Pleiotropic nature of bacteriophage tolerant mutants obtained in early-blocked asporogenous mutants of Bacillus subtilis 168

Summary Bacterial mutants tolerant to bacteriophages 15 and 29 were isolated from early-blocked asporogenous mutants ofBacillus subtilis 168. These mutants are able to adsorb phages but are not killed by them.Two classes of tolerant mutants were recognized:tolA mutants which are tolerant to 15 but not 29, andtolB mutants which are tolerant to both phages. Although the parental strain (spoA12) used for the isolation of thetol mutants is a pleiotropic negative mutant, alltol mutants which have been examined have regained some wild type traits. Genetic studies have shown that thesetol mutants are neither revertants nor suppressor mutants of thespoA gene. Thesetol mutants affect bothspoA andspoB mutations. These mutants do not sporulate and they do not lyse as rapidly as the parental strain. All these results are consistent with the hypothesis that they are all cell envelope mutants.     

Conditional lethal mutants of Bacillus subtilis dependent on kasugamycin for growth

Summary Mutants of Bacillus subtilis dependent on the antibiotic kasugamycin have been isolated and characterised. The mutant phenotype was the result of a kasugamycin resistance mutation mapping near leu, together with a mutation conferring dependence which mapped elsewhere on the chromosome. In some cases, the latter mutation caused spectinomycin dependence in a spectinomycin resistant strain. Four mutants had detectable alterations in ribosomal proteins, which were not, however, responsible for the phenotype. These alterations were in proteins BS3, BS7, BS9, and BL15. Some mutants had defects in ribosomal subunit assembly, or altered cell morphology associated with the mutant phenotype.     

Genetic properties of arsenate sensitive mutants of Bacillus subtilis 168

Summary Arsenate sensitive mutants were isolated from Bacillus subtilis strain 168 after treatment with N-methyl-N-nitro-N-nitrosoguanidine or ethyl methane sulfonate. Though all mutants are phenotypically identical, a high proportion (40%) of the induced mutations are of a multisite nature as they do not revert spontaneously and are poorly transformable to arsenate resistance with wild type DNA. On the basis of transformation efficiency, UV inactivation kinetics and cotransduction frequency of outside markers, four independently isolated multisite arsenate sensitive mutations are characterized as resulting from large deletions of homogenous size (24000±6000 base pairs). The arsenate resistance locus was mapped between phe and aroD on the B. subtilis chromosome by PBS1 mediated transduction. Mechanisms for the formation of such chromosomal deletions are discussed.Part of the dissertation of Alice Adams Lindahl, presented to New York University in partial fulfillment of requirements for the Ph. D. degree. A preliminary report of this work was presented at the NATO International Symposium, Mol, Belgium, August 1970.     

Isolation of acetyl esterase mutants of Bacillus subtilis 168.

Five mutants of Bacillus subtilis 168 defective in an intracellular esterase activity were identified. By polyacrylamide gel electrophoresis, four of the mutants were shown to lack esterase B activity, and the fifth lacked esterase A activity. All of the back-crossed esterase mutants were able to sporulate at wild-type frequency and produce exoprotease(s) and antibiotic(s). No difference in motility could be attributed to the esterase mutation. PBS1 transduction analysis showed all the esterase B mutations to be linked to the hisA marker.     

Isolation and Characterization of a Temperature-sensitive Sporulation Mutant of Bacillus subtilis

Temperature-sensitive sporulation mutants were isolated spontaneously from Bacillus subtilis 168 TT by a sequential transfer method. A representative mutant strain, ts32, was characterized in detail. The mutant grew normally at 30°C and 42°C, but did not sporulate at 42°C. Electron microscopic observation and physiological analysis showed that the mutant was blocked at stage 0-1 of sporulation. Genetic analysis suggested that the mutation was located at the spo0B locus on the B. subtilis chromosome. Temperature-shift experiments clearly showed that the spo0B gene product functions only at the beginning of sporulation.     

