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P Youngman

Publications and source records attributed to P Youngman.

At least 37 records · Page 2Linked to original sources
PubMed1996

SpoIIE mutants of Bacillus subtilis comprise two distinct phenotypic classes consistent with a dual functional role for the SpoIIE protein.

Mutations in the spoIIE locus of Bacillus subtilis block sporulation at the stage of asymmetric septation and prevent compartment-specific activation of the transcription factor delta F. Recent ultrastructural studies of spoIIE mutants led to the conclusion that cells blocked at the stage of asymmetric septation form primarily thick septal structures similar to those formed at the mid-cell site during vegetative growth, although in an earlier study Piggot (J. Bacteriol. 114:1241-1253, 1973) clearly detected a more complex range of phenotypes. We have examined the phenotypes of six spoIIE mutants, including one example of the previously studied null type, spoIIE21. We confirmed that the spoIIE21 mutant and two other null mutants exhibit the classic thick-septum phenotype. However, two of the missense mutants, the spoIIE64 and spoIIE71 mutants, were found to display a strikingly different phenotype characterized by the presence of only thin asymmetric septa, frequently at both polar positions, as noted by Piggot. This phenotype is essentially identical to those of spoIIA (delta F) and spoIIG (delta E) null mutants, which also form sporulation septa that appear structurally normal at the level of electron microscopy. Despite the formation of apparently normal asymmetric septa, spoIIE64 and spoIIE71 mutants are fully defective in activation of delta F-dependent gene expression. These results indicate that the functional roles performed by SpoIIE in septum assembly and sigma factor regulation are distinct and separable.

Bacillus subtilis↗
PubMed1994

Characterization of spo0A homologues in diverse Bacillus and Clostridium species identifies a probable DNA-binding domain.

Spo0A is a phosphorylation-activated transcription factor of Bacillus subtilis. It is a member of the response regulator superfamily of bacterial signal transduction proteins and controls many of the changes in gene expression that occur during the transition into stationary phase and during the initiation of sporulation. To identify the domains of Spo0A most critical for determining its structural and functional features, presumptive homologues of the spo0A gene were characterized in a collection of eight Bacillus species and six Clostridium species representing phylogenetically diverse members of these genera. An alignment of the partial or complete DNA sequences of these homologues revealed three regions of especially high conservation in the effector domain. We speculate that the most highly conserved of these corresponds to the recognition helix of a putative helix-turn-helix motif, and, therefore, represents the actual DNA-containing surface of the protein. In the case of homologues identified in Bacillus anthracis and Clostridium acetobutylicum and retrieved by polymerase chain reaction amplification, we confirmed by gene-disruption analysis that the homologue actually is required for initiation of sporulation. Apparent homologues of the B. subtilis spoIVB gene were also discovered immediately upstream from the spo0A homologues in all Bacillus and Clostridium species examined. The discovery of homologues of B. subtilis sporulation genes in these diverse species implies that the gene products required for specifying pathways of sporulation-specific gene activation and for determining key morphogenetic changes may be highly conserved and suggests that an approach similar to that undertaken here might be used as a general strategy to retrieve and compare their gene sequences. Exhaustive efforts to detect a spo0A-like gene in non-endospore formers, including close relatives of Bacillus such as Listeria and Staphylococcus, were uniformly unsuccessful, suggesting that regulation of gene activity during the transition into stationary phase mediated by Spo0A-like proteins may be exclusive to the endospore-forming bacteria.

Amino Acid Sequence↗
PubMed1994

Phosphorylation of Bacillus subtilis transcription factor Spo0A stimulates transcription from the spoIIG promoter by enhancing binding to weak 0A boxes.

