Whole-genome sequence of the plant-associated bacterium Pseudomonas granadensis CT364 isolated in Seville, Spain.

Pseudomonas sp. CT364 was isolated from olive tree rhizosphere in Seville (Spain). We report its complete genome sequence, acquired by co-assembling Illumina and Nanopore reads. The genome comprises a circular chromosome of 6.2 Mbp and a G + C content of 60.0%. Taxonomic analyses confirmed it to be Pseudomonas granadensis.

The antimicrobial polymer PHMB enters cells and selectively condenses bacterial chromosomes.

To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Because acquired resistance to PHMB has not been reported, selective chromosome condensation provides an unanticipated paradigm for antimicrobial action that may not succumb to resistance.

Antisense effects of PNAs in bacteria.

Peptide nucleic acids (PNAs) are a class of artificial DNA/RNA analogues that have unique physicochemical properties, which include a high chemical stability, resistance to nucleases and proteases and higher mismatch sensitivity than DNA. PNAs were initially anticipated to be useful for application in antisense and antigene therapies; however, their poor cellular uptake has limited their use for such purposes in the "real world". Recently, it has been shown that the addition of metal complexes to these oligonucleotide analogues could open up new avenues for their utilization in various research fields. Such metallo-constructs have shown great promise, for a diverse range of applications, most notably in the biosensing area. In this chapter, we report on the recent synthetic advances towards the preparation of these "(multi)-metallic PNAs" on the solid phase.

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. CONCLUSIONS: The results argue against DNA binding as the primary mechanism of action of berberine and support the hypothesis that its antibacterial properties are due to inhibition of the cell division protein FtsZ. In addition, the genetic approach used here provides a means to rapidly test the activity of other putative FtsZ inhibitors.

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.