No abstract

cPeptide phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA mimics that bind cRNA and inhibit bacterial gene expression. The PPMO (RFF) 3 RXB-AcpP (where R is arginine, F, phenylalanine, X is 6-aminohexanoic acid, B is ␤-alanine, and AcpP is acyl carrier protein) is complementary to 11 bases of the essential gene acpP (which encodes acyl carrier protein). The MIC of (RFF) 3 RXB-AcpP was 2.5 M (14 g/ml) in Escherichia coli W3110. The rate of spontaneous resistance of E. coli to (RFF) 3 RXB-AcpP was 4 ؋ 10 ؊7 mutations/cell division. A spontaneous (RFF) 3 RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF) 3 RXB-AcpP was 40 M (224 g/ml) for PR200.1. The MICs of standard antibiotics for PR200.1 and W3110 were identical. The sequence of acpP was identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF) 3 RXB or peptides with a similar composition or pattern of cationic and nonpolar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF) 3 RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped to sbmA. Genetic complementation of PR200.1 or RR3 with sbmA restored susceptibility to (RFF) 3 RXB-AcpP. Deletion of sbmA caused resistance to (RFF) 3 RXB-AcpP. We conclude that resistance to (RFF) 3 RXB-AcpP was linked to the peptide and not the phosphorodiamidate morpholino oligomer, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF) 3 R-XB PPMOs may be transported across the plasma membrane by SbmA.

The extracytoplasmic functioning sigma factor σE is known to play an essential role for Salmonella enterica serovar Typhimurium to survive and proliferate in macrophages and mice. However, its regulatory network is not well-characterized, especially during infection. Here we used microarray to identify genes regulated by σE in Salmonella grown in three conditions: a nutrient-rich condition and two others that mimic early and late intracellular infection. We found that in each condition σE regulated different sets of genes, and notably, several global regulators. When comparing nutrient-rich and infection-like conditions, large changes were observed in the expression of genes involved in Salmonella pathogenesis island (SPI)-1 type-three secretion system (TTSS), SPI-2 TTSS, protein synthesis, and stress responses. In total, the expression of 58% of Salmonella genes was affected by σE in at least one of the three conditions. An important finding is that σE up-regulates SPI-2 genes, which are essential for Salmonella intracellular survival, by up-regulating SPI-2 activator ssrB expression at the early stage of infection and down-regulating SPI-2 repressor hns expression at a later stage. Moreover, σE is capable of countering the silencing of H-NS, releasing the expression of SPI-2 genes. This connection between σE and SPI-2 genes, combined with the global regulatory effect of σE, may account for the lethality of rpoE-deficient Salmonella in murine infection.

The alternative sigma factor σE functions to maintain bacterial homeostasis and membrane integrity in response to extracytoplasmic stress by regulating thousands of genes both directly and indirectly. The transcriptional regulatory network governed by σE in Salmonella and E. coli has been examined using microarray, however a genome-wide analysis of σE–binding sites in Salmonella has not yet been reported. We infected macrophages with Salmonella Typhimurium over a select time course. Using chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq), 31 σE–binding sites were identified. Seventeen sites were new, which included outer membrane proteins, a quorum-sensing protein, a cell division factor, and a signal transduction modulator. The consensus sequence identified for σE in vivo binding was similar to the one previously reported, except for a conserved G and A between the -35 and -10 regions. One third of the σE–binding sites did not contain the consensus sequence, suggesting there may be alternative mechanisms by which σE modulates transcription. By dissecting direct and indirect modes of σE-mediated regulation, we found that σE activates gene expression through recognition of both canonical and reversed consensus sequence. New σE regulated genes (greA, luxS, ompA and ompX) are shown to be involved in heat shock and oxidative stress responses.