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Several species of aerobic sporulating bacilli recently isolated from soil, sewage, manure, and cheese, as well as authentic strains obtained from type culture collections, have been found to exhibit antagonistic activity against unrelated microorganisms. Cultures of these aerobic sporulating bacilli yield an alcohol-soluble, water-insoluble fraction,—tyrothricin,—which is bactericidal for most Gram-positive and Gram-negative microbial species. Two different crystalline products have been separated from tyrothricin. One, which may be called tyrocidine, is bactericidal in vitro for both Gram-positive and Gram-negative species; the other substance, gramicidin, is effective only against Gram-positive microorganisms. In general, tyrocidine behaves like a protoplasmic poison and like other antiseptics, loses much of its activity in the presence of animal tissues. Gramicidin on the contrary exerts a much more subtle physiological effect on the susceptible bacterial cells and, when applied locally at the site of the infection, retains in vivo a striking activity against Gram-positive microorganisms.

Prototrophic Bacillus subtilis cells can be formed in the presence of DNase as a result of cell fusion occurring in mixed populations of protoplasts derived from two parental strains which are both nutritionally-complementing and polyauxotrophic. No prototrophs ever appear from mixed nonprotoplasted bacteria, or from the auxotrophic parental protoplasts plated separately. The frequency of prototroph formation, which is appreciable only when the mixed protoplasts are exposed to polyethylene glycol treatment, may exceed 1 X 10(-4) of the total protoplast population initially present, which is 1 to 4 X 10(-3) of those protoplasts which reverted to the bacillary form. It is strongly dependent on the number and chromosomal location of the markers used in the selection of the prototrophs, and it is unaffected when either one of the parental strains bears the phage phi105 in the inducible prophage state. No auxotrophic bacteria, parental or otherwise, were found as segregants from repeatedly isolated protrotrophic clones growing in a nonselective medium. Unselected markers segregate among the selected recombinants. It is concluded that the observed formation of prototropic bacteria is due to protoplast fusion, a process which does not induce prophage development, and that the only stable products of the resulting diploid state are haploid recombinants.

The primary heterodiploid bacteria regenerated after Bacillus subtilis fusion, although generally noncomplementing diploids, behave in pedigree analysis as multipotential systems. Individual diploid colonies yielding complete reciprocal recombinant (RR) progeny—often accompanied by one or both parents—constitute 10-30% of the total recombinant-forming units. The RR (reciprocal for 8-11 genes) usually occur in equivalent numbers both among and within individual colonies. Novel for bacteria, they demonstrate that entire parental genomes brought together within a diploid protoplast are retained as two independent replicons able to undergo classical recombination characteristic of eukaryotic gametogenesis. Parental or recombinant genomes are also subject to multiple rounds of recombination without obligate segregation and often not reciprocal. Diploid recombinant clones, sharing streptomycin resistance but reciprocal for auxotrophic markers, have displayed a partial ability to make a facultative shift in chromosome expression. They have also produced two types of prototrophs: a stable one (presumably haploid and recombinant) and an unstable one, (diploid and temporarily complementing at low frequency). It follows that chromosome extinction may affect both parental and recombinant chromosomes and does not interfere with recombination. Analysis of the number and chromosomal distribution of crossovers in all recombinants and those from single diploid clones shows increased frequency of exchange in the regions of the replication origin and terminus, possibly a result of the association of these sites with the cell wall or membrane.

A general tendency for additivity prevailed in recombination frequencies for two-point fine-structure mapping of 14 mutants in the C cistron of Rhizobium meliloti phage 16-3, with little evidence of any marker effect. Intracistronic three-point mapping indicated that double crossovers are rare. Deletion mapping indicated that the two- and three-point mapping data gave the correct order of the mutations. A high frequency (5 to 8%) of c/c+ heterozygotic phage progenies was observed in standard crosses. This pattern implies formation of a relatively long region of heterozygosity. Together with the results of the three-point tests, it suggests certain properties of the branch migration and resolution steps envisioned in current mechanisms of recombination.

The DNA of a sulfonamide-resistant Pneumococcal strain (heterozygous for sulr-c) and that of three highly resistant and persistently heterozygous cd transformants, derived by introducing sulr-c marker into a stable sulfonamide resistant strain (sulr-d), were studied to analyze the genetic basis of their merodiploidy. The physical properties of the native and denatured DNA from the heterozygotes and the nonheterozygous strains were not distinguishable. The denaturability and the renaturability of biological activity for the heterozygous markers were essentially identical to those of the normal markers. The heterozygosity extends to the closely linked locus giving rise to four different configurations of cd and cd + transformants, characterized by their frequencies of segregation and donor-marker activities. The marker-activity ratios and the frequency of co-transfer of heterozygous markers were found to remain the same in each when the donor DNA was native, denatured or reannealed without fractionation or reannealed after remixing of resolved strands. Possible models were weighed against these observations and these considerations led to the suggestion that tandem duplication of a gene region may be responsible for the heterozygosity and instability of this region. A more detailed examination of this model will be presented in an accompanying paper.

