Construction of a supF-based system for detection of mutations in the chromosomal DNA of Arabidopsis.

The factors maintaining genomic integrity, which have been studied in detail in other species, have yet to be investigated in plants. Recent progress in gene-silencing technology has made it possible to produce transgenic plants with loss-of-function phenotypes for the effective analysis of these factors, even with the high redundancy of genes in plants. Therefore, a mutation-detection system for plants is necessary to estimate the biological function of a target gene for mutation frequencies and spectra. Here, we reported the development of a novel system to analyze mutations in the chromosomal DNA of plants. The supF gene of E. coli was used as a target for the mutation because it was possible to detect all mutational base changes. Based on the plasmid pTN30, which carries supF, we constructed a binary Ti vector for its introduction to Arabidopsis genomes. The system was validated by measuring mutations in both non-treated and mutagen-treated transgenic plants. DNA fragments including pTN30 were rescued from the plants, and introduced into E. coli KS40/pOF105 to isolate the supF mutant clones conferring both nalidixic acid and streptomycin resistance on transformants. We found that the mutation frequency was approximately three times higher with the ethyl methanesulfonate (EMS) treatment than without it and G:C to A:T transitions dominated, which was the most reasonable mutation induced by EMS. These results show that this system allowed for the rapid analysis of mutations in plants, and may be useful for analyzing plant genes related to the functions of genomic stability and monitoring environmental genotoxic substances.

Novel members of the phosphate regulon in Escherichia coli O157:H7 identified using a whole-genome shotgun approach.

Escherichia coli PhoB protein is the transcriptional activator of the phosphate (pho) regulon genes involved in phosphate utilization. To gain further insight into the potential roles of PhoB in the phosphate starvation response, we attempted to identify PhoB-regulated promoters using a random shotgun library of E. coli O157:H7 genomic fragments that were fused to a promoterless lacZ reporter gene on a low-copy-number plasmid. Using this approach, numerous chromosomal regions containing phosphate-starvation-inducible (psi) promoters, including nearly all known pho regulon promoters, were identified. ß-Galactosidase and electrophoretic mobility shift assays showed that transcription from the 22 identified psi promoters was directly regulated by PhoB. PhoB-binding sites within the promoter regions were identified by DNase I footprinting. The genes for yoaI, rpsG, galP, rnr, udp, sstT, ybiM, and vgrE were located downstream of these promoters, indicating that these genes are members of the pho regulon. Surprisingly, the other 14 promoters were located within sense or antisense strands of open reading frames (ORFs), and/or at a distance from ORFs. Our results suggest that PhoB has broader roles in gene regulation and RNA expression in E. coli strains than was previously supposed. Our shotgun-library cloning approach represents a powerful tool for identifying promoters activated or repressed by transcriptional regulators that respond to environmental stimuli.

Identification of LexA regulated promoters in Escherichia coli O157:H7.

In Escherichia coli (E. coli), most DNA damage-inducible (din) genes belong to the LexA regulon, whose products are related to functions such as DNA repair and induced mutagenesis. The E. coli K-12 cells have about 30 operons that are known to be members of the LexA regulon. LexA acts as a transcriptional repressor of these unlinked genes by binding to the specific DNA sequences located within the promoter regions. We developed a genetic screening method to isolate LexA dependent promoters. By using an applied whole-genome shotgun method with a lac-operon system, we isolated promoter candidates of din genes from the E. coli O157:H7 genome. We found that transcriptional repression from most of these promoters was dependent on lexA and purified LexA protein bound directly to the DNA fragments carrying them. Finally, we identified 16 and 5 promoters that regulated expression of previously known and novel LexA dependent genes, respectively. In addition to them, we also identified 2 antisense promoters which were considered to regulate expression of antisense RNAs for mRNAs of the ecs1779 and ecs2988 genes. All newly identified promoter regions contained DNA sequences similar to the consensus LexA binding sequence.

Identification of PhoB binding sites of the yibD and ytfK promoter regions in Escherichia coli.

By using a lacZ operon fusion genomic library of the Escherichia coli 0157:H7 Sakai, we identified phosphate-starvation-inducible (psi) promoters located upstream of the yibD and ytfK genes. They have been previously proposed to belong to the phosphate regulon (pho regulon) by Beak and Lee (2006), based on the DNA array and in vivo transcriptional experiments. However, the direct interaction of these promoters with the activator protein of the pho regulon, PhoB, has not been determined. We determined the binding regions of PhoB in these promoter regions by DNase I footprinting. Both regions contained two pho boxes similar to the consensus sequence for PhoB binding.

