MgLig4, a homolog of Neurospora crassa Mus-53 (DNA ligase IV), is involved in, but not essential for, non-homologous end-joining events in Magnaporthe grisea.

In many eukaryotic organisms, the non-homologous end-joining (NHEJ) system is a major pathway for the repair of DNA double-strand breaks (DSBs). DNA ligase IV is a component of the NHEJ system and is strictly required for the NHEJ system in Saccharomyces cerevisiae and in Neurospora crassa. To investigate the functions of DNA Ligase IV in Magnaporthe grisea, we generated deletion mutants of MGLIG4, which encodes a homolog of N. crassa DNA Ligase IV. Mutants (mglig4) showed no defects in asexual or sexual growth, and were fully pathogenic. Compared to the wild-type, mglig4 exhibited weak sensitivity to a DNA-damaging agent, camptothecin. In addition, the frequency of targeted-gene replacement was relatively elevated in mglig4, although this varied in a gene-dependent manner. Surprisingly, non-homologous integration of DNA was frequently observed in mglig4 transformants. Our results demonstrate that MgLig4 is involved in, but not essential for, the NHEJ system in M. grisea.

Self-activation of serine/threonine kinase AfsK on autophosphorylation at threonine-168.

A Hanks-type protein kinase AfsK autophosphorylates on threonine residue(s) and phosphorylates AfsR, a global regulator for secondary metabolism in Streptomyces coelicolor A3(2). Mass spectrometry of a tryptic digest of the autophosphorylated form of AfsK deltaC corresponding to the kinase catalytic domain (Met-1 to Arg-311) of AfsK, together with subsequent site-directed mutagenesis of the candidate amino acids, identified threonine-168 as a single autophosphorylation site. Threonine-168 is located in the activation loop that is known for some Ser/Thr kinases to modulate kinase activity on phosphorylation of one or more threonine residues within the loop. Consistent with this, mutant T168D, in which Thr-168 was replaced by Asp, became a constitutively active kinase; it phosphorylated AfsR to the same extent as AfsK deltaC produced in and purified from Escherichia coli cells during which a considerable population of it had been already phosphorylated intermolecularly. All these findings show that autophosphorylation or intermolecular phosphorylation of threonine-168 in AfsK accounts for the self-activation of its kinase activity.

Three chymotrypsin genes are members of the AdpA regulon in the A-factor regulatory cascade in Streptomyces griseus.

AdpA is a key transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus, activating a number of genes required for secondary metabolism and morphological differentiation. Of the five chymotrypsin-type serine protease genes, sprA, sprB, and sprD were transcribed in response to AdpA, showing that these protease genes are members of the AdpA regulon. These proteases were predicted to play the same physiological role, since these protease genes were transcribed in a similar time course during growth and the matured enzymes showed high end-to-end similarity to one another. AdpA bound two sites upstream of the sprA promoter approximately at positions -375 and -50 with respect to the transcriptional start point of sprA. Mutational analysis of the AdpA-binding sites showed that both AdpA-binding sites were essential for transcriptional activation. AdpA bound a single site at position -50 in front of the sprB promoter and greatly enhanced the transcription of sprB. The AdpA-binding site at position -40 was essential for transcription of sprD, although there was an additional AdpA-binding site at position -180. Most chymotrypsin activity excreted by S. griseus was attributed to SprA and SprB, because mutant deltasprAB, having a deletion in both sprA and sprB, lost almost all chymotrypsin activity, as did mutant deltaadpA. Even the double mutant deltasprAB and triple mutant deltasprABD grew normally and developed aerial hyphae and spores over the same time course as the wild-type strain.

Transcriptional control by A-factor of strR, the pathway-specific transcriptional activator for streptomycin biosynthesis in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers streptomycin production by inducing the transcription of strR, encoding the pathway-specific transcriptional activator, through signal transduction in the A-factor regulatory cascade in Streptomyces griseus. AdpA, one of the key transcriptional activators in the cascade, bound two upstream activation sites, approximately at nucleotide positions -270 and -50 with respect to the transcriptional start point of strR, as determined by gel mobility shift assays and DNase I footprinting. Transcriptional analysis of the strR promoter with mutated AdpA-binding sites showed that both sites were required for full transcriptional activation of strR by AdpA. Potassium permanganate footprinting showed that AdpA assisted RNA polymerase in forming an open complex at an appropriate position for transcriptional initiation of strR. Nine transcriptional units within the streptomycin biosynthesis gene cluster, including the strR-aphD operon, depended on StrR, indicating that StrR is the pathway-specific transcriptional activator for the whole gene cluster. Consistent with this, expression of strR under the control of a constitutively expressed promoter in an adpA null mutant caused the host to produce streptomycin.

AdpA, a central transcriptional regulator in the A-factor regulatory cascade that leads to morphological development and secondary metabolism in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is a microbial hormone that triggers aerial mycelium formation and secondary metabolism in Streptomyces griseus. A-factor produced in a growth-dependent manner switches on the transcription of adpA encoding a transcriptional activator by binding to the A-factor receptor protein (ArpA), which has bound the adpA promoter, and dissociating the DNA-bound ArpA from the DNA. AdpA then activates a number of genes with various functions required for morphological development and secondary metabolism, forming an AdpA regulon. AdpA, which contains a ThiJ/PfpI/DJ-1-like dimerization domain at its N-terminal portion and an AraC/XylS-type DNA-binding domain at its C-terminal portion, is a representative of a large subfamily of the AraC/XylS family. AdpA binds various positions with respect to the transcriptional start points of the target genes and recruits RNA polymerase to the specific promoter region, and facilitates the isomerization of the RNA polymerase-DNA complex into an open complex competent for transcriptional initiation. The AdpA-binding consensus sequence is 5'-TGGCSNGWWY-3' (S: G or C; W: A or T; Y: T or C; N: any nucleotide). The DNA-binding specificity of AdpA in conjunction with that of other AraC/XylS family members is also discussed.

DNA-binding specificity of AdpA, a transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus.

AdpA, belonging to the AraC/XylS family, is the key transcriptional activator for a number of genes of various functions in the A-factor regulatory cascade in Streptomyces griseus. It consists of a ThiJ/PfpI/DJ-1-like dimerization domain at its N-terminal portion and a DNA-binding domain with two helix-turn-helix motifs at its C-terminal portion, representing a large subgroup of the AraC/XylS family. Uracil interference assay and missing T and GA interference assays on several AdpA binding sites, followed by gel mobility shift assays on systematically mutated binding sites, revealed a consensus AdpA-binding sequence, 5'-TGGCSNGWWY-3' (S: G or C; W: A or T; Y: T or C; N: any nucleotide). A dimer of AdpA bound a site including the two consensus sequences, with a space of 13-14 bp, as an inverted repeat (type I) at various positions, for example more than 200 bp upstream (-200) and 25 bp downstream (+25) from the transcriptional start point of the target gene. In addition, AdpA also bound a site including the consensus sequence in a single copy (type II) at positions, in most cases, from -40 to -50 and from -50 to -60. For transcriptional activation, some genes required simultaneous binding of a dimer of AdpA to type I and II sites, but others required only a single type I or type II site. AdpA bound mutated type I sites with various distances between the two consensus sequences with significant affinities, although the optimal distances for AdpA to bind were 13-14 bp and 2 bp. The DNA-binding domain is therefore connected to the ThiJ/PfpI/DJ-1-like dimerization domain with a flexible linker. The DNA-binding specificity of AdpA in conjunction with that of other AraC/XylS family members is discussed.