Chromosomal location of mutations affecting sucrose metabolism in Bacillus subtilis Marburg

Summary Mutations affecting sucrose metabolism have been mapped by PBS1 transduction on the Bacillus subtilis chromosome in seven loci sacA, sacB, sacQ, sacR, sacS, sacT and sacU. sacA and sacB are presumed to be the structural genes of a sucrase and a levansucrase respectively. sacR, sacS and sacT correspond to groups of mutations leading to constitutive synthesis of sucrase or both sucrase and levansucrase. In sacQ, sacS and sacU are located either mutations increasing the level of synthesis of levansucrase specifically (sacQ ^h , sacS ^h , sacU ^h ) or mutations abolishing specifically the synthesis of levansucrase (sacU ^– ). sacA, sacS and sacT map to the left of purA16. sacQ is located to the left of thr5, sacB and sacR between cysB3 and hisA1 and sacU between uvr1 and gtaB.     

Mapping of mutations affecting synthesis of exocellular enzymes in Bacillus subtilis

Summary The sacU ^h , amyB and pap mutations are identical with respect to their pleiotropic phenotype and their genetic location. Strains bearing these mutations overproduce several exocellular enzymes: -amylase, lavansucrase and proteases, they are poorly or not at all transformable and most of them are devoid of flagella. These mutations are tightly linked to the sacU ^- mutations by transformation and therefore lie between the hisA1 and gtaB290 markers. It is possible that the sacU ^h , amyB and pap mutations on one hand and the sacU ^- mutations on the other are two different classes of alterations of the same regulatory gene controlling the synthesis of some exocellular enzymes and several other cellular functions. Furthermore an amy ^- mutation, leading to the lack of -amylase activity, was mapped between the lin2 and aroI906 markers which are not linked to the sacU locus.     

Macrolide and aminoglycoside antibiotic resistance mutations in the Bacillus subtilis ribosome resulting in temperature-sensitive sporulation

Summary Mutants of Bacillus subtilis resistant to various macrolide antibiotics have been isolated and characterized with respect to their sporulation phenotype and the electrophoretic mobility of their ribosomal proteins (r-proteins). Two types of major alterations of r-protein L17, one probably due to a small deletion, are found among mutants exhibiting high-level macrolide resistance. These mutants are all temperature-sensitive for sporulation (Spo^ts). Low-level resistance to some macrolides is found to be associated with minor alterations in r-protein L17. These mutations do not cause a defective sporulation phenotype. All of the macrolide resistance mutations map at the same locus within the Str-Spc region of the B. subtilis chromosome. Hence, changes in a single ribosomal protein can result in different sporulation phenotypes.Mutants resistant to the aminoglycoside antibiotics neomycin and kanamycin have been isolated. Approximately 5% of these are Spo^ts. Representative mutations, neo 162 and kan25, cause concomitant drug resistance and sporulation temperature-sensitivity and map as single-site lesions in the Str-Spc region of the chromosome. Strains bearing neo162 or kan25 are equally cross-resistant to several aminoglycoside antibiotics but show no resistance to streptomycin or spectinomycin. These mutations define a new B. subtilis drug resistance locus at which mutation can cause defective sporulation.     

Phenotypic reversion in some early blocked sporulation mutants of Bacillus subtilis. Genetic study of polymyxin resistant partial revertants

Summary A class of suppressor mutations restores, in pleiotropic sporulation mutants of B. subtilis (SPO mutants), the wild type level of resistance to Polymyxin, and, most often, other properties of the wild strain as well, but never the ability to sporulate. These suppressors, extracistronic, are active on mutations occurring in any one of the 5 genes in which SPO mutations have been found. The phenotype of the suppressed strains is dependent on both the suppressed (SPO) and the suppressive mutations. All these suppressors are located in a single locus and some of them are thermosensitive. The evidence suggests that a physiological compensation is at work in the partial revertants, so that the locus at which the suppressors are located was called cps X. Two hypotheses are discussed that might account for these observations.     

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.     

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.     

Erythromycin resistant mutations in Bacillus subtilis cause temperature sensitive sporulation.

