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D Karamata

Publications and source records attributed to D Karamata.

At least 37 records · Page 2Linked to original sources
PubMed1995

A periplasm in Bacillus subtilis.

The possibility of there being a periplasm in Bacillus subtilis, in the distinct cell compartment bounded by the cytoplasmic membrane and the thick cell wall, has been investigated quantitatively and qualitatively. Cytoplasmic, membrane, and protoplast supernatant fractions were obtained from protoplasts which were prepared isotonically from cells grown under phosphate limitation. The contents of the protoplast supernatant fraction represent an operational definition of the periplasm. In addition, this cell fraction includes cell wall-bound proteins, exoproteins in transit, and contaminating cytoplasmic proteins arising through leakage from, or lysis of a fraction of, protoplasts. The latter, measured by assay of enzyme markers and by radiolabeled RNA and protein, was found to represent 7.6% of total cell protein, yielding a mean of 9.8% +/- 4.8% for B. subtilis 168 protein considered periplasmic. Qualitatively, after subjection of all cell fractions to polyacrylamide gel electrophoresis, RNase and DNase, zymographs revealed that (i) each cell fraction had a unique profile of nucleases and (ii) multiple species and a major fraction of both nucleases were concentrated in the periplasm. We conclude that the operationally defined periplasmic fraction corresponds closely, both quantitatively and qualitatively, to the contents of the periplasm of Escherichia coli. We discuss evidence that the maintenance of the components of this surface compartment in B. subtilis is compatible with the thick negatively charged cell wall acting as an external permeability barrier.

Bacillus subtilis↗
PubMed1994

Specific labeling of diaminopimelate: a radioassay for the determination of the peptidoglycan cross-linking index.

A radioassay for the determination of the peptidoglycan cross-linking index (CLI) was devised. It is based on specific radioactive labeling of diaminopimelic acid (DAP) by diverting [14C]aspartate into the DAP pathway, while inhibiting incorporation of label into other cell wall components. Purified [14C]DAP-labeled cell walls were treated with fluorodinitrobenzene, hydrolyzed, and chromatographed by TLC. The radioactivity in well-separated mono dinitrophenyl-diaminopimelate (DNP-DAP) and DAP spots was counted and the CLI was determined from the ratio of DAP to the total of mono DNP-DAP and DAP counts. The method, suitable for bacteria such as Bacillus subtilis unable to incorporate exogenous DAP, can be applied to other systems. A CLI of 50.8 +/- 1.3% and 55.5 +/- 0.9% was obtained for B. subtilis 168 cells growing exponentially in rich and minimal medium, respectively. Comparison of these to results previously obtained on B. subtilis suggested the existence of a hitherto unreported peptidoglycan endopeptidase activity.

Bacillus subtilis↗
PubMed1994

Characterization of the chromosomes of Bacillus subtilis merodiploid strains by quantitative DNA-DNA hybridization.

The position of junctions and the extent of the duplicated chromosomal regions in Bacillus subtilis merodiploid strains were studied by quantitative DNA-DNA hybridization. We describe a method which allows (i) the identification of genes present in two copies per chromosome and (ii) the measurement of the amount of additional DNA in chromosomes with relatively large duplicated regions (about 10% or more). Analysis of previously described B. subtilis merodiploid strains GSY1127, GSY1800 and GSY1835 revealed that the duplicated segments represent 29 +/- 2%, 7 +/- 2% and 13 +/- 2% of the chromosome, respectively. Small discrepancies between these and previous genetic linkage data are discussed. Support for a role of prophage SP beta in the formation of merodiploid GSY1835 is provided. In conclusion, the described method confirmed the genetic maps of the merodiploids previously obtained by transduction and transformation crosses and showed that a duplication of a segment is not accompanied by large deletions of other chromosomal regions, providing direct evidence that a cell can accommodate genomes of substantially increased size.

Bacillus subtilis↗
PubMed1994

A rapid and simple method for Bacillus subtilis transformation on solid media.

