Biomedical subjects

M J Buttner

Publications and source records attributed to M J Buttner.

At least 19 recordsLinked to original sources

PubMed2003

A rare leucine codon in adpA is implicated in the morphological defect of bldA mutants of Streptomyces coelicolor.

Streptomycetes are mycelial bacteria that produce sporulating aerial hyphae on solid media. Bald (bld) mutants fail to form aerial mycelium under at least some conditions. bldA encodes the only tRNA species able to read the leucine codon UUA efficiently, implying the involvement of a TTA-containing gene in initiating aerial growth. One candidate for such a gene was bldH, because the bldH109 mutant of Streptomyces coelicolor resembles bldA mutants in some aspects. In the work reported here, adpAc, an S. coelicolor gene similar to the Streptomyces griseus A factor-regulated adpAg, was found to complement the bldH109 mutant partially at both single and multiple copies. The sequence of adpAc from the bldH109 mutant revealed a frameshift. A constructed in frame deletion of adpAc conferred a bald colony phenotype, and the mutant behaved like bldA mutants and bldH109 in its pattern of extracellular signal exchange. Both adpAc and adpAg contain a TTA codon. A TTA-free version of adpAc was engineered by replacing the TTA leucine codon with a cognate TTG leucine codon. The adpA(TTATTG) gene could partially restore aerial mycelium formation to a bldA mutant when it was followed in cis by the gene ornA, as in the natural chromosomal arrangement. This indicated that the UUA codon in adpAc mRNA is the principal target through which bldA influences morphological differentiation. It also implied that translational arrest at the UUA codon in adpAc mRNA caused a polar effect on the downstream ornA, and that the poor translation of both genes contributes extensively to the deficiency of aerial mycelium formation in bldA mutants. Unlike the situation in S. griseus, adpAc transcription does not depend on the host's -butyrolactone signalling system, at least in liquid cultures. In addition, sigma factor BldN, which is the homologue of an S. griseus sigma factor AdsA that is absent from adpAg mutants of S. griseus, was present in the constructed adpAc null mutant of S. coelicolor.

Bacterial Proteins↗

PubMed2001

Mutational analysis of RsrA, a zinc-binding anti-sigma factor with a thiol-disulphide redox switch.

In the Gram-positive bacterium, Streptomyces coelicolor A3(2), expression of the thioredoxin system is modulated by a sigma factor called sigmaR in response to changes in the cytoplasmic thiol-disulphide status, and the activity of sigmaR is controlled post-translationally by an anti-sigma factor, RsrA. In vitro, the anti-sigma factor activity of RsrA, which contains seven cysteines, correlates with its thiol-disulphide redox status. Here, we investigate the function of RsrA in vivo. A constructed rsrA null mutant had very high constitutive levels of disulphide reductase activity and sigmaR-dependent transcription, confirming that RsrA is a negative regulator of sigmaR and a key sensor of thiol-disulphide status. Targeted mutagenesis revealed that three of the seven cysteines in RsrA (C11, C41 and C44) were essential for anti-sigma factor activity and that a mutant RsrA protein containing only these three cysteines was active and still redox sensitive in vivo. We also show that RsrA is a metalloprotein, containing near-stoichiometric amounts of zinc. On the basis of these data, we propose that a thiol-disulphide redox switch is formed between two of C11, C41 and C44, and that all three residues play an essential role in anti-sigma factor activity in their reduced state, perhaps by acting as ligands for zinc. Unexpectedly, rsrA null mutants were blocked in sporulation, probably as a consequence of an increase in the level of free sigmaR.

Amino Acid Sequence↗

PubMed2001

BldD is a direct regulator of key developmental genes in Streptomyces coelicolor A3(2).

