Catabolism of N-Acetylneuraminic Acid, a Fitness Function of the Food-Borne Lactic Acid Bacterium Lactobacillus sakei, Involves Two Newly Characterized Proteins

Abstract

In silico analysis of the genome sequence of the meat-borne lactic acid bacterium (LAB) Lactobacillus sakei 23K has revealed a repertoire of potential functions related to the adaptation of this bacterium to the meat environment. Among these functions, the ability to use N-acetyl-neuraminic acid (NANA) as a carbon source could provide a competitive advantage for growth on meat in which this amino sugar is present. In this work, we proposed to analyze the functionality of a gene cluster encompassing nanTEAR and nanK (nanTEAR-nanK). We established that this cluster encoded a pathway allowing transport and early steps of the catabolism of NANA in this genome. We also demonstrated that this cluster was absent from the genome of other L. sakei strains that were shown to be unable to grow on NANA. Moreover, L. sakei 23K nanA, nanT, nanK, and nanE genes were able to complement Escherichia coli mutants. Construction of different mutants in L. sakei 23K ΔnanR, ΔnanT, and ΔnanK and the double mutant L. sakei 23K Δ(nanA-nanE) made it possible to show that all were impaired for growth on NANA. In addition, two genes located downstream from nanK, lsa1644 and lsa1645, are involved in the catabolism of sialic acid in L. sakei 23K, as a L. sakei 23K Δlsa1645 mutant was no longer able to grow on NANA. All these results demonstrate that the gene cluster nanTEAR-nanK-lsa1644-lsa1645 is indeed involved in the use of NANA as an energy source by L. sakei.

INTRODUCTION

Lactobacillus sakei is a lactic acid bacterium (LAB) first isolated from rice wine, but is commonly associated with the food environment. It belongs to the natural microbiota of raw meat and seafood stuffs and is also added as a starter culture for elaboration of fermented meat and fish products, notably raw fermented sausage in Western countries (1, 2). It is generally observed that the presence of L. sakei in meat limits the presence of spoilage bacteria (3). L. sakei is detected in the human digestive tract, albeit in the subdominant microbiota, and is considered to be a commensal bacterium (4, 5). The genome sequence of a strain isolated from dry sausage, L. sakei 23K, has been previously established (3), revealing several unexpected properties. These functions involved the transport and the use of alternative carbon sources such as N-acetylneuraminic acid, an efficient catabolism of nucleosides like inosine providing an additional energy source and conversion of arginine through the arginine deaminase pathway (reference 6 and references therein). Some of these functions have been shown to protect this species from acid stress and provide it a competitive advantage in meat (6). Not only one function but the combination of several functions would explain the adaptation of this species to the meat environment. In this work, we were interested in the ability of L. sakei to metabolize the nine-carbon amino sugar called N-acetyl-neuraminic acid (NANA) which is present in meat. L. sakei has the privilege to be generally recognized as safe (GRAS). Our group is interested in characterizing various functions with the aim to use several strains of L. sakei with an objective of biopreservation of the fresh meat.

Sialic acid is the generic name for a family of more than 40 naturally existing nine-carbon keto sugars derived from 2-keto-3-deoxy-5-acetamido-d-glycero-d-galacto-nonulosonic acid. N-acetylneuraminic acid, or Neu-5Ac (NANA), is the most common such sugar in the living world. Indeed, it is found in mammalian cells as a component of glycoproteins, and most abundantly in mucins, which are glycoproteins of very high molecular weight secreted by mucosa and some exocrine glands into the lumen of the respiratory, gastrointestinal, and reproductive tracts (7–10). It plays a crucial role in cellular interactions in eukaryotes and is also described as a key molecule in host/pathogen adhesion (7, 10–12). This major component, present in the mucous layer of the human digestive tract, can also be used as an energy source by the commensal bacteria of the human microbiota (8). In several pathogenic bacteria, sialic acid or its use is associated with virulence (8, 10, 13). Previous studies, mainly on human-pathogenic bacteria, showed that, after its internalization, sialic acid is either catabolized as a nutrient source or incorporated into polymers for display at the bacterial cell surface, in order to escape the bactericidal effect of serum, subsequently allowing bacterial proliferation within the host (12, 14–16). This phenomenon was particularly studied for the human-pathogenic bacterial species Neisseria meningitidis, Escherichia coli, Haemophilus influenzae, Haemophilus ducreyi, Pasteurella haemolytica, Pasteurella multocida, and the streptococci of group B (10, 12, 15, 17–20). The studies carried out on these pathogenic bacteria focused on the mechanisms of in vivo biosynthesis or ability to salvage sialic acid. In E. coli K1, it was shown that this strain has all the necessary functions for the uptake of NANA from its cellular environment and for its further metabolism both as a carbon source (by the two operons nanRATEK and nagAB) and for its polymerization at the surface to produce polysialic acid (PSA) (19, 21).