A new chemically-defined medium for RAC-certified and other strains of Bacillus subtilis

Summary Conventional chemically-defined media were found to be inadequate for extensive batch culture growth of an asporogenous mutant of Bacillus subtilis certified as an HV-1 host for recombinant DNA. We developed a chemically-defined medium for rapid and extensive growth of one such strain, BGSC-1S53. The medium contains no components requiring separate sterilization. It is also useful for the prototrophic 168 strain (from which 1S53 was derived), a triple auxotroph of 168, and a highly debilitated (HV-2) 168 strain (BGSC 1S76) upon addition of the specific growth factors required by these strains. Excellent growth was also obtained with W-23, a Bacillus subtilis strain unrelated to the 168 series.     

DNA Supercoiling and Osmoresistance in Bacillus subtilis 168

The importance of the DNA structure for the expression of the osmotic response (osmotolerance) was investigated in Bacillus subtilis 168. Plasmid pUB110 DNA was used as a reporter of the chromosomal DNA topology, and analyses were performed in chloroquine agarose gels. Plasmidic DNA obtained from cultures in Schaeffer medium (D) taken in those periods in which B. subtilis is able to express osmotolerance (early stationary phase or from germinating spores) or from adapted cultures to hyperosmotic medium (DN) presented a higher level of negative supercoiling than DNA samples from vegetative cultures, normally refractory to induction of osmotolerance. The involvement of the DNA gyrase was investigated through the sensitivity to novobiocin, an antibiotic inhibitor of its activity and the behavior of a gyrB1 mutant strain (RG1). In the wild-type strain, the addition of a sublethal concentration of novobiocin (0.5 μg/ml) to the hyperosmotic medium relaxed DNA and inhibited growth. Moreover, already growing cultures in DN medium and later submitted to the same antibiotic presented a relaxed DNA and stopped growing. The RG1 mutant strain submitted to similar novobiocin treatments displayed normal growth in DN novobiocin medium. These results pointed to the requirement of a highly negative supercoiled DNA structure involving the gyrase activity in osmotic response. Received: 9 May 1997 / Accepted: 18 June 1997     

Host-controlled modification and restriction in Bacillus subtilis: Bsu 168-system and BsuR-system in B. subtilis 168.

Summary A Bsu168-specific restriction deficient (r ₁₆₈ ^- ) mutant of Bacillus subtilis Marburg 168 was transformed to be BsuR-specific restriction proficient (r _R ⁺ ) with B. subtilis R DNA as efficiently as the Bsu168-specific restriction proficient (r ₁₆₈ ⁺ ) parental strain (hsrM ⁺, hsdR ^-).We constructed r _R ⁺ m _R ⁺ r ₁₆₈ ⁺ m ₁₆₈ ⁺ strain (ISMR 4), r _R ⁺ m _R ⁺ r ₁₆₈ ^- m ₁₆₈ ⁺ strain (ISR 11) and r _R ⁺ m _R ⁺ r ₁₆₈ ^- m ₁₆₈ ^- strain (ISR 6) from strain 101 (r ₁₆₈ ⁺ m ₁₆₈ ⁺ ), strain 1012 (r ₁₆₈ ^- m ₁₆₈ ⁺ ) and strain RM125 (r ₁₆₈ ^- m ₁₆₈ ^- ), respectively by transformation with B. subtilis R DNA, and tested their restriction and modification activities on phage 105C. The results show that the sites recognized by Bsu168-specific restriction and modification enzymes and the sites recognized by BsuR-specific ones are not overlapping.We conclude that the Bsu168-modification and restriction system and the BsuR-modification and restriction system are controlled independently by two distinct sets of genes in the r _R ⁺ m _R ⁺ transformant of r ₁₆₈ ⁺ m ₁₆₈ ⁺ strain B. subtilis 168.     

Spectinomycin-resistant mutants of Bacillus subtilis with altered sporulation properties.