A Pneumococcal mutant, sulr-c, resistant to sulfonamides, and three transformants bearing associated d or d + resistance markers have earlier been reported to be unstable and show distinct patterns and frequencies of segregating stable progeny lacking the c marker. Each of the four strains showed a characteristic dosage of the genes involved in the merodiploidy. Complementary strands of DNA's from these stable and unstable strains were resolved and homoduplex and heteroduplex hybrids made from the separated DNA strands were used as donors in genetic transformations. Activities of a normal marker (streptomycin resistance) and those involved in the heterozygosity (c, d and d +) were quantitatively measured. From those heterodyplexes made up of opposite strands derived from a heterozygote and a stable strain, the normal marker is transferred efficiently, but the heterozygous markers are not. On the other hand, if both strands of a heteroduplex are derived from different heterozygotic strains, all markers can be transferred with usual efficiency to a stable recipient strain. The lowered efficiency in the former type of heteroduplex is attributed to an inhomology resulting from a tandem duplication in the merodiploid strains, and a postulated DNA repair process stimulated by it while in the form of the donor duplex. The inhomology probably includes (a) a microheterogeneity between the c site and the wild type locus, and (b) a more extensive incompatibility attributable to an extra segment of genome in a tandem duplication covering the c and d sites. The first of these inhomologies produces a lowered efficiency of transfer from all configurations of the particular d allele associated with the mutant c marker, and therefore accounts for the characteristic transfer patterns even from the native merodiploid DNA's.

An enzyme of Pneumococcus responsible for the conversion of maltose to glucose and oligosaccharides was investigated. The genetic analysis of a group of mutants lacking this enzyme was accomplished by studying DNA-mediated transformations of the different strains. Transformation of the different mutants to maltose utilization by wild-type DNA occurred with different frequencies. Recombination of genetic material of the maltose-negative strains also gave rise to positive cells. Analysis of the recombinations indicated that the regions corresponding to the different markers are linked and arranged in a linear fashion. Some larger regions overlapped several smaller ones indicating that single mutations altered regions in the genetic material of different size. Heating the DNA used in transforming the mutant strains destroyed most rapidly the ability to transform the factors corresponding to the larger genetic regions. Factors corresponding to smaller regions were inactivated less rapidly. This is taken to indicate that a DNA molecule partly denatured by heat still retains part of its genetic activity. It is concluded, furthermore, that genetic properties of markers, such as size, linkage, and discreteness, are reflections of the actual distribution of determinants within DNA molecules.

Lactobacillus acidophilus R-26, which requires preformed deoxyriboside for its growth, is able to grow in a basal medium supplemented with nitrophenyl esters of thymidine 3′- and 5′-phosphates. Mutants unable to grow on the thymidine phosphate esters were isolated and classified thus far into two classes. One class of mutants that are unable to grow on the diesters have modified exonucleases, their extracts being unable to hydrolyze the specific nitrophenyl ester on which the strain did not grow. It appears therefore, that the known 3′- and 5′-exonucleases specifically hydrolyzing these esters can be used for the support of growth. A second mutagenization treatment of one of the mutants gave rise to a new strain unable to use or hydrolyze either diester for growth, although it could still grow on several deoxyribodinucleotides. Another class of mutants does not grow on deoxyribooligonucleotides but grows on monodeoxyribo-nucleosides and -nucleotides. These mutants have normal levels of exonuclease activities, so it appears possible that they bear a mutation affecting permeability to diesters or to molecules larger than mononucleotides.

Mutants of Lactobacillus acidophilus R-26, an organism which requires a source of deoxyribonucleoside for its growth, were selected for inability to grow in a basal medium supplemented with nitrophenyl esters of thymidine 3′- or 5′-monophosphates. These esters are specific substrates in vitro for exonucleases of the bacterium. Among the selected strains were found exonuclease-altered mutants, whose extracts failed to hydrolyze normally the 3′- or 5′-nitrophenyl diesters, respectively (Hotchkiss, Nozawa & Gabor, 1971). It is found that 5′-exonuclease mutants are sensitive to u.v.-irradiation, nitrosoguanidine and methyl methanesulfonate treatment. Spontaneous and nitrosoguanidine-induced mutability of these mutants is lower than that of the wild strain, indicating that the 5′-exonuclease function may be involved in the DNA repair system. By contrast, 3′-exonuclease mutants do not exhibit such sensitivity.

