Lmo0036, an ornithine and putrescine carbamoyltransferase in Listeria monocytogenes, participates in arginine deiminase and agmatine deiminase pathways and mediates acid tolerance

Abstract

Listeria monocytogenes is a foodborne pathogen causing listeriosis. Acid is one of the stresses that foodborne pathogens encounter most frequently. The ability to survive and proliferate in acidic environments is a prerequisite for infection. However, there is limited knowledge about the molecular basis of adaptation of L. monocytogenes to acid. Arginine deiminase (ADI) and agmatine deiminase (AgDI) systems are implicated in bacterial tolerance to acidic environments. Homologues of ADI and AgDI systems have been found in L. monocytogenes lineages I and II strains. Sequence analysis indicated that lmo0036 encodes a putative carbamoyltransferase containing conserved motifs and residues important for substrate binding. Lmo0036 acted as an ornithine carbamoyltransferase and putrescine carbamoyltransferase, representing the first example, to our knowledge, that catalyses reversible ornithine and putrescine carbamoyltransfer reactions. Catabolic ornithine and putrescine carbamoyltransfer reactions constitute the second step of ADI and AgDI pathways. However, the equilibrium of in vitro carbamoyltransfer reactions was overwhelmingly towards the anabolic direction, suggesting that catabolic carbamoyltransferase was probably the limiting step of the pathways. lmo0036 was induced at the transcriptional level when L. monocytogenes was subjected to low-pH stress. Its expression product in Escherichia coli exhibited higher catabolic carbamoyltransfer activities under acidic conditions. Consistently, absence of this enzyme impaired the growth of Listeria under mild acidic conditions (pH 4.8) and reduced its survival in synthetic human gastric fluid (pH 2.5), and corresponded to a loss in ammonia production, indicating that Lmo0036 was responsible for acid tolerance at both sublethal and lethal pH levels. Furthermore, Lmo0036 played a possible role in Listeria virulence.

Transcriptomic Analysis of the Adaptation of Listeria monocytogenes to Growth on Vacuum-Packed Cold Smoked Salmon

“…We also identified four upregulated genes with agmatine deiminase-related annotations (see Table S6 in the supplemental material); the fold changes of these genes ranged from 13.67 to 31.36 and were the highest among all upregulated genes. To further explore the functions of these genes, we reconstructed the reactions involved in the L. monocytogenes agmatine deiminase system, using previous studies on L. monocytogenes and other Gram-positive bacteria (45)(46)(47)(48). All four genes encoding the key enzymes involved in the breakdown of agmatine to CO 2 and NH 3 showed higher transcript levels in H7858 grown FIG 3 Schematic of galactitol-and mannose-specific phosphotransferase system (PTS), maltose-specific ATP-binding cassette (ABC) transporter system, catabolism reactions for each of the three molecules, and the nonoxidative branch of the pentose phosphate pathway in H7858.…”

“…The agmatine deiminase system in H7858 was constructed by connecting reactions associated with agmatine deiminase based on previous studies (45)(46)(47)(48). H7858 protein designations for enzymes involved in these reactions are shown in blue text.…”

Listeria monocytogenes aguA1, but Not aguA2, Encodes a Functional Agmatine Deiminase

“…Quantitative PCR was then performed in 20-l reaction mixtures containing SYBR quantitative PCR mix (TOYOBO) to measure transcriptional levels of aguA1 and aguA2 using an iCycler iQ5 real time PCR detection system (Bio-Rad) with specific primer pairs (supplemental Table S1). The housekeeping gene gyrB was used as an internal control for normalization as previously described (10). Relative transcription levels were quantified by the 2 Ϫ⌬⌬CT method and shown as relative fold changes in comparison with that at pH 7.0 (24).…”

“…Agmatine enters bacterial cells via an agmatine-putrescine antiporter (aguD), where it is hydrolyzed to N-carbamoylputrescine by agmatine deiminase (EC 3.5.3.12) (9). Putrescine carbamoyltransferase (EC 2.1.3.6), encoded by aguB, mediates phosphorolysis of N-carbamoylputrescine, producing putrescine and carbamoylphosphate (10). Finally, a carbamate kinase (EC 2.7.2.2) transfers the phosphate group from carbamoylphosphate to ADP (Fig.…”

Activation of PrfA results in overexpression of virulence factors but does not rescue the pathogenicity of Listeria monocytogenes M7

“…Quantitative real-time PCR was performed in 20 ml reaction mixtures containing SYBR green qPCR mix (TOYOBO) to detect the transcriptional levels of several virulence genes on the iCycler iQ5 real-time PCR system (Bio-Rad) with specific primer pairs ( Table S1). The housekeeping gene gyrB was selected as an internal control for normalization as previously described (Chen et al, 2011a(Chen et al, , 2013. The experiment was repeated three times.…”