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A Heat-sensitive Lysis Mutant of Bacillus subtilis 168 with a Low Activity of Pyruvate Carboxylase
Abstract
A mutant of Bacillus subtilis which grew in complex medium at 30 degrees C but lysed at 45 degrees C has been isolated. It could only grow on minimal medium at 45 degrees C with added aspartate (20 microgram ml-1) but lysed if lysine (20 microgram ml-1) was also present. The requirement for aspartate was due to a low activity of pyruvate carboxylase; the site of the mutation (pyc) was linked (16% cotransducible using phage PBSI) to the pyrD locus, and the order of markers deduced was: pyrD-cysC-pyc. This defect appeared to lead to decreased synthesis of mesodiaminopimelic acid (mesoA2pm), an amino acid unique to peptidoglycan and its precursors. At the restrictive temperature the mutant accumulated uridine-5'-diphosphate N-acetylmuramyl-L-alanyl-D-glutamate, since meso A2pm is the next amino acid to be added to the growing peptide chain of peptidoglycan. This resulted in an inhibition of peptidoglycan synthesis, determined as a reduced incorporation of N-acetyl[14C]glucosamine. Peptidoglycan synthesis was not decreased if the mutant was grown in media containing aspartate but lacking lysine. The sensitivity to lysine may arise because (i) at 45 degrees C the mutant was starved for aspartate and hence mesoA2pm even when aspartate was present, since aspartate utilization, as estimated by the incorporation of [3H]aspartate into trichloroacetic acid precipitable material, was relatively inefficient; and (ii) this diminished level of mesoA2pm synthesis from aspartate was further curtailed since lysine inhibits one of the aspartokinases in B. subtilis. Thus, addition of lysine allowed protein synthesis and hence autolysin production to proceed whilst peptidoglycan synthesis remained inhibited. When autolysis was blocked, either indirectly by stopping protein synthesis through starvation of aspartate and lysine, or directly by introducing a lyt mutation, then shifting the mutant to 45 degrees C did not result in lysis but growth still ceased.
... In Bacillus subtilis, genetic characterization of the LD-Azpm pathway cannot be approached through isolation of auxotrophic mutants since available strains are not endowed with an active LD-Azpm uptake system. Therefore, mutants with deficient enzymes in this pathway were searched for among stable L-forms (Ward, 19751, or conditional lethal, thermosensitive mutants (Buxton, 1978; Buxton & Ward, 1980). The latter approach resulted in the identification of only two classes of mutations; the first one was shown to affect dapE, a gene involved in deacylation of N-acetyl-LL-A,pm, whereas the second one was mistakenly (see below) assigned to pycA, the structural gene of pyruvate carboxylase. ...
... Shifting the latter strains from 30 "C to 45 "C results in a limited nephelometric density increase followed, at around 20 min, by a more or less massive lysis (Brandt & Karamata, 1987). We exploited the well-known phenomenon that lysis induced by interference with peptidoglycan synthesis can be suppressed by simultaneous inhibition of protein synthesis (Buxton, 1978; Kirby & Burnell, 1954; Meynell & Meynell, 1970). It is generally accepted that mutants affected in the synthesis of LD-A2pm are intrinsically also deficient in the formation of L-lysine or, depending on the exact site of the enzymic block (Fig. 2), of L-lysine and other amino acids belonging to the aspartate family. ...
... Therefore, the thermosensitive lysis phenotype of mutants deficient in LD-A,pm synthesis should not be expressed unless the growth medium contains all amino acids whose synthesis has been affected by their dap mutations. Supplementation with LD-A,pm is ineffective since B. suhtilis cells lack an active LD-A,pm uptake system (Ward, 1975; Buxton, 1978). Cultures of Iys+ derivatives harbouring lss mutations, grown in minimal media with or without L-lysine, were shifted from 30 "C to the non-permissive temperature, and their nephelometric density was followed. ...
Thermosensitive mutants of Bacillus subtilis deficient in peptidoglycan synthesis were screened for mutations in the meso-diaminopimelate (LD-A2pm) metabolic pathway. Mutations in two out of five relevant linkage groups, lssB and lssD, were shown to induce, at the restrictive temperature, a deficiency in LD-A2pm synthesis and accumulation of UDP-MurNAc-dipeptide. Group lssB is heterogeneous; it encompasses mutations that confer deficiency in the deacylation of N-acetyl-LL-A2pm and accumulation of this precursor. Accordingly, these mutations are assigned to the previously identified locus dapE. Mutations in linkage group lssD entail a thermosensitive aspartokinase 1. Therefore, they are most likely to affect the structural gene of this enzyme, which we propose to designate dapG. Mutation pyc-1476, previously reported to affect the pyruvate carboxylase, was shown to confer a deficiency in aspartokinase 1, not in the carboxylase, and to belong to the dapG locus, dapG is closely linked to spoVF, the putative gene of dipicolinate synthase. In conclusion, mutations affecting only two out of eight steps known to be involved in LD-A2pm synthesis were uncovered in a large collection of thermosensitive mutants obtained by indirect selection. We propose that this surprisingly restricted distribution of the thermosensitive dap mutations isolated so far is due to the existence, in each step of the pathway, of isoenzymes encoded by separate genes. The biological role of different aspartokinases was investigated with mutants deficient in dapE and dapG genes. Growth characteristics of these mutants in the presence of various combinations of aspartate family amino acids allow a reassessment of a metabolic channel hypothesis, i.e. the proposed existence of multienzyme complexes, each specific for a given end product.
