Extragenic Suppressors of Growth Defects inmsbB Salmonella

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°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°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.

PagP Activation in the Outer Membrane Triggers R3 Core Oligosaccharide Truncation in the Cytoplasm of Escherichia coli O157:H7

“…Mutation of pagP in this msbB-deficient background aggravates the antibiotic sensitivity (Table 2), which suggests that lipid A palmitoylation can partially compensate for the absence of myristate in lipid A. Previous studies of msbB mutants have revealed increased sensitivity to antibiotics in Salmonella but not in E. coli K-12 (46,47), and our observations confirm these latter findings (Table 2). We also observed slow growth and poor growth of msbB-and msbB/pagPdeficient E. coli O157:H7, respectively, on lactose MacConkey agar plates, which contain bile salts that select against organisms with a compromised OM permeability barrier (not shown).…”

EGFR‐targeted Chimeras of Pseudomonas ToxA released into the extracellular milieu by attenuated Salmonella selectively kill tumor cells

“…All strains are stored frozen at −80°C in 15% glycerol. Bacterial media used were LB (Miller, 1992) which contained 1% tryptone, 0.5% yeast extract, and 1% NaCl or LB plates containing 1.5% agar, LB-0 (LB no NaCl; Murray et al, 2001), and a novel media CTY containing 0.2% casamino acids, 0.2% tryptone, 0.1% yeast, and 0.9% NaCl adjusted to pH 7.4 with 0.2 M NaOH, which we found was less toxic to mammalian cells than LB, yet supported bacterial protein expression. Antibiotics used were 100 µg/mL ampicillin (Amp 100 ) except in CTY which was 50 µg/mL.…”

Optimizing the restored chemotactic behavior of anticancer agent Salmonella enterica serovar Typhimurium VNP20009

“…The gene msbB encodes an enzyme that adds terminal myristyl groups to lipid A (Somerville et al, 1996). Without the LPS modifying enzyme, strains of Salmonella are known to possess growth defects as well as EGTA and galactose-MacConkey medium sensitivity (Murray et al, 2001). In strain S. Typhimurium 14028s, these effects are partially suppressed by the Suwwan deletion, a spontaneous recombination event causing the excision of a 108-kilobase region of the genome (Murray et al, 2004).…”

“…Bacterial cell cultures were routinely grown in MSB (Murray et al, 2001) medium (1% tryptone, 0.5% yeast extract, 2 mM MgSO 4 , 2 mM CaCl 2 ) at 37°C.…”