1. 1. Pneumococcal DNA having transforming activities for resistance to streptomycin, aminopterin, canavanine, micrococcin and sulfonamides was chromatographed on columns of a polycarboxylate resin (IRC-50) and columns of methylated albumin-kieselguhr. Material determining the last three properties was eluted at constant proportion with DNA, whereas activities for the first two predominated in the earlier fractions. Transforming activity for the genetically linked pair, streptomycin-sulfonamide resistances, was distributed about a position intermediate between the peak positions of the single markers. 2. 2. The pneumococcal DNA is fractionated on IRC-50 according to both base composition and sedimentation coefficient. However, the various single genetic activities did not differ demonstrably in sedimentation coefficients as indicated by their distributions after sedimentation through sucrose gradients. 3. 3. A comparison of chromatographic behavior, temperature of critical heat inactivation and buoyant density in CsCl was made for several genetic markers. The ranking of transforming agents as to inferred guanine-cytosine contents was similar when based upon chromatographic behavior and buoyant density but different from that deduced from heat inactivation. On the basis of the first two properties, those molecules responsible for aminopterin and streptomycin resistance are the richest in guanine and cytosine, those transmitting sulfonamide, canavanine and micrococcin resistances are poorer in this regard. On the basis of heat inactivation, aminopterin resistance activity fulfils this expectation; however, streptomycin sulfonamide and canavanine resistances appear to be intermediate in guanine-cytosine, while micrococcin resistance appears to be lowest of all. 4. 4. Shear degradation of DNA results in a drop of several degrees in the temperature of critical heat inactivation of the agent for aminopterin resistance. Concomitant with this change, this agent drops in buoyant density and becomes the last activity to be eluted from columns. 5. 5. It appears that chromatographic behavior, buoyant density, and temperature of critical heat inactivation respond to different aspects of the same feature of DNA composition, presumably the guanine-cytosine content. It seems possible thate the heat inactivation may be dependent upon the base composition in a specific region of the particular DNA molecules involved, whereas the cromatographic behavior and buoyant density reflect the average base composition of the entire molecule.

Washed staphylococcal cells, while oxidizing glucose, are able to perform certain coupled assimilative reactions which are presumably a part of the complex of reactions involved in bacterial growth. When potassium phosphate is the principal growth requirement provided, other than glucose and oxygen, inorganic phosphate is accumulated by the cells, in proportion to the amount of glucose oxidized. There is a general increase and isotopic exchange in most of the cell phosphate constitutents; the latter can happen, but more slowly, without the glucose at 37 °C. The most unique effect of the presence of glucose is an increase in net amount and possibly also exchange rate of some of the phosphorus associated with the trichloroacetic acid-insoluble nucleoprotein fraction. When various incomplete mixtures of amino acids are provided to the respiring cell suspensions, amino nitrogen is taken up by the cells, in amounts increasing with the complexity of the mixture. The amino acids are found in the trichloroacetic acid-insoluble fraction, from which they are in general recoverable by hydrolysis, so it is concluded that protein synthesis has occurred by combination of the added amino acids and those previously present. This synthesis is also closely coupled to the oxidation of glucose, and, as is the case for phosphate assimilation, s less effective when other substrates are used, deaminative degradation being the other alternative. That synthesis of protein by whole cells is connected with phosphorylative processes seems to be indicated by the interrelation of the phosphate and amino nitrogen assimilation. The net accumulation of phosphate in the cells and particularly in the nucleoprotein fraction is diminished, more or less characteristically in proportion to the success with which amino acids are being incorporated. Both gramicidin and 2,4-dinitrophenol block phosphate assimilation and amino acid oxidation, but only the latter markedly inhibits protein synthesis from amino acids. It is concluded that protein synthesis from amino acids is a preferential alternative to phosphate accumulation brought about by some phosphorylated intermediate common to both processes, and that for each phosphorus atom not incorporated approximately two atoms of amino nitrogen can be utilized for protein formation.

