Cryptic Plasmids in Glutamic Acid-producing Bacteria

Seven filamentous (fil) mutants were isolated from B. subtilis, and the mutations were mapped by means of lysed-protoplast transformation. Five of the mutations were linked to aroD and the others to pyrD. rgn mutations, which lead to a decrease in autolysin(s) and the formation of filaments, were also linked to aroD, and the mapping order was rgn-dnaE-aroD. On comparison with other reported filamentous mutations (lyt-1, lyt-2 and lyt-152), fil-1, fil-3 to -6, rgn and the above lyt mutations were determined to be in the same locus. All of the seven fil strains lacked flagella and showed decreased aμtolysin activity. Among them, only mutants having arod- linked mutations showed low competency. Protease assay results indicated that rgn mutants produce a several times higher amount of the enzyme than the parent strain, and the initiation time for the production in rgn mutants was two hours earlier than in the parent strain.     

Mapping of the gene specifying DNA polymerase III of Bacillus subtilis

Summary polC, the gene specifying the structure of the replication-specific DNA polymerase III of B. subtilis, was mapped by exploiting azp-12, a mutation conferring resistance to azopyrimidine which determines a mutant, azopyrimidine-resistant enzyme. azp-12 was located in the area of the pyrA locus and is between spcB1 and recA1. azp-12 was linked by transformation to four other mutations which influence the in vitro behaviour of DNA polymerase III-polC25, polC26, mut-1(ts), and DNAF133; the close linkage of these five mutations strongly suggests that they are alleles of the same gene.     

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.     

Genetic mapping of regulatory mutations of Bacillus subtilis riboflavin operon

Summary Seven mutations leading to riboflavin overproduction inBacillus subtilis were found to be linked to the markerdnaF133 (145° on theB. subtilis genetic map) by transformation. Cotransfer indexes (42.5%–61.7%) suggest that theribC mutations are alleles of the same locus. Results of transduction and transformation crosses suggest the following order of markers:pyrD26 –ts-6 –dnaF133 –ribC –recA1.     

Mutagenesis during transformation of Bacillus subtilis I. An increase in “selfing” resulting from hybrid donor DNAs

Reverse mutations increase when competent Baciilus subtilis cells are transformed with high concentrations of homologous “selfer” DNA. A high proportion of the mutants were also transformants of linked genes. A stimulation in the appearance of reversed mutations occurred when homoduplex and heteroduplex “selfer” DNAs were used as donors. Digestions of native and hybrid DNAs with nuclease S1 from Aspergillus oryzae resulted in the preferential decrease of mutations as compared to a much smaller inactivation of single marker tranformation. Among various repair-deficient strains of B. subtilis, only polyA mutants showed a preferential effect of either suppressing or stimulating the frequency of reverse mutation induced by “selfer” DNA. The results are consistent with mutagenic errors occurring during gap-filling steps in the process of either mismatch repair or recombinational strand exchanges.     

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.     

Inactivation and reversion of multisite streptomycin resistance mutations of Pneumococcus

Summary Purified DNAs were prepared from pneumococcal strains bearing either a multisite or single site streptomycin resistance (str-r) marker. In addition, each DNA contained a single site erythromycin resistance marker (ery-r2) or a pair of closely linked ery-r markers (ery-r2 and ery-r6). These DNA preparations were subjected to inactivation by subcritical heating or nitrous acid. Regardless of the genetic size of the streptomycin resistance marker, it was inactivated in a manner identical to that of the ery-r2 marker and less rapidly than that of the linked pair of ery-r markers.Spontaneous reversions towards decreased resistance have been observed in cultures of multisite mutants. Genetic analysis of the revertants revealed that the multisite mutations had been replaced by mutations with altered properties of recombination.The simplest interpretation of the evidence is that these are point mutations of such a nature, or occurring in such a region, that recombination is inhibited in the region immediately adjacent to them. In this way they would appear genetically large but physically small.Supported by NSF grant GB-7295.     

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.     

Expression of luciferase genes from different origins in Bacillus subtilis

Summary A group of vectors for luciferase expression in Bacillus subtilis was constructed. So far, only bacterial luciferases have been expressed in Bacillus, but in this study we wanted also to express genes encoding eukaryotic luciferases to perform direct comparisons of the light levels produced by the two different systems in B. subtilis. The vectors constructed can replicate both in Escherichia coli and B. subtilis, and the luciferase expression is strictly regulated due to the dual plasmid system used. Nearly a 100-fold increase in light production compared to previous results was achieved when genes encoding bacterial luciferase were inserted into the constructs and transformed into B. subtilis. An additional tenfold increase in light production was obtained when luciferase genes from the North American firefly (Photinus pyralis) or a click beetle (Pyrophorus plagiophtalamus) were introduced in a similar fashion into B. subtilis. Measurement of the light emission was performed without disruption of bacterial cells in a real-time manner, which is a common feature when working with all of these constructions. Structures of the shuttle vector constructs and results from light emission measurements are presented.     

