Kengo Kanamaru

SdiA, an Escherichia coli homologue of quorum-sensing regulators, controls the expression of virulence factors in enterohaemorrhagic Escherichia coli O157:H7

The quorum‐sensing system in bacteria is a well‐known regulatory system that controls gene expression in a cell density‐dependent manner. A transcriptional regulator (LuxR homologue), signal synthase (LuxI homologue) and autoinducer (acyl homoserine lactone) are indispensable for this system in most Gram‐negative bacteria. In this study, we found that SdiA, an Escherichia coli LuxR homologue, is a negative regulator of the expression of virulence factors EspD and intimin in enterohaemorrhagic E. coli (EHEC) O157:H7. The expression of EspD and intimin was inhibited at the RNA level upon SdiA overexpression. SdiA has a DNA‐binding motif in its C‐terminal part and can bind to the promoter regions of the esp and eae genes in vitro. Extracellular factors, which accumulate in culture supernatants of O157:H7 at the stationary phase of growth and inhibit EspD and intimin synthesis, bind to the N‐terminal part of SdiA in vivo and in vitro. O157:H7 overproducing the N‐terminal part of SdiA exhibited hypertranscription of EspD and intimin, suggesting that the overproduced N‐terminal part had inhibited the activity of intact SdiA through titration of the extracellular factors. These results indicate that a quorum‐sensing system including the SdiA protein controls colonization by O157:H7.

A copper-transporting P-type ATPase found in the thylakoid membrane of the cyanobacterium Synechococcus species PCC7942.

P‐type ATPases constitute a large family of cation pumps that play crucial physiological roles in many organisms, including bacteria, plants and mammals. They are postulated to play important roles in a variety of environmental adaptation systems. Recently, we cloned two distinct putative P‐type ATPase genes (pacS and pacL) from a photosynthetic cyanobacterium, Synechococcus species PCC7942. in this study, one of the gene products (named PacS) was found to possess a putative metal‐binding motif (Gly‐Met‐X‐Cys‐X‐X‐Cys) in its N‐terminal portion. Thus we supposed that this ATPase may function as a metal pump. Indeed, the results of Northern blotting analysis showed that pacS‐mRNA specifically increases upon addition of copper or silver to the growth medium. The results of Western blotting analysis confirmed the view that PacS accumulates in copper‐treated Synechococcus cells. Thus we concluded that the expression of PacS ATPase is regulated in response to the change in concentration of external metals, namely copper and silver. Consistent with this, an insertional inactivation mutant of pacS exhibited hypersensitivity in terms of growth to these potentially toxic metals, it was also revealed that PacS was mainly located in the thylakoid membrane, in which the photosynthetic reactions take place. This P‐type ATPase in the thylakoid membrane is implicated as a copper‐transporting system that may be involved in copper‐homeostasis crucial to the photosynthetic thylakoid function.

Three new nuclear genes, sigD, sigE and sigF, encoding putative plastid RNA polymerase σ factors in Arabidopsis thaliana

Three new nuclear genes (sigD, sigE and sigF) of Arabidopsis thaliana, encoding putative plastid RNA polymerase σ factors, were identified and analyzed. Phylogenetic analysis revealed that higher plant σ factors fell into at least four distinct subgroups within a diverse protein family. In addition, Arabidopsis sig genes contained conserved chromosomal intron sites, indicating that these genes arose by DNA duplication events during plant evolution. Transcript analyses revealed two alternatively spliced transcripts generated from the sigD region, one of which is predicted to encode a σ protein lacking the carboxy‐terminal regions 3 and 4. Finally, the amino‐terminal sequence of the sigF gene product was shown to function as a plastid‐targeting signal using green fluorescent protein fusions.

Glutamyl-tRNA mediates a switch in RNA polymerase use during chloroplast biogenesis.

Chloroplast genes of higher plants are transcribed by two types of RNA polymerase that are encoded by nuclear (NEP (nuclear‐encoded plastid RNA polymerase)) or plastid (PEP (plastid‐encoded plastid RNA polymerase)) genomes. NEP is largely responsible for the transcription of housekeeping genes during early chloroplast development. Subsequent light‐dependent chloroplast maturation is accompanied by repression of NEP activity and activation of PEP. Here, we show that the plastid‐encoded transfer RNA for glutamate, the expression of which is dependent on PEP, directly binds to and inhibits the transcriptional activity of NEP in vitro. The plastid tRNAGlu thus seems to mediate the switch in RNA polymerase usage from NEP to PEP during chloroplast development.

DNA Microarray Analysis of Plastid Gene Expression in an Arabidopsis Mutant Deficient in a Plastid Transcription Factor Sigma, SIG2

The plastid genome of higher plants contains more than one hundred genes for photosynthesis, gene expression, and other processes. Plastid transcription is done by two types of RNA polymerase, PEP and NEP. PEP is a eubacteria-type RNA polymerase that is essential for chloroplast development. In Arabidopsis thaliana, six sigma factors (SIG1-6) are encoded by the nuclear genome, and postulated to determine the transcription specificity of PEP. In this study, we constructed a DNA microarray for all of the plastid protein-coding genes, and analyzed the effects of the sig2 lesion on the global plastid gene expression. Of the 79 plastid protein genes, it was found that only the psaJ transcript was decreased in the mutant, whereas transcripts of 47 genes were rather increased. Since many of the up-regulated genes are under the control of NEP, it was suggested that the NEP activity was increased in the sig2-1 mutant.

