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M D Yudkin

Publications and source records attributed to M D Yudkin.

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

Site of phosphorylation of SpoIIAA, the anti-anti-sigma factor for sporulation-specific sigma F of Bacillus subtilis.

Sigma F is regulated by an anti-sigma factor, SpoIIAB, and an anti-anti-sigma factor, SpoIIAA. SpoIIAB also functions as a phosphokinase which transfers phosphate from ATP to SpoIIAA; this phosphorylation is thought to be involved in the regulatory mechanism. By using [gamma-32P]ATP to phosphorylate SpoIIAA, cleaving the protein proteolytically, and analyzing the one resulting radiolabelled peptide by the Edman degradation procedure, we show that the site of phosphorylation in SpoIIAA is Ser-58.

Amino Acid Sequence↗
PubMed1994

Role of interactions between SpoIIAA and SpoIIAB in regulating cell-specific transcription factor sigma F of Bacillus subtilis.

Genetic experiments have suggested that sigma F, the first compartment-specific transcription factor in sporulating B. subtilis, is regulated by an anti-sigma factor SpoIIAB and an anti-anti-sigma factor SpoIIAA. Previously, we reported biochemical results demonstrating that SpoIIAB is both a phosphokinase whose substrate is SpoIIAA and an inhibitor of sigma F-directed transcription. We now show that in the presence of SpoIIAB and ATP or ADP, SpoIIAA can undergo two alternative reactions. When ATP is present, SpoIIAA is phosphorylated rapidly and completely to SpoIIAA-phosphate, and SpoIIAB is immediately released; but in the presence of ADP, SpoIIAA forms a long-lasting complex with SpoIIAB. ADP is an inhibitor of the phosphorylation by ATP. Furthermore, we have mutated SpoIIAA at residue Ser 58, the target for phosphorylation, to aspartate or alanine. SpoIIAAS58D, which apparently resembles SpoIIAA-phosphate, is unable to make a complex with SpoIIAB and is devoid of anti-anti-sigma F activity, whereas SpoIIAAS58A, which cannot be phosphorylated, makes complexes with SpoIIAB in the presence of ADP or ATP and has constitutive anti-anti-sigma F activity both in vivo and in vitro. It seems likely that the alternative reactions of SpoIIAA and SpoIIAB, involving ADP or ATP, regulate the anti-anti-sigma capacity of SpoIIAA and hence the activity of sigma F.

Adenosine Diphosphate↗
PubMed1993

Sigma F, the first compartment-specific transcription factor of B. subtilis, is regulated by an anti-sigma factor that is also a protein kinase.

The establishment of compartment-specific transcription in sporulating cells of B. subtilis is governed at the level of the activity of transcription factor sigma F. Genetic experiments have suggested that SpoIIAA and SpoIIAB, the other products of the sigma F operon, are involved in regulating sigma F activity. This activity is inhibited in the predivisional cell but specifically released from inhibition in the prespore about 1.5 hr after sporulation is induced. We now show that purified SpoIIAB inhibits transcription directed by sigma F in vitro. We note that the amino acid sequence of SpoIIAB shows some similarity to a group of bacterial histidine protein kinases, and we find that SpoIIAB is indeed a protein kinase that phosphorylates SpoIIAA on a serine residue. We suggest that this phosphorylation is responsible for the compartment-specific release of sigma F activity, perhaps through the formation of a tight complex between SpoIIAB and phosphorylated SpoIIAA.

Amino Acid Sequence↗
PubMed1993

Molecular and phenotypic characterization of promoter-proximal mutations in the spoIIA locus of Bacillus subtilis.