The NANA metabolism was first described in the nonpathogenic strain E. coli K-12 (22). Briefly, NANA from the surrounding environment is transported into the cytoplasm by a symporter (NanT) through the inner membrane. The N-acetylneuraminic acid lyase (NanA) first removes a pyruvate group, yielding N-acetylmannosamine (NAM), and NAM kinase (NanK) subsequently adds a phosphate group, which yields NAM-6P. The NAM-6P epimerase (NanE) transforms NAM-6P into N-acetylglucosamine-6P (NAG-6P), and the acetyl group and the amino moiety are cleaved, respectively, by NAG-6P deacetylase (NagA) and glucosamine-6P deaminase (NagB), generating fructose-6P, which may then enter the glycolysis pathway. In E. coli K-12, the expression of the genes involved in the catabolism of sialic acid is regulated by a repressor, encoded by nanR. NanR inactivation induces constitutive expression of the sialic acid catabolic genes (22, 23).

In H. influenzae, the transport, catabolism, and polymerization of NANA have been shown to be tightly controlled. Transcriptional analysis of the mutant siaR indicated that both the nan-nag and siaPT operons were upregulated (23). Regulatory mechanisms that determine routing of sialic acid into the transport, the catabolic, or the polymerization pathways have been described (13, 24, 25). Recently, it was shown that the repressor SiaR and CRP (Cyclic AMP [cAMP] Receptor Protein) cooperatively regulate the expression of the operons encoding the NANA tripartite ATP-independent periplasmic (TRAP) transporter SiaPT, also called SiaPQM, and the catabolic genes nanEKA and nagBA (10, 13, 24, 25). NANA utilization, whether to escape the bactericidal effect of serum or as an energy source, has also been described in bacteria such as Vibrio cholerae, Bacteroides fragilis, E. coli, H. influenzae, and Clostridium perfringens (21, 22, 26–29). Little is known regarding Gram-positive bacteria with respect to NANA metabolism except for C. perfringens (29).

Recently, the evolution and the sequence similarities of the genes involved in sialic acid metabolism among bacteria, based on in silico analysis of finished or unfinished genomes, have been described (14, 30). In this study, 1,902 genomes were investigated; the Nan cluster present in 46 species is predominantly confined to pathogens (33 species) and commensal bacteria (9 species). Several types of nan operons were found in many bacteria; while the NANA transporters and repressors were shown to vary, the core metabolic enzymes encoded by nanAEK were well conserved (30).

So far, no functional analysis of sialic acid metabolism has been reported in Gram-positive bacteria, including lactic acid bacteria. However, with regard to L. sakei, it is likely that NANA is an abundant source of energy, as it is found in the sialylated glycoforms of many glycoproteins present in meat, both in connective tissues and decorating the red blood cell membrane (11). The aim of the present study was an initial characterization of the Nan cluster found in L. sakei 23K and to explore its role in NANA metabolism.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

Bacterial strains and plasmids used in this study are described in Table 1. L. sakei strains were routinely grown at 30°C on MRS medium (37). For L. sakei, the chemically defined medium MCD (38) supplemented with 25 mM concentrations of various carbon sources was used. When necessary, erythromycin was used at 5 μg · ml⁻¹.

Table 1.