Summary Specitinomycin-resistant mutants of Bacillus subtilis show three different types of alterations in sporulation ability. Class 1 mutants can both grow and sporulate in the presence of spectinomycin. Class 2 mutants can grow in the presence of spectinomycin, but are unable to sporulate in either the presence or absence of spectinomycin. Class 3 mutants have a conditional phenotype, and are able to sporulate in the absence of spectinomycin, but not in its presence. The ability of these strains to produce alkaline phosphatase, a biochemical marker for early sporulation events, is correlated with the ability to sporulate in the presence or absence of antibiotic. All of the spectinomycin-resistance mutations could be genetically linked to the cysA marker, and a mutational alteration of a protein of the 30S ribosomal subunit has been identified in one of the Class 3 strains (Spc1–11). Fine-structure mapping of the spectinomycin resistance mutation of strain Spc 1–11 confirmed its location in the cluster of genes for ribosomal components on the B. subtilis genetic map. Genetic analysis indicated that the properties of the Class 1 and Class 2 mutants result from more than one mutation. The spectinomycin-resistance and altered sporulation properties of the two Class 3 mutants probably result from a single genetic lesion.     

Restriction of plasmid-mediated transformation in Bacillus subtilis 168.

Summary When plasmids carrying leucine genes of Bacillus subtilis 168 were isolated from a restriction and modification deficient (r^-m^-) strain and used for transformation of a restricting strain B. subtilis 168 leu recE4, the number of transformants was greatly reduced. Transformation of a rec ⁺ strain (transformation by integration of the donor DNA into the chromosome) with the plasmids was not affected irrespective of whether the recipient carried the r⁺ or r^- phenotype. These results show that the plasmid-mediated transformation is subject to the host controlled restriction and suggest that r^-m^- strains should be used for construction of recombinant DNA molecules in B. subtilis 168.     

Molecular basis for the differential anti-metabolite action of d-tyrosine in strains 23 and 168 of Bacillus subtilis

Summary The basis for the difference between strains 168 (d-tyrosine-sensitive) and 23 (d-tyrosine-resistant) of Bacillus subtilis at the molecular level is that of transport of d-tyrosine into the cell. Strain 23 does not incorporate significant amounts of d-tyrosine into whole cells. A mutant derivative was isolated from strain 23 which had an altered transport system permitting d-tyrosine uptake, a change which also led to inhibition of growth by d-tyrosine. Strain 168 is extremely sensitive to growth inhibition caused by low concentrations of the d-isomer of tyrosine. A mutant derivative of strain 168 selected for its d-tyrosine resistant phenotype had an altered transport system which no longer recognized the d-isomer of tyrosine. These mutants define at least one element of the tyrosine transport system in B. subtilis and provide a convenient phenotype for the eventual location of the chromosal map position.     

Physiological characterization of antibiotic resistant mutants of Bacillus subtilis

Summary Ten antibiotic resistance mutants inBacillus subtilis have been tested forin vitro resistance in cell-free amino acid incorporating systems. Seven of these mutants,str-1, ole-2, spc-2, mic-1, bry-2, nea-1, andlin-2 are shown to have alterations in ribosomal function.neo-2, kan-2 andery-1 did not showin vitro alterations. Extensive cross-resistance and some cross-sensitivity to ribosomal antibiotics are determined by these mutations.     