Eight mutations lying within the promoter-proximal one-fifth of the spoIIA locus of Bacillus subtilis were studied. Two of these mutations (spoIIAA42 and spoIIAA69) were previously characterized at the DNA level, five more (spo-562, spo-565, spo-567, spo-568, and spo-569) were isolated in our laboratory several years ago but not fully characterized, and the eight (an in-frame deletion confined to spoIIAA, the first gene in the spoIIA operon) was constructed for this study. DNA sequencing showed that spo-569 was a transitions in the -35 region of the spoIIA promoter; the remaining point mutations were all G:C to A:T transitions in spoIIAA, with spo-565 having two transitions, one of which was identical to that in spo-562. All the spoIIAA mutations except spo-562 led to the replacement of Gly residues. The incidence of sporulation, the rate of synthesis of sporulation-associated alkaline phosphatase, and the rate of expression of the forespore-specific genes gpr and spoIIIG were determined for isogenic strains carrying the eight mutations. All the mutations except spoIIAA42 and spo-569 (which were slightly leaky) made the strains asporogenous, and all except spo-562 and spo-569 abolished the synthesis of alkaline phosphatase and the expression of gpr and spoIIIG. spo-562 allowed alkaline phosphatase synthesis and gpr and spoIIIG expression to occur at about 15% of the wild-type rates but with normal kinetics. spo-59 allowed appreciable gpr and spoIIIG expression during exponential growth; we attribute this expression to transcription by RNA polymerase containing sigma G and suggest that a spo-569 strain makes insufficient SpoIIAB to inhibit sigma G in growing cells.

Amino Acid Sequence↗
PubMed1992

Structure and function of the spoIIIJ gene of Bacillus subtilis: a vegetatively expressed gene that is essential for sigma G activity at an intermediate stage of sporulation.

The spo-87 mutation is one of two sporulation mutations originally used to define the spo0J locus of Bacillus subtilis. We now show that it blocks sporulation after completion of prespore engulfment (stage III). Surprisingly, the operon is expressed vegetatively, probably from a sigma A-dependent promoter, and its expression is shut down at the transcriptional level at about the onset of sporulation. DNA sequencing reveals that the locus defined by spo-87, which we now designate spoIIIJ, consists of a bicistronic operon. However, only the first gene is essential for sporulation; the function of the second cistron is cryptic. The predicted SpoIIIJ product has an M(r) of 29,409. It probably forms a lipoprotein and is rich in basic and hydrophobic amino acids. Mutations in spoIIIJ abolish the transcription of prespore-specific genes transcribed by the sigma G form of RNA polymerase but not transcription of the spoIIIG gene encoding sigma G. The SpoIIIJ product could be involved in a signal transduction pathway coupling gene expression in the prespore to events in the mother cell, or it could be necessary for essential metabolic interactions between the two cells.

Amino Acid Sequence↗
PubMed1992

Activity of mutant sigma F proteins truncated near the C terminus.

sigma F, the product of the spoIIAC gene of Bacillus subtilis, is homologous in amino acid sequence throughout most of its length with several other sigma factors of B. subtilis and Escherichia coli. However, 8 residues from the C terminus the homology abruptly breaks down, suggesting that the C-terminal tail of the protein may be dispensable. It is known that an amber mutation at the 11th codon (wild-type glutamine 245) from the C terminus abolishes the function of the sigma factor. We have now placed chain-terminating codons at the ninth codon (wild-type lysine 247), the eighth codon (wild-type valine 248), or the seventh codon (wild-type glutamine 249) from the C terminus. We have tested the resulting mutants for their capacity to sporulate and for their ability to transcribe from a promoter (spoIIIG) that is normally read by RNA polymerase bound to sigma F (E sigma F). The results indicate that a mutant sigma F lacking the terminal 7 residues functions almost normally, which suggests that glutamine 249 is dispensable. By contrast, lysine 247 is crucial for the activity of sigma F: deletion of the 9 C-terminal residues totally inactivates the protein. When the terminal 8 residues were deleted, placing lysine 247 at the C terminus, the transcriptional activity of the factor is reduced by about 80%: we attribute this effect to neutralization of the positive charge of lysine 247 by formation of a salt bridge with the -COO- terminus.

Amino Acid Sequence↗
PubMed1991

Identification of the promoter and the transcriptional start site of the spoVA operon of Bacillus subtilis and Bacillus licheniformis.

The region upstream of the coding sequence of the spoVA operon was studied by several techniques to identify the promoter and to determine the start point for transcription of spoVA. The results of plasmid integration analysis in Bacillus subtilis showed that no more than 119 bp upstream of the coding sequence is needed for expression. A comparison of the sequence of this upstream region with the corresponding sequence from Bacillus licheniformis showed four stretches that were perfectly conserved, interspersed with poorly conserved stretches; the second and third of these conserved stretches appeared to represent the '-35' and '-10' regions of a promoter recognized by RNA polymerase containing sigma G. Primer extension analysis in B. subtilis revealed a spoVA transcript which had apparently initiated 6 bp downstream of the putative '-10' heptanucleotide CATACTA, that is, 27 bp upstream of the coding sequence. This transcript was observed 4 h and 5 h after the initiation of sporulation, but not at earlier times.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of the Bacillus licheniformis homologue of the sporulation locus spoIIA of Bacillus subtilis.

