Sumiko Inouye

A gene encoding a protein serine/threonine kinase is required for normal development of M. xanthus, a gram-negative bacterium

PCR reactions were carried out on the genomic DNA of M. xanthus, a soil bacterium capable of differentiation to form fruiting bodies, using oligonucleotides representing highly conserved regions of eukaryotic protein serine/threonine kinases. A gene (pkn1) thus cloned contains an ORF of 693 amino acid residues whose amino-terminal domain shows significant sequence similarity with the catalytic domain of eukaryotic protein serine/threonine kinases. The pkn1 gene was overexpressed in E. coli, and the gene product has been found to be autophosphorylated at both serine and threonine residues. The expression of pkn1 is developmentally regulated to start immediately before spore formation. When pkn1 is deleted, differentiation starts prematurely, resulting in poor spore production. These results indicate that the protein serine/threonine kinase plays an important role in the onset of proper differentiation.

Positive-negative KG cassettes for construction of multi-gene deletions using a single drug marker

Positive-negative KG cassettes were developed in order to create a number of independent deletion mutations on the bacterial chromosome using a single drug marker. These cassettes consist of a kanamycin-resistant (KmR) gene for positive screening and a galactokinase gene (galK) for negative screening. Both genes are in an operon driven by the native KmR promoter and are flanked by identical fragments of yeast chromosomal DNA approximately one kb in size. An internal region of a cloned target gene of a bacterium is replaced with a cassette, which is then transformed into the bacterium. The intact gene on the chromosome is replaced with the mutated gene by homologous recombination. From the KmR cells thus obtained, those cells which lose both KmR and galK genes by homologous recombination between the identical yeast DNA fragments are subsequently screened on plates containing 2-deoxygalactose, a non-metabolizable analogue of galactose. This method was applied to isolate a triple-deletion mutant of pkn3, pkn1, and pkn11 from Myxococcus xanthus.

FruA, a putative transcription factor essential for the development of Myxococcus xanthus

A new developmental gene, fruA, of Myxococcus xanthus was cloned using a one‐step cloning vector, TnV. DNA sequencing of the wild‐type allele of the fruA gene indicated that the fruA gene encodes a protein of 229 amino acid residues with a calculated molecular weight of 24 672. The deduced amino acid sequence of FruA protein showed similarity to those of many bacterial regulatory proteins carrying a DNA‐binding helix‐turn‐helix motif. The transcription‐initiation site of the fruA gene was determined by a primer‐extension experiment. Development of M. xanthus cells with a disrupted fruA gene stopped at the stage of mound formation. Although cells were able to aggregate to form mounds, myxospores were not formed. By Northern and Western blot analysis, it was found that the fruA expression was not detected during vegetative growth but initiated at around 6 h and reached the highest level at 12 h after the onset of development. Expression of the fruA gene was dependent on the expression of asg, bsg, csg, dsg, and esg genes, indicating that a series of intercellular signalling is necessary for the expression of the fruA gene. The effects of the fruA mutation on ‐galactosidase expression of various developmentally regulated genes fused with the lacZ gene were analysed; three developmental lacZ fusions (Ω4469, Ω4273 and Ω4500) were either poorly induced or not induced at all, while three other lacZ fusions (Ω4408, Ω4521 and Ω4455) expressed at the early stage of development were normally induced but were unable to be repressed at a later stage of development as in the wild‐type strain. Interestingly, in the fruA mutant, tps (the gene for protein S) was not activated. From these results together with analysis of the amino acid sequence of FruA, we propose that FruA is a putative transcription factor required for the development of M. xanthus.

Identification of a protein Ser/Thr kinase cascade that regulates essential transcriptional activators in Myxococcus xanthus development.

Pkn8 is a membrane‐associated protein Ser/Thr kinase (PSTK) of Myxoccocus xanthus that was previously found to associate with a novel cytoplasmic kinase, Pkn14. In the present study, MrpC, an essential transcription factor for fruA expression during fruiting body development, was identified using a genomic yeast two‐hybrid screen with Pkn14 as bait. Our biochemical studies demonstrated that purified Pkn8 and Pkn14 are active kinases and that Pkn8 is able to phosphorylate Pkn14 that forms a tetramer via its C‐terminal 41 residues. Moreover, Pkn14 phosphorylated purified MrpC, indicating that Pkn8 is a Pkn14 kinase and Pkn14 is an MrpC kinase. The pkn8 and pkn14 deletion strains (Δpkn8 and Δpkn14) developed into fruiting bodies significantly faster than that of the parent strain, DZF1. While mrpC expression was at a low level in DZF1 during vegetative growth, it was highly elevated in Δpkn8 and Δpkn14 during vegetative growth and development. Furthermore, FruA, usually induced at 6 h of development, was instead detected at the early stationary phase and accumulated faster during development in Δpkn8 and Δpkn14. Therefore, the developmental phenotype of Δpkn8 and Δpkn14 seems to be due to untimely FruA production mediated by elevated levels of MrpC in Δpkn8 and Δpkn14 during vegetative growth. As pkn14 expression was increased at the mid‐ and late‐log. phases in DZF1 but decreased during development, the Pkn8–Pkn14 kinase cascade appears to negatively regulate mrpC expression by phosphorylating MrpC during vegetative growth. This is the first demonstration of a functional PSTK cascade in prokaryotes. mrpC expression has been proposed to be activated by MrpA and MrpB which belong to a two‐component His‐Asp phosphorelay signal transduction system and that MrpC autoregulate its own expression (Sun H. and Shi W., 2001 J Bacteriol 183: 4786–4795). Therefore, M. xanthus seems to utilize both eukaryotic PSTK cascade and prokaryotic His‐Asp phosphorelay system to precisely regulate mrpC expression with specific timing during development.

