Growth Deficiencies of Neisseria meningitidis pfs and luxS Mutants Are Not Due to Inactivation of Quorum Sensing

Abstract

The activated methyl cycle (AMC) is a central metabolic pathway used to generate (and recycle) several important metabolites and enable methylation. Pfs and LuxS are considered integral components of this pathway because they convert S-adenosylhomocysteine (SAH) to S-ribosylhomocysteine (SRH) and S-ribosylhomocysteine to homocysteine (HCY), respectively. The latter reaction has a second function since it also generates the precursor of the quorum-sensing molecule autoinducer 2 (AI-2). By demonstrating that there was a complete lack of AI-2 production in pfs mutants of the causative agent of meningitis and septicemia, Neisseria meningitidis, we showed that the Pfs reaction is the sole intracellular source of the AI-2 signal. Analysis of lacZ reporters and real-time PCR experiments indicated that pfs is expressed constitutively from a promoter immediately upstream, and careful study of the pfs mutants revealed a growth defect that could not be attributed to a lack of AI-2. Metabolite profiling of the wild type and of a pfs mutant under various growth conditions revealed changes in the concentrations of several AMC metabolites, particularly SRH and SAH and under some conditions also HCY. Similar studies established that an N. meningitidis luxS mutant also has metabolite pool changes and growth defects in line with the function of LuxS downstream of Pfs in the AMC. Thus, the observed growth defect of N. meningitidis pfs and luxS mutants is not due to quorum sensing but is probably due to metabolic imbalance and, in the case of pfs inactivation, is most likely due to toxic accumulation of SAH.In all living organisms, both prokaryotic and eukaryotic, S-adenosyl-L-methionine (SAM) is an essential cofactor that is required for growth and is involved in several different pathways, including transmethylation and polyamine synthesis (16). In transmethylation in particular, SAM is the main methyl group donor used for the methylation of DNA, RNA, proteins, and numerous metabolites. Because of the range of cellular possesses that could be affected by a lack of SAM, the metabolic pathways which form SAM and maintain SAM levels are very important; one of these pathways is the activated methyl cycle (AMC) (Fig. 1A). When using SAM, methylases release a toxic product, S-adenosyl-L-homocysteine (SAH), which is removed by a one-step conversion to homocysteine (HCY) by an SAH hydrolase in eukaryotes, archaebacteria, and numerous eubacteria. However, many other eubacteria do not possess this enzyme and instead detoxify SAH in two steps. The enzyme Pfs (5Ј-methylthioadenosine/S-adenosylhomocysteine nucleosidase) generates S-ribosylhomocysteine (SRH) from SAH, which is then converted to HCY by a second enzyme, LuxS (for a review, see reference 30). HCY is further recycled into methionine and subsequently into SAM by a homolog of MetK (17). The LuxS-catalyzed reaction has recently received much attention because in addition to HCY it also generates 4,5-dihydroxy-2,3-pentane dione (DPD). This compound is the precursor of a diffusible sig...

Identification of a Key Amino Acid of LuxS Involved in AI-2 Production in Campylobacter jejuni

“…Previous work in other species has demonstrated elevated SRH concentrations in the culture supernatant of luxS mutant strains [40]. To test this we utilized the in vitro AI-2 synthesis reaction described above with the wildtype LuxS enzyme and the cell-free supernatant as the source of substrate.…”

Regulatory Mechanisms of the LuxS/AI-2 System and Bacterial Resistance

“…Guillermo et al first showed that hydroxylated pyrrolidine represents a SAH/MTA inhibitor and speculated that these compounds might be transitional analogs (76). However, inhibition of Pfs is fatal to cells because the accumulation of MTA and SAH is toxic to cells (77,78). Excess MTA levels in cells inhibit growth processes and DNA synthesis by indirectly preventing the synthesis of polyamines involved in these important processes (79,80).…”

Pfs promotes autolysis-dependent release of eDNA and biofilm formation in Staphylococcus aureus

“…As an important part of the S-adenosylmethionine (SAM) cycle, methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) function in a broad array of metabolic reactions, such as methylation reactions; the recycling pathway of adenine, sulfur, and methionine; and the synthesis of polyamine, vitamin, and the universal quorumsensing signal, autoinducer-2 (AI-2) [19,20]. It has been reported that Pfs inhibitors disrupt AI-2 production in Vibrio cholerae and Escherichia coli in a dose-dependent manner without affecting growth and biofilm formation in these organisms [21].…”