A cold-sensitive Listeria monocytogenes mutant has a transposon insertion in a gene encoding a putative membrane
Siqing Liu1, Darrell O Bayles, Tricia M Mason
1Bioproducts and Biocatalysis Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois 61790-4120, USA.
Abstract:
A cold-sensitive Listeria monocytogenes mutant designated cld-14 was obtained by transposon Tn917 mutagenesis. The gene interrupted by Tn917 in cld-14 was the L. monocytogenes LMOf2365_1485 homolog, which exhibits 45.7% homology to the Bacillus subtilis yqfF locus. LMOf2365_1485, here designated pgpH, encodes a putative integral membrane protein with a predicted molecular mass of 81 kDa. PgpH is predicted to contain a conserved N-terminal signal peptide sequence, seven transmembrane helices, and a hydrophilic C terminus, which likely extends into the cytosol. The Tn917 insertion in pgpH is predicted to result in production of a premature polypeptide truncated at the fifth transmembrane domain. The C terminus of PgpH, which is probably absent in cld-14, contains a highly conserved HD domain that belongs to a metal-dependent phosphohydrolase family. Strain cld-14 accumulated higher levels of (p)ppGpp than the wild type accumulated, indicating that the function of PgpH may be to adjust cellular (p)ppGpp levels during low-temperature growth. The cld-14pgpH(+) complemented strain was able to grow at a low temperature, like the parent strain, providing direct evidence that the activity of PgpH is important in low-temperature adaptation. Because of its predicted membrane location, PgpH may play a critical role in sensing the environmental temperature and altering cellular (p)ppGpp levels to allow the organism to adapt to low temperatures.
Insights
A newly identified protein, PgpH, is crucial for Listeria monocytogenes to adapt to cold temperatures. This membrane protein helps regulate cellular alarmones, enabling growth in low-temperature environments.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Physiology
Background:
- Listeria monocytogenes is a significant foodborne pathogen.
- Bacterial adaptation to low temperatures is critical for survival and pathogenesis.
- The precise mechanisms of cold adaptation in L. monocytogenes are not fully understood.
Purpose of the Study:
- To identify and characterize genes involved in cold-sensitive growth in Listeria monocytogenes.
- To elucidate the function of a novel membrane protein, PgpH, in bacterial low-temperature adaptation.
Main Methods:
- Transposon mutagenesis (Tn917) to generate cold-sensitive mutants.
- Gene identification and sequence homology analysis.
- Complementation studies to confirm gene function.
- Analysis of guanosine tetraphosphate ((p)ppGpp) levels.
Main Results:
- A cold-sensitive mutant, cld-14, resulted from Tn917 insertion in the LMOf2365_1485 gene (pgpH).
- PgpH encodes a putative 81 kDa integral membrane protein with seven transmembrane helices and a conserved HD domain.
- The mutant strain accumulated higher levels of (p)ppGpp, a stress alarmone, at low temperatures.
- Complementation restored wild-type cold growth, confirming PgpH's essential role.
Conclusions:
- PgpH is essential for Listeria monocytogenes adaptation to low temperatures.
- PgpH likely functions as a membrane-bound sensor that modulates (p)ppGpp levels in response to temperature changes.
- This mechanism allows L. monocytogenes to adjust its physiology for survival and growth in cold environments.
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