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.     

Genetic mapping and physiological consequences of metE mutations of Bacillus subtilis.

Three metE mutations of Bacillus subtilis, which cause cells to have a 25- to 200-fold decrease in L-methionine S-adenosyltransferase (EC 2.5.1.6) activity, were mapped between bioB and thr. The corresponding three metE mutants contained three- to fourfold less intracellular S-adenosylmethionine (SAM) but at least sevenfold more methionine than the metE+ strain when grown in synthetic medium. This indicates a strong feedback control of SAM on its synthesis. However, only the metE2 strain, with the lowest SAM concentration, grew at a slightly lower rate than the parent, which showed that an intracellular concentration of about 25 microM SAM was critical for growth at the normal rate. Neither DNA methylation (measured by bacteriophage luminal diameter 105 restriction) nor sporulation was affected at this low SAM concentration. Addition of methionine to the growth medium caused an increase in the pool of SAM in some but not all metE mutants. Coaddition of adenine did not change this result. However, the extent of sporulation (induced by mycophenolic acid) was decreased 50-fold in all mutants by the addition of methionine and adenine. Therefore, the combination of methionine and adenine suppresses sporulation regardless of whether it causes an increase in the level of SAM.     

Genetic mapping in Bacillus subtilis 168 of the aadK gene which encodes aminoglycoside 6-adenylyltransferase

Abstract Bacillus subtilis 168 has an aadK gene, which encodes aminoglycoside 6-adenylyltransferase, a streptomycin-modifying enzyme, on its chromosome. To characterize the aadK gene, we con tructed a B. subtilis 168 strain that carried the chloramphenicol resistance gene near the aadK on the chromosome and an aadK deletion mutant using an integration technique. The aadK gene was mapped between azlB and pheA on the chromosome of B. subtilis 168. The aadK deletion mutant was slightly more susceptible to streptomycin than the original strain. The result indicates that the aadK gene contributes low-level resistance to streptomycin in B. subtilis 168.     

Morphological stages of Bacillus subtilis sporulation and resistance to fusidic acid.

During spore development of Bacillus subtilis both protein synthesis and sporulation become resistant to the antibiotic fusidic acid. This resistance develops at the time when asymmetric prespore septa are formed. Simultaneously ribosomes lose their ability to bind fusidic acid, as demonstrated by their affinity chromatography with the immobilized drug. Mutants resistant to fusidic acid during growth are oligosporogenous; their sporulation development is blocked before septum formation. These results indicate that normal ribosomes are needed for prespore septation sporulation; only after septation can protein synthesis be maintained, throughout the development period, by fusidate resistant ribosomes.     

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.     

Genetic mapping by means of protoplast fusion in Bacillus subtilis

Summary A new mapping method involving protoplast fusion in Bacillus subtilis is described. Protoplasts from an isogenic standard marker strain containing purA and from a strain containing both purB and the marker, x, to be mapped were fused with polyethylene glycol, and purA ⁺ purB ⁺ fusants were selected. After isolation of single colonies and determination of unselected markers, marker x was mapped between two standard markers. This method was fully applicable to PBS1-resistant strains (e.g., lyt strains). The results obtained by protoplast fusion, conventional transformation and/or lysed protoplast transformation indicated that a lyt strain, Ni15, contained two new autolysin-minus mutations (lyt-151 and lyt-152). The properties of lyt-15 are also discussed.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - SMM 0.5 M sucrose, 0.02 M MgCl₂, 0.02 M maleate buffer, pH 6.5     

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.     

Genetic mapping of a linked cluster of ribosomal ribonucleic acid genes in Bacillus subtilis.

A ribosomal ribonucleic acid gene set consisting of genes for 16S, 23S, 5S, and 4S ribonucleic acid species has been genetically mapped to a position between the markers recG13 and abrB74 on the Bacillus subtilis chromosome and designated rrnA. A ribosomal mutation, ksgA, was found to be linked to rrnA. This places rrnA in a region of the chromosome where ribosome-related genes occur but that is not directly adjacent to the major cluster of ribosome-related markers.     

Genetic location of two mutations affecting the lysyl-transfer ribonucleic acid synthetase of Bacillus subtilis

Two mutations (lysS1 and lysS2), each independently resulting in a thermosensitive, lysyl-transfer RNA synthetase (l-lysine: tRNA ligase [adenosine 5'-monophosphate] EC 6.1.1.6), have been mapped on the Bacillus subtilis chromosome between purA16 (adenine requirement) and sul (sulfanilamide resistance). They are linked by transformation with sul (70 to 74% cotransfer) in the order purA16-lysS1-lysS2-sul. The mutant loci are either in the same gene or in two closely linked genes. They are not linked to the tryptophanyl-tRNA synthetase structural gene or to the lys-1 locus.     

Characterization and mapping of temperature-sensitive division initiation mutations of Bacillus subtilis.