Cells of Bacillus subtilis strains 168 and W23 deprived of an amino acid or a base on a given solid medium were found to develop competence. We describe a rapid and simple method of genetic transformation of this organism consisting in spreading a sample containing 1 microgram DNA and 10(7) exponentially growing cells of an auxotrophic mutant onto plates devoid of the required amino acid or base. After overnight incubation, about 100-200 prototrophic transformants per plate were obtained, i.e. a frequency of about 10(-5), as compared to 10(-4) routinely obtained by the method of transformation in liquid medium with frozen competent cells. Plasmids and other chromosomal or plasmid-borne markers, which cannot be directly selected for, were transferred by congression. The dependence of the transformation efficiency on cell density, medium richness, incubation time and the nature of transforming DNA was investigated. We conclude that the development of competence accompanies amino acid or base starvation of cells under appropriate physiological conditions.

Bacillus subtilis↗
PubMed1994

Lytic enzymes associated with defective prophages of Bacillus subtilis: sequencing and characterization of the region comprising the N-acetylmuramoyl-L-alanine amidase gene of prophage PBSX.

Prophage induction in Bacillus subtilis strains 168, S31 and W23 is accompanied by synthesis of two endolysins. The synthesis of those of strain 168, with molecular masses of 32 and 34 kDa, was shown to be controlled by the repressor of the defective phage PBSX. The 32 kDa protein corresponds to an N-acetylmuramoyl-L-alanine amidase, and plays the major role in PBSX-mediated lysis. Its structural gene, xlyA, is the last in the PBSX late operon, whose four most distal open reading frames have been cloned and sequenced. Analysis of the nucleotide sequence suggests that the two open reading frames preceding xlyA, designated xhlA and xhlB, encode polypeptides whose combined action could play the role of a holin. The open reading frame upstream of xhlA, designated xepA, encodes an exoprotein. The phage amidase, although endowed with a signal peptide, is apparently, like Xep, exported by a holin-like mechanism which does not involve the cleavage of the signal peptide. The presence on the B. subtilis chromosome of other, similar, genes, and their possible widespread occurrence, is discussed.

Amino Acid Sequence↗
PubMed1994

Analysis of Bacillus subtilis tag gene expression using transcriptional fusions.

Five of the genes known to encode the synthesis of poly(glycerol phosphate), the major teichoic acid of Bacillus subtilis 168, are organized in two divergently transcribed operons (a divergon), denoted tagAB and tagDEF. To monitor their expression, the 399 bp intergenic region separating the first structural genes of these operons was fused, in both orientations, to a lacZ reporter gene, allowing measurement of promoter activity under specific physiological conditions. Under all experimental conditions, tagA and tagD appeared coordinately expressed, the level of tagD being always higher than that of tagA. No influence of the chromosomal context was observed. Phosphate limitation was accompanied by reduced tag gene expression. Following the onset of sporulation, expression of tag genes diminished rapidly and was essentially abolished by stage II. During germination, the activity of tag genes was detectable before the rise in culture turbidity associated with spore outgrowth. In contrast to tagC (dinC), the expression of which is DNA-damage-inducible, the induction of SOS functions had no effect on tagA and tagD gene expression. The biological significance of these results is discussed.

Bacillus subtilis↗
PubMed1994

The gene of the N-acetylglucosaminidase, a Bacillus subtilis 168 cell wall hydrolase not involved in vegetative cell autolysis.

lytD, the structural gene of the Bacillus subtilis 168 N-acetylglucosaminidase was localized at 310 degrees, next to the tagABC operon. Sequence analysis revealed a monocistronic operon encoding a 95.6 kDa protein endowed with an export signal, the cleavage of which yields the monomer polypeptide (92.8 kDa) of the dimeric active form of the enzyme. Transcription is initiated at a sigma-D (sigma D)-dependent promoter and ends at a terminator common to lytD and the divergently transcribed tagABC operon. In addition, we report the sequence of the adjacent upstream ORF, transcribed in the same direction as lytD, which shows significant homology to phosphomannose isomerase-encoding genes. Cell separation, motility, autolysis, cell wall turnover and growth were not affected in strains devoid of the N-acetylglucosaminidase. A mutant deficient in the two most abundant autolysins, i.e. the LytC amidase and the glucosaminidase, exhibited the phenotype of the amidase-deficient strains, revealing their non-requirement for growth. This conclusion raises two fundamental questions: how does the cell undo the highly cross-linked peptidoglycan so as to be able to grow, and what is the role of the considerable amount of autolysin normally present? Possible answers to these questions are discussed.