Summary All of several hundred erythromycin resistant (ery^R) single site mutants ofBacillus subtilis W168 are temperature sensitive for sporulation (spo^ts). The mutants and wild type cells grow vegetatively at essentially the same rates at both permissive (30° C) and nonpermissive (47° C) temperatures. In addition, cellular protein synthesis, cell mass increases and cell viabilities are similar in mutant and wild type strains for several hours after the end of vegetative growth (47° C). In the mutants examined, the temperature sensitive periods begin when the sporulation process is approximately 40% completed, and end when the process is 90% complete. At nonpermissive temperatures, the mutants produce serine and metal proteases at 50% of the wild type rate, accumulate serine esterase at 16% of the wild type rate, and do not demonstrate a sporulation related increase in alkaline phosphatase activity.The ery^R and spo^ts phenotypes cotransform 100%, and cotransduce 100% using phage PBS1. Revertants selected for ability to sporulate normally at 47° C (spo⁺), simultaneously regain parental sensitivity to erythromycin. No second site revertants are found.Ribosomes from ery^R spo^ts strains bind erythromycin at less than 1% of the wild type rate. A single 50S protein (L17) from mutant ribosomes shows an altered electrophoretic mobility. Ribosomes from spo⁺ revertants bind erythromycin like parental ribosomes and their proteins are electrophoretically identical to wild type. These data indicate that the L17 protein of the 50S ribosomal subunit fromBacillus subtilis may participate specifically in the sporulation process.     

Cloning and sequencing the degS-degU operon from an alkalophilic Bacillus brevis

The sacU region from an alkalophilic Bacillus brevis was cloned and sequenced. The two open reading frames of the degS-degU operon encode polypeptides that gave calculated molecular masses of 43.8 kDa and 27.0 kDa, respectively. Sequence comparisons at the amino acid level to the B. subtilis degS-degU genes showed 74% and 84% similarity, respectively. On a multicopy vector the B. brevis degS-degU genes were found to cause hypersecretion of several extracellular enzymes in a B. subtilis rec^– strain as well as in a B. subtilis sacU(HY) strain.     

Inducible Secretion of a Cellulase from Clostridium thermocellum in Bacillus subtilis

A host-vector system for inducible secretion during the logarithmic growth phase in Bacillus subtilis has been developed. The B. subtilis levansucrase gene promoter and the region encoding its signal sequence have been used. The endoglucanase A of Clostridium thermocellum was used as a model protein to test the efficiency of the system. Effective inducible secretion of the endoglucanase A was observed when either the levansucrase signal sequence or its own signal sequence was used. Expression of the endoglucanase A in different genetic backgrounds of B. subtilis showed that its regulation was similar to that of levansucrase, and high enzyme activity was recovered from the culture supernatant of a hyperproducing B. subtilis sacU(Hy) strain. The molecular weight of 46,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the secreted endoglucanase A is compatible with the calculated molecular weight of the mature polypeptide.     

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.     

Chromosomal Location of Pleiotropic Negative Sporulation Mutations in BACILLUS SUBTILIS

Genetic analysis by PBS-1 transduction and transformation of a large group of pleiotropic negative sporulation mutants has shown that mutations of this phenotype may be located in five genetically distinct regions. The first group of mutant sites, spoA mutations, is located in the terminal region of the chromosome and linked to the lys-1 marker by PBS-1 transduction. The second group, spoB mutations, is located between phe-1 and the attachment site for the lysogenic bacteriophage ϕ 105. Fine structure analysis of the mutant sites within the spoB locus has been accomplished. A third location for mutants of this phenotype, spoE mutants, was found between the metC3 and ura-1 markers. Two mutants were found at this site and both were capable of sporulation, in contrast to the rest of the pleiotropic sporulation mutants. A fourth chromosomal site, spoH mutations, was found near the ribosomal and RNA polymerase loci. A large group of mutant sites, spoF mutations, was found to be linked to each other by recombination index analysis in transformation but unlinked to any of the known auxotrophic mutations comprising the chromosomal map. All mutants analyzed showing a pleiotropic negative phenotype were found to map within one of these five regions. Interspecific transformation with Bacillus amyloliquefaciens as donor has shown that all of the pleiotropic negative sporulation mutations are conserved relative to a selected group of auxotrophic markers. The degree of conservation in decreasing order is: spoH > spoF = spoB > spoA.     

