Concurrent growth rate and transcript analyses reveal essential gene stringency in Escherichia coli

BACKGROUND: Genes essential for bacterial growth are of particular scientific interest. Many putative essential genes have been identified or predicted in several species, however, little is known about gene expression requirement stringency, which may be an important aspect of bacterial physiology and likely a determining factor in drug target development. METHODOLOGY/PRINCIPAL FINDINGS: Working from the premise that essential genes differ in absolute requirement for growth, we describe silencing of putative essential genes in E. coli to obtain a titration of declining growth rates and transcript levels by using antisense peptide nucleic acids (PNA) and expressed antisense RNA. The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL(50)). When applied to four growth essential genes, both RNA silencing methods resulted in MTL(50) values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required. The established antibacterial targets murA and fabI were less stringently required. CONCLUSIONS: RNA silencing can reveal stringent requirements for gene expression with respect to growth. This method may be used to validate existing essential genes and to quantify drug target requirement.Peer reviewe

Genetic Evidence for Inhibition of Bacterial Division Protein FtsZ by Berberine

Background: Berberine is a plant alkaloid that is widely used as an anti-infective in traditional medicine. Escherichia coli exposed to berberine form filaments, suggesting an antibacterial mechanism that involves inhibition of cell division. Berberine is a DNA ligand and may induce filamentation through induction of the SOS response. Also, there is biochemical evidence for berberine inhibition of the cell division protein FtsZ. Here we aimed to assess possible berberine mechanism(s) of action in growing bacteria using genetics tools. Methodology/Principal Findings: First, we tested whether berberine inhibits bacterial growth through DNA damage and induction of the SOS response. The SOS response induced by berberine was much lower compared to that induced by mitomycin C in an SOS response reporter strain. Also, cell filamentation was observed in an SOS-negative E. coli strain. To test whether berberine inhibits FtsZ, we assessed its effects on formation of the cell division Z-rings, and observed a dramatic reduction in Z-rings in the presence of berberine. We next used two different strategies for RNA silencing of ftsZ and both resulted in sensitisation of bacteria to berberine, visible as a drop in the Minimum Inhibitory Concentration (MIC). Furthermore, Fractional Inhibitory Concentration Indices (FICIs) showed a high level of synergy between ftsZ silencing and berberine treatment (FICI values of 0.23 and 0.25 for peptide nucleic acid- and expressed antisense RNA-based silencing of ftsZ, respectively). Finally, over-expression of ftsZ led to a mild rescue effect in berberine-treated cells

Plasmids used in this study.

<p>*antisense target locations are indicated relative to the start codon.</p

Effect of berberine on <i>E. coli</i> morphology.

<p>Bacteria (wild-type and the SOS-negative strain) were either untreated or treated with 1.5 mM berberine. Scale bars are 10 µm.</p

Structure of PNA used in this study.

<p>*antisense target locations are indicated relative to the start codon.</p

<i>Escherichia coli</i> strains used in this study.

<p><i>Escherichia coli</i> strains used in this study.</p

Z-ring formation and cell morphology in berberine-treated <i>E. coli</i>.

<p>Representative cells untreated and treated with 3 mM berberine; FtsZ was tagged with YFP and bacterial DNA was visualised through staining with DAPI. Scale bars are 10 µm.</p

Over-expression of <i>ftsZ</i> and its effect on <i>E. coli</i> growth in the presence and absence of berberine.

<p>Bacteria (<i>E. coli</i> DH5α carrying plasmid pBAD-ftsZ) were induced using a concentration range of L-arabinose (0–0.01%) to allow over-expression of the FtsZ protein and grown in the absence or presence of berberine. The growth rate in the absence of L-arabinose or berberine was set as 1. As a control, <i>E. coli</i> DH5α without the plasmid was treated with berberine in the same range of L-arabinose concentrations.</p

Bacterial SOS response in the presence of berberine.

<p>The doses of each compound were chosen as indicated percentage of the MIC (4 mM, 15 nM and 400 nM for berberine, mitomycin C and triclosan, respectively). Mitomycin C and triclosan were included as positive and negative controls for SOS induction. The Relative Fluorescence Units (RFUs) indicate the level of SOS reporter expression in the SOS-negative <i>E. coli</i> strain SS996.</p

Chemical structures of antibacterial compounds used in this study.

<p>Chemical structures of antibacterial compounds used in this study.</p