Acetylglucosaminidase↗
PubMed1993

Sequencing and analysis of the divergon comprising gtaB, the structural gene of UDP-glucose pyrophosphorylase of Bacillus subtilis 168.

Nucleotide sequencing revealed that gtaB, the structural gene of UDP-glucose pyrophosphorylase (EC 2.7.7.9), is part of a divergon-like genetic entity. The latter consists of two monocistronic operons gtaB and orfX, transcribed from a 245 bp regulatory region, each encoding an acidic protein with a molecular mass of 33.0 and 42.6 kDa, respectively. gtaB is transcribed from a distal PA promoter, and a proximal PB promoter which is negatively controlled by the Sin protein. Sin-mediated transcriptional attenuation and enhancement of PB and PD, respectively, suggest that these promoters control functions which antagonize each other. Transcription of orfX is mediated by a PA promoter. The regulatory region comprises four ATGAAA hexamers, present as two inverse repeats. Protein GtaB exhibits high homology to analogous prokaryotic enzymes, while OrfX shows 55.4% homology with the product of Escherichia coli o389, which is part of a regulatory unit involved in sugar processing. Mutations gtaB515 and gtaBg100, which define different bacteriophage adsorption patterns, were sequenced. They are transitions leading to substitution of amino acids which occupy conserved positions, and are thus likely to be part of an enzyme active site. The nature of the possible receptors for defective bacteriophages PBSY and PBSZ is discussed.

Alleles↗
PubMed1992

Endogenous synthesis of peptidoglycan in eukaryotic cells; a novel concept involving its essential role in cell division, tumor formation and the biological clock.

Degradation products of peptidoglycan, the universal bacterial cell wall constituent, were previously found in animal tissues and urine. Reassessment and quantitative analysis of available data lead to an original concept, i.e. that eukaryotic cells synthesize peptidoglycan. We present a model in which this endogenously synthesized peptidoglycan is essential for the processes of eukaryotic cell division and sleep induction in animals. Genes for peptidoglycan metabolism, like those for lysine biosynthesis in plants, are probably inherited from endosymbiotic bacteria, the ancestors of mitochondria and chloroplasts. Corollaries of this concept, i.e. roles for peptidoglycan metabolism in tumor formation and in the biological clock, are supported by abundant evidence. We propose that many interactions between bacteria and eukaryotes are conditioned by their common genetic heritage.

Animals↗
PubMed1992

Identification of the structural genes for N-acetylmuramoyl-L-alanine amidase and its modifier in Bacillus subtilis 168: inactivation of these genes by insertional mutagenesis has no effect on growth or cell separation.

The region of the Bacillus subtilis 168 chromosome that contains the structural genes for the major vegetative cell autolysin, (N-acetyl-muramoyl-L-alanine amidase), and its modifier protein has been cloned. Insertional mutagenesis with integrative plasmids carrying small DNA fragments from this region has revealed that both genes are located on a 4 kb fragment; they are organised in one transcription unit, the modifier being transcribed first. Studies of derivatives in which either the amidase or the modifier or both proteins are inactivated have revealed that amidase-deficient strains are not affected in growth, cell separation, transformability or sporulation. Observed phenotypic differences were altered kinetics of, cell wall turn-over and a reduced rate of, autolysis of native cell wall preparations. A residual amidase activity, about 3% of that of the wild-type strain, was found in strains devoid of the major amidase. A new, distinct cell wall-bound protein, designated CWBP49', with the same molecular weight as the amidase, was identified in mutants devoid of the latter enzyme.

Bacillus subtilis↗
PubMed1992

Ploidy of Bacillus subtilis exfusants: the haploid nature of cells forming colonies with biparental or prototrophic phenotypes.

To investigate the relationship between DNA content and cell volume, we have attempted to repeat the construction of stable Bacillus subtilis diploid cells through protoplast fusion. Colonies with a biparental phenotype and those with a prototrophic phenotype were identified among exfusants of a cross between two polyauxotrophic strains. The ploidy of cells constituting such colonies was assessed by protoplast self-fusion, determination of the DNA to dry weight ratio of exponentially growing cells, and by quantitative DNA-DNA hybridization. Within the precision of these methods, all colonies were found to consist of haploid cells. A previously described non-complementing diploid was also found to be haploid. Therefore, the genetic evidence in favour of diploidy, based on continuing segregation of cells with a parental or recombinant phenotype, cannot be accounted for except by the maintenance of such cells as a minority population in mixed colonies through cross-feeding. Reconstruction experiments with mixtures of whole parental cells confirm that biparental colonies are indeed mixed colonies which arise either by sticking of parental cells or through coincidence, i.e. their plating within a distance of about 0.4 mm. The previously reported experimental results can be accounted for in the light of our results.