Activation of the spoIIG promoter at the onset of sporulation in Bacillus subtilis requires the regulatory protein, Spo0A, which binds to two sites in the promoter, sites 1 and 2. Phosphorylation of Spo0A is essential for the initiation of sporulation. Therefore, we examined the role of Spo0A phosphorylation in spoIIG promoter activation. Phosphorylation of Spo0A stimulated transcription from the spoIIG promoter in vitro. In DNAse I footprinting experiments with the spoIIG promoter, we found that phosphorylation of Spo0A increased its affinity for site 2 more than for site 1, which is the site to which nonphosphorylated Spo0A binds most avidly. This result could not be explained by increased cooperativity between Spo0A bound at sites 1 and 2 because the increased affinity for site 2 by phosphorylated Spo0A was also observed with a deletion derivative of the spoIIG promoter containing only site 2. We have located Spo0A-binding sequences in the spoIIG promoter by DMS protection assays and mutational analysis, and found that site 1 contains one higher-affinity binding sequence whereas site 2 contains two weaker-binding sites. Two substitutions in site 2 of the spoIIG promoter that change the sequence to be more like an optimal Spo0A-binding site were found to increase promoter activity. Moreover, phosphorylation of Spo0A was not required in vivo for activation of the spoIIG promoter containing these strong binding sites. The results suggest that the primary role for phosphorylation of Spo0A is to increase its affinity for specific sites rather than to activate an activity of Spo0A that acts on RNA polymerase at promoters.

Bacillus subtilis↗
PubMed1993

Physical and functional characterization of the Bacillus subtilis spoIIM gene.

The spoIIM locus of Bacillus subtilis is the most recently discovered of six genetic loci in which mutations can prevent the synthesis of a normal asymmetric septum or prevent migration of the septal structure to engulf the forespore compartment of the sporangium. Ultrastructure studies of a spoIIM mutant confirmed a block prior to the completion of engulfment. Introduction of a spoIIM mutation into a panel of strains containing lacZ fusions belonging to different regulatory classes allowed us to determine that the spoIIM gene product is required for the efficient expression of genes transcribed by sigma G-associated RNA polymerase but is not required for the expression of sigma F-controlled genes, including spoIIIG, which encodes sigma G. The results of complementation studies, gene disruption analysis, and DNA sequencing revealed that the spoIIM locus contains a single sporulation-essential gene encoding a polypeptide with a predicted molecular mass of 24,850 Da. The predicted spoIIM gene product is highly hydrophobic and very basic, and it does not exhibit significant homology to sequence files in several major data bases.

Amino Acid Sequence↗
PubMed1993

Evidence that the spoIIM gene of Bacillus subtilis is transcribed by RNA polymerase associated with sigma E.

We have investigated the temporal and spatial regulation of spoIIM, a gene of Bacillus subtilis whose product is required for complete septum migration and engulfment of the forespore compartment during sporulation. The spoIIM promoter was found to become active about 2 h after the initiation of sporulation. The effects of mutations on the expression of a spoIIM-lacZ fusion were most consistent with its utilization by sigma-E-associated RNA polymerase (E sigma E). A unique 5' end of the in vivo spoIIM transcript was detected by primer extension analysis and was determined to initiate at the appropriate distance from a sequence conforming very closely to the consensus for genes transcribed by E sigma E. A partially purified preparation of E sigma E produced a transcript in vitro that initiated at the same nucleotide as the primer extension product generated from in vivo RNA. Ectopic induction of sigma E synthesis during growth resulted in the immediate and strong expression of a spoIIM-lacZ fusion, but an identical fusion was completely unresponsive to induced synthesis of either sigma F or sigma G under similar conditions. The results of plasmid integration-excision experiments in which the spoIIM gene was reversibly disrupted by a temperature-sensitive integrational vector suggested that spoIIM expression is required in the forespore compartment, but direct examination of subcellular fractions enriched for mother cell or forespore material indicated that spoIIM expression cannot be confined to the forespore. We conclude that spoIIM is a member of the sigma E regulon and that it may be transcribed exclusively by E sigma E. We discuss the implications of this conclusion for models in which activation of sigma E in the mother cell is proposed to be a part of the mechanism responsible for initiating separate programs of gene activity in the two sporangium compartments.

Bacillus subtilis↗
PubMed1992

Transcriptional activation of the Listeria monocytogenes hemolysin gene in Bacillus subtilis.