BldD is a transcription factor required for aerial hyphae formation in the filamentous bacterium Streptomyces coelicolor. Three targets of BldD regulation were discovered by a number of means, including examination of bld gene interdependence, selective enrichment of chromosomal DNA fragments bound by BldD and searching the promoter regions of known developmental genes for matches to a previously characterized BldD binding site. The three BldD targets identified were the developmental sigma factor genes, whiG and bldN, and a previously uncharacterized gene, designated bdtA, encoding a putative transcription factor. In each target gene, the sequences bound by BldD were characterized by electrophoretic mobility shift and DNase I footprinting assays, and their alignment suggested AGTgA (n)m TCACc as a consensus BldD operator. The in vivo effect of mutation in bldD on the expression of these three target genes was assessed using S1 nuclease protection assays. In each case, target gene expression was upregulated during early colony development in the bldD background, suggesting that, in the wild type, BldD acts to repress premature expression of whiG, bldN and bdtA during vegetative growth.

Bacterial Proteins↗

PubMed2001

A connection between stress and development in the multicellular prokaryote Streptomyces coelicolor A3(2).

Morphological changes leading to aerial mycelium formation and sporulation in the mycelial bacterium Streptomyces coelicolor rely on establishing distinct patterns of gene expression in separate regions of the colony. sigmaH was identified previously as one of three paralogous sigma factors associated with stress responses in S. coelicolor. Here, we show that sigH and the upstream gene prsH (encoding a putative antisigma factor of sigmaH) form an operon transcribed from two developmentally regulated promoters, sigHp1 and sigHp2. While sigHp1 activity is confined to the early phase of growth, transcription of sigHp2 is dramatically induced at the time of aerial hyphae formation. Localization of sigHp2 activity using a transcriptional fusion to the green fluorescent protein reporter gene (sigHp2-egfp) showed that sigHp2 transcription is spatially restricted to sporulating aerial hyphae in wild-type S. coelicolor. However, analysis of mutants unable to form aerial hyphae (bld mutants) showed that sigHp2 transcription and sigmaH protein levels are dramatically upregulated in a bldD mutant, and that the sigHp2-egfp fusion was expressed ectopically in the substrate mycelium in the bldD background. Finally, a protein possessing sigHp2 promoter-binding activity was purified to homogeneity from crude mycelial extracts of S. coelicolor and shown to be BldD. The BldD binding site in the sigHp2 promoter was defined by DNase I footprinting. These data show that expression of sigmaH is subject to temporal and spatial regulation during colony development, that this tissue-specific regulation is mediated directly by the developmental transcription factor BldD and suggest that stress and developmental programmes may be intimately connected in Streptomyces morphogenesis.

Bacterial Proteins↗

PubMed2001

Defining the disulphide stress response in Streptomyces coelicolor A3(2): identification of the sigmaR regulon.

In the Gram-positive, antibiotic-producing bacterium Streptomyces coelicolor A3(2), the thiol-disulphide status of the hyphae is controlled by a novel regulatory system consisting of a sigma factor, sigmaR, and its cognate anti-sigma factor, RsrA. Oxidative stress induces intramolecular disulphide bond formation in RsrA, which causes it to lose affinity for sigmaR, thereby releasing sigmaR to activate transcription of the thioredoxin operon, trxBA. Here, we exploit a preliminary consensus sequence for sigmaR target promoters to identify 27 new sigmaR target genes and operons, thereby defining the global response to disulphide stress in this organism. Target genes related to thiol metabolism encode a second thioredoxin (TrxC), a glutaredoxin-like protein and enzymes involved in the biosynthesis of the low-molecular-weight thiol-containing compounds cysteine and molybdopterin. In addition, the level of the major actinomycete thiol buffer, mycothiol, was fourfold lower in a sigR null mutant, although no candidate mycothiol biosynthetic genes were identified among the sigmaR targets. Three sigmaR target genes encode ribosome-associated products (ribosomal subunit L31, ppGpp synthetase and tmRNA), suggesting that the translational machinery is modified by disulphide stress. The product of another sigmaR target gene was found to be a novel RNA polymerase-associated protein, RbpA, suggesting that the transcriptional machinery may also be modified in response to disulphide stress. We present DNA sequence evidence that many of the targets identified in S. coelicolor are also under the control of the sigmaR homologue in the actinomycete pathogen Mycobacterium tuberculosis.