Construction of a recombinant plasmid composed of B. subtilis leucine genes and a B. subtilis (natto) plasmid: its use as cloning vehicle in B. subtilis 168.

Summary Recombinant plasmids composed of Bacillus subtilis 168 leucine genes and a B. subtilis (natto) plasmid have been constructed in a recombination deficient (recE4) mutant of Bacillus subtilis 168. The process involved EcoRI fragmentation and ligation of a B. subtilis (natto) plasmid and a composite plasmid RSF2124-B · leu in which B. subtilis 168 leucine genes are linked to the R-factor RSF2124. A constructed plasmid (pLS102) was found to be composed of an EcoRI fragment derived from the vector plasmid and two tandemly repeated EcoRI fragments carrying the leucine genes. A derivative plasmid (pLS101 or pLS103) consisting of one molecule each of the EcoRI fragments was obtained by in vivo intramolecular recombination between the repeated leucine gene fragments in pLS102. pLS103 was cleaved once with BamNI, SmaI and HpaI. Insertion of foreign DNA (Escherichia coli plasmid pBR322) into the BamNI site inactivated leuA but not the leuC function which thus can serve as selective marker if the plasmid is used as vector in molecular cloning. The penicillin resistance carried in pBR322 was not functionally expressed in B. subtilis cells. By partial digestion of pLS103 with HindIII followed by ligation with T4-induced ligase, pLS107 was obtained which contained only one EcoRI site. However, insertion of exogenous DNA (pBR322) into this EcoRI site inactivated both leuA and leuC functions.     

Saturation mutagenesis and self-inducible expression of trehalose synthase in Bacillus subtilis

Trehalose is a nonreducing disaccharide synthesized by trehalose synthase (TreS), which catalyzes the reversible interconversion of maltose and trehalose. We aimed to enhance the catalytic conversion of maltose to trehalose by saturation mutagenesis, and constructed a self-inducible TreS expression system by generating a robust Bacillus subtilis recombinant. We found that the conversion yield and enzymatic activity of TreS was enhanced by saturation mutations, especially by the combination of V407M and K490L mutations. At the same time, these saturation mutations were contributing to reducing by-products in the reaction. Compared to WT TreS, the conversion yield of maltose to trehalose was increased by 11.9%, and the k_cat/K_m toward trehalose was 1.33 times higher in the reaction catalyzed by treS^V407M-K490L. treS^V407M-K490L expression was further observed in the recombinant B. subtilis W800N(Δσ^F) under the influence of P_srfA, P_cry3Aa, and P_srfA-cry3Aa promoters without an inducer. It was shown that P_srfA-cry3Aa was evidently a stronger promoter for treS^V407M-K490L expression, with the intracellular enzymatic activity of recombinant treS^V407M-K490L being over 5,800 U/g at 35 hr in TB medium. These results suggested the combination of two mutations, V407M and K490L, was conducive for the production of trehalose. In addition, the self-inducible TreS^V407M/K490L mutant in the B. subtilis host provides a low-cost choice for the industrial production of endotoxin-free trehalose with high yields.     

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.     

Development of natto with germination-defective mutants of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> (<Emphasis Type="Italic">natto</Emphasis>)

The effects of cortex-lysis related genes with the pdaA, sleB, and cwlD mutations of Bacillus subtilis (natto) NAFM5 on sporulation and germination were investigated. Single or double mutations did not prevent normal sporulation, but did affect germination. Germination was severely inhibited by the double mutation of sleB and cwlD. The quality of natto made with the sleB cwlD double mutant was tested, and the amounts of glutamic acid and ammonia were very similar to those in the wild type. The possibility of industrial development of natto containing a reduced number of viable spores is presented.     

Expression of a xylanase gene of Bacillus pumilus in Escherichia coli and Bacillus subtilis

