Quorum-dependent expression of rsmX and rsmY, small non-coding RNAs, in Pseudomonas syringae

Pseudomonas syringae pathovars are known to produce N-acyl-homoserine lactones (AHL) as quorum-sensing molecules. However, many isolates, including P. syringae pv. tomato DC3000 (PtoDC3000), do not produce them. In P. syringae, psyI, which encodes an AHL synthase, and psyR, which encodes the transcription factor PsyR required for activation of psyI, are convergently transcribed. In P. amygdali pv. tabaci 6605 (Pta6605), there is one nucleotide between the stop codons of both psyI and psyR. However, the canonical stop codon for psyI in PtoDC3000 was converted to the cysteine codon by one nucleotide deletion, and 23 additional amino acids extended it to a C-terminal end. This resulted in overlapping of the open reading frame (ORF) for psyI and psyR. On the other hand, stop codons in the psyR ORF of P. syringae 7 isolates, including pv. phaseolicola and pv. glycinea, were found. These results indicate that many pathovars of P. syringae have genetically lost AHL production ability by the mutation of their responsible genes. To examine whether PtoDC3000 modulates the gene expression profile in a population-dependent manner, we carried out microarray analysis using RNAs prepared from low- and high-density cells. We found the expressions of rsmX and rsmY remarkably activated in high-density cells. The activated expressions of rsmX and rsmY were confirmed by Northern blot hybridization, but these expressions were abolished in a ΔgacA mutant of Pta6605. These results indicate that regardless of the ability to produce AHL, P. syringae regulates expression of the small noncoding RNAs rsmX/Y by currently unknown quorum-sensing molecules

PsyR, a transcriptional regulator in quorum sensing system, binds lux box-like sequence in psyI promoter without AHL quorum sensing molecule and activates psyI transcription with AHL in Pseudomonas syringae pv. tabaci 6605

Quorum sensing (QS) is a mechanism for bacterial cell-cell communication using QS signals. N-acyl-homoserine lactones (AHLs), QS signals in Pseudomonas syringae pv. tabaci (Pta) 6605, are synthesized by an AHL synthase (PsyI) and recognized by the cognate transcription factor PsyR. To reveal the role of PsyR in virulence, we generated a psyR mutant and complemented strains of Pta 6605 and found that the psyR mutant is remarkably reduced in AHL production and ability to cause disease and propagate in host tobacco leaves. The phenotypes of complemented strains were restored to that of the wild type (WT). Because the psyR mutant lost nearly all AHL production, we investigated the function of PsyR in the transcription of psyI and production of AHL. Electrophoretic mobility shift assays suggested that the recombinant PsyR protein binds the promoter region of psyI but not psyR without AHL. The addition of AHL did not significantly affect this binding. The binding core sequence of this region was identified as a 20-bp lux box-like sequence. To reveal the function of PsyR and AHL on psyI transcription, we constructed a psyI promoter::lacZYA chimeric reporter gene, and inserted it into the WT and psyI mutant of Pta 6605. beta-galactosidase activity increased in a bacterial density-dependent manner in the WT and also in a psyI mutant after the addition of exogenous AHL. These results indicate that the solo PsyR binds the lux box in the psyI promoter and activates transcription in the concomitant presence of AHL

Insertion and Deletion Mismatches Distant from the Target Position Improve Gene Correction with a Tailed Duplex

A 5-tailed duplex (TD) DNA corrects a base-substitution mutation. In this study, the effects of insertion and deletion (indel) mismatches distant from the target position on the gene correction were examined. Three target plasmid DNAs with and without indel mismatches approximate to 330 bases distant from the correction target position were prepared, and introduced into HeLa cells together with the TD. The indel mismatches improved the gene correction efficiency and specificity without sequence conversions at the indel mismatch site. These results suggested that the gene correction efficiency and specificity are increased when an appropriate second mismatch is introduced into the TD fragment

Combination of Zinc and All-Trans Retinoic Acid Promotes Protection against Listeria monocytogenes Infection.

Zinc (Zn) is the second most abundant transition metal after iron. It plays a vital role in living organisms and affects multiple aspects of the immune system. All-trans retinoic acid (atRA) is an isomeric form of the vitamin A or retinol. It possesses the greatest biological activity of Vitamin A. Vitamin A and related retinoids influence many aspects of immunity. In this study, we demonstrated that treatment with a combination of Zn and atRA contributes to host resistance against infection by Listeria monocytogenes. Pretreatment with Zn and atRA enhanced resistance against L. monocytogenes infection in mice and treatment with both Zn and atRA showed a higher protective effect than treatment with either alone. Supplementation with Zn, atRA or their combination decreased the number of L. monocytogenes present in target organs. In vitro, supplementation increased the bacterial uptake by macrophage cells and reduced the replication of L. monocytogenes. Our results suggest that the combination of Zn and atRA has a great bacteriostatic impact on L. monocytogenes and its infection

Importance of the 1+7 configuration of ribonucleoprotein complexes for influenza A virus genome packaging

インフルエンザウイルスの遺伝の仕組みを解明. 京都大学プレスリリース. 2018-01-10.The influenza A virus genome is composed of eight single-stranded negative-sense RNAs. Eight distinct viral RNA segments (vRNAs) are selectively packaged into progeny virions, with eight vRNAs in ribonucleoprotein complexes (RNPs) arranged in a specific “1+7” pattern, that is, one central RNP surrounded by seven RNPs. Here we report the genome packaging of an artificially generated seven-segment virus that lacks the hemagglutinin (HA) vRNA. Electron microscopy shows that, even in the presence of only seven vRNAs, the virions efficiently package eight RNPs arranged in the same “1+7” pattern as wild-type virions. Next-generation sequencing reveals that the virions specifically incorporate host-derived 18S and 28S ribosomal RNAs (rRNAs) seemingly as the eighth RNP in place of the HA vRNA. These findings highlight the importance of the assembly of eight RNPs into a specific “1+7” configuration for genome packaging in progeny virions and suggest a potential role for cellular RNAs in viral genome packaging

Number of <i>L</i>. <i>monocytogenes</i> in the target organs of Zn- and atRA-supplemented mice.

<p>Mice were injected intraperitoneally with Zn, atRA or a combination of both compounds. Twenty-four hours after the last dose, the mice were infected intravenously with 10⁴/100 μl per mouse. CFU was measured in the liver and spleen 48 h after infection. The experiment was performed three times with four mice per group each time. Control mice received corn oil alone. Data are mean ± SD values of 12 mice per group. Statistically significant differences, compared with each group, are indicated by asterisks.</p

Survival of lethally infected mice supplemented with Zn and atRA.

<p>Panel A shows the administration procedure. At the end of atRA and Zn administration, mice were inoculated intravenously with 10⁵/100 μl <i>L</i>. <i>monocytogenes</i> per animal. Groups of four mice per compound were checked daily for survival. Survival was observed until day 5 post-infection.</p