Temperature-dependent expression of internalin and internalin-like genes

Listeria monocytogenes, the etiological agent of listeriosis, is capable of growth and survival at temperatures ranging from 2 to 48oC, reflecting the diverse environments and host species inhabited by this organism. L. monocytogenes expresses up to 29 proteins, termed internalins, whose structure indicates they make good candidates for facilitating bacterial-host cell interactions. Considering the ubiquitous nature of this organism, we speculated that environmental temperature might serve as an important biological signal in controlling their expression. We therefore employed a subgenomic microarray to investigate the expression profiles of 24 members of the internalin gene family identified in L. monocytogenes 10403S. Competitive hybridization was performed between RNA extracted from 10403S grown to early stationary phase at 37oC, and 10403S grown to early stationary phase at 16oC, 30oC and 42oC. The data reveal that internalin genes can be divided into four broad categories such that (i) four internalin genes inlC2, inlD, lmo0331 and lmo0610 show temperature-dependent expression similar to sigB and the σB-dependent gene opuCA; (ii) three internalin genes inlA, inlB and inlC show temperature-dependent expression similar to the PrfA-dependent gene plcA; (iii) five internalin-like genes inlG, inlJ, lmo0327, lmo0514 and lmo1290 show unique temperature-dependent expression and (iv) twelve internalin genes show no difference in expression under the conditions investigated in this study. Our data also shows that the expression of many housekeeping genes can vary considerably under different temperatures, and normalization of both microarray and qRT-PCR data using housekeeping genes should be based on comprehensive experimental validation. Keywords: Listeria monocytogenes, internalins, temperature, sub-genomic microarrays To enhance the relative proportion of cells in log phase, for all experiments, each strain was initially grown in BHI at 37oC with shaking (200 rpm) to OD600=0.4, then diluted 1:100 into fresh BHI and grown to OD600=0.4. . A log phase culture of L. monocytogenes 1040S was diluted 1:100 in prewarmed BHI (16oC, 30oC, 37oC and 42oC) and subsequrently incubated with shaking (200 rpm) at the respective temperature until early stationary phase, defined as OD600=1.0 + 3 hrs for cells grown at 30 oC, 37 oC and 42 oC. For cells cultured at 16oC, early stationary phase was defined as OD600=1.0 + 6 hrs. Following incubation, RNA was stabilized by the addition of two volumes of RNAprotect™ (Qiagen, CA, USA). Bacterial cells were then harvested by centrifugation and stored at –80oC for no longer than 24 h before RNA isolation. For each growth condition described above, three independent RNA isolations were performed on different days to provide distinct biological replicates. Each set of independent RNA samples was used to perform competitive hybridization microarray experiments. For each set of biological replicates, RNA from cells cultured at 37oC was hybridized to RNA from cells cultured at 16oC, 30oC and 42oC