Amino Acid Sequence↗

PubMed2000

Different alleles of the response regulator gene bldM arrest Streptomyces coelicolor development at distinct stages.

whiK was one of five new whi loci identified in a recent screen of NTG-induced whi mutants and was defined by three mutants, R273, R318 and R655. R273 and R318 produce long, tightly coiled aerial hyphae with frequent septation. In contrast, R655 shows a more severe phenotype; it produces straight, undifferentiated aerial hyphae with very rare short chains of spores. Subcloning and sequencing showed that whiK encodes a member of the FixJ subfamily of response regulators, with a C-terminal helix-turn-helix DNA-binding domain and an apparently typical N-terminal phosphorylation pocket. Unexpectedly, a constructed whiK null mutant failed to form aerial mycelium, showing that different alleles of this locus can arrest Streptomyces coelicolor development at very distinct stages. As a consequence of the null mutant phenotype, whiK was renamed bldM. The bldM null mutant fits into the extracellular signalling cascade proposed for S. coelicolor and is a member of the bldD extracellular complementation group. The three original NTG-induced mutations that defined the whiK/bldM locus each affected the putative phosphorylation pocket. The mutations in R273 and in R318 were the same, replacing a highly conserved glycine (G-62) with aspartate. The more severe mutant, R655, carried a C-7Y substitution adjacent to the highly conserved DD motif at positions 8-9. However, although bldM has all the highly conserved residues associated with the phosphorylation pocket of conventional response regulators, aspartate-54, the putative site of phosphorylation, is not required for bldM function. Constructed mutant alleles carrying either D-54N or D-54A substitutions complemented the bldM null mutant in single copy in trans, and strains carrying the D-54N or the D-54A substitution at the native chromosomal bldM locus sporulated normally. bldM was not phosphorylated in vitro with either of the small-molecule phosphodonors acetyl phosphate or carbamoyl phosphate under conditions in which a control response regulator protein, NtrC, was labelled efficiently.

Alleles↗

PubMed2000

Generation of a non-sporulating strain of Streptomyces coelicolor A3(2) by the manipulation of a developmentally controlled ftsZ promoter.

The differentiation of Streptomyces aerial hyphae into chains of unigenomic spores occurs through the synchronous formation of multiple FtsZ rings, leading to sporulation septa. We show here that developmental control of ftsZ transcription is required for sporulation in Streptomyces coelicolor A3(2). Three putative ftsZ promoters were detected in the ftsQ-ftsZ intergenic region. In addition, some readthrough from upstream promoter(s) contributed to ftsZ transcription. S1 nuclease protection assays and transcriptional fusions of the ftsZ promoter region to the egfp gene (for green fluorescent protein) provided evidence that ftsZ2p is a developmentally controlled promoter that is specifically upregulated in sporulating aerial hyphae. This upregulation required all the six early regulatory sporulation genes that were tested: whiA, B, G, H, I and J. The DNA sequence of the promoter indicated that it is not part of the developmental regulon that is controlled by the RNA polymerase sigma factor sigma(WhiG). A strain in which the ftsZ2p promoter was inactivated grew normally during vegetative growth and formed aerial mycelium, but was deficient in sporulation septation. Thus, ftsZ2p was dispensable for vegetative growth, but was required for the strain to make sufficient FtsZ to support developmentally controlled multiple cell divisions in aerial hyphae.

Bacterial Proteins↗

PubMed2000

sigma(BldN), an extracytoplasmic function RNA polymerase sigma factor required for aerial mycelium formation in Streptomyces coelicolor A3(2).

Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the gray polyketide spore pigment, and such white (whi) mutants have been used to define 13 sporulation loci. whiN, one of five new whi loci identified in a recent screen of NTG (N-methyl-N'-nitro-N-nitrosoguanidine)-induced whi strains (N. J. Ryding et al., J. Bacteriol. 181:5419-5425, 1999), was defined by two mutants, R112 and R650. R650 produced frequent spores that were longer than those of the wild type. In contrast, R112 produced long, straight, undifferentiated hyphae, although rare spore chains were observed, sometimes showing highly irregular septum placement. Subcloning and sequencing showed that whiN encodes a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors and that the sigma factor has an unusual N-terminal extension of approximately 86 residues that is not present in other sigma factors. A constructed whiN null mutant failed to form aerial mycelium (the "bald" phenotype) and, as a consequence, whiN was renamed bldN. This observation was not totally unexpected because, on some media, the R112 point mutant produced substantially less aerial mycelium than its parent, M145. The bldN null mutant did not fit simply into the extracellular signaling cascade proposed for S. coelicolor bld mutants. Expression of bldN was analyzed during colony development in wild-type and aerial mycelium-deficient bld strains. bldN was transcribed from a single promoter, bldNp. bldN transcription was developmentally regulated, commencing approximately at the time of aerial mycelium formation, and depended on bldG and bldH, but not on bldA, bldB, bldC, bldF, bldK, or bldJ or on bldN itself. Transcription from the p1 promoter of the response-regulator gene bldM depended on bldN in vivo, and the bldMp1 promoter was shown to be a direct biochemical target for sigma(BldN) holoenzyme in vitro.

Amino Acid Sequence↗

PubMed2000

WhiD and WhiB, homologous proteins required for different stages of sporulation in Streptomyces coelicolor A3(2).

The whiD locus, which is required for the differentiation of Streptomyces coelicolor aerial hyphae into mature spore chains, was localized by map-based cloning to the overlap between cosmids 6G4 and D63 of the minimal ordered library of Redenbach et al. (M. Redenbach et al., Mol. Microbiol. 21:77-96, 1996). Subcloning and sequencing showed that whiD encodes a homologue of WhiB, a protein required for the initiation of sporulation septation in S. coelicolor. WhiD and WhiB belong to a growing family of small (76- to 112-residue) proteins of unknown biochemical function in which four cysteines are absolutely conserved; all known members of this family are found in the actinomycetes. A constructed whiD null mutant showed reduced levels of sporulation, and those spores that did form were heat sensitive, lysed extensively, and were highly irregular in size, arising at least in part from irregularity in septum placement. The whiD null mutant showed extreme variation in spore cell wall deposition; most spores had uniformly thin (20- to 30-nm) walls, but spore chains were frequently observed in which there was irregular but very pronounced (up to 170 nm) cell wall thickening at the junctions between spores. whiD null mutant spores were frequently partitioned into irregular smaller units through the deposition of additional septa, which were often laid down in several different planes, very close to the spore poles. These "minicompartments" appeared to be devoid of chromosomal DNA. Two whiD promoters, whiDp1 and whiDp2, were identified, and their activities were analyzed during development of wild-type S. coelicolor on solid medium. Both promoters were developmentally regulated; whiDp1 and whiDp2 transcripts were detected transiently, approximately at the time when sporulation septa were observed in the aerial hyphae.

Amino Acid Sequence↗

PubMed1999

RsrA, an anti-sigma factor regulated by redox change.

SigR (sigma(R)) is a sigma factor responsible for inducing the thioredoxin system in response to oxidative stress in the antibiotic-producing, Gram-positive bacterium Streptomyces coelicolor A3(2). Here we identify a redox-sensitive, sigma(R)-specific anti-sigma factor, RsrA, which binds sigma(R) and inhibits sigma(R)-directed transcription in vitro only under reducing conditions. Exposure to H(2)O(2) or to the thiol-specific oxidant diamide caused the dissociation of the sigma(R)-RsrA complex, thereby allowing sigma(R)-dependent transcription. This correlated with intramolecular disulfide bond formation in RsrA. Thioredoxin was able to reduce oxidized RsrA, suggesting that sigma(R), RsrA and the thioredoxin system comprise a novel feedback homeostasis loop that senses and responds to changes in the intracellular thiol-disulfide redox balance.

