Death of Bacillus subtilis Auxotrophs Due to Deprivation of Thymine, Tryptophan, or Uracil

Pritikin, William B. (University of California, Los Angeles), and W. R. Romig. Death of Bacillus subtilis auxotrophs due to deprivation of thymine, tryptophan, or uracil, J. Bacteriol. 92:291-296. 1966.-Auxotrophic mutants of Bacillus subtilis 168 that require either tryptophan, uracil, or thymine died rapidly when deprived of any of these compounds. Phage PBS1 was produced by infected B. subtilis 168 (thy try-2) deprived of thymine. Phage PBS1 was not produced by infected B. subtilis 168 (try-2) deprived of tryptophan or infected B. subtilis 168-15 (try-2 ura) deprived of uracil. B. subtilis 168 thy try-2 and 168-15 could be transduced by phage PBS1 after prolonged deprivation of tryptophan or uracil, respectively. When B. subtilis 168-15 was transduced to uracil independence by phage PBS1, the uracil-independent transductants became immune to uracil-less death within 10 min of exposure to phage, and began to multiply within 2 hr after exposure to phage at an incubation temperature of 46 C.     

Contrasting Effects of Polycations on Plaquing Efficiency of Encephalomyocarditis Virus Variants

Polycation treatment of L cell monolayers affected plaquing efficiency of both the r(+) and r variants of the encephalomyocarditis virus. Plaque formation by r(+) variant was decreased markedly by three structurally different types of synthetic basic polymers, diethylaminoethyl dextran, hexadimethrene (polybrene), and basic polyamino acids. In contrast, these same substances increased substantially the number of plaques formed by the r variant. The effect on the two variants was observed when polycations were applied to the cells before or simultaneously with the introduction of virus. The molar concentration and size of the polymer proved important. Thus, basic polyamino acids of low molecular weight were significantly more inhibitory for the r(+) variant than were those of high molecular weight. On the other hand, plaquing efficiency of the r variant was increased by relatively large polyamino acids, but not by polymers of small size. Basic polyamino acids inhibited r(+) plaque formation to a greater degree at low than at high pH values. However, plaquing efficiency of the r variant in polycation-treated cultures was not affected by changes in pH. Basic polymers appear to bind to cell membranes and affect either attachment or uptake of the viruses. The evidence suggests that the substances influence by different mechanisms the interaction of the r(+) and r variants with cells.     

Improved translational efficiency of subtilisin YaB gene with different initiation codons in Bacillus subtilis and alkalophilic Bacillus YaB

The ale gene specifying the subtilisin YaB produced by alkalophilic Bacillus YaB, has an unusual start codon UUG. Changing this codon to AUG and GUG increasedexpression of the ale gene in B. subtilis DB104 and in an ale deficient mutant strain YaB-DEC4. The relative translational efficiency order of the threeinitiation codons is AU G > GU G > UUG in B. subtilis DB104 and in YaB-DEC4. These data suggest that the preferred initiation codon is AUG for ale gene expression in Bacillus .     

Engineering subtilisin E for enhanced stability and activity in polar organic solvents

We examined the effect of a novel disulfide bond engineered in subtilisin E from Bacillus subtilis based on the structure of a thermophilic subtilisin-type serine protease aqualysin I. Four sites (Ser163/Ser194, Lys170/Ser194, Lys170/Glu195, and Pro172/Glu195) in subtilisin E were chosen as candidates for Cys substitutions by site-directed mutagenesis. The Cys170/Cys195 mutant subtilisin formed a disulfide bond in B. subtilis, and showed a 5-10-fold increase in specific activity for an authentic peptide substrate for subtilisin, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, compared with the single-Cys mutants. However, the disulfide mutant had a 50% decrease in catalytic efficiency due to a smaller k(cat) and was thermolabile relative to the wild-type enzyme, whereas it was greatly stabilized relative to its reduced form. These results suggest that an electrostatic interaction between Lys170 and Glu195 is important for catalysis and stability in subtilisin E. Interestingly, the disulfide mutant was found to be more stable in polar organic solvents, such as dimethylformamide and ethanol, than the wild-type enzyme, even under reducing conditions; this is probably due to the substitution of uncharged Cys by charged surface residues (Lys170 and Glu195). Further, the amino-terminal engineered disulfide bond (Gly61Cys/Ser98Cys) and the mutation Ile31Leu were introduced to enhance the stability and catalytic activity. A prominent 3-4-fold increase in the catalytic efficiency occurred in the quintet mutant enzyme over the range of dimethylformamide concentration (up to 40%).     