Identification and characterization of novel phosphate regulon genes, ecs0540-ecs0544, in Escherichia coli O157:H7.

In response to environmental phosphate limitation, the transcriptional activator PhoB of Escherichia coli (E. coli) activates transcription of the phosphate regulon (pho regulon) genes that are involved in phosphate utilization. At least 31 of pho regulon genes have been identified and well characterized in E. coli by numerous studies using non-pathogenic K-12 derivative strains. In this study, we searched for PhoB-regulated promoters from a lacZ-fused genomic library of the E. coli O157:H7 Sakai in an attempt to find novel pho regulon genes in the strain. A promoter region located upstream of a gene cluster (ecs0540-ecs0544) that mapped within one of the strain-specific chromosomal regions of the E. coli O157:H7 was identified. By further in vivo analysis with various subclones of the 5'-flanking region, it was suggested that the ecs0540 transcription was regulated by at least two promoters, an upstream PhoB-regulated promoter and a downstream constitutive promoter. S1 mapping and footprinting experiments revealed two transcription start sites and a sequence similar to the consensus sequence of PhoB binding, respectively. Bioinformatic analysis of the ecs0540-ecs0544 genes showed that these genes were highly homologous to the Escherichia fergusonii (E. fergusonii) siiCA-DA operon encoding a 718 kDa giant protein (SiiEA) and its cognate type I secretion system. In addition, a highly repetitive region and motifs that are shared among RTX (repeats in toxin) toxin family were found in the amino acid sequence of these giant proteins. Our finding is the first example of a member of the pho regulon identified in the O157:H7 strain-specific chromosomal region.

Transcription regulation by feast/famine regulatory proteins, FFRPs, in archaea and eubacteria.

Feast/famine regulatory proteins (FFRPs) comprise a single group of transcription factors systematically distributed throughout archaea and eubacteria. In the eubacterial domain in Escherichia coli, autotrophic pathways are activated and heterotrophic pathways are repressed by an FFRP, the leucine-responsive regulatory protein (Lrp), in some cases in interaction with other transcription factors. By sensing the concentration of leucine, Lrp changes its association state between hexadecamers and octamers to adapt the autotrophic or heterotrophic mode. The lrp gene is regulated so that the concentration of Lrp decreases in the presence of rich nutrition. In the archaeal domain a large part of the metabolism of Pyrococcus OT3 is regulated by another FFRP, FL11. In the presence of rich nutrition, the metabolism is released from repression by FL11; transcription of fl11 is terminated by FL11 forming octamers in interaction with lysine. When the nutrient is depleted, the metabolism is arrested by a high concentration of FL11; FL11 disassembles to dimers in the absence of lysine, and repression of transcription of fl11 is relaxed. Common characteristics of the master regulations by FL11 and Lrp hint at the prototype regulation once achieved in the common ancestor of all extant organisms. Mechanisms of discrimination by FFRPs between DNA sequences and also between co-regulatory molecules, mostly amino acids, and variations of transcription regulations observed with archaea and eubacteria are reviewed.

Expression of Escherichia coli DcuS-R two-component regulatory system is regulated by the secondary internal promoter which is activated by CRP-cAMP.

The DcuS-R two-component system of Escherichia coli senses C4-dicarboxylates of the medium and regulates expression of the genes related to utilization of them. It is known that phospho-DcuR induces expression of genes such as the dcuB-fumB operon, the frdABCD operon, and the dctA gene. We analyzed promoters of the dcuS-R operon to elucidate the transcriptional regulation system. We found a novel internal promoter within the dcuS gene that is regulated by the transcriptional regulator, CRP-cAMP, in both aerobic and anaerobic conditions.

NMR dynamics distinguish between hard and soft hydrophobic cores in the DNA-binding domain of PhoB and demonstrate different roles of the cores in binding to DNA.

The transcription factor PhoB contains an N-terminal regulatory domain and a C-terminal DNA-binding/transactivation domain. The DNA-binding/transactivation domain alone can bind specifically to DNA and consequently activate transcription. It consists of an N-terminal four-stranded beta-sheet and a winged helix domain, containing a three-helix bundle and a C-terminal beta-hairpin. The second and third helices, together with the beta-hairpin, contact DNA and the loop between the second and third helices is responsible for the transactivation. Here, we have examined the backbone and side-chain dynamics of the DNA-binding domain in its DNA-free and bound forms by NMR. The side-chain dynamics identified two apparent hydrophobic cores: one, a soft hydrophobic core, shows inherently flexible dynamics on the pico-to nanosecond timescale and maintains the DNA-binding and transactivation surfaces; the other is a hard hydrophobic core formed between the N-terminal beta-sheet and the three-helix bundle, which maintains the other non-functional surface. Upon binding to DNA, the flexibility of the soft core decreases but remains more flexible than the hard core. The winged helix domain itself has inherent flexibility in the DNA-binding and transactivation functions. However, the back surface of both functional surfaces seems to be covered by the N-terminal beta-sheet in order to mask a possible function arising from the inherent flexibility of the winged helix domain.

