Development of Competence in Thymine-starved Bacillus subtilis with Chromosomes Arrested at the Terminus

Tryptophan- and thymine-requiring cells of Bacillus subtilis, emerging from an amino acid starvation treatment which causes arrest of the chromosomes at the terminus, were not transformable. During subsequent incubation in a thymineless medium supplemented with amino acids, the cultures developed competence while retaining chromosome arrest. The competent subpopulation apparently shares the synchronous chromosome arrest of the bulk population. This was shown by different methods. The principal method was marker frequency analysis of the deoxyribonucleic acid extracted from a population enriched for competent cells by a column-chromatographic method. It is concluded that development of the competent state can occur in nondividing cells, and that the presence of a replication fork actively engaged in synthesis of deoxyribonucleic acid is not required for the development of this state.     

Congression of Unlinked Markers and Genetic Mapping in the Transformation of BACILLUS SUBTILIS 168

A thorough examination of cotransformation of two unlinked genetic markers in Bacillus subtilis 168 shows that the two recombinational events do not occur randomly. The cotransformation frequency is dependent on the distance between the two markers as well as on the order in which they replicate in the competent cell. These results indicate that uptake and/or integration of DNA molecules bearing these genetic markers is enhanced at the time these markers replicate in the competent cell.     

The influence of glutamic acid and arginine on the competence of Bacillus subtilis growing in a chemostat

Using a new method to get stable steady state levels of competence in B. subtilis, we have obtained in our experimental conditions 0.1% competence as determined by the frequency of transfection with phage SPP1 DNA. The effect of growth rate (DT) and certain amino acids on the competence levels was also determined.     

Transport of Donor Deoxyribonucleic Acid into the Cell Interior of Thymine-starved Bacillus subtilis with Chromosomes Arrested at the Terminus

The chromosomes of a tryptophan(-), thymine(-) double auxotroph of Bacillus subtilis were uniformly aligned at the chromosome terminus by an amino acid starvation treatment. By subsequent incubations, the starved culture was rendered competent, while its state of synchronous chromosome arrest was maintained by thymine starvation. The competent, chromosome-arrested cells were transformed for three unlinked markers, located in two different chromosome regions. Shortly after addition of deoxyribonucleic acid, the cell walls were removed with lysozyme in a medium containing deoxyribonuclease and no thymine, and the protoplasted culture was assayed for single and double transformants. It was found that markers both near and distant from the terminus entered freely into the cell interior. There was no important difference in the relative frequency of entry of different markers between synchronously arrested cells and nonsynchronized control cultures. It is concluded that entry of a given marker into the cell interior can occur even if the replication site of the chromosome is stationary at a location distant from the locus of the resident homolog of the entering marker. A mechanism of donor deoxyribonucleic acid entry involving homology at the replication fork is excluded.     

Relationship Between Competence for Transfection and for Transformation

Deoxyribonucleic acid (DNA) extracted from phage SPP1 is highly infectious on Bacillus subtilis competent cells; the efficiency of infection is 5 x 10(3) to 6 x 10(3) phage equivalents per plaque-forming unit. This DNA was used to study the relationship between competence for transfection and for transformation. The experiments were concerned with the frequency of infection and transformation in mutants exhibiting different levels of competence, the effect of periodate on competence for infection and for transformation, the competition between phage and bacterial DNA, the transformation of cells preinfected with phage DNA, and the infection of cells pretreated with bacterial DNA. The data show that B. subtilis cells competent for transformation are also competent for transfection and vice versa; transfection with phage DNA represents, therefore, a simple way to measure the total number of competent cells in a culture. The fraction of competent cells, determined by SPP1 DNA infection, varied from 10(-2) to 7 x 10(-2).     

Intercellular Effects on Development of Competence in Bacillus subtilis

The intercellular transfer of competence during growth under the conditions specified by the transformation procedure of Spizizen was investigated with Bacillus subtilis 168. The rate of competence development as assayed uniformly in medium B was not affected by variations in the cell concentration, although the first appearance of transformants occurred earlier with high cell densities in medium A, approximately in proportion to the onset of the stationary phase in the culture. Growth in the presence of Pronase enhanced the frequency of transformation, but did not detectably alter the kinetics of competence development. The rate of competence increase in physiologically noncompetent cultures was not changed by mixing with competent cultures either in medium A or in medium B; however, an early appearance of transformants was noted in mixed cultures in which the proportion of competent to noncompetent cells prevented exponential growth of the noncompetent strain. These experiments indicate that the normal development of competence in B. subtilis is not mediated by a soluble or loosely bound protein factor capable of transmitting competence directly via cell contact. The onset of competence is thus a function of internal physiological changes which are induced by the overall metabolic state of the culture.     