Cloning and expression of subtilisin amylosacchariticus gene

The gene encoding subtilisin Amylosacchariticus from Bacillus subtilis var. amylosacchariticus was isolated and the entire nucleotide sequence of the coding sequence was determined. The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprising 275 residues. There were discrepancies in 10 amino acids between the sequence elucidated from the nucleotide sequence and the published protein sequence (Kurihara et al. (1972) J. Biol. Chem. 247, 5619-5631). The nucleotide sequence was highly homologous to that of subtilisin E gene from B. subtilis 168, with discrepancies at 12 nucleotides out of 1,426 nucleotides we sequenced. Ten of them were found in mature subtilisin coding sequence, which resulted in two amino acid changes and another one was in the putative promoter region between two genes. The productivity of subtilisin in culture broth of B. subtilis var. amylosacchariticus was much higher than that of B. subtilis 168. The enzyme gene was inserted in a shuttle vector pHY300PLK, with which B. subtilis ISW1214 was transformed. The proteolytic activity found in the culture broth of the transformed bacterium was 20- and 4-fold higher than those of the host strain and B. subtilis var. amylosacchariticus, respectively. Subtilisin Amylosacchariticus was easily purified to a crystalline form from culture filtrate of cloned B. subtilis, after a single step of chromatography on CM-cellulose.     

Kinetic and molecular properties of Bacillus subtilis IBTC-3 subtilisin

Bacillus subtilis IBTC-3 subtilisin was purified by gel filtration on Sephadex G 75 and affinity chromatography on bacitracin-CNBr-Sepharose 4B and characterized. Its molecular mass of 27 kDa was determined by SDS-PAGE, and isoelectric pH of 8.4 by chromatofocusing. FT-Raman and FT-IR spectroscopy studies revealed fragments with alpha-helix and irregular secondary structures within the polypeptide chain. The beta-sheet conformation was observed only in second-derivatives of FT-RS and FT-IR spectra, in the range of the amide II, III, and I bands. Tyr residues were shown to be hydrogen bonded and CSCH(3) groups adopted two conformations (P(H)-T and P(C)-G conformers). Kinetic properties of B. subtilis IBTC-3 subtilisin in hydrolysis of ethyl esters of amino acid derivatives were compared with that of alkaline peptidase from Bacillus alcalophilus PB92. The first enzyme displayed the highest affinity for NAc-Phe-OEt, both in hydrolysis (K(m) of 0.22 mM) and in synthesis (K(m) of 0.85 mM), whereas PB92 peptidase preferred Tyr derivatives (NAc-Tyr-OEt, K(m) of 0.043 and 0.75 mM, respectively). In contrast to the latter enzyme, B. subtilis IBTC-3 subtilisin catalyzed hydrolysis and synthesis of Bz-Arg-OEt.     

Isolation of Bacillus subtilis genes from a charon 4A library.

A library of Bacillus subtilis chromosomal deoxyribonucleic acid (DNA) was constructed, using lambda charon 4A as a cloning vector. Partially cleaved Bacillus subtilis DNA was prepared by partial methylation with EcoRI methylase, followed by complete EcoRI endonuclease digestion. More than 95% of the phage particles carried B. subtilis DNA inserts. When this library was screened for transforming activity, using competent cells, 70% of the genetic markers tested were found in a sample of 1,710 plaques. Cloned genetic loci were found to be about 100-fold more efficient in transforming activity than chromosomal DNA. Intact phage particles containing the pheA locus were found to be able to transform competent recipients with approximately the same efficiency as phage DNA. Transformation by intact particles was insensitive to deoxyribonuclease.     

Behavior of a temperate bacteriophage in differentiating cells of Bacillus subtilis.

During the first 6 hr of sporulation, infection of Bacillus subtilis by by phi105 wild type or the clear-plaque mutant phi105 c30 was nonproductive, but phage DNA was trapped inside developing spores. After infection with either wild-type or mutant phage at early times of sporulation (T1-T3), phage DNA entered the developing spores in a heat-stable form, which may represent integration of the phage DNA into the host chromosome. Phage DNA in carrier spores produced by infection at later times (T4-T6) was much more heat sensitive. Spore preparations containing either phi105 wild type or phi105 c30 carrier spores gave rise to a spontaneous burst of phage during outgrowth, although the fraction of carried wild-type phage that chose lysis over lysogeny at germination has not been determined. Heat induction of the thermoinducible lysogen 3610 (phi105 cts23) was also abortive during sporulation. Furthermore, induction neither prevented eventual spore formation nor resulted in the conversion of prophage DNA to the carrier state; during outgrowth, the previously induced lysogenic spores remained stable lysogens. However, if the sporulating lysogenic cells were plated immediately after induction, they did not form colonies at high efficiency, as though transfer to fresh medium allowed sufficient phage expression to kill the host.     