Bacillus subtilis↗
PubMed1992

Sequencing and analysis of the Bacillus subtilis lytRABC divergon: a regulatory unit encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and its modifier.

The regulatory unit of Bacillus subtilis strain 168 encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and of its modifier has been sequenced, and found to be a divergon consisting of divergently transcribed operons lytABC and lytR. Proteins LytA, LytB and LytC are endowed with export signal peptides. Mature LytA is a 9.4 kDa, highly acidic polypeptide whose deduced amino acid sequence points to a lipoprotein. LytB and LytC, the modifier and the amidase, are highly basic. After cleavage of the signal sequence their molecular masses are 74.1 and 49.9 kDa, respectively. These two proteins share considerable homology in their N-terminal moieties and have three GSNRY consensus motifs, characteristic of nearly all amidases. The C-terminal moiety of LytB exhibits homology to the product of spoIID. LytR is a 35 kDa protein which acts as an attenuator of the expression of both lytABC and lytR operons. Transcription of the lytABC operon proceeds from two promoters: PD, identified as P28-7 (Gilman et al., 1984), and an upstream PA. The former only is subject to LytR attenuation. Translational initiation of lytB and lytC is directed by UUG start codons, suggesting that lytA, B and C undergo coupled translation. Transcription of lytR is initiated at two start sites, one of which corresponds to a highly intense PA promoter whereas the other does not seem to share much homology with any of the known promoter consensus sequences. Both promoters are attenuated by LytR. It is confirmed that the synthesis of the amidase is controlled at least in part by SigD, i.e. that it belongs to the fla regulon and that its activity, or part of it, is co-regulated with flagellar motility. The role of the mutations conferring the Sin, Fla and Ifm phenotypes in the expression of the lytABC operon is discussed.

Amino Acid Sequence↗
PubMed1992

CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase, which is involved in the synthesis of the major wall teichoic acid in Bacillus subtilis 168, is encoded by tagF (rodC).

Assays of CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase (CGPTase) (EC 2.7.8.12) in membranes isolated from Bacillus subtilis 168 wild type and 11 strains bearing conditional lethal thermosensitive mutations in tagB, tagD, or tagF revealed that CGPTase deficiency was associated only with mutant tagF alleles. In vitro, thermosensitivity of CGPTase strongly suggests that the structural gene for this enzyme is tagF. We discuss apparent discrepancies between biochemical evidence favoring a membrane location for TagF and a previous report that suggested a cytoplasmic location based on sequence analysis.

Bacillus subtilis↗
PubMed1991

N-acetylmuramoyl-L-alanine amidase assay based on specific radioactive labeling of muropeptide L-alanine: quantitation of the enzyme activity in the autolysin deficient Bacillus subtilis 168, flaD strain.

A sensitive and highly reproducible assay for N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) was devised, based on specific and homogeneous L-[14C]alanine labeling of the substrate, the peptidoglycan. The method involves partial purification of both the enzyme and the substrate and monitoring the muropeptide cleavage by coupling fluorodinitrobenzene to freed L-alanine NH2 groups. After acid hydrolysis of the substrate, the resulting DNP-L-alanine and L-alanine are separated by TLC, and radioactive counts in relevant spots are determined. Application of the method to the autolysin-endowed strain and an autolysis-deficient flaD-bearing mutant has revealed (i) that the N-acetylmuramoyl-L-alanine amidase behaves like an endoenzyme with an apparent Kcat(s-1) of 40, and (ii) that the residual enzyme activity in the flaD bearing strain amounts to 2.5 (+/- 0.1)% of that of the parent strain.

Alanine↗
PubMed1991

A conditional-lethal mutant of bacillus subtilis 168 with a thermosensitive glycerol-3-phosphate cytidylyltransferase, an enzyme specific for the synthesis of the major wall teichoic acid.

