Since 1995, new virulent strains of Vibrio parahaemolyticus have emerged and spread throughout the world. These “pandemic” strains have four strain specific genomic islands (GIs), which are considered to be potential factors of the pandemicity. We investigated the origin and function of 24 genes in the so-called VPaI-1, one of the four GIs, by searching homologs in various species in Bacteria and Archaea. Of these 24 genes, two are found only in Vibrio vulnificus CMCP6 and Shewanella sp. MR-7. The genomic segment (~8 kb) encompassing the two genes shows the synteny among the three species. Since many of the Shewanella species can grow at 4°C, these two genes may be candidates of adaptation to temperature stress. Further, we found a candidate for a swarming gene, which is reported as the V. cholerae virulence gene. Based on these findings, we hypothesized the emergence of pandemicity and discuss the mechanism for how these strains spread throughout the world.
The initiation of bacterial chromosome DNA replication and its regulation are critical events. DnaA is essential for initiation of DNA replication and is conserved throughout bacteria. In Escherichia coli, hydrolysis of ATP-DnaA is promoted by Hda through formation of a ternary complex with DnaA and DnaN, ensuring the timely inactivation of DnaA during the replication cycle. In Bacillus subtilis, YabA also forms a ternary complex with DnaA and DnaN, and negatively regulates the initiation step of DNA replication. However, YabA shares no structural homology with Hda and the regulatory mechanism itself has not been clarified. Here, in contrast to Hda, we observed that dnaA transcription was stable during under- and overexpression of YabA. ChAP-chip assays showed that the depletion of YabA did not affect DNA binding by DnaA. On the other hand, yeast two-hybrid analysis indicated that the DnaA ATP-binding domain interacts with YabA. Moreover, mutations in YabA interaction-deficient mutants, isolated by yeast two-hybrid analysis, are located at the back of the ATP-binding domain, whereas Hda is thought to interact with the ATP-binding pocket itself. The introduction into B. subtilis of a dnaAY144C mutation, which disabled the interaction with YabA but did not affect interactions either with DnaA itself or with DnaD, resulted in over-initiation and asynchronous initiation of replication and disabled the formation of YabA foci, further demonstrating that the amino acid on the opposite side to the ATP-binding pocket is important for YabA binding. These results indicate that YabA indeed regulates the initiation of DNA replication by a different mechanism from that used by Hda in the E. coli RIDA system. Interestingly, all DnaA mutants deficient in YabA binding also displayed reduced DnaD binding in yeast two-hybrid assays, suggesting that YabA can inhibit replication initiation through competitive inhibition of DnaD binding to DnaA.
The cbbX gene is generally encoded in proteobacterial genomes and red-algal plastid genomes. In this study, we found two distinct cbbX genes of Cyanidioschyzon merolae, a unicellular red alga, one encoded in the plastid genome and the other encoded in the cell nucleus. The phylogenetic tree inferred from cbbX genes and strongly conserved gene organization (rbcLS-cbbX) suggests that the plastid-encoded cbbX gene of C. merolae came from an ancestral proteobacterium by horizontal gene transfer. On the other hand, the nuclear-encoded cbbX gene of C. merolae was classified in another cluster together with the nucleomorph-encoded cbbX gene of Guillardia theta. Furthermore, expression of the two cbbX genes were regulated differently in response to extracellular CO2 concentration. Our results imply that cbbX gene in the plastid genome was copied and transferred to the cell nucleus after horizontal gene transfer of RuBisCo operon from ancestral β-proteobacteria at comparatively early stage, and that each cbbX evolved in different ways.