Intracellular proteolytic activity during sporulation ofBacillus megaterium

Intracellular proteolytic activity increased during incubation of the sporogenic strain ofBacillus megaterium KM in a sporulation medium together with excretion of an extracellular metalloprotease. The exocellular protease activity in a constant volume of the medium reached a 100-fold value with respeot to the intracellular activity. Maximal values of the activity of both the extracellular and intracellular enzyme were reached after 3 – 5 h of incubation. After 7 h 20 – 50% cells formed refractile spores. The intracellular proteolytic system hydrolyzed denatured proteinsin vitro at a rate up to 150 μg mg_-1 h_-1 and native proteins at a rate up to 70 μg mg^-1 h^-1. Degradation of proteinsin vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 μg mg^-1 h^-1 and the inactivation of beta-galactosidase at a rate of 70 μg mg^-1 h^-1. The intracellular proteolytic activity was inhibited to 65 – 88% by EDTA, to 23 – 76% by PMSF. Proteolysis of denatured proteins was inhibited both by EDTA and PMSF more pronouncedly than proteolysis of native proteins; 50 – 65% of the activity were localized in protoplasts. Another strain ofBacillus megaterium (J) characterized by a high (up to 90%) and synchronous sporulation activity was found to behave in a similar way, but the rate of protein turnover in this strain was almost twice as high. The asporogenic strain ofBacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3 – 4 h. The intraeellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.     

Stepwise Introduction of Regulatory Genes Stimulating Production of α-Amylase into Bacillus subtilis : Construction of an α-Amylase Extrahyper Producing Strain

The production of extracellular α-amylase in Bacillus subtilis is probably regulated by many genetic elements, such as amyR, tmrA7, pap, amyB and sacU. Additional genetic elements, C-108 and A-2 for production of the α-amylase were found in D-cycloserine and ampicillin resistant mutants (C108 and A2) of B. subtilis 6160, respectively. Strain C108 increased the production of α-amylase about 5 times and protease about 80 times compared to parental 6160 strain. Strain A2 showed a nearly 6-fold increased α-amylase production.

These genetic elements displayed a synergistic effect with other genetic factors in production of extracellular α-amylase when these elements were transferred by DNA mediated transformation. By stepwise introduction of these and other genetic elements into B. subtilis 6160 by transformation and mutation, strains with higher α-amylase producing activity were obtained. The finally obtained strain, T2N26, produced about 1,500-2,000 times more α-amylase than parental 6160 strain.     

Genetic mapping of cadmium resistance mutations inBacillus subtilis

Bacillus subtilis, which accumulates cadnium via the manganese transport system, may acquire cadmium resistance by chromosomal mutations that reduce Cd²⁺ uptake without affecting Mn²⁺ transport. A cadmium resistance mutation,cdr-1, maps at about 40° on theB. subtilis chromosome. The deduced map order wasarol-narB-mtlB-cdr-dal-purB. Thecdr mutations in four other, independently isolated Cd²⁺-resistant mutants demonstrating reduced Cd²⁺ uptake also mapped betweenaroI anddal.     

Characterization of glutamine requiring mutants of Bacillus subtilis

Mutants of $\text{B. subtilis}$ 168 which exhibited an absolute requirement for glutamine have been isolated and characterized. Of the two mutants studied in detail, one had normal levels of glutamine synthetase and sporulated normally, the other had reduced glutamine synthetase and was asporogenic. Both mutants were mapped close to the $\text{thy}$ A region of the chromosome by PBS1 transduction.A study of spontaneous revertants selected for glutamine prototrophy (or the sporulation character in the case of the asporogenic mutant) led to the conclusion that there is a relationship between the glutamine requirement and sporulation. However, the influence of glutamine could not be entirely explained by the catalytic properties of glutamine synthetase.     

Early blocked asporogenous mutants of Bacillus subtilis 168. I. Isolation and characterization of mutants resistant to antibiotic(s) produced by sporulating Bacillus subtilis 168

Summary A number of mutants (abs)-resistant to antibiotic(s) produced by sporulating Bacillus subtilis 168 have been isolated from an early blocked asporogenous mutant (spoA12). At least four classes were recognized according to their phenotypic properties. Genetic analysis has shown that these mutants were neither partial revertants nor suppressor mutants of the spoA gene. Both nonsense and missense mutants of the spoA gene are reverted partially by a secondary mutation which is resistant to antibiotic of B. subtilis 168. Another asporogenous mutant, spoB, whose locus is closely linked to pheA, is also affected by the same abs mutation. The nature of abs mutants is discussed.     