... Constitutive acetolactate synthase 250 Glutamate requirement 3-Aminotyrosine resistance; part of.or very close to tyrA locus, see tyrA Control of amylase synthesis; probably identical to sacQ and pap, see sacQ 25 Amylase structural gene, also called amyA 25 ...
Among the earliest knowledge about microbes is the fact that cells, suspended and incubated under unfavourable conditions for growth and anabolism, disintegrate. Indeed autolysates of yeasts and other cells were some of the earliest sources of soluble enzymes, and did trojan work in the heroic days of the investigations of intermediary carbohydrate metabolism. With the development of the electron microscope and consequent ultrastructural awareness, cell walls were recognized, isolated and their chemistry studied. Autolysis could then be seen to be due to dissolution of the walls. As the chemical structure of the polymers making up the walls was elucidated, it became possible to designate the bonds specifically hydrolysed by various autolytic enzymes and thus to account for the solubilization of the walls and the disintegration of the cells. Since the supportive polymers in microbial cell walls are often either complex, such as, for example, are the peptidoglycans of bacteria or, consist of a number of polymers interacting to provide mechanical support for the cell as in some microfungi, autolysins with a variety of bond specificities are often to be found in the same cell. It is not always possible to distinguish which autolysin is responsible for any particular effect, or to exclude the necessity for the combined action of more than one enzyme.
The ctaBCDEF genes coding for cytochrome c oxidase were found to reside adjacent to a regulatory gene ctaA at 127° on the Bacillus subtilis chromosome. The structural genes for subunits I and II, ctaD and ctaC, were deleted by gene-replacement using a phleomycin-resistance marker. The mutant was unable to oxidize N,N,N′,N′-tetramethyl-p-phenylene-diamine and oxidized cytochrome c at a significantly lower rate. Absorption spectra of the mutant and wild-type membranes confirmed the presence of two haem A-containing enzymes in B. subtilis. Another mutant, with a spontaneous deletion upstream from CtaC, was found to express neither of these enzymes. Radioactive haem-labelling was used to identify sub-unit ii, which contains a haem C, and cytochrome c-550 among the membrane-bound c-type cytochromes of B. subtilis.
The ctaBCDEF genes coding for cytochrome c oxidase were found to reside adjacent to a regulatory gene ctaA at 127 degrees on the Bacillus subtilis chromosome. The structural genes for subunits I and II, ctaD and ctaC, were deleted by gene-replacement using a phleomycin-resistance marker. The mutant was unable to oxidize N,N,N',N'-tetramethyl-p-phenylene-diamine and oxidized cytochrome c at a significantly lower rate. Absorption spectra of the mutant and wild-type membranes confirmed the presence of two haem A-containing enzymes in B. subtilis. Another mutant, with a spontaneous deletion upstream from ctaC, was found to express neither of these enzymes. Radioactive haem-labelling was used to identify subunit II, which contains a haem C, and cytochrome c-550 among the membrane-bound c-type cytochromes of B. subtilis.
Cytochrome aa3 is one of two terminal oxidase complexes in the Bacillus subtilis electron transport chain. A novel genetic strategy was devised which permitted the isolation of B. subtilis mutants lacking cytochrome aa3 by selection for streptomycin-resistant clones which failed to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine. Two mutations were studied intensively. Spectroscopic examination showed that each mutant lacked cytochrome aa3; they were also asporogenous and unable to grow on lactate as the sole carbon and energy source. These mutations were mapped to a locus designated ctaA, located at 127 degrees between pyrD and metC on the B. subtilis chromosome. Both ctaA mutations were closely linked by transformation to the pycA locus. The ctaA locus and a portion of the pycA locus were cloned from a B. subtilis integration library constructed in Escherichia coli. A recombinant plasmid containing a 4.0-kilobase insert of B. subtilis DNA could transform both ctaA mutants to CtaA+. Gene disruption and complementation experiments with subcloned fragments revealed that the ctaA locus consisted of a single transcriptional unit about 1.35 kilobase pairs in size. The nucleotide sequence of the ctaA transcriptional unit contains a single open reading frame capable of coding for a protein with a predicted molecular weight of 34,065. The predicted protein is extremely hydrophobic, with several probable membrane-spanning domains. No sequence similiarity was found between ctaA and the highly conserved procaryotic and mitochondrial oxidase polypeptides. Cloning and sequence analysis of two ctaA mutations revealed that one allele is a nonsense mutation in the carboxy terminus and the other is a missense mutation in the amino terminus; this indicates that the pleiotropic phenotype conferred by each mutation was caused by loss of CtaA or of its activity. Genetic evidence suggests that the ctaA gene product is required as an accessory protein in the genetic expression, posttranslational biogenesis, or both, of the cytochrome aa3 complex and during an early stage of sporogenesis.