Two temperature-sensitive, filamenting mutants of Bacillus subtilis (ts1 and ts12) have been shown to be defective in the initiation of septation. Recombination index mapping showed that these mutations mapped in two different but closely linked genes. A third proposed initiation mutation, tms-12, probably maps in the same gene as ts12. Another proposed initiation mutation was not linked with these genes by transformation, indicating that there was a minimum of three genes involved in the initiation of division. PBS1 transduction mapping located these three genes close to the pyr cluster.     

Genetic and biochemical characterization of kirromycin resistance mutations in Bacillus subtilis.

Spontaneous mutations causing resistance to the EF-Tu-specific antibiotic kirromycin have been isolated and mapped in Bacillus subtilis. Three-factor transductional and transformational crosses have placed the kir locus proximal to ery-1 and distal to strA (rpsL) and several mutations affecting elongation factors EF-G and EF-Tu, in the order: cysA strA [fus-1/ts-6(EF-G)] [ts-5(EF-Tu)] kir ery-1 spcA. Purified EF-Tu from mutant strains is more resistant to kirromycin as measured by in vitro protein synthesis and also shows a more acidic isoelectric point than wild-type EF-Tu. This indicates that the kir locus is the genetic determinant (tuf) for EF-Tu and that there is a single active gene for this enzyme in B. subtilis.     

Genetic and chemical studies of ribosomes from spectinomycin resistant mutants of Bacillus subtilis

Summary Spectinomycin resistant mutants were isolated fromBacillus subtilis ATCC 6633. Some of these mutants had an altered specific 30s ribosomal protein on CMC chromatography, the site of alteration residing in a different tryptic peptide fragment from one mutant to another on Dowex 50×8 chromatography.This work was supported by a grant from the Mitsubishi Foundation and grants from the Ministry of Education of Japan.     

Genetic and physiological characterization of Bacillus subtilis mutants resistant to purine analogs.

Bacillus subtilis mutants defective in purine metabolism have been isolated by selecting for resistance to purine analogs. Mutants resistant to 2-fluoroadenine were found to be defective in adenine phosphoribosyltransferase (apt) activity and slightly impaired in adenine uptake. By making use of apt mutants and mutants defective in adenosine phosphorylase activity, it was shown that adenine deamination is an essential step in the conversion of both adenine and adenosine to guanine nucleotides. Mutants resistant to 8-azaguanine, pbuG mutants, appeared to be defective in hypoxanthine and guanine transport and normal in hypoxanthine-guanine phosphoribosyltransferase activity. Purine auxotrophic pbuG mutants grew in a concentration-dependent way on hypoxanthine, while normal growth was observed on inosine as the purine source. Inosine was taken up by a different transport system and utilized after conversion to hypoxanthine. Two mutants resistant to 8-azaxanthine were isolated: one was defective in xanthine phosphoribosyltransferase (xpt) activity and xanthine transport, and another had reduced GMP synthetase activity. The results obtained with the various mutants provide evidence for the existence of specific purine base transport systems. The genetic lesions causing the mutant phenotypes, apt, pbuG, and xpt, have been located on the B. subtilis linkage map at 243, 55, and 198 degrees, respectively.     

Genetic transformation with cell wall-associated deoxyribonucleic acid in Bacillus subtilis

Cell walls from bacillus subtilis 168 were prepared by conventional methods and found to contain deoxyribonucleic acid (DNA). In transformation assays, after autolysis, it was found that two major regions of the chromosome were selectively enriched in the wall preparations. One region clustered around the replication origin and is represented by the markers purA16, ts8132, thiC5, sacA321, and hisA1. The other region included the replication terminus with representative loci metB10, citK5, gltA292, and pyrA1. All other (internal) loci which were examined showed no statistical enrichment. The two areas of enrichment were similar to but more extensive than those reported for membrane-DNA complexes. The wall preparations also contained protein and lipid, indicating a possible membrane involvement. Analyses of the cell walls revealed that the fatty acid composition of the membrane component was not typical of the for B. subtilis protoplast membranes or for lipoteichoic acids. In addition, radioiodination of cell wall autolysates, followed by gel electrophoresis and autoradiography, demonstrated the presence of proteins not readily detectable in bulk protoplast membranes or on the surfaces of intact cells. These data suggest that a unique component of the membrane and regions of the B. subtilis genome involved in DNA replication events are tightly associated with cell walls. The binding of DNA-membrane complexes to the "rigid" cell wall and the replication of the wall could be a mechanism by which the segregation of growing chromosomes occurs.     

Erythromycin resistant mutants of Bacillus subtilis

Summary Erythromycin resistant (ery ^r) mutants were isolated from Bacillus subtilis ATCC 6633. The composition of ribosomal proteins were analyzed for thirteen such ery ^r-mutants with chromatography on a carboxymethyl cellulose (CMC) column. The 50s subunit from all of the ery ^r-mutants was found to contain the altered 50d protein. The ribosomes prepared from the ery ^r-mutants did not show in vitro alteration of the ability to combine with erythromycin.