Effect of pps disruption and constitutive expression of srfA on surfactin productivity,spreading and antagonistic properties of Bacillus subtilis 168 derivatives

Aims: To analyse the effects of plipastatin operon disruption and constitutive expression of surfactin operon in Bacillus subtilis 168 on surfactin productivity, in vitro invasive growth and antagonism against fungi. Methods and Results: The srfA native promoter was replaced by the constitutive promoter P_repU in B. subtilis 168 after integration of a functional sfp gene. Moreover, the plipastatin synthesis was further disrupted in the B. subtilis 168 derivatives. In liquid media, an earlier and higher expression of P_repU, than that found with P_srfA, led to a specific surfactin production fivefold higher after 6 h of culture. On solid media, not only the invasive growth and the haemolytic activity but also the antifungal activity of the constitutive strains were improved when compared to the parental strain BBG111. As expected, the disruption of the plipastatin operon strongly reduced in vitro antifungal properties but, interestingly, enhanced specific surfactin production (1·47 g g^?1 of biomass), spreading behaviour and haemolytic activity of the strains. Conclusions: This work demonstrates for the first time the interdependency of surfactin and plipastatin regarding their biosynthesis as well as their influence on the biological activities of the producing strain. Significance and Impact of the Study: The constitutive overproduction of surfactin enhances the invasive growth and the in vitro antagonistic activity of the mutant strain. Both properties are known to play an important role in the biocontrol of plant diseases. Plipastatin operon disruption increases the surfactin productivity of mutant strains. These mutants are interesting for use in continuous bioprocesses for surfactin production or in bioremediation.     

Autolytic enzyme-deficient mutants of Bacillus subtilis 168.

Mutants of Bacillus subtilis strain 168 have been isolated that are at least 90 to 95% deficient in the autolytic enzymes N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase. These mutants grow at normal rates as very long chains of unseparated cells. The length of the chains is directly related to the growth rates. They are nonmotile and have no flagella, but otherwise appear to have normal cell morphology. Their walls are fully sysceptible to enzymes formed by the wild type and have the same chemical composition as the latter. Cell wall preparations from the mutants lyse at about 10% of the rate of those from the isogenic wild type, with the correspondingly small liberation of both the amino groups of alanine at pH 8.0 and of reducing groups at pH 5.6. Likewise, Microcococcus luteus walls at pH 5.6 and B. subtilis walls at pH 8 are lysed only very slowly by LiCl extracts made from the mutants as compared with rates obtained with wild-type extracts. Thus, the activity of both autolytic enzymes in the mutants is depressed. The frequencies of transformation, the isolation of revertants, and observations with a temperature-sensitive mutant all point to the likelihood that the pleiotropic, phenotypic properties of the strains are due to a single mutation. The mutants did not produce more protease or amylase than did the wild type. They sporulate and the spores germinate normally. The addition of antibiotics to exponentially growing cultures prevents wall synthesis but leads to less lysis than is obtained with the wild type. The bacteriophage PBSX can be induced in the mutants by treatment with mitomycin C.     

Identification and characterization of the zosA gene involved in copper uptake in Bacillus subtilis 168

dl-Penicillamine, a copper-specific metal chelator, remarkably suppressed the growth of Bacillus subtilis 168 when added to a synthetic medium under Cu²⁺ limitation. DNA microarray and screening of 2,602 knockout mutants showed that the zosA gene was de-repressed in the presence of 0.1% dl-penicillamine, and that the zosA mutant was sensitive to dl-penicillamine medium. The zosA mutant delayed the growth under Cu-limitation even without the chelator, and the sensitivity to dl-penicillamine was reversed by induction using 0.3 mM IPTG and the Pspac promoter inserted directly upstream of the zosA gene. Furthermore, the zosA mutant showed elevated tolerance of excessive Cu²⁺ but not of excessive Zn²⁺ added to LB and synthetic media. Homology modeling of the ZosA protein suggested that the protein can fold itself into essential domains for constituting a metal transporting ATPase. Our study suggests that zosA is a candidate gene involved in copper uptake.     

