In vivo evidence that S-adenosylmethionine and fatty acid synthesis intermediates are the substrates for the LuxI family of autoinducer synthases - PubMed (original) (raw)
In vivo evidence that S-adenosylmethionine and fatty acid synthesis intermediates are the substrates for the LuxI family of autoinducer synthases
D L Val et al. J Bacteriol. 1998 May.
Abstract
Many gram-negative bacteria synthesize N-acyl homoserine lactone autoinducer molecules as quorum-sensing signals which act as cell density-dependent regulators of gene expression. We have investigated the in vivo source of the acyl chain and homoserine lactone components of the autoinducer synthesized by the LuxI homolog, TraI. In Escherichia coli, synthesis of N-(3-oxooctanoyl)homoserine lactone by TraI was unaffected in a fadD mutant blocked in beta-oxidative fatty acid degradation. Also, conditions known to induce the fad regulon did not increase autoinducer synthesis. In contrast, cerulenin and diazoborine, specific inhibitors of fatty acid synthesis, both blocked autoinducer synthesis even in a strain dependent on beta-oxidative fatty acid degradation for growth. These data provide the first in vivo evidence that the acyl chains in autoinducers synthesized by LuxI-family synthases are derived from acyl-acyl carrier protein substrates rather than acyl coenzyme A substrates. Also, we show that decreased levels of intracellular S-adenosylmethionine caused by expression of bacteriophage T3 S-adenosylmethionine hydrolase result in a marked reduction in autoinducer synthesis, thus providing direct in vivo evidence that the homoserine lactone ring of LuxI-family autoinducers is derived from S-adenosylmethionine.
Figures
FIG. 1
Effect of fatty acids and a fadD disruption on AAI synthesis. Shake-flask RB cultures of the parental strain JM83-TI (▪) and a fadD derivative of this strain (JMD6-TI [▴]) were grown at 37°C from a starting OD600 of 0.1. The optical density was monitored at regular intervals; after 1 h, the strain JM83-TI culture was divided into two flasks, and oleate was added to one of the flasks (•) to a final concentration of 0.1%. The expression of the TraI enzyme in each of the cultures was induced at mid-log phase by the addition of IPTG to 1 mM (indicated by the arrows), and samples for AAI assays were removed after 0, 1, 2, and 4 h of induction. (A) Optical density. (B) Concentration of AAI detected in medium from each of the cultures.
FIG. 2
Effect of diazoborine or cerulenin on AAI synthesis. A shake-flask RB culture of strain JM83-TI was grown at 30°C while the optical density was monitored at regular intervals. At mid-log phase, the culture was divided into five separate flasks. Three of the flasks contained an inhibitor: diazoborine (100 μg/ml [▴]), cerulenin (100 μg/ml [▾]), or nalidixic acid (100 μg/ml [•]). After these cultures had been exposed to the inhibitors for 30 min, IPTG was added (0.5 mM; indicated by the arrows) to each of these flasks and to one of the flasks lacking an inhibitor (⧫). The final flask (▪) was used as the minus-IPTG control. Duplicate samples were taken for AAI bioassays after 0, 1, and 4 h of IPTG induction. (A) Optical density. (B) Concentration of AAI detected in medium from each of the cultures.
FIG. 3
Effect of diazoborine on AAI synthesis in strain JMAC-TI. An RB-oleate (0.1%) culture of JMAC-TI was grown at 37°C from a starting OD600 of 0.1, and the OD600 was monitored until mid-log phase. The culture was then divided into three flasks, containing either no inhibitor (▪), diazoborine (100 μg/ml [▴]), or nalidixic acid (100 μg/ml [•]). After 15 min of incubation with the inhibitors, IPTG was added (indicated by the arrows) to a final concentration of 0.5 mM, and the concentrations of AAI in the media were determined after 0, 1, and 3 h of IPTG induction. (A) Optical density. (B) Concentration of AAI detected in medium from each of the cultures.
FIG. 4
Effect of AdoMet hydrolase expression in strain C41 on AAI synthesis. Shake-flask RB cultures of a positive control strain (C41PC-TI [▪ and □]) and two isolates of C41 containing the T3 AdoMet hydrolase expression plasmid pACETT3 (C41T3-TIa [▴ and ▾] and C41T3-TIb [• and ○]) were grown at 37°C from a starting OD600 of 0.1. The optical density was monitored at regular intervals (A), and the cells were induced with 0.5 mM IPTG (indicated by the arrows) after being washed twice with minimal E medium to remove any AAI resulting from background TraI expression. Duplicate 15-ml culture samples were used to assay the intracellular AdoMet levels (B) and the AAI concentrations in the media (C) after 0, 1, and 3 h of IPTG induction.
FIG. 5
Effect of AdoMet hydrolase expression in strain LEPRT7 on AAI synthesis. Shake-flask RB cultures of a positive control strain (LEPPC-TI [▪ and □]) and an isolate of LEPRT7 containing the T3 AdoMet hydrolase expression plasmid pACETT3 (LEPT3-TI [▴ and ▾]) were grown at 30°C from a starting OD600 of 0.1. The cultures were grown to mid-log phase in the presence of 0.5 mM IPTG. The cells were washed twice with minimal E salts medium, resuspended in fresh RB medium containing 0.5 mM IPTG, and incubated at 37°C, and the optical density was monitored at regular intervals (A). Duplicate 15-ml samples were used to assay the intracellular AdoMet levels (B) and the AAI concentrations in the medium (C) after 0, 0.5, 2, and 4 h of heat induction.
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