The nucleotide sequence of a 7-kilobase segment of the Bacillus subtilis chromosome containing the entire coding regions for the enzymes catalyzing the first three steps of diaminopimelate synthesis as well as dipicolinate synthase has been determined. This group of functionally related genes, termed the dap operon, were arranged in the order orfY, orfX, asd, dapG, and dapA and were bracketed by potential rho-independent transcription terminators. The asd locus could complement the growth defect of Escherichia coli strains with an asd deletion. Disruption of the dapG locus led to the loss of aspartokinase I, with a phenotype similar to that of the temperature-sensitive dapG mutants described earlier (Roten, C. A. H., Brandt, C., and Karamata, D. (1991) J. Gen. Microbiol. 137, 951-962). The amino acid sequences of the deduced products of the asd, dapG, and dapA loci had high degrees of similarity with those of other aspartate semialdehyde dehydrogenases, aspartokinases, and dihydrodipicolinate synthases, respectively. Disruption of orfX had no effect on growth but caused a sporulation defect, characterized by low sporulation frequencies and heat-sensitive spores, which could be cured by supplementation with dipicolinate, similar to the phenotype of mutants defective in spoVF, the putative structural gene for dipicolinate synthase. Two other open reading frames, upstream of spoVF, encoded the 380 COOH-terminal residues of a protein homologous to mitochondrial processing proteases and an 85-residue polypeptide of unknown function. Transcription initiation sites associated with the orfY-orfX-asd-dapG-dapA gene cluster were mapped by primer extension. The results indicate that during vegetative growth, the three distal genes of the dap operon, asd, dapG, and dapA, are transcribed as a unit and orfY and orfX are not expressed, whereas at stage 5 of sporulation two separate transcripts are produced, one comprising all five genes, the other just the three distal genes of the operon.
A group of temperature-sensitive lysis mutants of Escherichia coli K-12 was studied. Mutants impaired in the synthesis of uridine diphosphate-N-acetylmuramyl (UDP-MurNAc)-pentapeptide or in the synthesis of murein amino acids were found. Their rate of murein synthesis at the restrictive temperature was decreased. A large number of mutants did not differ from the parent strain with respect to the rate of murein synthesis and the precursor pattern. The behavior of these mutants is discussed. It was impossible to accumulate UDP-MurNAc-pentapeptide in E. coli by the antibiotics penicillin and vancomycin. The hypothesis is put forward that the amount of this murein precursor is regulated by feedback inhibition.
B. megaterium strain NCIB7581 grew at 30°C in a simple chemically defined medium without biotin. At 37°C the bacteria grew more slowly in the same medium and were deformed, especially during the earlier stages of growth, as filaments or with swollen ends. Addition of biotin restored the normal appearance and rate of growth at 37°C. The organisms synthesized more biotin at 30°C (11 ng l-1 in a stationary phase culture) than at 37°C (1.4 ng l-1). Addition of either aspartate, malate, fumarate or acetate to minimal medium also restored normal morphology and accelerated the growth rate in biotin-deficient medium at 37°C. Neither acetate nor aspartate increased synthesis of biotin at 37°C. After growth in medium containing only 0.2% glucose almost every organism in the culture was able to sporulate at 30°C whether or not biotin was added. About 50 times fewer spores were formed at 37°C without biotin; the yield of spores increased about 20-fold at this temperature when biotin, aspartate or dipicolinate was added.
A radical revision of the genetic map of E. coli K12 has been desirable for some time. Since the last review of mapping data, 200 new loci have been reported in the literature. In addition, an evaluation of the time of entry data now available indicates that the lengths of several major intervals on the map are significantly different from those that had been given in previous maps. In this review, the results of a basic recalibration of the map, derived mainly from time of entry and cotransductional data, are presented. Over 650 loci have been reviewed and assigned map locations. In addition, problems encountered in evaluation of the available data are discussed and changes in nomenclature are tabulated. The authors also include a discussion of the possible significance of the nonrandom distribution of gene loci on the map.
A thermosensitive sporulation mutant was used to determine the order of sporulation operonsin the urs region of the Bacillus subtilis chromosome. Data from three-factor transformation crosses and three- and four-factor transduction crosses established the order metC-SPO-96(SpoII)-spo-85(SpoV)-spo-279(SpoII)-furA-ura-cysC-spo-NG1.67(SpoIII). Previously, furA was thought to lie to the right of ura and cysC to the left (Dubnau, 1970; Young and Wilson, 1972).
Temperature sensitive mutants of E. coli JE1011 (derived from E. coli K-12), not forming cell walls at 42° were isolated and the genes of four of those mutants were mapped. Two cell wall-ts genes were located between ara and lac and the other two close to argH on the chromosomes.
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