Summary A hybrid plasmid, pOXN29 (10.4 Mdal), coding the xylanase (xynA) and -xylosidase (xynB) genes of Bacillus pumilus IPO was constructed by the ligation of pBR322 and a 7.7 Mdal PstI-fragment of chromosomal DNA as reported in our previous paper (Panbangred et al. 1983). A deletion plasmid of pOXN29, pOXN293 (9.2 Mdal), which contains xynA and xynB, was ligated with pUB110 at an EcoRI site, and used to transform B. subtilis MI111. Two selected clones of B. subtilis as xylanase hyper-producers contained plasmids pOXW11 (4.2 Mdal) and pOXW12 (4.0 Mdal), both consisting of only pUB110, xynA, and its flanking regions, as the result of spontaneous deletion. These B. subtilis clones produced 2.7–3.0 times as much xylanase as B. pumilus. Escherichia coli and B. subtilis clones harbouring the hybrid plasmids synthesized xylanase and -xylosidase constitutively, whereas both enzymes were induced by xylose in B. pumilus.Xylanase synthesized by B. subtilis harbouring pOXW11 or pOXW12 was excreted into the medium like that of B. pumilus IPO, but xylanase synthesized in E. coli harbouring pOXN29, 293 or pOXW1 coding xynA was intracellular. In a previous investigation (Panbangred et al. 1983), xylanase was found to be located in the cytoplasm, not the periplasm nor the membrane fraction in E. coli cells harbouring pOXN29 derivatives. In spite of the abnormal location of xylanase synthesized in E. coli, the signal peptide was processed in the same way as in B. pumilus, with the same molecular weight and the same amino terminal sequences of xylanase prepared from E. coli cells and B. pumilus culture fluid.     

Proteases of the genus Bacillus. II. Alkaline proteases

The alkaline proteases of B. subtilis NRRL B3411, B. pumilis, and B. licheniformis have been isolated by fractionation followed by ion exchange chromatography and their homogeneity demonstrated. General enzyme properties of the B. sublitis NRRL B3411 alkaline protease have been studied and attempts made to differentiate a group of alkaline proteases. It is clear that the alkaline proteases known as Subtilisins or Subtilopeptidases are not, exclusive to B. subtilis but are common to many Bacilli and therefore the generic name Bacillopeptidases has been proposed. It is clear too that on the basis of the effect of pH on activity, amino acid composition, esterase activity, and immunological cross-reactions the Bacillopeptidases can be divided into two groups or types: (a) Bacillopcptidase A (Subtilisin A or Subtilopeptidase A) which includes Subtilisin Carlsberg, B. licheniformis, and B. pumilis alkaline proteases; ( b ) Bacillopeptidase B (Subtilisin B or Subtilopeptidase B) which includes B subtilis NRRL B3411, Subtilisin Novo, Subtilisin BPN' (Nagarse), alkaline protease Daiwa Kasei, and (probably) B. subtilis var. amylosacchariticus. At present, no further differentiation is possible and whether or not the enzymes within group A or B are identical remains an open question. Methods for examination of crude enzyme mixtures or fermentation beers are described and from the examination of a number of crude enzymes and fermentation beers it appears that organisms producing Bacillopeptidase A do not produce neutral protease or amylase, while organisms producing Bacillopeptidase B produce a neutral protease and amylase as well.     

Gene-directed mutagenesis on the chromosome of Bacillus subtilis 168

Summary We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B. subtilis chromosome. The procedure depends on the accuracy and high frequency of homologous recombination between the B. subtilis chromosome and the DNA taken up by the cell. The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst. The utility of the method has been demonstrated using leuB and pro of B. subtilis as target gene and catalyst, respectively, and mutations such as leuB: : cat, leuB ^–, and pro: : neo constructed in vitro on the cloned DNA fragments. Transformation in sequential steps as (leuB ⁺ pro⁺)(leuB: : cat pro ⁺) (leuB ^– pro: : neo)(leuB ^– pro ⁺) resulted in a leuB ^– single mutant without affecting other regions of the B. subtilis chromosome (gene-directed mutagenesis). We also demonstrate that other single mutations such as metD ^– and pro ^–, as well as the double mutation leuB ^– pro ^– can be introduced by the same procedure. In principle, true isogenies with multiple mutations can be constructed by the method described in this paper. Furthermore, the procedure should be generally applicable to any organisms in which homologous recombination is proficient.     

Genetic characterization of a mutation that enhances paraquat tolerance in the fern Ceratopteris richardii

Summary Three nuclear mutations that affect tolerance to the herbicide paraquat have been selected in the fern Ceratopteris richardii. Two of the mutations, pq2 and pq45 are allelic and confer low and moderate tolerance, respectively. A third mutation, pqa6, is not linked to the other two and significantly enhances the level of tolerance when in combination with either pq2 or pq45. The pqa6 mutation does not independently confer tolerance in the absence of the other mutations.     

Identification and Immunochemical Location of UMP Kinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Phosphorylation of CMP and UMP is accomplished in Bacillus subtilis, as in Escherichia coli, by two different enzymes exhibiting characteristic structural and catalytic properties. UMP kinase from B. subtilis is an oligomer whose activity is strictly dependent on GTP. The B. subtilis enzyme is unstable in the absence of UTP, which acts as an allosteric inhibitor. Antibodies raised against recombinant B. subtilis UMP kinase recognized the protein both in soluble extract and in immunoelectron microscopy. UMP kinase from B. subtilis has a peripheral distribution which is related most probably to its role in the synthesis of membrane sugar components and its putative role in cell division.