Highly efficient gene expression of a fibrinolytic enzyme (subtilisin DFE) in Bacillus subtilis mediated by the promoter of α-amylase gene from Bacillus amyloliquefaciens

Subtilisin DFE is a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4. The promoter and signal peptide-coding sequence of alpha-amylase gene from B. amyloliquefaciens was cloned and fused to the sequence coding for pro-peptide and mature peptide of subtilisin DFE. This hybrid gene was inserted into the Escherichia coli/Bacillus subtilis shuttle plasmid vector, pSUGV4. Recombinant subtilisin DFE gene was successfully expressed in B. subtilis WB600 with a fibrinolytic activity of 200 urokinase units ml(-1).     

Complementation by Restricted Phage T1

The ability of restricted phage T1hr to complement stocks of T1am mutants carrying the P1 modification has been tested in mixed infection of Escherichia coli B(P1). Of the 18 genes tested, 15 could be complemented to give successful infection of approximately 10 to 25% of the cells, and 2 other genes consistently gave at least 5% complementation. The progeny phage produced in these infections was predominantly of the hr(+)am genotype. A mutant in gene 12 could be complemented very poorly, if at all. With one double mutant stock, the complementation obtained was nearly as good as the more poorly complemented of the two corresponding single mutants.     

Transforming activity of phage T4r+ DNA, treated with ultraviolet light, nitrous acid, hydroxylamine and visible light in the presence of methylene blue]

The efficiency of phages T4rIIB-638v+ and T4rIIB-638v- transformation by native and denatured DNA treated with UV, nitrous acid, hydroxylamine and visible light in the presence of methylene blue is studied. A greater transformation efficiency of UV-irradiated T4r+ phage native and denatured DNA was observed in the v+ recipient as compared with v- recipients. Denatured donor DNA treated with nitrous acid has higher transformation activity in spheroplasts infected with T4v+ phage than in those infected with T4v- phage. Native donor DNA, treated with methylene blue and visible light-irradiated, developed a decrease of the transformation activity in T4v- phage-infected spheroplasts as compared with T4v+ phage-infected spheroplasts. Hydroxylamine treatment of native and denatured donor DNA did not reveal any differences in the transforming activity for v+ and v- recipients. Denatured donor DNA was more resistant to the effect of hydroxylamine than native DNA.     

Influence of growth condition on the concentration of potassium in Bacillus subtilis var. niger and its possible relationship to cellular ribonucleic acid, teichoic acid and teichuronic acid

1. Mg(2+)-limited Bacillus subtilis var. niger, growing in a chemostat in a simple salts medium, contained considerably more potassium and phosphorus than Mg(2+)-limited Aerobacter aerogenes growing in a similar medium at corresponding dilution rates. 2. Growth of the bacillus in a K(+)-limited environment did not lower the cellular potassium and phosphorus contents, the molar proportions of cell-bound magnesium, potassium, RNA (as nucleotide) and phosphorus being approximately constant at 1:13:5:13 (compared with 1:4:5:8 in Mg(2+)-limited or K(+)-limited A. aerogenes). 3. Growth of B. subtilis in a phosphate-limited environment caused the cellular phosphorus content to be lowered to a value similar to that of Mg(2+)-limited A. aerogenes, but the potassium content was not correspondingly lowered; the molar potassium:magnesium ratio varied from 14 to 17 with changes in dilution rate from 0.4 to 0.1hr.(-1). 4. Whereas over 70% of the cell-bound phosphorus of Mg(2+)-limited or K(+)-limited A. aerogenes was contained in the nucleic acids, these polymers accounted for less than 50% of the phosphorus present in similarly limited B. subtilis; much phosphorus was present in the walls of the bacilli, bound in a teichoic acid-type compound composed of glycerol phosphate and glucose (but no alanine). 5. Phosphate-limited B. subtilis cell walls (from organisms grown at a dilution rate of 0.2hr.(-1)) contained little phosphorus and no detectable amounts of teichoic acid, but 40% of the cell-wall dry weight could be accounted for by a teichuronic acid-type compound; this contained a glucuronic acid and galactosamine, neither of which could be detected in the walls of Mg(2+)-limited B. subtilis grown at a corresponding rate. 6. It is suggested that the high concentration of potassium in growing B. subtilis (compared with A. aerogenes) results from the presence of large amounts of anionic polymer (teichoic acid or teichuronic acid) in the bacillus cell walls.     

