Temporal transcriptomic analysis of the Listeria monocytogenes EGD-e sigmaB regulon

Abstract

Background: The opportunistic food-borne gram-positive pathogen Listeria monocytogenes can exist as a free-living microorganism in the environment and grow in the cytoplasm of vertebrate and invertebrate cells following infection. The general stress response, controlled by the alternative sigma factor, sigmaB, has an important role for bacterial survival both in the environment and during infection. We used quantitative real-time PCR analysis and immuno-blot analysis to examine sigmaB expression during growth of L. monocytogenes EGD-e. Whole genome-based transcriptional profiling was used to identify sigmaB-dependent genes at different growth phases.

Results: We detected 105 sigmaB-positively regulated genes and 111 genes which appeared to be under negative control of sigmaB and validated 36 sigmaB-positively regulated genes in vivo using a reporter gene fusion system.

Conclusion: Genes comprising the sigmaB regulon encode solute transporters, novel cell-wall proteins, universal stress proteins, transcriptional regulators and include those involved in osmoregulation, carbon metabolism, ribosome- and envelope-function, as well as virulence and niche-specific survival genes such as those involved in bile resistance and exclusion. Ten of the sigmaB-positively regulated genes of L. monocytogenes are absent in L. innocua. A total of 75 sigmaB-positively regulated listerial genes had homologs in B. subtilis, but only 33 have been previously described as being sigmaB-regulated in B. subtilis even though both species share a highly conserved sigmaB-dependent consensus sequence. A low overlap of genes may reflects adaptation of these bacteria to their respective environmental conditions.

Figure 1

(A) Copy number of the sigB gene in L. monocytogenes EGD-e grown in BHI medium for 3 h, 4 h, 8 h and 16 h. Data shown here is representative of three independent biological replicates. (B) Immuno-blot analysis quantifying σ^B from L. monocytogenes EGD-e at different growth phases. Proteins were isolated from cultures of L. monocytogenes EGD-e grown for 3 h (lane 1), 4 h (lane 2), 8 h (lane 3) and 16 h (lane 4) in BHI at 37°C and σ^B was detected using rabbit polyclonal anti-serum produced against S. aureus COL σ^B. The L. monocytogenes ΔsigB deletion mutant (lane 5) was used as negative control and S. aureus COL (lane 6) was used as positive control for specific binding of the antibody. Molecular masses (in kilodaltons) of prestained SDS-PAGE standard marker (Bio-Rad) are indicated on the left and σ^B is marked on the right.

Figure 2

Chromosomal distribution and orientation of σ^B-differential regulated genes. The first circle represents the scale in kb starting with the origin of replication at position 0. The second circle shows the distribution of coding sequences (CDS) of the leading and lagging strand in gray including σ^B-dependent up regulated genes in green and σ^B-dependent down regulated genes in red. The third circle indicates the COG classification for each gene and distribution of COG classes within the genome. Colour scheme and categories were chosen to the convention of the COG database. The innermost circle displays the GC skew ([G+C]/[G-C]), with values greater than zero in purple and less than zero in orange. For GC skew analysis we applied a sliding window of 1000 bp with an overlap of 500 bp. The figure was drawn using the GenomeViz software [70].

Figure 3

Functional COG categories of σ^B-differential regulated genes of L. monocytogenes EGD-e obtained from temporal transcriptome profiling experiments. Functional categories were determined using COG for L. monocytogenes provided by NCBI [62]. The figure was drawn using the Augur software [71].