Transcriptomic analysis of multi-drug resistant Escherichia coli K-12 strain in response to Lavandula angustifolia essential oil.

To better understand the synergistic antibacterial activity between piperacillin and Lavandula angustifolia essential oil (LEO) against multidrug-resistant Escherichia coli, we performed microarray transcriptomic analysis of LEO when used alone and in combination with piperacillin against the non-treated control. In total, 90 genes were differentially expressed after the combination of LEO and piperacillin treatment. Among the up-regulated genes, nfsB, nemA, fruA, nfsB, nemA are known to control microbial metabolism and nitrotoluene degradation, which were observed only in the LEO-piperacillin combinatory treatment. Four candidate genes from the microarray result, srIA, srID, waaR and nfsB, were validated by qRT-PCR as these genes showed differential expression consistently in the two methods. Biochemical pathway analysis showed that there was upregulation of genes involved in several biological processes including fructose and mannose metabolism, phosphotransferase system (PTS), lipopolysaccharide biosynthesis and nitrotoluene degradation. Genes involved in microbial metabolism in diverse environments were found both up- and down-regulated in LEO-piperacillin combinatory treatment. Our study provides new information concerning the transcriptional changes that occur during the LEO and piperacillin interaction against the multidrug-resistant bacteria and contributes to unravel the mechanisms underlying this synergism.

Long inverted repeat transiently stalls DNA replication by forming hairpin structures on both leading and lagging strands.

Long inverted repeats (LIRs), often found in eukaryotic genomes, are unstable in Escherichia coli where they are recognized by the SbcCD (the bacterial Mre11/Rad50 homologue), an endonuclease/exonuclease capable of cleaving hairpin DNA. It has long been postulated that LIRs form hairpin structures exclusively on the lagging-strand template during DNA replication, and SbcCD cleaves these hairpin-containing lagging strands to generate DNA double-strand breaks. Using a reconstituted oriC plasmid DNA replication system, we have examined how a replication fork behaves when it meets a LIR on DNA. We have shown that leading-strand synthesis stalls transiently within the upstream half of the LIR. Pausing of lagging-strand synthesis at the LIR was not clearly observed, but the pattern of priming sites for Okazaki fragment synthesis was altered within the downstream half of the LIR. We have found that the LIR on a replicating plasmid was cleaved by SbcCD with almost equal frequency on both the leading- and lagging-strand templates. These data strongly suggest that the LIR is readily converted to a cruciform DNA, before the arrival of the fork, creating SbcCD-sensitive hairpin structures on both leading and lagging strands. We propose a model for the replication-dependent extrusion of LIRs to form cruciform structures that transiently impede replication fork movement.

A novel mode of nuclease action is revealed by the bacterial Mre11/Rad50 complex.

The Mre11/Rad50 complex is a central player in various genome maintenance pathways. Here, we report a novel mode of nuclease action found for the Escherichia coli Mre11/Rad50 complex, SbcC2/D2 complex (SbcCD). SbcCD cuts off the top of a cruciform DNA by making incisions on both strands and continues cleaving the dsDNA stem at ∼10-bp intervals. Using linear-shaped DNA substrates, we observed that SbcCD cleaved dsDNA using this activity when the substrate was 110 bp long, but that on shorter substrates the cutting pattern was changed to that predicted for the activity of a 3'-5' exonuclease. Our results suggest that SbcCD processes hairpin and linear dsDNA ends with this novel DNA end-dependent binary endonuclease activity in response to substrate length rather than using previously reported activities. We propose a model for this mode of nuclease action, which provides new insight into SbcCD activity at a dsDNA end.

Inhibitory activities of microalgal extracts against Epstein-Barr virus DNA release from lymphoblastoid cells.

This study aimed to assess the inhibitory activities of methanol extracts from the microalgae Ankistrodesmus convolutus, Synechococcus elongatus, and Spirulina platensis against Epstein-Barr virus (EBV) in three Burkitt's lymphoma (BL) cell lines, namely Akata, B95-8, and P3HR-1. The antiviral activity was assessed by quantifying the cell-free EBV DNA using real-time polymerase chain reaction (PCR) technique. The methanol extracts from Ankistrodesmus convolutus and Synechococcus elongatus displayed low cytotoxicity and potent effect in reducing cell-free EBV DNA (EC(50)<0.01 µg/ml) with a high therapeutic index (>28000). After fractionation by column chromatography, the fraction from Synechococcus elongatus (SEF1) reduced the cell-free EBV DNA most effectively (EC(50)=2.9 µg/ml, therapeutic index>69). Upon further fractionation by high performance liquid chromatography (HPLC), the sub-fraction SEF1'a was most active in reducing the cell-free EBV DNA (EC(50)=1.38 µg/ml, therapeutic index>14.5). This study suggests that microalgae could be a potential source of antiviral compounds that can be used against EBV.