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l-Malate was found to protect inducible C4-dicarboxylate transport in Bacillus subtilisagainst the inhibitory effects of the irreversible sulfhydryl reagent N-ethylmaleimide. This effect served as the basis for “specifically labeling” B. subtiliswith N-ethylmaleimide according to a procedure originally employed by Fox and Kennedy (Fox, C.F. andKennedy, E. P. (1965) Proc. Nat. Acad. Sci. U.S.A.54, 891) for N-ethylmaleimide labeling the M-protein component of the lactose transport system of Escherichia coli. By means of this method, three proteins were detected in the membranes of l-malate-induced cells, but not of uninduced cells. These proteins were protected against reaction with N-ethylmaleimide by the presence of saturating amounts of l-malate.

The rates of deoxyribonucleic acid (DNA) synthesis during the division cycles of the Escherichia colistrains B/r, K-12 3000, 15T−, and 15 have been measured in synchronous cultures, under several conditions of slow growth. These synchronous cultures were obtained by sucrose gradient centrifugation of exponentially growing cultures, after which the smallest cells were removed from the gradient and allowed to grow. Sucrose gradient centrifugation did not adversely affect the cell cycle, since an experiment in which an exponentially growing culture was pulsed with [3H]thymidine prior to the periodic separation and assay of the smallest cells resulted in the same conclusions, as given below. In the strains of E. colithat were studied, a decreased rate of [3H]thymidine incorporation was seen late in the cell cycle, prior to cell division. No decrease in the rate of [3H]thymidine incorporation was seen at or near the beginning of the cell cycle. Thus, all these strains appear to regulate DNA synthesis in a similar fashion during slow growth. In addition, a correlation between the appearance of cells with visible cross-walls and the start of a new round of DNA synthesis was seen, indicating that these two events might be related.

A procedure has been described for the centrifugal fractionation of the cellular particulate components of CNS tissue infected with poliomyelitis virus. A study of the distribution of infectivity among these components revealed that approximately four-fifths of the virus is found free in the submicroscopic particle fraction. The validity of the conclusion that the virus is located in this fraction of the intact neuron is discussed.

A heat-labile protein required for division accumulates during the duplication cycle of Escherichia coli. Its formation appears to commence shortly after the cell divides, and it reaches a maximal amount shortly before the next division. A plausible mechanism for timing cell division depends on building up the critical amount of this protein. Completion of deoxyribonucleic acid (DNA) replication is also necessary for division to occur, but it does not uniquely initiate division. The evidence for these conclusions comes from heat-shock experiments; heating to 45 C for 15 min delays division increasingly with the age of a cell. A heat shock given near the end of a cycle delays division for about 30 min, whereas at the beginning of the cycle it hardly affects division. The net result is synchronization of cell division. The effect of heat is increased in bacteria which have incorporated p-fluoro-phenylalanine into their proteins. When the incorporation is early and the heat shock is late in the cycle, division is delayed by about 30 min, indicating that the division protein is synthesized early even though its sensitivity is not observed until later. At any time in the cell cycle, heat shock simply delays total protein and DNA synthesis (3H-thymidine uptake) for approximately 14 min. DNA replication and cell division are thus discoordinated, since DNA replication is not synchronized by the treatment.

Synchronous cell division in an arginine auxotroph and a histidine auxotroph of Escherichia coliwas obtained after starving for the required amino acid for 1 hr. However, cell division was not synchronized after starvation for 1 hr in another arginine auxotroph. This difference is proposed to depend on differences in the concentrations of polyamines in the cells. During amino acid starvation the ratio of putrescine concentration to spermidine concentration decreased in all strains, but it recovered afterward more rapidly in the third strain than in the other two. The cells divided when the ratio returned to normal in the Arg−mutants. Added putrescine permitted some of the cells of the first two mutants to divide sooner after amino acid starvation and thus eliminated synchrony. Spermidine added alone had no effect, but, when it was added together with putrescine, it restored synchronous division. Synchrony was established in the third mutant by adding spermidine after arginine starvation. Thus, both the variations in polyamine content and the effects of added polyamines suggest that the polyamines are essential in permitting cell division. We suggest that the molar ratio of putrescine to spermidine can be a critical factor for cell division. This effect of polyamines seems to be specific for cell division. Amino acid starvation does not induce delays in subsequent mass increase or deoxyribonucleic acid synthesis. Possible mechanisms of polyamine action are discussed.

A method is described for location of proteins in bacteria. It depends upon two techniques. One technique is the inactivation of the protein by a reagent which is incapable of penetrating the bacterial membrane (permeability barrier). Proteins inside this membrane cannot be inactivated unless the cells are disrupted; proteins on or outside the membrane can be inactivated. The second technique depends upon inactivation of the protein by specific antibody. Antibody should not penetrate the external bacterial wall, and therefore should only inactivate proteins that are on the wall surface. Thus, proteins can be localized inside the membrane, in the wall-membrane area, or outside the wall. One reagent developed for use with the first technique is diazo-7-amino-1,3-naphthalene-disulfonate. It inactivated β-galactoside transport, but not β-galactosidase of intact Escherichia coli. Similarly, it inactivated sulfate binding and transport but not uridine phosphorylase activity of Salmonella typhimurium. This indicates that the sulfate-binding protein is on or outside the cell membrane, and that uridine phosphorylase is inside the cell. The organic mercurial compounds used also showed that the sensitive parts of the sulfate and α-methylglucoside transport systems are less reactive than the sensitive part of the β-galactoside system. Antibody to the sulfate-binding protein inactivated the purified protein but did not inactivate this protein when intact bacteria were employed. Thus, it appears that the sulfate-binding protein does not protrude outside the cell wall. The conclusion that the binding protein is located in the wall-membrane region is supported by its release upon spheroplast formation or osmotic shock, and also by its ability to combine with sulfate in bacteria which cannot transport sulfate into the cell.