Amino Acid Sequence↗

PubMed1999

A putative two-component signal transduction system regulates sigmaE, a sigma factor required for normal cell wall integrity in Streptomyces coelicolor A3(2).

The extracytoplasmic function (ECF) sigma factor, sigmaE, is required for normal cell wall integrity in Streptomyces coelicolor. We have investigated the regulation of sigmaE through a transcriptional and mutational analysis of sigE and the surrounding genes. Nucleotide sequencing identified three genes located downstream of sigE; orf202, cseB and cseC (cse, control of sigE ). cseB and cseC encode a putative response regulator and a putative transmembrane sensor histidine protein kinase respectively. Although most sigE transcription appeared to be monocistronic, sigE was also transcribed as part of a larger operon, including at least orf202. sigE null mutants are sensitive to cell wall lytic enzymes, have an altered peptidoglycan muropeptide profile, and on medium deficient in Mg2+ they overproduce actinorhodin, sporulate poorly and form crenellated colonies. A constructed cseB null mutant appeared to have the same phenotype as a sigE null mutant, which was accounted for by the observed absolute dependence of the sigE promoter on cseB. It is likely that the major role of cseB is to regulate sigE transcription because expression of sigE alone from a heterologous promoter suppressed the cseB mutation. Mg2+ suppresses the CseB/SigE phenotype, probably by stabilizing the cell envelope, and sigE transcript levels were consistently higher in Mg2+-deficient cultures than in high Mg2+-grown cultures. We propose a model in which the CseB/CseC two-component system modulates activity of the sigE promoter in response to signals from the cell envelope.

Amino Acid Sequence↗

PubMed1999

Evidence that the extracytoplasmic function sigma factor sigmaE is required for normal cell wall structure in Streptomyces coelicolor A3(2).

The sigE gene of Streptomyces coelicolor A3(2) encodes an RNA polymerase sigma factor belonging to the extracytoplasmic function (ECF) subfamily. Constructed sigE deletion and disruption mutants were more sensitive than the parent to muramidases such as hen egg white lysozyme and to the CwlA amidase from Bacillus subtilis. This correlated with an altered muropeptide profile, as determined by reverse-phase high-performance liquid chromatography analysis of lytic digests of purified peptidoglycan. The sigE mutants required high levels of magnesium for normal growth and sporulation, overproducing the antibiotic actinorhodin and forming crenellated colonies in its absence. Together, these data suggest that sigE is required for normal cell wall structure. The role of sigmaE was further investigated by analyzing the expression of hrdD, which is partially sigE dependent. The hrdD gene, which encodes the sigmaHrdD subunit of RNA polymerase, is transcribed from two promoters, hrdDp1 and hrdDp2, both similar to promoters recognized by other ECF sigma factors. The activities of hrdDp1 and hrdDp2 were reduced 20- and 3-fold, respectively, in sigE mutants, although only hrdDp1 was recognized by EsigmaE in vitro. Growth on media deficient in magnesium caused the induction of both hrdD promoters in a sigE-dependent manner.

Amidohydrolases↗

PubMed1999

New sporulation loci in Streptomyces coelicolor A3(2).

Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the grey polyketide spore pigment, and such white (whi) mutants had been used to define eight sporulation loci, whiA, whiB, whiD, whiE, whiG, whiH, whiI, and whiJ (K. F. Chater, J. Gen. Microbiol. 72:9-28, 1972; N. J. Ryding, Ph.D. thesis, University of East Anglia, 1995). In an attempt to identify new whi loci, we mutagenized S. coelicolor M145 spores with nitrosoguanidine and identified 770 mutants with colonies ranging from white to medium grey. After excluding unstable strains, we examined the isolates by phase-contrast microscopy and chose 115 whi mutants with clear morphological phenotypes for further study. To exclude mutants representing cloned whi genes, self-transmissible SCP2*-derived plasmids carrying whiA, whiB, whiG, whiH, or whiJ (but not whiD, whiE, or whiI) were introduced into each mutant by conjugation, and strains in which the wild-type phenotype was restored either partially or completely by any of these plasmids were excluded from further analysis. In an attempt to complement some of the remaining 31 whi mutants, an SCP2* library of wild-type S. coelicolor chromosomal DNA was introduced into 19 of the mutants by conjugation. Clones restoring the wild-type phenotype to 12 of the 19 strains were isolated and found to represent five distinct loci, designated whiK, whiL, whiM, whiN, and whiO. Each of the five loci was located on the ordered cosmid library: whiL, whiM, whiN, and whiO occupied positions distinct from previously cloned whi genes; whiK was located on the same cosmid overlap as whiD, but the two loci were shown by complementation to be distinct. The phenotypes resulting from mutations at each of these new loci are described.

Genes, Bacterial↗

PubMed1999

A novel gene: sawD related to the differentiation of streptomyces ansochromogenes.

A 1.3 kb DNA fragment was cloned from a total DNA library of Streptomyces ansochromogenes using Southern hybridization. Nucleotide sequencing analysis indicated that the 1320 bp DNA fragment contained a complete open reading frame (ORF). In search of databases, the deduced product of ORF containing 213 amino acids is homologous to the serine protease of Caulobacter cresceatus, and a conserved serine-catalytic active site (GPSAG) exists. The gene was designated as sawD. The function of this gene was studied with the strategy of gene disruption, and the result showed that the sawD may be related to sporulation and especially to the spore septation in Streptomyces ansochromogenes. The preliminary result indicated that sawD mutant could produce abundant pigment in contrast with the wild type, it seems that sawD gene may be involved in pigment biosynthesis, and this gene is also dispensable for biosynthesis of nikkomycin in Streptomyces ansochromogenes.

Amino Acid Sequence↗

PubMed1998

sigmaR, an RNA polymerase sigma factor that modulates expression of the thioredoxin system in response to oxidative stress in Streptomyces coelicolor A3(2).

We have identified an RNA polymerase sigma factor, sigmaR, that is part of a system that senses and responds to thiol oxidation in the Gram-positive, antibiotic-producing bacterium Streptomyces coelicolor A3(2). Deletion of the gene (sigR) encoding sigmaR caused sensitivity to the thiol-specific oxidant diamide and to the redox cycling compounds menadione and plumbagin. This correlated with reduced levels of disulfide reductase activity and an inability to induce this activity on exposure to diamide. The trxBA operon, encoding thioredoxin reductase and thioredoxin, was found to be under the direct control of sigmaR. trxBA is transcribed from two promoters, trxBp1 and trxBp2, separated by 5-6 bp. trxBp1 is transiently induced at least 50-fold in response to diamide treatment in a sigR-dependent manner. Purified sigmaR directed transcription from trxBp1 in vitro, indicating that trxBp1 is a target for sigmaR. Transcription of sigR itself initiates at two promoters, sigRp1 and sigRp2, which are separated by 173 bp. The sigRp2 transcript was undetectable in a sigR-null mutant, and purified sigmaR could direct transcription from sigRp2 in vitro, indicating that sigR is positively autoregulated. Transcription from sigRp2 was also transiently induced (70-fold) following treatment with diamide. We propose a model in which sigmaR induces expression of the thioredoxin system in response to cytoplasmic disulfide bond formation. Upon reestablishment of normal thiol levels, sigmaR activity is switched off, resulting in down-regulation of trxBA and sigR. We present evidence that the sigmaR system also functions in the actinomycete pathogen Mycobacterium tuberculosis.