Since transformation of Pneumococcus seems to be effected largely by native double stranded DNA (Marmur and Lane, 1960; Roger and Hotchkiss, 1961) which is relatively stiff and rod-like, but yields within the transforming cells 50% degradation to small fragments plus a single stranded half (Lacks, 1962) of considerable integrity and length (Fox and Allen, 1964), it seems likely that entry is endwise. Degradation is so selective, virtually all-or-none, that presumably it is occasioned by an exonuclease attacking either the 5' or conversely the 3' ends; therefore, only one of the strands will survive to be genetically utilized, the particular one depending upon which end of the double stranded DNA makes contact with the cell. It is generally assumed that bacterial chromosomes are long, double strands of DNA interrupted principally by the progression of one, or very few, replication points. The continuity of the molecule may also be interrupted by single strand gaps, or small amounts of non-nucleotide matter; but there is little evidence for this. It is often further assumed that all segments of the chromosome may be considered randomly subject to shear, hydrolysis, etc., despite manifold evidences that there are structural modulations building up messages and that there are specific points involved with punctuation, functional repression, high rates of mutation, and sites for methylation or rare base incorporation. In recovering DNA from bacteria, it is possible to treat the cells and extracts so gently that crude DNA of high particle weight is obtained (Davison, 1959; Berns and Thomas, 1965; Kelly and Pritchard, 1965). These products may be, biologically speaking, quite pure, being devoid of active viruses, enzymes, or ribosomes; but chemically they are impure DNA preparations. The very easily accomplished shear on manipulation of these crude preparations, bringing them down to the in vitro stable size approximating 107 molecular weight-not nearly so subject to further shear-is also for the present taken to be random, since this makes certain calculations possible. Nevertheless, it is predicted and confirmed that shear tends to give successive breaks near the midpoints of long molecules (Burgi and Hershey, 1961; Thomas, 1966), and this would give random products only if the original chromosome had random ends. Moreover, if the gentle shear occurs at predetermined interruptions or weak spots (physical

Washed staphylococcal cells separated from peptone-broth cultures containing penicillin G did not differ markedly from cells not exposed to penicillin in their rate of oxygen, phosphate, glutamic acid, or amino nitrogen utilization. Washed normal staphyloccal cells, respiring in solutions containing glucose and various mixtures of amino acids, utilized the amino acids with an increase in the cellular protein nitrogen. Similar cells under the same conditions, but exposed to penicillin G, utilized oxygen, phosphate, and amino acids at essentially the same rates, but there was no increase in the protein nitrogen of the cells. Penicillin-treated washed cells, when utilizing amino acids, produced increased amounts of extracellular substances containing non-amino nitrogen in quantities approximately equivalent to the amino acid nitrogen utilized. The non-amino fraction could be tentatively identified as polypeptide, which was produced, instead of cellular protein, when penicillin was present.

4 general topics are presented and discussed informally: 1) control of population (numbers and quality); 2) environmental control and public health; 3) genetic engineering; and 4) family institutions. Participants are Jacques Bergier Johan Bjorksten Robert Cancro Yusuf Ali Eraj Arnold J. Friedhoff Alexander Hollaender Lawrence R. Kegan John Norton Moore N. Henry Moss Stuart Mudd Larry Ng Ruth Sager William Simon Richard Wakefield Walter N. Walmsley and James Wellesley-Wesley. A biological bill of rights for mankind which was later adopted in modified form was read into the working groups report. Among the biological rights are a pure and adequate food supply an upbringing that lays the foundation for healthy psychological development an equitable psychological environment and adequate medical care. Among the other recommendations are that decisionmakers at every level attend to developing and implementing strategies for dealing with expected population problems; that research be done on the genetic and behavioral effects of low levels of environmental agents; that a greater emphasis in medical research and practice be put on prevention of disease and managing the problems of normal life such as maturation and aging; and that research be continued into the range of functions performed by the family with exploration of alternative models of family organization that could fulfill these functions. In view of the urgency and complexity of the problems of environment and health care the Working Group recommends the establishment of an interdisciplinary commission to function on a continuing basis to study both environmental and health care problems and make policy recommendations.

1. Azoprotein antigens containing glucuronic and galacturonic acids give rise in rabbits to specific antibodies. The immune sera show no serological crossing with antigens containing glucose or galactose. 2. The galacturonic acid antigen reacts in antipneumococcus horse serum Type I in high dilutions. 3. Azoprotein antigens containing galacturonic acid, benzene sulfonic and carboxylic acids precipitate in antipneumococcus horse sera of various types but not in normal horse serum. The mechanism underlying these cross reactions is discussed.