Dynamics of molecular markers linked to the resistance loci in a mosquito-Plasmodium system

Models on the evolution of resistance to parasitism generally assume fitness tradeoffs between the costs of being parasitized and the costs associated with resistance. This study tested this assumption using the yellow fever mosquito Aedes aegypti and malaria parasite Plasmodium gallinaceum system. Experimental mosquito populations were created by mixing susceptible and resistant strains in equal proportions, and then the dynamics of markers linked to loci for Plasmodium resistance and other unlinked neutral markers were determined over 12 generations. We found that when the mixed population was maintained under parasite-free conditions, the frequencies of alleles specific to the susceptible strain at markers closely linked to the loci for resistance (QTL markers) as well as other unlinked markers increased significantly in the first generation and then fluctuated around equilibrium frequencies for all six markers. However, when the mixed population was exposed to an infected blood meal every generation, allele frequencies at the QTL markers for resistance were not significantly changed. Small population size caused significant random fluctuations of allele frequencies at all marker loci. Consistent allele frequency changes in the QTL markers and other unlinked markers suggest that the reduced fitness in the resistant population has a genome-wide effect on the genetic makeup of the mixed population. Continuous exposure to parasites promoted the maintenance of alleles from the resistant Moyo-R strain in the mixed population. The results are discussed in relation to the proposed malaria control strategy through genetic disruption of vector competence.     

Mechanism of Transfection with Deoxyribonucleic Acid from the Temperate Bacillus Bacteriophage φ105

Bacteriophage phi105 is a temperate phage for the transformable Bacillus subtilis 168. The infectivity of deoxyribonucleic acid (DNA) extracted from mature phi105 phage particles, from bacteria lysogenic for phi105 (prophage DNA), and from induced lysogenic bacteria (vegetative DNA) was examined in the B. subtilis transformation system. About one infectious center was formed per 10(8) mature DNA molecules added to competent cells, but single markers could be rescued from mature DNA by a superinfecting phage at a 10(3)- to 10(4)-fold higher frequency. Single markers in mature DNA were inactivated at an exponential rate after uptake by a competent cell. Prophage and vegetative DNA gave about one infectious center per 10(3) molecules added to competent cells. Infectious prophage DNA entered competent cells as a single molecule; it gave a majority of lytic responses. Single markers in sheared prophage DNA were inactivated at the same rate as markers in mature DNA. Prophage DNA was dependent on the bacterial rec-1 function for its infectivity, whereas vegetative DNA was not. The mechanism of transfection of B. subtilis with viral DNA is discussed, and a model for transfection with phi105 DNA is proposed.     

Quantitive Autoradiographic Study of Competence and Deoxyribonucleic Acid Incorporation in Bacillus subtilis

The relationships among deoxyribonucleic acid (DNA) uptake, transformation, and autoradiographic labeling were investigated. It is shown that: (i) autoradiography is a good method for measuring the total fraction of competent cells able to incorporate transforming DNA; in our best experiment that fraction was 11.5%. (ii) Computation of the fraction of competent cells in a culture of Bacillus subtilis, by comparing the frequencies of single and double transformants for two unlinked markers, gives values which are somewhat different from those obtained by auto-radiography. (iii) On the average, a competent cell of B. subtilis irreversibly takes up from 0.9 x 10(-10) to 1.4 x 10(-10) mug of DNA; this amount represents about 1/55 to 1/35 of the bacterial chromosome weighing 5 x 10(-9) mug, and corresponds to three to six DNA molecules having a molecular weight of 1.6 x 10(7).     

Development of competence of Haemophilus influenzae

Spencer, Hugh T. (The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Md.), and Roger M. Herriott. Development of competence of Haemophilus influenzae. J. Bacteriol. 90:911-920. 1965.-A chemically defined nongrowth medium was developed for the induction of competence of Haemophilus influenzae by a stepdown procedure. Cells grown logarithmically in Heart Infusion Broth became competent after being transferred to a medium which consisted of amino acids, sodium fumarate, and inorganic salts. Chloramphenicol (2 mug/ml) or l-valine (1 mug/ml) in the nongrowth medium inhibited development of competence. The inhibitory action of l-valine was reversed by comparable concentrations of l-isoleucine. Kinetic studies of the development of competence showed a variable capacity of competent cells to take up deoxyribonucleic acid and reaffirmed earlier findings that competence was not transmissible in H. influenzae. Addition of nicotinamide adenine dinucleotide, thiamine, calcium pantothenate, uracil, and hypoxanthine to the medium for competence resulted in a minimal growth medium in which reduced levels of competence were developed.     