NMR-derived three-dimensional solution structure of protein S complexed with calcium.

BACKGROUND Protein S is a developmentally-regulated Ca(2+)-binding protein of the soil bacterium Myxococcus xanthus. It functions by forming protective, multilayer spore surface assemblies which may additionally act as a cell-cell adhesive. Protein S is evolutionarily related to vertebrate lens beta gamma-crystallins. RESULTS The three-dimensional solution structure of Ca(2+)-loaded protein S has been determined using multi-dimensional heteronuclear NMR spectroscopy. (Sixty structures were calculated, from which thirty were selected with a root mean square difference from the mean of 0.38 A for backbone atoms and 1.22 A for all non-hydrogen atoms.) The structure was analyzed and compared in detail with X-ray crystallographic structures of beta gamma-crystallins. The two internally homologous domains of protein S were compared, and hydrophobic cores, domain interfaces, surface ion pairing, amino-aromatic interactions and potential modes of multimerization are discussed. CONCLUSIONS Structural features of protein S described here help to explain its overall thermostability, as well as the higher stability and Ca2+ affinity of the amino-terminal domain relative to the carboxy-terminal domain. Two potential modes of multimerization are proposed involving cross-linking of protein S molecules through surface Ca(2+)-binding sites and formation of the intramolecular protein S or gamma B-crystallin interdomain interface in an intermolecular content. This structural analysis may also have implications for Ca(2+)-dependent cell-cell interactions mediated by the vertebrate cadherins and Dictyostelium discoideum protein gp24.

A protein Ser/Thr kinase cascade negatively regulates the DNA‐binding activity of MrpC, a smaller form of which may be necessary for the Myxococcus xanthus development

The developmental process of Myxococcus xanthus is achieved by the expression of a specific set of genes under the influence of developmental signals. MrpC is a member of the CRP family of transcription regulators, essential for fruA expression during development. The Pkn8‐Pkn14 protein kinase cascade negatively regulates mrpC expression (H. Nariya and S. Inouye, 2005. Mol Microbiol 58: 367–379). Elevated levels of mrpC in pkn8 and pkn14 deletion strains (Δpkn8 and Δpkn14) induce untimely FruA production during vegetative growth resulting in significantly faster fruiting body development. mrpC expression is presumably activated by MrpA and MrpB which belong to a two‐component His‐Asp phosphorelay system and is proposed to require MrpC on the basis of the genetic analysis. In the present study, we demonstrate that MrpC binds to at least eight sites in the upstream region of its promoter. Based on analysis of MrpC binding sites in the mrpC and fruA promoter regions, there are two types of MrpC‐specific binding sequences. Importantly, MrpC‐binding activity was greatly reduced upon its phosphorylation by Pkn14. MrpC2, a transcription activator for fruA expression, lacks the N‐terminal 25 residues of MrpC and exhibited four‐ and eightfold greater binding activity to the mrpC and fruA promoter regions respectively. Pkn14 was not able to phosphorylate MrpC2 and phosphorylates MrpC at Thr residue(s), thus Thr‐21 and/or Thr‐22 is (are) the likely site(s) of MrpC phosphorylation. MrpC2 was not detected in a lonD mutant in which fruA expression is low. Thus, the LonD protease essential for development may play an important role for the activation of MrpC‐binding activity through its proteolytic processing to MrpC2, required for developmental progression. MrpC2, only detectable during development in DZF1, was present at high levels during vegetative growth in Δpkn8 and Δpkn14, thus MrpC phosphorylation may inhibit its proteolytic processing. Based on these results, we propose a mechanism by which two transcription factors essential to development, MrpC and FruA, are regulated during the M. xanthus life cycle.

Nucleoside diphosphate kinase from Escherichia coli; its overproduction and sequence comparison with eukaryotic enzymes

The gene encoding nucleoside diphosphate (NDP) kinase of Escherichia coli was identified by polymerase chain reaction using oligodeoxyribonucleotide primers synthesized on the basis of consensus sequences from Myxococcus xanthus and various eukaryotic NDP kinases. The gene (ndk), mapped at 54.2 min on the E. coli chromosome, was cloned and sequenced. The E. coli NDP kinase was found to consist of 143 amino acid residues that are 57, 45, 45, 42, 43, and 43% identical to the M. xanthus, Dictyostelium discoideum, Drosophila melanogaster, mouse, rat, and human enzymes, respectively. The ndk gene appears to be in a monocistronic operon and, when cloned in a pUC vector, NDP kinase was overproduced at a level of approx. 25% of total cellular proteins. The protein could be labeled with [gamma-32P]ATP and migrated at a 16.5 kDa when electrophoresed in SDS-polyacrylamide gel, which is in good agreement with the Mr of the purified E. coli NDP kinase previously reported.