Toxic effects of zinc and iron on the routine oxygen consumption of Tilapia sparrmanii (Cichlidae)

1. The routine oxygen consumption of Tilapia sparrmanii without the addition of any toxicants over a 72 hr period showed a decrease for the first 48 hr, but stabilised thereafter.2. Addition of zinc (98 mg l⁻¹) resulted in a drastic decrease of oxygen consumption for 3 hr. The routine oxygen consumption showed a significant decrease for the first 24 hr, while the second and third 24 hr revealed significant differences with great individual variance.3. The decrease in oxygen consumption observed after exposure to zinc, could be caused by gill damage as well as the internal action of zinc.4. An increase in oxygen consumption was noted for almost 3 hr after addition of iron (88mg l⁻¹). During the first-, second- and third 24 hr the oxygen consumption increased significantly, compared to the control values.5. The increase in routine oxygen consumption of T. sparrmanii when compared to control values after exposure to iron, could be attributed to stress and possible gill changes.6. The study revealed that after acute (72 hr) exposure to sublethal concentrations of zinc and iron, the routine oxygen consumption of T. sparrmanii was altered.     

Inactivation of Escherichia coli by Near-Ultraviolet Light and 8-Methoxypsoralen: Different Responses of Strains B/r and K-12

A series of Escherichia coli K-12 AB1157 strains with normal and defective deoxyribonucleic acid repair capacity were more resistant to treatment with 8-methoxypsoralen (8-MOP) and near-ultraviolet light (NUV) than a comparable series of strains from the B/r WP2 family although sensitivities to 254-nm ultraviolet light were closely similar. The difference was most marked with strains deficient in both excision and postreplication repair (uvrA recA). The hypothesis that the internal level of 8-MOP was lower in K-12 than B/r uvrA recA derivatives was ruled out on the basis of fluorometric determinations of 8-MOP content and the similar inactivation curves for phage T3 treated intracellularly within the two strains. The demonstration of liquid holding recovery with AB2480 but not WP100 (both recA uvrA strains) and the somewhat greater resistance of the former strain to inactivation by captan revealed the presence in the K-12 strain of a deoxyribonucleic acid repair system independent of the recA(+) and uvrA(+) genes. The presence of this repair system did not, however, affect the survival of T3 phage treated with 8-MOP plus NUV and probably has a relatively small effect on survival of AB2480 under normal conditions. Experiments in which 8-MOP monoadducts were converted to cross-links by a second NUV exposure in the absence of 8-MOP indicated that the level of potentially cross-linkable monoadducts immediately after 8-MOP + NUV is about eightfold lower in K-12-than in B/r-derived strains. It is therefore suggested that the photoproduct yield in the former is well below that in the latter. In agreement with this is the observation that, during the first 10 min after treatment, deoxyribonucleic acid synthesis was just over five times more sensitive to inhibition by 8-MOP plus NUV in WP100 than in AB2480. We assume that 8-MOP in K-12 bacteria is hindered in some way from adsorbing to cellular (though not to phage T3) deoxyribonucleic acid. Consistent with this, 8-MOP has been shown to act as an inhibitor of a component of repair of 254-nm ultraviolet light damage in WP2 but not in AB1157.     

HIV-infected Langerhans cells preferentially transmit virus to proliferating autologous CD4+ memory T cells located within Langerhans cell-T cell clusters

Langerhans cells (LC) are likely initial targets for HIV following sexual exposure to virus and provide an efficient means for HIV to gain access to lymph node T cells. The purpose of this study was to examine the nature of the CD4(+) T cell that becomes infected by HIV-infected LC. We infected human LC within tissue explants ex vivo and then, 3 days later, cocultured HIV-infected LC with different subsets of autologous CD4(+) T cells. Using multicolor flow cytometric analyses of LC-CD4(+) T cell cocultures, we documented that HIV-infected LC preferentially infected memory (as compared with naive) CD4(+) T cells. Proliferating and HIV-infected CD4(+) memory T cells were more frequently detected in conjugates of LC and autologous CD4(+) T cells, suggesting that T cells become activated and preferentially get infected through cluster formation with infected LC, rather than getting infected with free virus produced by single HIV-infected LC or T cells. p24(+) Memory CD4(+) T cells proliferated well in the absence of superantigen; by contrast, p24(+) T cells did not divide or divided only once in the presence of staphylococcal enterotoxin B, suggesting that virus production was rapid and induced apoptosis in these cells before significant proliferation could occur. These results highlight that close interactions between dendritic cells, in this case epidermal LC, and T cells are important for optimal HIV replication within specific subsets of CD4(+) T cells. Disrupting cluster formation between LC and memory CD4(+) T cells may be a novel strategy to interfere with sexual transmission of HIV.     