Effect of lysogeny on transfection and transfection enhancement in Bacillus subtilis.

Strains of Bacillus subtilis 168 lysogenic for bacteriophage phi105 transfer with deoxyribonucleic acid (DNA) isolated from bacteriophage SPO2 at a higher efficiency than non-lysogenic strains. This enhancement of transfection was not the result of recombination between bacteriophages SPO2 and phi105. Superinfection marker rescue increased transfection with DNA from bacteriophage phi105 occurred simultaneously with the addition of the transfecting DNA. Again, this enhancement of transfection was not the result of recombination but rather a protection of the transfecting DNA by the superinfecting bacteriophage. The ability of the superinfecting bacteriophage to protect the transfecting DNA from inactivation was maximal when the bacteria were just becoming competent. Bacteriophage phi1 cannot replicate after the transfection of competent bacteria lacking a functional DNA replication system, whereas bacteriophage phi1 was able to replicate after infection of competent bacteria grown under comparable conditions. These observations support the hypothesis that GAPase and an inducible repair system play an important role in the development of competence.     

Transformation and Transfection in Lysogenic Strains of Bacillus subtilis 168

Strains of Bacillus subtilis lysogenic for either temperate bacteriophage phi105 or SPO2 were reduced to less than 1.0% of the level of transformation of the nonlysogenic strains. Strains lysogenic for both phi105 and SPO2 are virtually nontransformable, indicating that the effect of lysogeny is additive. Lysogenic cultures transfected at essentially wild-type levels with deoxyribonucleic acid (DNA) isolated from bacteriophages phi29 and SPO1. The residual transformation and transfection achieved by the lysogenic cultures changed dramatically during growth in SPII medium, whereas nonlysogenic strains remained competent for 5 hr in SPII medium. Despite a marked reduction in transformation, lysogenic cultures initially irreversibly bound as much DNA as nonlysogenic cultures. After 60 min in SPII medium, there was a rapid decrease in the capacity of lysogenic cells to bind DNA irreversibly. These results, as discussed, indicate that the inhibition of transformation is probably due to an alteration of the cell surface or a differential inactivation of bacterial genes after lysogenic conversion.     

A membrane protein with similarity to N-methylphenylalanine pilins is essential for DNA binding by competent Bacillus subtilis.

In a cloned copy of comG open reading frame 3 (ORF3), an in-frame deletion was generated by site-directed in vitro mutagenesis, removing the coding sequence for 15 amino acids from the central portion of this pilin-related protein. The mutagenized ORF3 was incorporated into the Bacillus subtilis chromosome, replacing the wild-type ORF3. The presence of the deleted ORF3 in the chromosome, as confirmed by Southern analysis, was associated with the complete loss of competence by the mutant strain. The ability of the mutant cells to bind exogenous radiolabeled DNA was reduced to the level of nonspecific binding of DNA by noncompetent cells. The chromosomal ORF3 mutation was partially complemented in trans by a plasmid-encoded wild-type ORF3 copy under PSPAC control upon induction of the PSPAC promoter. Using antiserum raised against a synthetic 14-mer oligopeptide deduced from the ORF3 sequence, an immunoreactive band of approximately the expected molecular size was obtained in Western blot (immunoblot) experiments with extracts of cells containing the plasmid-encoded inducible gene. A signal was also detected when cells harboring the chromosomal wild-type or mutant ORF3 in single copy were grown in competence medium. This signal was detected only in the light-buoyant-density (competent) cell fraction and only after the transition from the exponential to the stationary growth phase. In cell fractionation experiments with competent cell extracts, the immunoreactive protein was found in both the NaOH-insoluble and -soluble membrane fractions and was sensitive to proteinase K treatment of either protoplasts or whole cells.     

Transfection of Bacillus subtilis protoplasts by bacteriophage phi do7 DNA.

DNA from the Bacillus subtilis temperate bacteriophage phi do7 was found to efficiently transfect B. subtilis protoplasts; protoplast transfection was more efficient than competent cell transfection by a magnitude of 10(3). Unlike competent cell transfection, protoplast transfection did not require primary recombination, suggesting that phi do7 DNA enters the protoplast as double-stranded molecules.     

