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Extragenic suppressors of growth defects in msbB Salmonella
S R Murray1, D Bermudes, K S de Felipe
1Department of Biology, Yale University, New Haven, Connecticut 06520, USA.
Abstract:
Lipid A, a potent endotoxin which can cause septic shock, anchors lipopolysaccharide (LPS) into the outer leaflet of the outer membrane of gram-negative bacteria. MsbB acylates (KDO)(2)-(lauroyl)-lipid IV-A with myristate during lipid A biosynthesis. Reports of knockouts of the msbB gene describe effects on virulence but describe no evidence of growth defects in Escherichia coli K-12 or Salmonella. Our data confirm the general lack of growth defects in msbB E. coli K-12. In contrast, msbB Salmonella enterica serovar Typhimurium exhibits marked sensitivity to galactose-MacConkey and 6 mM EGTA media. At 37 degrees C in Luria-Bertani (LB) broth, msbB Salmonella cells elongate, form bulges, and grow slowly. msbB Salmonella grow well on LB-no salt (LB-0) agar; however, under specific shaking conditions in LB-0 broth, many msbB Salmonella cells lyse during exponential growth and a fraction of the cells form filaments. msbB Salmonella grow with a near-wild-type growth rate in MSB (LB-0 containing Mg(2+) and Ca(2+)) broth (23 to 42 degrees C). Extragenic compensatory mutations, which partially suppress the growth defects, spontaneously occur at high frequency, and mutants can be isolated on media selective for faster growing derivatives. One of the suppressor mutations maps at 19.8 centisomes and is a recessive IS10 insertional mutation in somA, a gene of unknown function which corresponds to ybjX in E. coli. In addition, random Tn10 mutagenesis carried out in an unsuppressed msbB strain produced a set of Tn10 inserts, not in msbB or somA, that correlate with different suppressor phenotypes. Thus, insertional mutations, in somA and other genes, can suppress the msbB phenotype.
Insights
The msbB gene knockout in Salmonella Typhimurium causes significant growth defects and cell lysis, unlike in E. coli. Compensatory mutations in somA and other genes can suppress these msbB phenotypes.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- Lipid A is essential for anchoring lipopolysaccharide (LPS) in Gram-negative bacteria.
- The msbB gene is involved in lipid A biosynthesis, with previous studies showing minimal impact on bacterial growth.
- Understanding msbB function is crucial for insights into bacterial membrane structure and virulence.
Purpose of the Study:
- To investigate the phenotypic consequences of msbB gene knockout in Salmonella Typhimurium.
- To identify genetic factors that can suppress the growth defects associated with msbB mutations.
- To elucidate the role of msbB in bacterial physiology and adaptation.
Main Methods:
- Comparative growth analysis of wild-type and msbB mutant strains in various media conditions.
- Microscopic examination of cell morphology under stress conditions.
- Genetic screening for spontaneous suppressor mutations using insertional mutagenesis (IS10 and Tn10).
Main Results:
- msbB Salmonella Typhimurium exhibited severe growth defects, including cell elongation, bulging, lysis, and filamentation, particularly in specific media.
- Growth was restored in the presence of divalent cations (Mg2+, Ca2+) or through compensatory mutations.
- Suppressor mutations were identified in the somA gene (ybjX in E. coli) and other unlinked loci, indicating diverse genetic pathways can rescue msbB phenotypes.
Conclusions:
- The msbB gene plays a critical, previously underestimated role in Salmonella Typhimurium growth and cell envelope integrity.
- The identification of suppressor mutations highlights the complex genetic network regulating bacterial viability and adaptation.
- Targeting msbB or related pathways could offer novel strategies for combating Gram-negative bacterial infections.
