P-labeled ribosomes did not correspond to the rate of decrease in the viable cell count. 3. 3. Ribosomal degradation was accompanied by a conformational change as revealed by the alteration of the s-value of the two major ribosomal particles in sedimentation analysis at 0.1 mM MgCl2. 4. 4. The degradation products were excreted into the surrounding media almost quantitatively. The main products were found to be bases, nucleosides and P1.

Unusual Mode of Inhibition of Citrobacter freundii β-Lactamases by Ceftriaxone

Several Citrobacter freundii β-lactamases, discernible by their substrate profiles and sensitivities to inhibition by newer cephalosporins, were inhibited by ceftriaxone in an unusual manner. Inhibition was noncompetitive at low concentrations of cephaloridine but became competitive at concentrations above 600 μM, which is close to the Km (570 μM) for cephaloridine.

In vitro potentiation of cephalosporins by alafosfalin against urinary tract bacteria.

Potentiating activity of alafosfalin was examined in detail with 8 cephalosporins and mecillinam against 164 urinary bacteria representing 8 genera. Alafosfalin was generally comparable in activity to cefamandole and mecillinam but superior to other cephalosporins tested. When the minimal fractional inhibitory concentration indices were compared, synergism was observed with all of the beta-lactams tested for all species except Streptococcus faecalis. Marked synergism was observed with Escherichia coli, Citrobacter, and enterobacter and, among the drugs tested, with cefamandole and mecillinam. Although alafosfalin is inactive against Pseudomonas aeruginosa, potentiation was seen with cefsulodin. The most favorable combination ratio of alafosfalin/cephalosporin to elicit potentiation was determined for each species and occurred in a rather narrow range of combinations (1:4 to 4:1 in general).