Combinatorial selection for replicable RNA by Qß replicase while maintaining encoded gene function.

Construction of a complex artificial self-replication system is challenging in the field of in vitro synthetic biology. Recently, we developed a translation-coupled RNA replication system, wherein an artificial genomic RNA replicates with the Qß RNA replicase gene encoded on itself. The challenge is to introduce additional genes into the RNA to develop a complex system that mimics natural living systems. However, most RNA sequence encoding genes are not replicable by the Qß replicase owing to its requirement for strong secondary structures throughout the RNA sequence that are absent in most genes. In this study, we establish a new combinatorial selection method to find an RNA sequence with secondary structures and functional amino acid sequences of the encoded gene. We selected RNA sequences based on their in vitro replication and in vivo gene functions. First, we used the α-domain gene of ß-galactosidase as a model-encoding gene, with functional selection based on blue-white screening. Through the combinatorial selection, we developed more replicable RNAs while maintaining the function of the encoded α-domain. The selected sequence improved the affinity between the minus strand RNA and Qß replicase. Second, we established an in vivo selection method applicable to a broader range of genes by using an Escherichia coli strain with one of the essential genes complemented with a plasmid. We performed the combinatorial selection using an RNA encoding serS and obtained more replicable RNA encoding functional serS gene. These results suggest that combinatorial selection methods are useful for the development of RNA sequences replicable by Qß replicase while maintaining the encoded gene function.

Toward Network Biology in E. coli Cell.

E. coli has been a critically important model research organism for more than 50 years, particularly in molecular biology. In 1997, the E. coli draft genome sequence was published. Post-genomic techniques and resources were then developed that allowed E. coli to become a model organism for systems biology. Progress made since publication of the E. coli genome sequence will be summarized.

Identification of essential genes and synthetic lethal gene combinations in Escherichia coli K-12.

Here we describe the systematic identification of single genes and gene pairs, whose knockout causes lethality in Escherichia coli K-12. During construction of precise single-gene knockout library of E. coli K-12, we identified 328 essential gene candidates for growth in complex (LB) medium. Upon establishment of the Keio single-gene deletion library, we undertook the development of the ASKA single-gene deletion library carrying a different antibiotic resistance. In addition, we developed tools for identification of synthetic lethal gene combinations by systematic construction of double-gene knockout mutants. We introduce these methods herein.

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.

The DNA-recognition mode shared by archaeal feast/famine-regulatory proteins revealed by the DNA-binding specificities of TvFL3, FL10, FL11 and Ss-LrpB.

The DNA-binding mode of archaeal feast/famine-regulatory proteins (FFRPs), i.e. paralogs of the Esherichia coli leucine-responsive regulatory protein (Lrp), was studied. Using the method of systematic evolution of ligands by exponential enrichment (SELEX), optimal DNA duplexes for interacting with TvFL3, FL10, FL11 and Ss-LrpB were identified as TACGA[AAT/ATT]TCGTA, GTTCGA[AAT/ATT]TCGAAC, CCGAAA[AAT/ATT]TTTCGG and TTGCAA[AAT/ATT]TTGCAA, respectively, all fitting into the form abcdeWWWedcba. Here W is A or T, and e.g. a and a are bases complementary to each other. Apparent equilibrium binding constants of the FFRPs and various DNA duplexes were determined, thereby confirming the DNA-binding specificities of the FFRPs. It is likely that these FFRPs recognize DNA in essentially the same way, since their DNA-binding specificities were all explained by the same pattern of relationship between amino-acid positions and base positions to form chemical interactions. As predicted from this relationship, when Gly36 of TvFL3 was replaced by Thr, the b base in the optimal DNA duplex changed from A to T, and, when Thr36 of FL10 was replaced by Ser, the b base changed from T to G/A. DNA-binding characteristics of other archaeal FFRPs, Ptr1, Ptr2, Ss-Lrp and LysM, are also consistent with the relationship.

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.

Feast/famine regulation by transcription factor FL11 for the survival of the hyperthermophilic archaeon Pyrococcus OT3.

Transcriptional repressor FL11 from the hyperthermophilic archaeon, Pyrococcus OT3, was crystallized in its dimer form in complex with a DNA duplex, TGAAAWWWTTTCA. Chemical contacting of FL11 to the terminal 5 bps, and DNA bending by propeller twisting at WWW confirmed specificity of the interaction. Dimer-binding sites were identified in promoters of approximately 200 transcription units coding, for example, H+-ATPase and NAD(P)H dehydrogenase. In the presence of lysine, four FL11 dimers were shown to assemble into an octamer, thereby covering the fl11 promoter. In the "feast" mode, when P. OT3 grows on amino acids, the FL11 octamer will terminate transcription of fl11, as was shown in vitro, thereby derepressing transcription of many metabolic genes. In the "famine" mode in the absence of lysine, approximately 6000 FL11 dimers present per cell will arrest growth. This regulation resembles global regulation by Escherichia coli leucine-responsive regulatory protein, and hints at a prototype of transcription regulations now highly diverged.

A structural code for discriminating between transcription signals revealed by the feast/famine regulatory protein DM1 in complex with ligands.

Feast/famine regulatory proteins (FFRPs) comprise the largest group of archaeal transcription factors. Crystal structures of an FFRP, DM1 from Pyrococcus, were determined in complex with isoleucine, which increases the association state of DM1 to form octamers, and with selenomethionine, which decreases it to maintain dimers under some conditions. Asp39 and Thr/Ser at 69-71 were identified as being important for interaction with the ligand main chain. By analyzing residues surrounding the ligand side chain, partner ligands were identified for various FFRPs from Pyrococcus, e.g., lysine facilitates homo-octamerization of FL11, and arginine facilitates hetero-octamerization of FL11 and DM1. Transcription of the fl11 gene and lysine synthesis are regulated by shifting the equilibrium between association states of FL11 and by shifting the equilibrium toward association with DM1, in response to amino acid availability. With FFRPs also appearing in eubacteria, the origin of such regulation can be traced back to the common ancestor of all extant organisms, serving as a prototype of transcription regulations, now highly diverged.

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.

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.

A SELEX study of the DNA-binding specificity of archaeal FFRPs: 2. FL4 (pot1613368).

The DNA-binding specificity of a transcription factor, the FFRP FL4 (pot1613368) from Pyrococcus sp. OT3, was studied. Using SELEX (systematic evolution of ligands by exponential environment) experiments, from a set of fragments, ∼150 bps, of the genomic DNA of P. OT3, seven were selected as containing binding sites. Thirteen bases identified as shared by the seven selected fragments with the least mismatches, 2.71 on average, was ATGAA AAAGTCAT. This sequence was closely related with another sequence, ATGAA[AAA/TTT]TTCAT, in the 5-3-5 arrangement, i.e. NANBNCNDNE [AAA/TTT]NENDNCNBNA , where, e.g. NA was the base complementary to NA. The average number of mismatches found between this sequence and the seven fragments was 3.14. A sequence, TTGAA ATT TACAA, resembling the sequence ATGAA[AAA/TTT]TTCAT and also another 5-3-5 sequence, TTGAA[AAA/TTT]TTCAA, was found upstream of the fl4 gene, which is potentially recognized by FL4 for auto-regulation. Thus it is likely that an ideal binding-site of FL4 is ATGAA[AAA/TTT]TTCAT or TTGAA[AAA/TTT]TTCAA. In this abstract, the sequences were highlighted in Italic at 3, and with bold characters at 5 and 5. When two sequences compared were the same at some positions, there they were underlined.

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.

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.