The prfA gene of Listeria monocytogenes was recently reported to be required for expression of hly, which encodes a pore-forming hemolysin essential for pathogenicity (M. Leimeister-Wachter, C. Haffner, E. Domann, W. Goebel, and T. Chakraborty, Proc. Natl. Acad. Sci. USA 87:8336-8340, 1990). We demonstrate here that a hly-lacZ fusion introduced into Bacillus subtilis is strongly activated when the prfA gene product is supplied in trans under the control of an isopropyl-beta-D-thiogalactopyranoside-inducible promoter, Pspac. Moreover, the PrfA-dependent activation of hly is abolished by point mutations in a 14-bp DNA palindromic sequence present in the 5' upstream region of hly. This indicates that PrfA is both necessary and sufficient for hly transcriptional activation and establishes the palindrome as the likely target sequence for PrfA interaction. The presence of a palindrome in the upstream regions of three additional L. monocytogenes genes clustered near hly suggests that PrfA may serve as a transcriptional activator for a major virulence regulon of L. monocytogenes. In addition, the ability of PrfA to activate its target promoters effectively in B. subtilis suggests that further analysis of this regulon and perhaps other aspects of L. monocytogenes gene regulation might be carried out in part through reconstruction experiments in B. subtilis.

Bacillus subtilis↗
PubMed1992

Spo0A controls the sigma A-dependent activation of Bacillus subtilis sporulation-specific transcription unit spoIIE.

The spoIIE operon is a developmentally regulated transcription unit activated in the second hour of sporulation in Bacillus subtilis. Its promoter has an unusual structure, containing sequences which conform perfectly to the consensus for vegetative promoters recognized by sigma A-associated RNA polymerase (E sigma A), but with a spacing of 21 bp between the apparent -10 and -35 elements instead of the 17- or 18-bp spacing typical of promoters utilized by E sigma A. Mutations introduced into the apparent -10 element affected transcription in a manner consistent with its functioning as a polymerase recognition sequence. The deleterious effect of one -10 mutation was also suppressed in an allele-specific manner by a mutation in sigA known to suppress analogous -10 mutations in conventional vegetative promoters recognized by E sigma A. Similar suppression experiments failed to provide evidence for a direct interaction between E sigma A and the "-35-like" element, however, and DNase I protection experiments suggested instead that the Spo0A protein binds to a site overlapping this -35-like hexamer. Moreover, the effects of mutations within the -35-like hexamer on the binding of Spo0A in vitro paralleled their effects on transcription in vivo. We suggest that spoIIE belongs to a class of early-intermediate sporulation genes whose transcription by E sigma A is activated by the Spo0A protein.

Bacillus subtilis↗
PubMed1990

Novel mutations that alter the regulation of sporulation in Bacillus subtilis. Evidence that phosphorylation of regulatory protein SpoOA controls the initiation of sporulation.

Sporulation in Bacillus subtilis is a complex developmental process that occurs in response to nutrient deprivation. To identify components of the mechanism that allows cells to monitor their nutritional status and to understand how this sensory information is transduced into a signal to activate specific sporulation genes, we have isolated mutants that are able to sporulate efficiently under nutritional conditions that strongly inhibit sporulation in wild-type bacteria, a phenotype we refer to as Coi (control of initiation). Four coi mutations were found to be within the coding sequence of spoOA, a gene in which null mutations prevent the initiation of sporulation and a gene whose product shares a domain of homology with phosphorylation-activated proteins that play signal transduction roles in bacteria. All four of the spoOA mutations were within this conserved domain and in close proximity to the presumptive phosphoacceptor site. The wild-type and one of the mutant SpoOA proteins were purified and shown to be competent to accept phosphoryl groups from a phosphohistidine within a bacterial signal transduction kinase (CheA). The mutant SpoOA protein exhibited enhanced phosphoacceptor activity compared with the wild-type. This property of the mutant protein, together with additional genetic information, supports a model for regulation of sporulation initiation by control of the phosphorylation level of SpoOA.

Alleles↗
PubMed1990

Bacillus subtilis expressing a haemolysin gene from Listeria monocytogenes can grow in mammalian cells.