Feast/famine regulatory proteins (FFRPs): Escherichia coli Lrp, AsnC and related archaeal transcription factors.

Feast/famine regulatory proteins comprise a diverse family of transcription factors, which have been referred to in various individual identifications, including Escherichia coli leucine-responsive regulatory protein and asparagine synthase C gene product. A full length feast/famine regulatory protein consists of the N-terminal DNA-binding domain and the C-domain, which is involved in dimerization and further assembly, thereby producing, for example, a disc or a chromatin-like cylinder. Various ligands of the size of amino acids bind at the interface between feast/famine regulatory protein dimers, thereby altering their assembly forms. Also, the combination of feast/famine regulatory protein subunits forming the same assembly is altered. In this way, a small number of feast/famine regulatory proteins are able to regulate a large number of genes in response to various environmental changes. Because feast/famine regulatory proteins are shared by archaea and eubacteria, the genome-wide regulation by feast/famine regulatory proteins is traceable back to their common ancestor, being the prototype of highly differentiated transcription regulatory mechanisms found in organisms nowadays.

Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V cholerae.

BACKGROUND: Vibrio parahaemolyticus, a gram-negative marine bacterium, is a worldwide cause of food-borne gastroenteritis. V parahaemolyticus strains of a few specific serotypes, probably derived from a common clonal ancestor, have lately caused a pandemic of gastroenteritis. The organism is phylogenetically close to V cholerae, the causative agent of cholera. METHODS: The whole genome sequence of a clinical V parahaemolyticus strain RIMD2210633 was established by shotgun sequencing. The coding sequences were identified by use of Gambler and Glimmer programs. Comparative analysis with the V cholerae genome was undertaken with MUMmer. FINDINGS: The genome consisted of two circular chromosomes of 3288558 bp and 1877212 bp; it contained 4832 genes. Comparison of the V parahaemolyticus genome with that of V cholerae showed many rearrangements within and between the two chromosomes. Genes for the type III secretion system (TTSS) were identified in the genome of V parahaemolyticus; V cholerae does not have these genes. INTERPRETATION: The TTSS is a central virulence factor of diarrhoea-causing bacteria such as shigella, salmonella, and enteropathogenic Escherichia coli, which cause gastroenteritis by invading or intimately interacting with intestinal epithelial cells. Our results suggest that V parahaemolyticus and V cholerae use distinct mechanisms to establish infection. This finding explains clinical features of V parahaemolyticus infections, which commonly include inflammatory diarrhoea and in some cases systemic manifestations such as septicaemia, distinct from those of V cholerae infections, which are generally associated with non-inflammatory diarrhoea.

Crystallization and preliminary X-ray diffraction studies on the DNA-binding domain of the transcriptional activator protein PhoB from Escherichia coli.

PhoB is a transcriptional factor that activates more than 30 genes of the pho regulon in response to phosphate starvation. Crystals of its C-terminal domain (PhoBC) were obtained in two forms. The first crystal form, obtained from phosphate solution, belongs to space group P2(1), with unit-cell parameters a = 30.7, b = 105.9, c = 30.9 A, beta = 110.3 degrees. The second form, crystallized from PEG solution, belongs to the same space group, but has a smaller unit cell (a = 30.6, b = 37.5, c = 44.4 A, beta = 109.4 degrees ). Crystals of selenomethionyl-derivatized PhoBC were obtained using the conditions for the second crystal form. Diffraction data from wild-type PhoBC (2.0 A resolution) and MAD data sets from selenomethionyl-derivative PhoBC (3.0 A resolution) have been collected at 100 K with a synchrotron-radiation source. MAD data analysis is in progress.

Filamentous bacteriophages of vibrios are integrated into the dif-like site of the host chromosome.

The dif site is located in the replication terminus region of bacterial chromosomes, having a function of resolving dimeric chromosomes formed during replication. We demonstrate that filamentous bacteriophages of vibrios, such as f237 (Vibrio parahaemolyticus) and CTXphi (V. cholerae), are integrated into the dif-like site of host chromosome.