CbbX is believed to be a transcriptional regulator of the subunit genes (rbcL and rbcS) of RuBisCO (Ribulose 1,5-bisphosphate carboxylase/oxygenase) as well as possibly a molecular chaperon of RuBisCO subunit assembly. The unicellular red alga Cyanidioschyzon merolae strain 10D possesses two distinct cbbX genes; one is part of the plastid genome and the other is found in the cell nucleus, whereas the RuBisCO operon (rbcL-rbcS-cbbX) is located only on the plastid genome. We examined the role of CbbX proteins of C. merolae in the expression of the RuBisCO operon. First, His-tagged nuclear and plastid CbbX proteins were produced in Escherichia coli and purified by affinity column chromatography. Both proteins showed binding activity to upstream of the coding region of rbcL. Yeast two hybrid analysis showed direct interaction between nuclear and plastid CbbX proteins but no interaction were found among CbbX, RbcL and RbcS. Then the transcription initiation site of the RuBisCO operon of C. merolae was determined. Next, in order to examine the role of CbbX in vivo, we constructed a plasmid carrying the promoter region of the RuBisCO operon fused to Escherichia colilacZ, and introduced it into E. coli cells into which a cloned nuclear or plastid cbbX gene under IPTG inducible promoter control was also introduced. Expression of LacZ in the transformed E.coli was observed. Enforced expression of either one of the cbbX genes resulted in a remarkable reduction of lacZ expression suggesting that CbbXs are rather transcriptional regulators than the molecular chaperon of RuBisCO. We discuss the mechanism by which the nuclear and plastid CbbX proteins regulate the RuBisCO operon of C. merolae.
The 3-ketoacyl-ACP synthase (KAS) II is a fatty-acid-related enzyme which catalyzes the elongation of 16:0-acyl carrier protein (ACP) to 18:0-ACP in plastids. The fatty acid biosynthesis 1-1 (fab1-1) mutant of Arabidopsis thaliana is partially deficient in its activity of Arabidopsis thaliana3-ketoacyl-ACP synthase 2 (AtKAS2), and its phenotype has been intensively studied in connection with the chilling resistance and fatty acid composition. In this study, we used the T-DNA insertion mutant of AtKAS2 to examine its possible role in plant development. Reverse transcription (RT)-PCR showed that the AtKAS2 gene was expressed in various plant organs, except for roots, and was highly expressed in siliques. The fusion of β-glucuronidase (GUS) to the AtKAS2 promoter demonstrated that the promoter was active in various tissues such as embryos, stomatal guard cells, inflorescences and pollen grains. We were not able to identify atkas2 homozygous mutant adult plants in heterozygous mutant progeny. Phenotypic and genetic analyses showed that disruption of the AtKAS2 by T-DNA insertion caused embryo lethality, and the development of the embryos was arrested at the globular stage. Taken together, our results suggest that AtKAS2 is required for embryo development in Arabidopsis during the transition from the globular to the heart stage.
Viviparous 1 (Vp1) of maize is known to encode a transcription factor VP1 that controls seed germination. Hexaploid wheat possesses three Vp1 homoeologues (TaVp1): TaVp-A1, TaVp-B1 and TaVp-D1. In this study, we attempted to characterize the molecular properties of TaVp1 in a highly dormant wheat cultivar, Minamino-komugi (Minamino). The seeds of Minamino showed much higher sensitivity to the inhibitory effect of ABA on germination than those of non-dormant cultivars, Sanin-1 and Tozan-18. The sequence analyses of cDNAs also revealed that some of TaVp-A1 transcripts and TaVp-D1 transcripts were spliced incorrectly, presumably resulting in production of truncated or deleted proteins. Most TaVp-B1 transcripts were spliced correctly, but some had an additional 3-bp (AAG) insertion in the B3 domain, which may not affect their function. RT-PCR analyses showed that TaVp1 was highly expressed in Minamino embryos in maturing seeds but much less in roots and leaves of seedlings. The level of TaVp1 mRNA was high when the embryos were treated with ABA but markedly decreased in water-imbibed mature embryos whose dormancy had been broken. Expression analyses of the individual homoeologues showed that the level of TaVp-A1 transcripts was highest in embryos of DAP 20 but much lower in the matured embryos. TaVp-B1 was highly expressed in developing and maturing seed embryos, while TaVp-D1 mRNA existed at lower levels in developing embryos but increased as the seeds were matured. These results suggest that the majority of TaVp1, especially TaVp-B1, are properly spliced and may function as a transcription factor playing an important role on dormancy in Minamino. By employing an efficient transient expression system using diploid wheat seeds, we confirmed the dual function of TaVP-B1: the activation of Em expression and the repression of α-amylase expression.