Intracellular parasites can be classified into those that reside within a host vacuole and those which grow directly in the host cytoplasm. Members of the latter group, which includes Rickettsia, Shigellae, Trypanosoma cruzi, and Listeria monocytogenes, possess haemolytic activity associated with the ability to enter the host cytoplasm. Therefore mutants of L. monocytogenes lacking a pore-forming haemolysin, listeriolysin O, do not escape from the endosomal compartment and consequently fail to become established in the cytoplasm. To examine the role of listeriolysin O, we cloned the structural gene for the L. monocytogenes haemolysin, hlyA, into an asporogenic mutant of Bacillus subtilis under the control of an IPTG-inducible promoter. After being internalized by the macrophage-like cell line J774, haemolytic B. subtilis disrupted the phagosomal membrane and grew rapidly within the macrophage cytoplasm. These results show that a single gene product is sufficient to convert a common soil bacterium into a parasite that can grow in the cytoplasm of a mammalian cell.

Bacillus subtilis↗
PubMed1990

Insertional mutagenesis of Listeria monocytogenes with a novel Tn917 derivative that allows direct cloning of DNA flanking transposon insertions.

To carry out efficient insertional mutagenesis in Listeria monocytogenes and to facilitate the characterization of disrupted genes, two novel derivatives of Tn917 were constructed, Tn917-LTV1 and Tn917-LTV3. The derivatives (i) transpose at a significantly elevated frequency, (ii) generate transcriptional lacZ fusions when inserted into a chromosomal gene in the appropriate orientation, and (iii) allow the rapid cloning in Escherichia coli of chromosomal DNA flanking transposon insertions. The rapid cloning of DNA flanking insertions is possible because the transposon derivatives carry ColE1 replication functions, a cluster of polylinker cloning sites, and antibiotic resistance genes selectable in E. coli (bla in the case of Tn917-LTV1; neo and ble in the case of Tn917-LTV3). The enhanced transposition frequency of Tn917-LTV1 and Tn917-LTV3 (about 100-fold in Bacillus subtilis) is believed to be due to the fortuitous placement of vector-derived promoters upstream from the Tn917 transposase gene. In L. monocytogenes, Tn917-LTV3 transposed at a frequency of 8 x 10(-4) when introduced on a pE194Ts-derived vector and generated at least eight different auxotrophic mutations. Two nonhemolytic insertion mutants of L. monocytogenes were isolated, and DNA flanking the transposon insertions was cloned directly into E. coli, making use of the ColE1 rep functions and neo gene carried by Tn917-LTV3. Both insertions were shown to be within hlyA, the L. monocytogenes hemolysin structural gene. Although Tn917-LTV1 and Tn917-LTV3 were constructed specifically for genetic analysis of L. monocytogenes, their enhanced transposition frequency and convenience for cloning of DNA adjacent to sites of insertions make them the transposon derivatives of choice for insertional mutagenesis in any gram-positive bacteria that support replication of pE194Ts.

Bacillus subtilis↗
PubMed1989

Genetic evidence that RNA polymerase associated with sigma A factor uses a sporulation-specific promoter in Bacillus subtilis.

The construction of allele-specific suppressor mutations has enabled us to demonstrate that a sporulation-specific transcription unit in Bacillus subtilis, the spoIIG operon, is transcribed by a form of RNA polymerase associated with sigma A, the principal sigma factor in vegetative cells. The spoIIG operon encodes sporulation-specific factor sigma E, and its transcription is directed from a promoter that is activated about 1 hr after the onset of endospore formation. This promoter contains sequences that are similar to those found at the -10 and -35 regions of promoters that are used by sigma A-associated RNA polymerase, but these sigma A-like recognition sequences are separated by 22 base pairs rather than the typical 17 or 18 base pairs. We have found that substitution of an arginyl residue for the glutamyl residue at position 196 of sigma A (Glu-196----Arg) suppresses the deleterious effect of a thymidine-to-cytidine base substitution at position -11 in the spoIIG promoter. This suppression was allele-specific, since it did not suppress the effects of base substitutions in other positions in the spoIIG promoter or the effects of a thymidine-to-guanosine change at -11. These results support a model in which a form of RNA polymerase containing sigma A is utilized in an unusual manner to activate the transcription of the spoIIG operon well after the onset of endospore formation.