The spoIIA locus of Bacillus licheniformis was cloned into the phage vector phi 105J9; selection was based on the ability of the clone to complement mutations in both the first and the last of the spoIIA genes of B. subtilis. The B. licheniformis DNA was subcloned into M13 and sequenced; it includes three genes of identical lengths to those of B. subtilis. The average interspecies difference in nucleotide sequence is 24%; C occurs at the third position of codons 55% more often in the B. licheniformis than in the B. subtilis sequence. The average difference in predicted amino acid sequence is 11%, but the distribution of these differences is far from random, and there are several long stretches of amino acid sequence that are identical in the two organisms. The distribution of non-conservative amino acid changes between the species is also strikingly non-random; no such changes are found in those regions in which missense mutations are known in B. subtilis. Each species has an open reading frame 5' of the spoIIAA gene, but the predicted amino acid sequence, and the distribution of differences between the two species in both nucleotides and predicted amino acids, suggest that these open reading frames are not expressed. Both species have regions 3' of the open reading frames which resemble rho-independent terminators of transcription, but that in B. licheniformis is longer and could form a more elaborate secondary structure than that in B. subtilis.

Amino Acid Sequence↗
PubMed1989

Mutations that alter the helix-turn-helix region of the spollAC protein: a Bacillus subtilis sporulation-specific sigma factor.

It has previously been shown that spollAC561, a mutation that diminishes the incidence of sporulation by more than six orders of magnitude, alters the residue at position 13 of the helix-turn-helix region of the sporulation-specific sigma factor encoded by spollAC from valine to methionine (Yudkin, 1987b). We have now found that four spontaneous revertants, which sporulate at an incidence of 30-60%, all contain transitions within the codon that was altered by spo-561. The mutant methionine is replaced by isoleucine in two revertants, and by threonine in the other two.

Amino Acids↗
PubMed1988

Phenotypic and genetic characterization of mutations in the spoIVC locus of Bacillus subtilis.

The spoIVC locus of Bacillus subtilis was analysed. Fourteen spoIVC mutants isolated following nitrosoguanidine mutagenesis were used along with two previously characterized spoIVC mutants to construct a fine structure genetic map of the locus. The recombination index (RI) measured between extreme mutations was 0.26; no recombination could be detected between four of the mutations. Complementation analysis showed that all the mutations fall into two cistrons. The RI between extreme mutations in cistron A was about 0.17 and that between extreme mutations in cistron B was about 0.05. In respect of biochemical markers, the spoIVC mutations all produced similar phenotypes, irrespective of their location. However, in both cistrons oligosporogenous and asporogenous mutations mapped close together.

Bacillus subtilis↗
PubMed1987

Effect of precisely identified mutations in the spoIIAC gene of Bacillus subtilis on the toxicity of the sigma-like gene product to Escherichia coli.

Yudkin (1986) has shown that the spoIIAC gene of Bacillus subtilis cannot be cloned in Escherichia coli in such an orientation that it is expressed. This toxicity of the gene product has been attributed to its close homology with the sigma subunit of the E. coli RNA polymerase. The effect of six individual mutations in spoIIAC has now been studied. All six mutant genes could be cloned in E. coli in an orientation that does not allow expression. When in the orientation that permits expression, one mutant gene could not be cloned, and a second substantially hampered growth; both mutations lie in the region that is believed to encode the DNA-binding domain of the protein. By contrast, two missense mutations in the region of the gene thought to encode the domain that binds to the core RNA polymerase rendered the protein harmless in E. coli, as did two nonsense mutations.

Bacillus subtilis↗
PubMed1987

Structure and function in a Bacillus subtilis sporulation-specific sigma factor: molecular nature of mutations in spoIIAC.