Amino Acid Sequence↗

PubMed1998

The Streptomyces coelicolor sporulation-specific sigma WhiG form of RNA polymerase transcribes a gene encoding a ProX-like protein that is dispensable for sporulation.

In the non-motile mycelial organism Streptomyces coelicolor A3(2), the sporulation gene whiG encodes a protein that closely resembles RNA polymerase sigma factors such as sigma D of Bacillus subtilis, which mainly control motility and chemotaxis genes. Here, we show that the whiG gene product, purified from an Escherichia coli strain carrying an expression construct, could activate E. coli core RNA polymerase in vitro to transcribe a sigma D-dependent motility-related promoter from B. subtilis. Such RNA polymerase holoenzyme preparations could also transcribe from an S. coelicolor promoter, PTH4, previously shown to require an intact whiG gene for in-vivo transcription. The in-vivo dependence on whiG was therefore shown to be direct. Unusually, the initiation of PTH4 transcription in vitro depended on the provision of appropriate dinucleotides. The whiG-dependent PTH4 transcription unit consisted of a single gene, orfTH4. Sequence comparisons suggested that the gene product was a member of a small group of proteins that include the B. subtilis and E. coli ProX proteins. Though none of these proteins shared more than about 30% of extended primary sequence identity, they had similar size and hydropathy profiles, and could be aligned end to end to reveal a mosaic of similarities. The ProX proteins of B. subtilis and E. coli are implicated in glycine betaine transport in response to hyperosmotic stress. However, disruption of orfTH4 did not cause any obvious phenotypic changes in growth or development on media of varying osmotic strengths.

Amino Acid Sequence↗

PubMed1998

Specific peptide-activated proteolytic cleavage of Escherichia coli elongation factor Tu.

Phage exclusion is a form of programmed cell death in prokaryotes in which death is triggered by infection with phage, a seemingly altruistic response that limits multiplication of the phage and its spread through the population. One of the best-characterized examples of phage exclusion is the exclusion of T-even phages such as T4 by the e14-encoded Lit protein in many Escherichia coli K-12 strains. In this exclusion system, transcription and translation of a short region of the major head coat protein gene late in phage infection activates proteolysis of translation elongation factor Tu (EF-Tu), blocking translation and multiplication of the phage. The cleavage occurs between Gly-59 and Ile-60 in the nucleotide-binding domain. In the present work, we show that a 29-residue synthetic peptide spanning the activating region of the major head coat protein can activate the cleavage of GDP-bound EF-Tu in a purified system containing only purified EF-Tu and purified Lit protein. Lit behaves as a bona fide enzyme in this system, cleaving EF-Tu to completion when present at substoichiometric amounts. Two mutant peptides with amino acid changes that reduce the activation of cleavage of EF-Tu in vivo were also greatly reduced in their ability to activate EF-Tu cleavage in vitro but were still able to activate cleavage at a high concentration. Elongation factor G, which has the same sequence at the cleavage site and a nucleotide-binding domain similar to EF-Tu, was not cleaved by this system, and neither was heat-inactivated EF-Tu, suggesting that the structural context of the cleavage site may be important for specificity. This system apparently represents an activation mechanism for proteolysis that targets one of nature's most evolutionarily conserved proteins for site-specific cleavage.

Amino Acid Sequence↗

PubMed1998

Initiation of aerial mycelium formation in Streptomyces.

In the past two years, the isolation of extracellular factors involved in the initiation of aerial mycelium formation, the identification of metabolic defects in certain developmental mutants, and the characterisation of three further bld genes and several gamma-butyrolactone receptor genes have led to new ideas about the mechanisms that initiate aerial mycelium formation in Streptomyces. The emerging picture suggests the integration of numerous signals from both inside and outside the cell.

Gene Expression Regulation, Bacterial↗