Alleles↗
PubMed1989

Temporal and spatial control of the mother-cell regulatory gene spoIIID of Bacillus subtilis.

Gene expression during endospore formation in Bacillus subtilis is compartmentalized between the mother-cell and forespore chambers of the sporangium, which follow separate pathways of cellular differentiation. The earliest acting regulatory gene so far identified in the mother-cell line of gene expression is spoIIID, whose product is required for the transcription of the composite gene (sigK) encoding the mother-cell RNA polymerase sigma-factor sigma K and for the chromosomal rearrangement that gives rise to the composite gene. Here we report the nucleotide sequence of spoIIID and studies on the temporal, spatial, and genetic control of its expression during sporulation. We show that the deduced spoIIID gene product, a 93-residue-long polypeptide, is a previously identified transcription factor that is known to activate the promoter for the sigK gene in vitro. Expression of spoIIID is largely confined to the mother-cell chamber of the sporangium and is turned on at, or shortly before, the time (hour 3 of sporulation) that the mother-cell chromosome is rearranged and transcription of the sigK gene commences. This gene expression depends strongly on the sporulation sigma-factor sigma E and partially on the spoIIID gene product, itself. We conclude that the timing and compartmentalization of the rearrangement and transcription of the sigK gene and, hence, of subsequent gene activation in the mother cell, are, in part, direct consequences of the temporal and spatial control of spoIIID gene expression.

Amino Acid Sequence↗
PubMed1989

xylE functions as an efficient reporter gene in Streptomyces spp.: use for the study of galP1, a catabolite-controlled promoter.

We describe the development of a convenient and sensitive reporter gene system for Streptomyces spp. based on the use of a promoterless copy of the xylE gene of Pseudomonas putida. The xylE gene product is a catechol dioxygenase, which converts the colorless substrate catechol to an intensely yellow hydroxymuconic semialdehyde. A promoterless copy of xylE was placed under the transcriptional control of galP1, a glucose-repressed and galactose-induced promoter from Streptomyces lividans, and its expression was examined in bacterial colonies on agar plates or in liquid cultures grown in the presence of glucose or galactose as the sole carbon source. On plates, colonies of bacteria grown on galactose turned bright yellow within a few minutes of being sprayed with a solution of catechol, whereas colonies on glucose-containing plates remained white or only slightly colored, even after extensive incubation. Activity of galP1-xylE fusions was conveniently measured in crude cell extracts with a simple colorimetric assay and was shown to faithfully reflect intracellular RNA levels, as determined by quantitative dot blots. Moreover, differences in expression levels of xylE fusions driven by mutant galP1 promoters were readily apparent in color reactions on plates. The properties of xylE as a reporter gene thus make it suitable not only for quantitatively monitoring expression of regulated promoters in Streptomyces spp. but also for recovering mutations that alter the expression levels of promoters of interest.

Base Sequence↗
PubMed1988

A new cloning system for Bacillus subtilis comprising elements of phage, plasmid and transposon vectors.

A new cloning system for Bacillus subtilis was devised which makes use of a combination of Tn917-containing phage SP beta derivatives and Tn917-containing Escherichia coli-B. subtilis shuttle plasmids. This system allows the initial cloning of genes in single copy, via 'prophage transformation', with a selection for complementation of mutational defects in B. subtilis hosts and permits subsequent transfer of the cloned material by homologous recombination to low-copy and high-copy vectors that replicate in both B. subtilis and E. coli. Because cloned sequences are adjacent to pB322-derived DNA in the recombinant phages, inserts can also be 'rescued' directly from the phage DNA after digestion with appropriate restriction enzymes, circularization of the fragments by ligation and transformation of an E. coli recipient. Two genomic libraries of B. subtilis chromosomal Sau3A-generated partial-digest fragments in the size ranges of 5-8 kb and 8-10 kb were constructed and screened for the complementation of mutations aroI906, cysA14, dal-1, glyB133, metC3, purA16, purB33, thrA5, trpC2 and recE4. In all cases, specialized transducing phages carrying inserts that complemented the selected markers were recovered. Inserts complementing the dal-1 and trpC2 mutations could be transferred from recombinant phages to Tn917-containing plasmids by homologous recombination without in vitro subcloning. Another insert complementing the purB33 mutation was rescued directly into E. coli from a recombinant phage DNA.