Bacterial strains and plasmids

Strain or plasmid	Relevant characteristic(s)a	Source or reference
L. sakei strains
23K	nan+ wild-type plasmid-cured strain	3
LV5	nan+ wild type	31 (and references therein)
300	nan+ wild type	31 (and references therein)
LTH675	Wild-type plasmid-free strain lacking nan	31 (and references therein)
LTH677	Wild-type strain lacking nan	31 (and references therein)
64	Wild-type strain lacking nan	31 (and references therein)
RV6030	23K Δlsa1645	This study
RV6031	23K Δ(nanA-nanE)	This study
RV6032	23K ΔnanK	This study
RV6033	23K ΔnanT	This study
RV6034	23K ΔnanR	This study
E. coli strains
DH5α	Recipient strain for plasmid construction	Invitrogen
MG1655	nan+ wild-type E. coli K-12 strain	32
JW25113	nan+ strain	33
JW3192	JW25113 ΔnanE, Kanr	34
JW3193	JW25113 ΔnanT, Kanr	34
JW3194	JW25113 ΔnanA, Kanr	34
JW3195	JW25113 ΔnanR, Kanr	34
JW5538	JW25113 ΔnanK, Kanr	34
Plasmids
pRV610	Shuttle vector, Apr, Ermr	35
pRV386	pRV610::nanRAETK	This study
pRV392	pRV610::nanRAETK lsa1644 lsa1645	This study
pRV300	Integrative vector for L. sakei, Apr, Ermr	36
pRV398	pRV300::Δ(nanA-nanE), Apr, Ermr	This study
pRV400	pRV300::Δlsa1645, Apr, Ermr	This study
pRV716	pRV300::ΔnanR, Apr, Ermr	This study
pRV717	pRV300::ΔnanK, Apr, Ermr	This study
pRV718	pRV300::ΔnanT, Apr, Ermr	This study

^aAp, ampicillin; Erm, erythromycin; Kan, kanamycin.

E. coli strains were grown at 37°C under conditions of agitation on Luria broth (LB) or M63 or M9 minimal medium (39) supplemented with the appropriate carbon source at a concentration of 25 mM. The main difference between M63 medium and M9 medium is that M63 does not contain a sodium source. When mentioned, ampicillin and kanamycin were used at 100 μg · ml⁻¹ and 30 μg · ml⁻¹ in E. coli, respectively.

Growth on NANA.

NANA was purchased from Sigma. As this product is very expensive, we evaluated growth on NANA as a sole carbon source on small plates (5-ml diameter) on MCD agar for L. sakei and on M9 or M63 agar for E. coli strains. Overnight cultures on minimal media_glucose were harvested and washed. A drop of 5 μl was spotted on the plates and then incubated.

DNA isolation and amplification.

PCR was carried out using Taq DNA polymerase purchased from Fermentas by using a conventional protocol comprising an initial denaturation step (4 min at 94°C), followed by 25 amplification cycles (30 s at 95°C; 1 min at 55°C; 2 min at 72°C) and a final elongation step (5 min at 72°C). Isolation of plasmid DNA from E. coli or L. sakei using commercially available reagents (Qiagen) and chromosomal DNA extraction from L. sakei were performed as previously described (40).

Plasmid construction.

The shuttle vector pRV610 (35) is able to replicate in both E. coli and L. sakei, and it was used to clone the gene cluster encompassing nanTEAR and nanK (nanTEAR-nanK). A 5,659-bp fragment was obtained by PCR amplification of strain 23K chromosomal DNA with primers olsnan1 and olsnan3 (see Table S1 in the supplemental material), thereby introducing an XbaI and a SacI restriction site at the two extremities of the amplification product. The PCR product was then cloned at the XbaI and SacI sites of pRV610, generating the plasmid pRV386 (Table 1).

A PCR fragment encompassing lsa1644 and lsa1645 (lsa1644-lsa1645) was obtained by amplification of 23K chromosomal DNA using primers olsnan12 and olsnan13 (see Table S1 in the supplemental material), which both bore an extension with a SacI site. The PCR product was cloned downstream from nanK at the SacI site of pRV386, generating pRV392 (Table 1). The pRV386 and pRV392 plasmids were transformed in E. coli DHα, extracted, and verified by restriction digestion and sequence analysis.

Transformation of E. coli and L. sakei.

E. coli mutants from the Keio collection (34) were made chemically competent (39), and electrocompetent cells of E. coli DH5α were obtained as previously described (41). Transformants were selected on an LB agar plate containing 100 μg · ml⁻¹ ampicillin with or without 30 μg · ml⁻¹ kanamycin depending on the strain used. Transformation of L. sakei was carried out by electroporation as previously described (42). Transformants were selected on MRS agar containing 5 μg · ml⁻¹ of erythromycin. Screening of the potential transformants was routinely done by PCR amplification on total DNA.

Construction of the L. sakei mutants.