Identification of an activator protein required for the induction of fruA, a gene essential for fruiting body development in Myxococcus xanthus

Myxococcus xanthus exhibits social behavior and multicellular development. FruA is an essential transcription factor for fruiting body development in M. xanthus. In the present study, the upstream promoter region was found to be necessary for the induction of fruA expression during development. A cis-acting element required for the induction was identified and was located between nucleotides –154 and –107 with respect to the transcription initiation site. In addition, it was found that two binding sites exist within this element of the fruA promoter. By using DNA affinity column chromatography containing the cis-acting element, a fruA promoter-binding protein was purified. The purified protein was shown by N-terminal sequence analysis to be identical to MrpC, a protein identified previously by transposon insertion mutagenesis as an essential locus for fruiting body development [Sun, H. & Shi, W. (2001) J. Bacteriol. 183, 4786–4795]. Furthermore, fruA mRNA was not detectable in the mrpC::km strain, demonstrating that MrpC is essential for fruA expression. Moreover, mutational analysis of the binding sites for MrpC in the fruA promoter indicates that binding of MrpC activates transcription of fruA in vivo. This report provides evidence for a direct molecular interaction involved in temporally regulated gene expression in M. xanthus.

Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus

The Myxococcus xanthus gene, pkn9, encodes a protein that contains significant homology with eukaryotic Ser/Thr protein kinases. The pkn9 gene was singled out of a previously identified family of kinase genes by amplification techniques that displayed differences in kinase gene expression during selected periods of the M. xanthus life cycle. Pkn9 was constitutively expressed during vegetative growth and upregulated during the aggregation stage of early development. It consists of 589 amino acids, and its N‐terminal 394 residues show 38% identity with both Pkn1 and Pkn2 of M. xanthus. This region also shows 29, 25 and 29% identity with myosin light‐chain kinase, protein kinase C, and cAMP‐dependent protein kinase, respectively. A 22‐residue hydrophobic transmembrane domain separates the kinase domain from the 173‐residue C‐terminal domain that resides on the outside of the inner membrane. The C‐terminal domain contains two sets of tandem repeats of 13 and 10 residues which have no known function. When expressed in Escherichia coli under the T7 promoter, Pkn9 was found to be phosphorylated on serine and threonine residues. Disruption of the pkn9 kinase catalytic subdomains I–III by the insertion of a kanamycin‐resistance gene resulted in slightly delayed, smaller and more‐crowded fruiting bodies, while spore formation was normal. Total deletion of the pkn9 gene caused severely reduced progression through development resulting in light loose mounds that become slightly more compact over time. Development progressed further at the centre than at the edge of the spot, and spore formation was significantly reduced. Two‐dimensional gel analysis revealed that both the disruption and the deletion of pkn9 prevented the expression of five membrane proteins (KREP9‐1‐4). These results suggest that the loss of Pkn9 kinase activity caused altered fruiting‐body formation, the absence of the KREP9 proteins in the membrane, and reduced spore production.

Reciprocal regulation of the differentiation of Myxococcus xanthus by Pkn5 and Pkn6, eukaryotic‐like Ser/Thr protein kinases

Myxococcus xanthus contains a large family of genes encoding eukaryotic‐like serinehhreonine kinases. Among them, two genes, pkn5 and pkn6, are divergently located on the chromosome and share a 46 bp promoter region between their transcription initiation sites, as determined by RNA protection. Pkn5, consisting of 380 amino acid residues, is a soluble protein in the cytoplasm, while Pkn6, consisting of 710 amino acid residues, is a transmembrane protein. Its membrane topology was determined using the Pkn6‐PhoA fusion protein in Escherichia coli, which has a single transmembrane domain with the N‐terminal domain in the cytoplasm and the C‐terminal domain outside the cytoplasmic membrane. Both proteins, when expressed in E. coli, were autophosphorylated: Pkn5 only at Ser, and Pkn6 at both Ser and Thr. In M. xanthus, both genes are expressed constitutively throughout the life cycle, with slight increases at an early stage of development. Most strikingly, a pkn5‐deletion strain forms fruiting bodies much faster than the wild‐type strain, while a pknb‐deletion strain develops slower than the wild‐type strain. These results, together with the fact that the pkn5‐deletion strain is able to form fruiting bodies on semi‐rich media, suggest that Pkn5 and Pkn6 have reciprocal roles in M. xanthus growth and development. Furthermore, Pkn6 may be a transmembrane sensor of external signals for development, while Pkn5 is a kinase that negatively regulates M. xanthus development.