Identification of a putative Bacillus subtilis rho gene.

Transposon Tn917 mutagenesis of Bacillus subtilis BD99 followed by selection for protonophore resistance led to the isolation of strain MS119, which contained a single Tn917 insertion in an open reading frame whose deduced amino acid sequence was 56.6% identical to that of the Escherichia coli rho gene product. The insertional site was near the beginning of the open reading frame, which was located in a region of the B. subtilis chromosome near the spoOF gene; new sequence data for several open reading frames surrounding the putative rho gene are presented. The predicted B. subtilis Rho protein would have 427 amino acids and a molecular weight of 48,628. The growth of the mutant strain was less than that of the wild type on defined medium at 30 degrees C. On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type on defined medium at 30 degrees C. On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type at 30 degrees C but was much slower at lower temperatures; sporulation occurred and competence was developed in cells of the mutant grown at 30 degrees C. To determine whether the protonophore resistance and sensitivity to low growth temperature resulted from the insertion, a chloramphenicol resistance cassette was inserted into the wild-type B. subtilis rho gene of strain BD170; the resulting derivative displayed the same phenotype as MS119.     

Isolation and characterization of mutants of the Bacillus subtilis oligopeptide permease with altered specificity of oligopeptide transport

Bacterial oligopeptide permeases are members of the large family of ATP binding cassette transporters and typically import peptides of 3 to 5 amino acids, apparently independently of sequence. Oligopeptide permeases are needed for bacteria to utilize peptides as nutrient sources and are sometimes involved in signal transduction pathways. The Bacillus subtilis oligopeptide permease stimulates competence development and the initiation of sporulation, at least in part, by importing specific signaling peptides. We isolated rare, partly functional mutations in B. subtilis opp. The mutants were resistant to a toxic tripeptide but still retained the ability to sporulate and/or become competent. The mutations, mostly in the oligopeptide binding protein located on the cell surface, affected residues whose alteration appears to change the specificity of oligopeptide transport.     

The Entire Population of Thermus thermophilus Cells Is Always Competent at Any Growth Phase

Thermus thermophilus mutants carrying unlinked double auxotrophic markers were transformed with wild type chromosomal DNA at various growth phases. The percentages of competent cells in the total population were calculated based on the results of transformation efficiencies for single or double markers. It was concluded that all the T. thermophilus cells at any growth phase were competent.     

Identification of Bacillus subtilis NRRL B-3275 as a Strain of Bacillus pumilus

The physiological and biochemical properties of a species of Bacillus previously identified as B. subtilis NRRL B-3275 (B-3275) were compared with those of seven strains of B. pumilus and five strains of B. subtilis. The biotin requirement of B-3275, its inability to hydrolyze starch, and its failure to reduce nitrate indicate that the organism is more closely related to the B. pumilus strains than to those of B. subtilis. Hybridization of deoxyribonucleic acid (DNA) from B-3275 with that of the strains of B. pumilus showed a binding efficiency (compared with the homologous reaction) of 58 to 99%, depending on the strain. Hybridization with the DNA from any of the strains of B. subtilis did not exceed 24%. DNA from B-3275 was unable to transform two amino acid auxotrophic markers to prototrophy in a highly competent strain of B. subtilis 168. We conclude that B-3275 is a strain of B. pumilus which we designate as B. pumilus NRRL B-3275.     

Modulation of competence for genetic transformation in Streptococcus pneumoniae

The spontaneous development of competence by cultures of Streptococcus pneumoniae in casein hydrolysate medium was strongly dependent on the initial pH of the culture medium. Cells growing in cultures beginning with a wide range of initial pH values (6.8 to 8.0) all developed competence, as measured by [3H]DNA uptake, [3H]DNA degradation and genetic transformation; but the initial pH of the medium affected both the timing of the occurrence of competence and the number of times the culture became competent. In cultures grown in media of lower initial pH, competence occurred only once, at high population densities, while in more alkaline media a succession of competence cycles occurred, beginning at lower cell densities. The critical population density required for the initiation of competence varied tenfold over the pH range studied. Successive competence cycles in an alkaline medium were not equivalent: while the percentage of competent cells in the first competence cycle was high (approximately 80%), that in the second competence cycle was lower (approximately 12%). Correspondingly, competence-specific proteins were less prominent in the labelled-protein pattern of the second competence cycle than in that of the first. These features of the physiology of competence control make it possible to adjust the expression of competence to suit various experimental requirements.