The first high frequency of recombination-like conjugal transfer from an integrated origin of transfer sequence in Bacillus subtilis 168

Bacillus subtilis 168 was developed as a genome vector to manipulate large DNA fragments. The system is based on the inherent natural transformation (TF) activity. However, DNA size transferred by TF is limited up to approximately 100 kb. A conjugal transfer system capable of transferring DNA fragments considerably larger than those transferred by TF was developed. A well-defined oriT¹¹⁰ sequence and a cognate relaxase gene from the pUB110 plasmid were inserted into the xkdE gene of the B. subtilis genome. Transfer of antibiotic resistance markers distant from the oriT¹¹⁰ locus to the recipient B. subtilis occurred only in the presence of pLS20, a helper plasmid that provides a type IV secretion system. Marker transmission was consistent with the orientation of oriT¹¹⁰ and required a recA-proficient recipient. The first conjugal transfer system of genomic DNA should provide a valuable alternative genetic tool for editing the B. subtilis genome.     

Genetic analysis of a class of polymyxin resistant partial revertants of stage O sporulation mutants of Bacillus subtilis: map of the chromosome region near the origin of replication.

Summary A group of extracistronic mutations restored polymyxin resistance to stage O sporulation mutants of Bacillus subtilis, as well as resistance to antibioticfrom the wild type sporulating strain, and to phages 2 and 15,without restoring the ability to sporulate.The spoOA mutants containing these partial revertant mutations fell into two major classes: Those that grew well in minimal media and produced little antibiotic;and those that grew poorly and produced high levels of antibiotic.The partial revertant mutations were located near the origin of replication of the chromosome on the genetic map,in a locus called abrB.A detailed map of this region was constructed using data from two and three factor transduction and transformation crosses.The following order of markers was obtained: purA 16-spo-CM1-novA1-gua-1-(dnaH151-ts8132)-recG13-abrB-pac-tms-26-lysS-cysA14. The abrBlocus was separated by at least 50% recombination in transformation from the known loci on either side of it.Similar partial revertant mutations from other laboratories (cpsX, abs, and tol markers)also mapped at the abrB locus. The mutations at this locus were closely linked by transformation (recombination index <0.1 in most cases)suggesting that they comprise a single gene.     

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

Summary A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA)·poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid.Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such as thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec ^- recipient strain of B. subtilis was used.Abbreviations Ap^R resistance to ampicillin - Tc^R resistance to tetracycline - Cm^R resistance to chloramphenicol - CCC covalently closed circular duplex - Mdal magadalton     

Early-blocked asporogenous mutants of Bacillus subtilis are lysogenized at reduced frequency by temperate bacteriophages.

The establishment of lysogeny in early-blocked asporogenous (Spo-) mutants of Bacillus subtilis 168, which were also defective in the production of antibiotics (Abs-), by temperate phage phi105 or SPO2 was studied. It was found that the frequency of lysogenization of Spo-Abs-mutants was 10 to 20% that of the wild-type bacteria. There was no difference in the efficiency of plating and the burst size of phi105 between wild-type and mutant strains. Phi105 lysogens of mutant strains were as stable as those of the wild type. Several rifampin-resistant mutants defective in the production of antibiotics were isolated. They were also defective in spore formation and lysogenized by phi105 at reduced frequency.     

Restriction and modification in Bacillus subtilis 168. Regulation of hsrM(nonB) expression in spoOA mutants and effects on permissiveness for phi15 and phi105 phages

Summary Gene hsrM (nonB) of Bacillus subtilis 168, causing non-permissiveness to phage SP10 (Saito et al. 1979) and reduced plating efficiency of unmodified phage 105, is responsible for non-permissiveness of B. subtilis 168 for phages 15 and PZA. Upon transformation to sporulation deficiency (allele spoOA) B. subtilis 168 becomes permissive for 15 and PZA and loses the ability to restrict 105. spoOA str-1 double transformants of B. subtilis 168, however, retain the restriction 168 and non-permissiveness for 15 and PZA phages, in spite of their Spo^– phenotype. Therefore it appears that a functional product of the spoOA gene is required for expression of gene hsrM in wild-type bacteria, but is not essential in streptomycin-resistant bacteria. Phage genomes (PZA) were trapped in spores of the restriction deficient strain with much higher efficiency than in the wild-type.