A biochemical analysis was undertaken of thermosensitive mutants of Bacillus subtilis 168 harbouring mutations in several tag genes, involved in the synthesis of the major wall teichoic acid, poly(glycerol phosphate), poly(groP). Incorporation of a pulse of [2-3H]glycerol into whole cells, following shift to the restrictive growth temperature, was used to assess synthesis of this polymer and to seek evidence of accumulation of a specific precursor. The rate of incorporation into poly(groP) was strongly decreased in all mutants; glycerol uptake was diminished by 80% or more for a strain harbouring mutation tagB1 (formerly tag-1) and one bearing tagD11 (formerly tag-11). The pool of CDP-glycerol (CDP-gro), a specific precursor of poly(groP), was increased, relative to the wild-type, for all mutations except tagD11, where the pool of CDP-gro was reduced. Cytoplasmic extracts, assayed at the permissive temperature for glycerol-3-phosphate cytidylyltransferase (gro-PCT), the enzyme synthesizing CDP-gro, revealed wild-type activities for all mutations except tagD11. Gro-PCT activity in the latter strain was 100-fold lower and, unlike that in all other mutant strains, highly thermolabile. This thermosensitivity suggests that tagD encodes gro-PCT. The identification, in a gene encoding a poly(groP)-specific enzyme, of a mutation conferring a thermosensitive growth phenotype renders explicit the conclusion that synthesis of this teichoic acid is essential for the growth of B. subtilis.

Bacillus subtilis↗
PubMed1991

Genes concerned with synthesis of poly(glycerol phosphate), the essential teichoic acid in Bacillus subtilis strain 168, are organized in two divergent transcription units.

Insertional mutagenesis has revealed that a 22 kbp segment from the hisA region of the Bacillus subtilis 168 chromosome (310 degrees on the genetic map) contains at least six independent transcription units, all apparently devoted to production of cell envelope components. Genes concerned with synthesis of poly(glycerol phosphate), poly(groP), an essential cell wall polymer in B. subtilis 168, are organized in two divergently transcribed operons denoted tagABC and tagDEF. Nucleotide sequence analysis indicates that three of these six genes encode extremely basic polypeptides. The deduced products of the tagABC operon may be involved in poly(groP) assembly and export, whereas those of the tagDEF operon, which are very hydrophilic, are more likely to be implicated in poly(groP) precursor biosynthesis. The first gene of the tagDEF operon encodes glycerol-3-phosphate cytidylyltransferase (Pooley et al., 1991, Journal of General Microbiology 137, 921-928) and its deduced product has significant homology with cholinephosphate cytidylyltransferase from yeast. There is also substantial homology between the deduced products of tagB in the tagABC operon and tagF in the tagDEF operon.

Amino Acid Sequence↗
PubMed1991

Genetic and biochemical characterization of Bacillus subtilis 168 mutants specifically blocked in the synthesis of the teichoic acid poly(3-O-beta-D-glucopyranosyl-N-acetylgalactosamine 1-phosphate): gneA, a new locus, is associated with UDP-N-acetylglucosamine 4-epimerase activity.

The resistance spectrum to bacteriophage phi 3T of different Bacillus subtilis 168/W23 strains hybrid for wall teichoic acids suggested that poly(3-O-beta-D-glucopyranosyl-N-acetylgalactosamine 1-phosphate), a so-called minor teichoic acid of strain 168, forms part of the receptor for this phage, and a serologically related group of phages. A representative sample of 25 mutants specifically resistant to phi 3T, obtained from a mutagenized culture by direct selection, were all found to have a greatly reduced galactosamine content. Relevant mutations in these strains were shown by PBS1 transduction and transformation to belong to two linkage groups; a minority, associated with an atypical colony morphology, were localized between sacA and purA, whereas the majority mapped between gtaB and tagB1 (formerly tag-1), a region containing all known genes involved in the synthesis of the major wall teichoic acid, poly(glycerol phosphate). The former mutations mapped in a new locus, gneA, characterized by a deficiency in UDP-N-acetylglucosamine 4-epimerase, while the latter ones, as well as the previously identified pha-3 (Estrela et al., 1986, Journal of General Microbiology 132, 411-415), map is a locus named gga. They are likely to affect membrane-bound enzymes involved in the synthesis of the galactosamine-containing teichoic acid. A possible biological role of this polymer is discussed.

Adsorption↗