Catabolite-Induced Repression of Sporulation in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

In response to nutrient limitations, Bacillus subtilis cells undergo a series of morphological and genetic changes that culminate in the formation of endospores. Conversely, excess catabolites inhibit sporulation. It has been demonstrated previously that excess catabolites caused a decrease in culture medium pH in a process that required functional AbrB. Culture medium acidification was also shown to inhibit Ï^H-dependent sporulation gene expression. The studies reported here investigate the effects of AbrB-mediated pH sensing on B. subtilis developmental competence. We have found that neither addition of a pH stabilizer, MOPS (pH 7.5), nor null mutations in abrB blocked catabolite repression of sporulation. Moreover, catabolite-induced culture medium acidification was observed in cultures of catabolite-resistant sporulation mutants, crsA47, rvtA11, and hpr-16, despite their efficient sporulation. These results suggest that AbrB-mediated pH sensing is not the only mechanism regulating catabolite repression of sporulation. The AbrB pathway may function to channel cells toward genetic competence, as opposed to other postexponential differentiation pathways.     

The contribution of the cell wall to a transmembrane calcium gradient could play a key role in Bacillus subtilis protein secretion

A weak Ca²⁺-binding site (K_a= 0.8× 10³ M^?1, at pH7) was identified in the mature part of levansucrase. An amino acid substitution (Thr-236 →lle) in this site alters simultaneously the affinity for calcium, the folding transition and the efficiency of the secretion process of levansucrase. Moreover, the ability of the Bacillus subtilis cell wall to concentrate calcium ions present in the culture medium was studied. We confirm the results of Beveridge and Murray who showed that the concentration factor is about 100 to 120 times. This property preserves a high concentration of Ca²⁺ (>2 mM) on the external side of the cytoplasmic membrane, even in the absence of further Ca2+ supplementation in the growth medium. Such local conditions allow the spontaneous unfolding folding transition of levansucrase en route for secretion. Since several exocellular proteins of B. subtilis are calcium-binding proteins, we propose that the high concentration of calcium ion in the microenvironment of the cell wall may play a key role in the ultimate step of their secretion process.     

ScoC Mediates Catabolite Repression of Sporulation in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Sporulation in Bacillus subtilis can be triggered by carbon catabolite limitation. Conversely, carbon source excess can repress the production of extracellular enzymes, motility, and sporulation. Recent studies have implicated a pH-sensing mechanism, involving AbrB, the TCA cycle, Spo0K, and Ï^H in controlling the catabolite repression of sporulation gene expression. In an accompanying paper, we demonstrate that the AbrB-dependent pH-sensing mechanism may not be the only means by which carbon catabolites affect sporulation. In the studies reported here, we have examined the molecular basis underlying the catabolite repression phenotype of mutations in the hpr (scoC), rpoD (crsA47), and spo0A (rvtA11) loci. Loss of function mutations in hpr (scoC) restored sporulation gene expression and sporulation in the presence of excess catabolite(s), suggesting that Hpr (ScoC) has a pivotal role in mediating catabolite repression. Moreover, hpr gene expression increased substantially in the presence of excess catabolite(s), further supporting the involvement of Hpr (ScoC) in the carbon catabolite response system. We suggest that alterations in the phosphorelay response to catabolites may be one mechanism by which catabolite-resistant mutants such as crsA and rvtA are able to sporulate in the presence of excess glucoseReceived: 12 November 2002 / Accepted: 13 December 2002     

The relationship of serine protease activity to RNA polymerase modification and sporulation in Bacillus subtilis

The isolation and properties of a single site temperature sensitive protease mutant of Bacillus subtilis are described. Numerous criteria suggest that the mutation resides in the structural gene coding for a basic serine protease. The mutation has been mapped between aroD and lys-1 on the Bacillus subtilis chromosome. This protease exists as an intracellular and extracellular enzyme. The mutant cells are temperature sensitive for sporulation, antibiotic production, and the sporulation-specific alteration in DNA-dependent RNA polymerase β subunit. Several types of evidence indicate a direct involvement of this enzyme in a limited proteolytic cleavage of vegetative RNA polymerase β subunit, which produces the lower molecular weight β subunit found in sporulating cells. The derangement in this process is sufficient to account for the stoppage of sporulation at stage 0 when the mutant cells are grown at the non-permissive temperature.