Sixty-four mutants were isolated from the LT-2 wild-type strain of Salmonella typhimuriumby selecting for chromate resistance. The majority of lesions were shown to lie in the cysAgene. (i) The mutants cannot take up sulfate, a finding which verifies the role of cysAin sulfate transport. In addition, 52 sulfate-transport mutants isolated without chromate selection were defective in the cysAgene. (ii) Most had less than 25% of the binding activity of the wild-type strain. (iii) Most had normal sulfite reductase (H2S-nicotinamide adenine dinucleotide phosphate oxidoreductase, EC 1.8.1.2) activity. (iv) Their sulfate-binding protein (binder) appears electrophoretically and immunologically normal. (v) Amber cysAmutants also make apparently normal binder in small amounts. (vi) All classical cysAmutants tested, including two with long deletions, had normal binding activity. From these observations, it is suggested that the cysAgene does not code for the binder. But many mutations in this gene reduce the binding activity in some unknown way. Other mutants, identified as cysBmutants, had neither binding nor uptake activities and their sulfite reductase activities were similarly reduced, thus confirming the regulatory role of the cysBgene. When binder was detectable, it had wild-type properties. No mutations in the binder gene were found among more than 100 mutants examined. Thus, sulfate binding has not been established as a part of sulfate transport. However, the production of binder is intimately connected with cysA, the established sulfate transport gene, and is regulated by the same mechanism that regulates both transport and the rest of the cysteine biosynthetic pathway.

Inhibition by ultraviolet light of β-galactosidase and alkaline phosphatase synthesis was investigated in both ultraviolet (UV)-sensitive and UV-resistant (wild-type) Escherichia coli, with the objective of determining the sensitivity of various targets. Kinetics of enzyme formation by unmated bacteria and in mating systems, in which the donor provided the specific genetic material and the recipient the cytoplasm, permit the following conclusions regarding the sensitivity of various targets. Catabolite repression resulting from UV damage causes most of the inhibition of β-galactosidase formation. When it is largely eliminated by a step-down in nutrition, the principal target in UV-sensitive bacteria appears to be the structural gene (lacZ+), but damage to the cytoplasm is also important. Transitory inhibition by inactivation of messenger ribonucleic acid is also observed. In wild-type bacteria, repair reduces the importance of lesions in deoxyribonucleic acid sufficiently that cytoplasmic damage appears to be at least as important. Repair occurs within 10 min, as shown by recovery of enzyme-synthesizing ability. Caffeine and proflavine prevent recovery. Newly mated bacteria respond to irradiation very differently than do unmated bacteria. The β-galactosidase or alkaline phosphatase structural gene (lacZ+or phoP+) is much more inhibited after it is transferred than it is in unmated bacteria. This sensitivity seems to depend on a sensitive state of the injected material, rather than on a different physiological condition of the entire zygote. Irradiation of recipient uvr+bacteria much more strongly inhibited expression of injected genes than if the F−was uvrs. Studies on mating systems are not very useful for learning about the function of unmated bacteria.

Riley, Monica(University of California, Berkeley) and Arthur B. Pardee. Nutritional effects on frequencies of bacterial recombination. J. Bacteriol. 83:1332–1335. 1962.—The number of recombinant colonies obtained from mated Escherichia coliis highly dependent on the composition of the medium, both during and after mating, and of the agar medium on which the bacteria are plated. Many bacteria appear to participate in mating, but the efficiency with which they carry out subsequent events leading to recombinant colony formation depends on the physiological state of the zygotes and the challenge to which they are subjected on the selective plates. These findings are reminiscent of the factors which influence the frequency of production of mutants from irradiated bacteria, the survival of bacteria irradiated with ultraviolet light, lysogenization by bacteriophage, or transformation by deoxyribonucleic acid. As a practical measure, care must be taken to hold conditions of plating constant to obtain reproducible numbers of recombinant colonies in mating experiments. For instance, traces of nutrients in agar can greatly affect recombination frequencies.

The uptake of several dicarboxylic acids by Bacillus subtilishas been surveyed. Gratuitous conditions for induction of l-malate uptake were achieved under which the addition of l-malate to the medium resulted in increased uptake of l-malate by the cells without significantly influencing the growth rate.

The active transport process, so important in cell function, has been studied in the past with intact cells. Models which have arisen from this work all depend on: first, a specific protein to recognize the substrate; second, translocation of the substrate across the cell membrane; third, release of substrate within the cell and restoration of the system to its initial state. These steps are adequate for facilitated transport, but in active transport an energy input is required to maintain a concentration gradient. Parts of transport systems have been isolated recently. A protein which specifically recognizes ß-galactosides has been partially purified. In another case, a protein that appears to be the recognition part of the sulfate transport system of Salmonella typhimurium has been crystallized, and many of its properties have been described. The role of this protein in recognition and in translocation is discussed. Also proteins that phosphorylate a variety of sugars as they enter the cell's interior provide a mechanism for concentrating sugars as their phosphates, against a gradient.

Experiments have been performed on the apyrase activity of E. coli, strain B. Although the dependence on pH and substrate is similar to that of rat tissue, the bacterial extracts are inhibited by Ca++ and stimulated by Mg++. In bacterial extracts the rate of phosphate release decreases in the course of the reaction, possibly owing to product inhibition. With multiple bacteriophage infection, the apyrase activity of the intact cells increased several fold, and the activity of extracts increased about 30 per cent. It is suggested that the changes could be attributed to an increase in the amount of enzyme although other alternatives cannot be precluded at present.