Concurrent changes in transducing efficiency and content of transforming deoxyribonucleic acid in Bacillus subtilis bacteriophage SP-10

Taylor, Martha J. (Fort Detrick, Frederick, Md.), and Curtis B. Thorne. Concurrent changes in transducing efficiency and content of transforming deoxyribonucleic acid in Bacillus subtilis bacteriophage SP-10. J. Bacteriol. 91:81-88. 1966.-Spores of Bacillus subtilis W-23-S(r) infected with transducing phage SP-10 served as convenient inocula for broth cultures from which transducing phage was harvested. Methods are described for producing highly infected spores. The inoculum level of infected spores in nutrient broth-yeast extract-glucose medium affected the transducing efficiency of SP-10 in lysates of these cultures. Phage in lysates of cultures inoculated with about 10(5) or fewer spores per milliliter transduced 20- to 350-fold more efficiently than did phage in lysates from cultures inoculated with 10(6) to 10(7) spores per milliliter. Transduction frequencies in the order of 10(-5) per plaque-forming unit were obtained routinely, and some infected-spore preparations yielded phage that gave frequencies as high as 10(-4). The combination of inoculum level and incubation time required to produce the best transducing phage had to be determined empirically for each batch of infected spores. Several possible explanations for the difference between lysates having high (HTE) and those having low (LTE) transducing efficiency were ruled out by special experiments. The hypothesis is presented that some cultural condition resulting from a relatively low inoculum of phage-infected spores favors the incorporation by phage particles of bacterial deoxyribonucleic acid (DNA) in the manner required for the production of transducing phage. Support for this hypothesis is a demonstration, through transformation experiments with DNA extracted from HTE and LTE phage particles, that populations of HTE phage particles yielded significantly more (7 to 27 times) transforming activity per microgram of DNA than did populations of LTE phage.     

GENETIC TRANSFER OF THE ABILITY TO GROW AT 55 C IN BACILLUS SUBTILIS

McDonald, William C. (U.S. Army Chemical Corps, Fort Detrick, Frederick, Md.) and Thomas S. Matney. Genetic transfer of the ability to grow at 55 C in Bacillus subtilis. J. Bacteriol. 85:218-220. 1962.-Two strains of Bacillus subtilis, 168 and P1, were found to grow at 55 C (55(+)) on complete media; strain 168S(r) failed to grow at temperatures above 50 C (55(-)). When 168S(r) bacteria were grown in the presence of deoxyribonucleic acid extracted from 168, the ability to grow at 55 C was transformed at a frequency of 10(-4). An incubation period of 3 to 4 hr at 37 C was necessary for phenotypic expression of the 55(+) character. Only 10 to 20% of the 55(+) transformants retained the high-level streptomycin resistance (S(r)) of the recipient, indicating close linkage between the S(s) and 55(+) loci.     

Genetics of Sensitivity of Salmonella Species to Colicin M and Bacteriophages T5, T1, and ES18

Nearly all of 62 strains of Salmonella paratyphi B were sensitive to colicin M and phage T5 but resistant to phages T1 and ES18 and to colicin B. All tested S. typhimurium strains were resistant to colicin M and phage T5, and many were sensitive to phage ES18. A rough S. typhimurium LT2 strain given the tonA region of Escherichia coli or S. paratyphi B became sensitive to colicin M and phage T5. We infer that the tonA allele of S. paratyphi B, like that of E. coli, determines an outer membrane protein that adsorbs T5 and colicin M but not phage ES18, whereas the S. typhimurium allele determines a protein able to adsorb only ES18. The partial T1 sensitivity of a rough LT2 strain with a tonA allele from E. coli or S. paratyphi B and also the tonB(+) phentotype of an E. coli B trp-tonB Delta mutant carrying an F' trp of LT2 origin showed that S. typhimurium LT2 has a tonB allele like that of E. coli with respect to determination of sensitivity to colicins and phage T1. Rough S. paratyphi B, although T5 sensitive, remained resistant to T1 even when given F' tonB(+) of E. coli origin. Classes of Salmonella mutants selected as resistant to colicin M included: T5-resistant mutants, probably tonA(-); mutants unchanged except for M resistance, perhaps tolerant; and Exb(+) mutants, producing a colicin inhibitor (presumably enterochelin). Some Exb(+) mutants were resistant to a bacteriocin inactive on E. coli but active on all tested S. paratyphi B and S. typhimurium strains (and on nearly all other tested Salmonella). A survey showed sensitivity to colicin M in several other species of Salmonella.