The Gram-positive bacterium Listeria monocytogenes transitions from an environmental organism to an intracellular pathogen following its ingestion by susceptible mammalian hosts. Bacterial replication within the cytosol of infected cells requires activation of the central virulence regulator PrfA followed by a PrfA-dependent induction of secreted virulence factors. The PrfA-induced secreted chaperone PrsA2 and the chaperone/protease HtrA contribute to the folding and stability of select proteins translocated across the bacterial membrane. L. monocytogenes strains that lack both prsA2 and htrA exhibit near-normal patterns of growth in broth culture but are severely attenuated in vivo. We hypothesized that, in the absence of PrsA2 and HtrA, the increase in PrfA-dependent protein secretion that occurs following bacterial entry into the cytosol results in misfolded proteins accumulating at the bacterial membrane with a subsequent reduction in intracellular bacterial viability. Consistent with this hypothesis, the introduction of a constitutively activated allele of prfA (prfA*) into ⌬prsA2 ⌬htrA strains was found to essentially inhibit bacterial growth at 37°C in broth culture. ⌬prsA2 ⌬htrA strains were additionally found to be defective for cell invasion and vacuole escape in selected cell types, steps that precede full PrfA activation. These data establish the essential requirement for PrsA2 and HtrA in maintaining bacterial growth under conditions of PrfA activation. In addition, chaperone function is required for efficient bacterial invasion and rapid vacuole lysis within select host cell types, indicating roles for PrsA2/HtrA prior to cytosolic PrfA activation and the subsequent induction of virulence factor secretion. The Gram-positive bacterium Listeria monocytogenes habitually exists in soil and decomposing plant matter (1-3) but can cause severe invasive disease in animals and humans following the ingestion of contaminated food (4, 5). The successful transition of L. monocytogenes from the outside environment to life within the mammalian host is dependent upon the activation of PrfA, a transcriptional activator which regulates the majority of the gene products associated with bacterial virulence (6-9). Full activation of PrfA occurs following entry of L. monocytogenes into the cytosol of infected-host cells, with PrfA-dependent gene products facilitating the major steps of L. monocytogenes pathogenesis that include intracellular replication, actin-based bacterial motility, and spread to adjacent cells (10, 11). Following cell entry, the escape of L. monocytogenes from host cell vacuoles is mediated by three PrfA-dependent gene products: the cholesterol-dependent poreforming cytolysin listeriolysin O (LLO) and two phospholipases (PlcA and PlcB) (12-15). Within the cytosol, L. monocytogenes recruits and polymerizes host cell actin through the expression of a PrfA-dependent bacterial surface protein known as ActA, enabling bacterial movement into adjacent cells (16,17). Activation of PrfA within the cytosol ha...

In addition to nucleotide variation, many bacteria also undergo changes at a much larger scale via rearrangement of their genome structure around long repeat sequences. These rearrangements result in genome fragments shifting position and/or orientation in the genome without necessarily affecting the underlying nucleotide sequence. To date, scalable techniques have not been applied to genome structure (GS) identification, so it remains unclear how extensive this variation is and the extent of its impact upon gene expression. However, the emergence of multiplexed, long-read sequencing overcomes the scale problem, as reads of several thousand bases are routinely produced that can span long repeat sequences to identify the flanking chromosomal DNA, allowing GS identification. Genome rearrangements were generated in Salmonella enterica serovar Typhi through long-term culture at ambient temperature. Colonies with rearrangements were identified via long-range PCR and subjected to long-read nanopore sequencing to confirm genome variation. Four rearrangements were investigated for differential gene expression using transcriptomics. All isolates with changes in genome arrangement relative to the parent strain were accompanied by changes in gene expression. Rearrangements with similar fragment movements demonstrated similar changes in gene expression. The most extreme rearrangement caused a large imbalance between the origin and terminus of replication and was associated with differential gene expression as a factor of distance moved towards or away from the origin of replication. Genome structure variation may provide a mechanism through which bacteria can quickly adapt to new environments and warrants routine assessment alongside traditional nucleotide level measures of variation.

In addition to nucleotide variation, many bacteria also undergo changes at a much larger scale via rearrangement of their genome structure (GS) around long repeat sequences. These rearrangements result in genome fragments shifting position and/or orientation in the genome without necessarily affecting the underlying nucleotide sequence. To date, scalable techniques have not been applied to GS identification, so it remains unclear how extensive this variation is and the extent of its impact upon gene expression. However, the emergence of multiplexed, long‐read sequencing overcomes the scale problem, as reads of several thousand bases are routinely produced that can span long repeat sequences to identify the flanking chromosomal DNA, allowing GS identification. Genome rearrangements were generated in Salmonella enterica serovar Typhi through long‐term culture at ambient temperature. Colonies with rearrangements were identified via long‐range PCR and subjected to long‐read nanopore sequencing to confirm genome variation. Four rearrangements were investigated for differential gene expression using transcriptomics. All isolates with changes in genome arrangement relative to the parent strain were accompanied by changes in gene expression. Rearrangements with similar fragment movements demonstrated similar changes in gene expression. The most extreme rearrangement caused a large imbalance between the origin and terminus of replication and was associated with differential gene expression as a factor of distance moved toward or away from the origin of replication. Genome structure variation may provide a mechanism through which bacteria can quickly adapt to new environments and warrants routine assessment alongside traditional nucleotide‐level measures of variation.