Conditions are described in which the pneumococcal mutant strain sulr-c, resistant to the drug sulfanilamide, gives rise to sensitive segregants resistant to nitrobenzoic acid at a frequency constant with time. This segregant frequency is markedly enhanced upon exposure of the cells to doses of ultraviolet light or mitomycin C that permit survival of 50% to 90% of the cells. Treatment with acridine orange diminishes the segregant frequency. From the known influences of these three agents on genetic recombination, we propose that a recombination event is necessary in the generation of segregants.—During a period of incubation following treatment with ultraviolet light or mitomycin C, cell division resumes and the original segregant frequency is restored. Thus potential segregants are either unable to replicate in the absence of selection, or they are under-represented among the cells dividing soon after treatment.—If the sulr-c mutation is introduced into a mutant pneumococcal strain lacking an ATP-dependent exonuclease activity and deficient in recombination with transforming DNA, segregant frequencies are unaffected. This fact may indicate limits upon the type of recombination event responsible for segregation.

The population problem is an imbalance between numbers of people and limited resources. The resources of food, energy, and living space have been well documented as finite in a world with population expanding geometrically. Terrorism, breakdown of social order, and other social symptoms may also stem from this overcrowding. The economically more affluent nations are attempting to deal with unregulated fertility by developing and spreading information on means of conception control and granting equality of status to women to compensate for the reduction in the childbearing role. Social systems are very complex and changing the desire for children is quite difficult. In addition, people have a great capacity for ignoring potential disaster when surrounded by it. Liberty of the individual and control must be kept in balance. It is recommended that more effort be made to understand the motivations behind human reproduction and what social incentives exist to reduce family size. There is also need for contraceptive technology and for more effective dissemination thereof. Values and attitudes need to be changed and the mechanisms of communication, the institutional and organizational structures operating in the field of reproductive behavior, and the role of policy determination by the nation need to be studied. There is need for fertility control in conjunction with mortality control. The committee believes that basic minimum components of the quality of life may be imposed upon a people if necessary in order to preserve the diversity of cultural and individual differences.

The altered growth of a pneumococcal mutant containing marker M, which confers ability to utilize mannitol, and a modifier gene is reported. The modifier gene is closely linked to the M gene and imposes a requirement for growth in 0.4% glucose before growth in mannitol medium. The tentative position of the M gene of pneumococcus is ery-r str-r M sul-rd.

THE enzyme system known as nitrogenase is responsible for conversion of elementary nitrogen in microbial-plant symbiotic nitrogen fixing associations. A method has been developed for the isolation of nitrogenase mutants of Rhizobium meliloti for the study of genes regulating the synthesis of nitrogenase. This type of ineffective mutant cannot be selected directly since nitrogenase is only synthesised in bacteroids within the plant host.

Publisher Summary The chapter discusses the procedure for fusion of bacterial protoplasts. Bacterial protoplasts, being in some respects comparable to animal cells, have been recently investigated in regard to their ability to undergo fusion. This process has in fact been observed in the light microscope, and followed by time-lapse photography by Stahelin, working with protoplasts of Bacillus anthracis. This chapter presents an overview of the searches undertaken with protoplasts from Bacillus species, submitted to a fusogenic treatment. The chapter presents the rationale for a fusion experiment. Successful protoplast fusion with genetically marked bacteria is reported only with polyauxotrophic mutants derived from Bacillus subtilis strain 168, or from an asporogenous subline of Bacillus megaterium strain KM. In the former species, a mutation for rifamycin resistance introduced into both parental strains has made it possible practically to eliminate any risk of contamination. There are presently no contraindications to the use of any other grampositive bacterial species.

Efficiently regenerated single colonies from mixed multiply auxotrophic Bacillus subtilis protoplasts, fused with polyethylene glycol, reveal colonies carrying each of the parent types (biparentals) and recombinant colonies. The latter appear in high yields (up to 1% of certain recombinant classes); the yield of biparentals may be as large as 10%, in the range of the indicated physical fusion events. Many of the biparentals are diploids although, contrary to expectation, they are not complementing prototrophs, but show precisely the phenotype of one (either one) of the parent strains. Extensive pedigree analysis and subcloning of diploid lines show that they can propagate with varying stability on the appropriate parental selective medium to reproduce diploid progeny, parental segregants, and late-appearing recombinants, including some prototrophs. Thus, the principal product of intertype protoplast fusion is a diploid carrying two chromosomes, only one of which is expressed in each particular clone. The extinction of one parental genome is especially well demonstrated when it includes suppression of a normally dominant antibiotic sensitivity marker. In transformation experiments, DNA made from a selected diploid clone was able to transfer several of the unexpressed genes. The structural or topological character of DNA associated with the chromosome extinction remains unexplained.

The search for a general model or description of genetic recombination has stirred many investigators over the years, and has been recently intensified as we seem to be coming within reach of some manipulable components of the system. It seems to me that this search, though conceptual, has had its own evolution like any material system.