The spoIIAC gene was cloned from chromosomal DNA of seven spoIIAC mutants of Bacillus subtilis, and the complete sequence of the gene was determined for each mutant. Three of the mutants proved to have chain-terminating mutations (one of which, previously shown to be suppressible by sup-3, was identified as amber); these led in every case to complete failure either to manufacture spores or to synthesize two enzymes normally associated with stage II of sporulation. The four remaining mutations were missense, and these corresponded to a phenotype in which a few spores are formed and about half the wild-type quantities of the two enzymes are made. Of the four missense mutations, two were near the promoter-distal end of the gene, in a region believed to correspond to the DNA-binding domain of the sigma factor that spoIIAC encodes. The remaining two mutations were in the region of the gene that is thought to correspond to the domain of the protein that interacts with core RNA polymerase.

Bacillus subtilis↗
PubMed1985

Effects of transition mutations in the regulatory locus spoIIA on the incidence of sporulation in Bacillus subtilis.

We have determined the changes in DNA sequence corresponding to three mutations in the promoter-proximal open reading frame of spoIIA, a locus that regulates sporulation in Bacillus subtilis. All three mutations prevent the synthesis of two sporulation-associated enzymes, but they differ in their effects on spore incidence. We now find that mutation spo-42, which allows spores to be produced at a low incidence, is a transition that changes Gly95 to Asp in the protein encoded by the open reading frame. Mutation spo-69, which blocks sporulation entirely, consists of two transitions: these change Gly62 to Asp and Ala1 16 to Thr. Mutation sas-1, which partially suppresses spo-69, is also a transition: this changes residue 62 (which had become Asp as a result of the spo-69 mutation) to Asn.

Bacillus subtilis↗
PubMed1984

Tryptophanase synthesis in Escherichia coli: the role of indole replacement in supplying tryptophan and the nature of the constitutive mutation tnaR3.

The properties of the tryptophanase constitutive mutation tnaR3 have been investigated. It has previously been reported that mutants carrying tnaR3 grow poorly on medium that selects for constitutive expression of tryptophanase. Our results now show that this poor growth can be explained by the inability of tryptophanase to catalyse the synthesis of L-tryptophan from indole, pyruvate and ammonia at a rate sufficient to allow normal growth. Improved media for the characterization of tryptophanase constitutive mutants are described. The mutation tnaR3 rendered tryptophanase synthesis constitutive (at a different rate that at 37 degrees C is 30% of the fully induced wild-type) and not further inducible. Diploid studies showed that tnaR3 is cis-dominant, but no evidence was found for any effect in trans. In addition to rendering tryptophanase synthesis constitutive, tnaR3 affects the response of tryptophanase synthesis to different growth temperatures.

Escherichia coli↗
PubMed1982

Location of the gene for the low-affinity tryptophan-specific permease of Escherichia coli.

L-Tryptophan uptake was assayed under conditions in which the aroT gene had been inactivated by deletion and the product of the aroP permease was competitively inhibition. A mutant carrying a deletion from bgl through tnaA showed negligible L-tryptophan uptake, in contrast to a strain possessing an intact tna region or to strains carrying point mutations in tna. The ability to take up L-tryptophan was not restored by lysogenizing the tna-deleted strain with lambda tna+.

Amino Acid Transport Systems↗
PubMed1980

Identification of a positive regulatory protein in Escherichia coli: the product of the cysB gene.

From the specialized transducing bacteriophage lambdacysB, recombinant phages lambdacysB242 and lambdacysB257 have been obtained, each of which carries an amber mutation in the cysB cistron. A comparison of polyacrylamide gel electrophoretic profiles of labelled extracts from uv-irradiated bacteria that had been infected with lambdacysB+ or with lambdacysB-amber phages, led to the identification of a 39,000-dalton polypeptide as the product of the cysB gene. The native protein was purified to near radiochemical purity and was found to be an oligomer with an isoelectric point close to pH 7.

Bacterial Proteins↗
PubMed1980

Characterization of lambda cysB and of two derivatives carrying cysB amber mutations.

The specialized transducing phage lambda cysB (Borck et al., 1976) was found to carry about 5 kilobases of Escherichia coli DNA. It was shown to have an intact cysB gene but none of the known neighbouring genetic loci. The phage (which is known to be deficient in its site-specific recombination functions) was shown to integrate into the chromosome of bacterial recipients at the cysB locus. Excision from this site occasionally generated recombinant phages that had exchanged their cysB allele for the one originally present in the host. In this way lambda cysB derivatives were prepared from lysogens of two strains carrying the amber mutations cysB242 and cysB257; these phases were proved by several tests to contain the expected cysB amber mutations.

Alleles↗