Roles of Chloroplast RNA Polymerase Sigma Factors in Chloroplast Development and Stress Response in Higher Plants

Chloroplast transcription in higher plants is performed by two types of RNA polymerases, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP). PEP is a eubacteria-type multisubunit enzyme whose catalytic core subunits are encoded by the chloroplast genome, whereas NEP is the nuclear encoded T7 phage-type single subunit enzyme. PEP is critical for the biogenesis and maintenance of chloroplasts, and is finely tuned by the nuclear encoded sigma subunits. Of the six Arabidopsis sigma subunits, SIG2 is involved in the transcription of several chloroplast tRNA genes, including trnE encoding tRNA-Glu. SIG2 possibly couples translation and pigment synthesis in chloroplasts. On the other hand, SIG5 is induced by various stresses and contributes to repair of damaged photosystem II (PSII) through transcription of the psbD and psbC genes. Thus target genes and the physiological role of each sigma subunit are becoming clearer.

Chloroplast development in Arabidopsis thaliana requires the nuclear-encoded transcription factor Sigma B

Development of plastids into chloroplasts, the organelles of photosynthesis, is triggered by light. However, little is known of the factors involved in the complex coordination of light‐induced plastid gene expression, which must be directed by both nuclear and plastid genomes. We have isolated an Arabidopsis mutant, abc1, with impaired chloroplast development, which results in a pale green leaf phenotype. The mutated nuclear gene encodes a sigma factor, SigB, presumably for the eubacterial‐like plastid RNA polymerase. Our results provide direct evidence that a nuclear‐derived prokaryotic‐like SigB protein, plays a critical role in the coordination of the two genomes for chloroplast development.

The cyanobacterium, Synechococcus sp. PCC7942, possesses two distinct genes encoding cation-transporting P-type ATPases

P‐type (or E1 E2‐type) ATPases comprise a large family of prokaryotic and eukaryotic proteins capable of transporting a variety of cations, and function in a wide variety of cellular processes. The present study was carried out to search for genes encoding P‐type ATPases in the phototrophie cyanobacterium, Synechococcus sp. PCC7942. We succeeded in cloning two genes each encoding P‐type ATPases from this bacterium. It was found that Synechococcus at least, two distinct P‐type ATPases; one belongs to the family of typical prokaryotic P‐type ATPases and the other markedly resembles eukaryotic P‐type ATPases. An insertion mutant lacking either of these two ATPase‐genes was constructed. The results showed that the growth of these mutants is hypersensitive to osmotic stress upon addition of NaCl or sorbitol to the medium.

Cyclic GMP acts as a common regulator for the transcriptional activation of the flavonoid biosynthetic pathway in soybean

Cyclic GMP (cGMP) is an important signaling molecule that controls a range of cellular functions. So far, however, only a few genes have been found to be regulated by cGMP in higher plants. We investigated the cGMP-responsiveness of several genes encoding flavonoid-biosynthetic enzymes in soybean (Glycine max L.) involved in legume-specific isoflavone, phytoalexin and anthocyanin biosynthesis, such as phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate:CoA ligase, chalcone synthase, chalcone reductase, chalcone isomerase, 2-hydroxyisoflavanone synthase, 2-hydroxyisoflavanone dehydratase, anthocyanidin synthase, UDP-glucose:isoflavone 7-O-glucosyltransferase, and isoflavone reductase, and found that the majority of these genes were induced by cGMP but not by cAMP. All cGMP-induced genes were also stimulated by sodium nitroprusside (SNP), a nitric oxide (NO) donor, and illumination of cultured cells with white light. The NO-dependent induction of these genes was blocked by 6-anilino-5,8-quinolinedione, an inhibitor of guanylyl cyclase. Moreover, cGMP levels in cultured cells were transiently increased by SNP. Consistent with the increases of these transcripts, the accumulation of anthocyanin in response to cGMP, NO, and white light was observed. The treatment of soybean cotyledons with SNP resulted in a high accumulation of isoflavones such as daidzein and genistein. Loss- and gain-of-function experiments with the promoter of chalcone reductase gene indicated the Unit I-independent activation of gene expression by cGMP. Together, these results suggest that cGMP acts as a second messenger to activate the expression of genes for enzymes involved in the flavonoid biosynthetic pathway in soybean.

Analysis of wild-type and mutant plant nitrate reductase expressed in the methylotrophic yeast Pichia pastoris.

Recombinant Arabidopsis thaliana NADH:nitrate reductase (NR; EC 1.6.6.1) was produced in the methylotrophic yeast Pichia pastoris and purified to near-electrophoretic homogeneity. Purified enzyme had the spectral and kinetic properties typical of highly purified NR from natural plant sources. Site-directed mutagenesis altering several key residues and regions was carried out, and the mutant enzyme forms were expressed in P. pastoris. When the invariant cysteine residue, cysteine-191, in the molybdo-pterin region of the A. thaliana NIA2 protein was replaced with serine or alanine, the NR protein was still produced but was inactive, showing that this residue is essential for enzyme activity. Deletions or substitutions of the conserved N terminus of NR retained activity and the ability to be inactivated in vitro when incubated with ATP. Enzyme with a histidine sequence appended to the N terminus was still active and was easily purified using metal-chelate affinity chromatography. These results demonstrate that P. pastoris is a useful and reliable system for producing recombinant holo-NR from plants.