Bacillus subtilis↗
PubMed1988

Identification of the promoter for a spore coat protein gene in Bacillus subtilis and studies on the regulation of its induction at a late stage of sporulation.

The cotA (pig) gene of Bacillus subtilis encodes a 65,000 Mr protein that is a component of the spore coat and is responsible for the brown pigment characteristic of colonies in which cells are undergoing sporulation. To study developmental regulation of the cotA gene, we identified its promoter and studied its transcription in a large number of mutants blocked at various stages of sporulation and germination. Deletion analysis showed that induction and efficient transcription of cotA required DNA sequences extending no more than 55 base pairs (bp) upstream (and no more than 130 bp downstream) from the 5' terminus of cotA mRNA. Transcription from the cotA promoter was found to be switched on at approximately the time (4 to 5 h after the onset of sporulation) of spore coat synthesis and deposition. Strikingly, this transcription was substantially inhibited in almost all asporogenous mutants blocked prior to the developmental stage (V) of spore coat formation. cotA transcription was also impaired in several stage V mutants but not in other stage V mutants or in mutants blocked in germination. The germination mutant gerE caused a several-fold overexpression of cotA. The dependence of cotA expression on so many genes required at early to intermediate stages of sporulation suggests that transcription of this spore coat gene is somehow coupled (directly or through several intervening steps) to a morphological or physiological feature(s) of the developing sporangium.

Amino Acid Sequence↗
PubMed1988

Characterization of the promoter region of the Bacillus subtilis spoIIE operon.

Mutations that define the spoIIE locus of Bacillus subtilis block sporulation at an early stage and recently were shown to prevent the proteolytic processing of sigma E (sigma 29) into its active form, an event that is believed to control critical changes in gene expression during the second hour of development. By taking advantage of two Tn917-mediated insertional mutations in spoIIE, we have cloned DNA spanning the locus. Gene disruption experiments with subcloned fragments transferred to integrational vectors revealed that the locus consisted of a single transcription unit about 2.5 kilobase pairs in size. Transcriptional lacZ fusions were used to show that expression of this transcription unit initiated at 1.5 h after the end of log-phase growth and depended upon the products of all spo0 loci. Expression was directed by a single promoter whose position was determined by high-resolution S1 protection mapping. A deletion analysis of the promoter region was also carried out, with novel integrational vectors based on derivatives of coliphage M13. The results indicated that a region of DNA extending from 183 to 118 base pairs upstream from the start point of transcription was required for full activity of the spoIIE promoter. The presumptive RNA polymerase-binding region of the promoter exhibited striking similarity to the spoIIG promoter and featured perfect but unusually spaced -10 and -35 consensus sequences for sigma A (sigma 43)-associated RNA polymerase.

Bacillus subtilis↗
PubMed1988

The promoter for a sporulation gene in the spoIVC locus of Bacillus subtilis and its use in studies of temporal and spatial control of gene expression.

We have identified the transcription start site and regulatory region governing the expression of a sporulation gene in the spoIVC locus of Bacillus subtilis. Efficient expression and developmental regulation of this gene was controlled from a promoter region that extended no more than 110 base pairs upstream and no more than 4 base pairs downstream from the start site of transcription, on which basis we infer that spoIVC is regulated at the level of transcription initiation. Using a transcriptional fusion of the spoIVC gene to the lacZ gene of Escherichia coli, we found that spoIVC expression was turned on at the third to fourth hour of sporulation (at about the developmental stage [IV] that its products are required in spore formation) and that this transcription was largely restricted to the mother cell chamber of the sporangium. Mutations in many different spo genes (causing blocks at stages 0 to V) were found to influence (negatively and positively) the level of spoIVC expression. Our results distinguish the mode of spoIVC regulation from that of previously studied sporulation genes and indicate that it is representative of a new regulon of mother cell-specific gene expression.

Bacillus subtilis↗