Abscisic acid (ABA) signaling includes positive and negative regulators in the signaling pathway. ABA-insensitive five (ABI5) binding protein (AtAFP), one of the negative regulators found in Arabidopsis, is involved in the proteolysis of a positive regulator, ABI5 (bZIP-type transcription factor). Three wheat orthologs (TaAFPs) of AtAFP were isolated. TaAFPs have a nuclear localization domain in the middle of the deduced amino acid sequence and an ABI5 binding domain in the C-terminal region as AtAFP. Three TaAFPs were located on the short arms of chromosomes 2A, 2B, and 2D of wheat, and based on their chromosomal locations, they were named TaAFP-A, TaAFP-B, and TaAFP-D. In comparison to AtAFP, which was activated in developing seeds and the early stage of germination, TaAFPs were expressed in a greater variety of tissues, such as flag leaves, roots, and leaves of seedlings, and developing grains. TaAFP-B was expressed predominantly in all tissues examined; TaAFP-A and TaAFP-D responded to ABA and stresses, such as salt and dehydration. These three TaAFPs may differentiate their roles in ABA signaling during wheat evolution.
In reforestation programs the genetic composition and diversity of populations that could be used as sources of planting material needs to be carefully considered to maximize the chances of successful establishment. For such purposes genetic analyses that include the identification of functional genes are required. In this study, we constructed a cDNA library from inner bark of Quercus mongolica (which is widely distributed in Japan) and collected 3385 ESTs. After constructing 2140 unigenes, 274 microsatellites were found within them. The most frequent microsatellite had AG motif (48%) and the next most common was AAG motif (12%). There were no CG repeats in the unigenes. In total, 20 EST-SSR markers were developed, polymorphisms of which were described by using eight individuals from eight populations over the species’ distributional range. The number of alleles per locus (Na) and observed heterozygosity (Ho) ranged from 2 to 12, and from 0.25 to 1.00, respectively. Cross-species amplification was successful for 19 loci in eight individuals of Q. serrata and for 20 loci in eight individuals of Q. dentata, with values of Na and Ho comparable to those of Q. mongolica. The EST-SSR markers characterized in this study should facilitate the analysis of genetic diversity in future studies.
One mechanism of eukaryotic signaling is protein phosphorylation by protein tyrosine phosphatases (PTPs). Here we have identified the PTP Receptor-Type IV (PTPR4) family, including one form of PTPα and two forms of PTPε (PTPε M and PTPε C) in flounder. The existence of PTPε C has not been reported in non-mammalian animals. Semi-quantitative RT-PCR revealed independent expression patterns and levels of PTPα and the two forms of PTPε in various tissues. The sequence of PTPε C was identical to that of PTPε M except for its 5’-terminal regions. Southern blot analysis proved that there existed only one PTPε gene in flounder genome, indicating that the two isoforms of PTPε might have been derived from alternative splicing of the single gene. Phylogenetic analysis of PTP domain D2 and part of D1 of PTPR4 showed that flounder was first joint with other teleost fish and then tetrapods, and also provided evidence that the gene duplication from the ancestor gene to PTPα and PTPε occurred before the divergence of Gnathastomata and Agnatha. These results showed that the functional evolution of protein phosphorylation is promoted by not only genome duplication, but also elaborate regulation of gene expression.
MeCP2, a methyl-CpG binding domain (MBD) protein, is known to bind to methylated CpG sites via a conserved MBD, leading to transcriptional repression. However, studies in cell-free system for gene repression and MeCP2 binding have suggested that DNA methylation-independent repression also occurs in living cells. It has been difficult to characterize the target genes of MeCP2 because a limited number have been identified to date. In this context, we screened for MeCP2 target genes using knockdown (KD) experiments combined with microarray gene expression analyses. Of the 49 genes that showed a more than three-fold increase in expression in two independent KD experiments conducted with different siRNA sets, unexpectedly, half (24 genes) did not contain promoter CpG islands (CGIs). Of seven selected genes that did contain CGIs, only two were methylated at the CGI, bound MeCP2 before KD, and reduced MeCP2 after KD. For three, MeCP2 was observed to bind to the unmethylated CGI before KD, and for one MeCP2 was reduced after KD. Another two genes neither had DNA methylation nor bound MeCP2 before KD. Gene ontology analysis suggested that MeCP2 represses a certain group of genes. These results suggest that in addition to the canonical gene repression function, MeCP2 can repress gene expression by binding to unmethylated DNA in particular genes in living cells.