For the construction of L. sakei 23K ΔnanR, Δ(nanA-nanE), ΔnanT, ΔnanK, and Δlsa1645, the method described previously involving two successive crossovers was performed (36, 43). Briefly, two PCR fragments were first amplified using specific primers (see Table S1 in the supplemental material) to generate the upstream and downstream regions of the gene to be deleted. The primers used were carefully designed in order to keep the frame in the final product with extensions bearing restriction enzyme sites. The two PCR fragments were digested, and then ligated, and a second run of PCR amplification using the ligated DNA as a template was performed using the two external primers to produce a fragment with the expected internal deletion. This fragment was then introduced into the L. sakei integrative plasmid pRV300 (36). Five plasmids, pRV398[Δ(nanA-nanE)], pRV400(Δlsa1645), pRV716(ΔnanR), pRV717(ΔnanK), and pRV718(ΔnanT), were obtained in this way. These plasmids were used to transform L. sakei 23K, and correct insertion at the targeted locus was checked by PCR amplification of genomic DNA with appropriate primers. The second event of homologous recombination was performed as previously described (43) by screening for erythromycin-sensitive bacteria, which yielded around 50% of appropriately deleted strains. All potential deletion mutants were verified in comparison to the wild-type strain by PCR on genomic DNA using appropriate primers (see Table S1 in the supplemental material).

RESULTS

The gene repertoire of L. sakei 23K may allow NANA utilization.

The genome sequence of L. sakei 23K (3) revealed three loci comprising genes putatively involved in NANA catabolism: a first locus that encompassed two divergent operons, one on the lagging strand annotated nanTEAR and a nanK gene on the leading strand (Fig. 1), and two loci comprising nagA (lsa1588) and nagB (lsa0417) genes, located elsewhere on the chromosome. Neither the genes putatively involved in NANA polymerization nor the putative sialidase was detected in the genome. Three potential promoters that closely matched the consensus sequence deduced from previously characterized L. sakei genes were identified. The first one is likely to initiate transcription of the nanR gene. Two potential −10 boxes (TATAAT) were present at, respectively, 39 bp and 139 bp upstream from the nanR start codon, although no obvious −35 box was found. The second promoter, comprising −35 (TTGACA) and −10 (TATAGT) consensus boxes, was identified 110 bp upstream from the nanT gene. The third promoter, potentially directing the expression of the nanK gene, was localized 362 bp upstream from nanK start codon. In addition, a bidirectional putative consensus cre (catabolite repression element)-like box was present 17 bp upstream from the start codon of the nanK gene (TGATAACGCTTACA) and 154 bp upstream from the nanT start codon (TGTAAGCGTTATCA). These cre-like boxes were similar to those described previously in L. sakei (6, 44). A second cre-like box was also present 62 bp upstream from the nanE start codon gene (TGTAAGCGGTTAATACA). Such elements are generally present upstream from genes regulated by the carbon catabolite protein A (CcpA) as described previously (6, 44, 45). Another striking observation is the presence of a conserved box of 14 bp [(A/C)TAATTTTTAATT(A/T)] found 136, 90, and 112 bp upstream from the start codon of nanR, nanT, and nanK genes, respectively. Moreover, this 14-bp box was not found elsewhere on the chromosome. In addition to these genes, which are commonly found for the catabolism of sialic acid in bacteria, we also investigated the two genes located downstream from nanK, lsa1644 and lsa1645 (Fig. 1). Sequence analysis revealed that the L. sakei NanR belongs to the RpiR family. These proteins are usually involved in regulation of the expression of various genes. While most are repressors, these regulators can also activate expression of genes (46). NanR was also found in other species in association with nan gene clusters, such as in Pediococcus pentosaceus ATCC 25745, in two strains of Lactobacillus salivarius (UCC118 and NIAS840), in Carnobacterium sp. AT7, and in three staphylococcal strains, Staphylococcus aureus strain Newman, S. saprophyticus ATCC 15305, and S. lugdunensis HKU09-01. Like the L. sakei NanR protein, all share the same two domains, that is, a helix turn helix (HTH) motif, which suggests a capacity to bind to DNA, and a sugar isomerase domain in the carboxy-terminal region. L. sakei NanT presents high similarity to members of the sodium solute symporter family, which is mostly found in lactobacilli. The LSA1644 protein is similar to a putative sugar-phosphate isomerase/epimerase also present in two Lactobacillus plantarum strains (Lp_3566 in WFCS1 and JDM1_2850 in JDM1) within nan gene clusters and in Carnobacterium sp. AT7, though with no link to the nan genetic cluster (CAT7_09150:86511 … 87272). The LSA1645 protein has several transmembrane domains, and has been identified as a putative Na⁺/H⁺ antiporter. This protein is similar to many Na⁺/H⁺ antiporters found in Gram-positive bacteria, though two L. plantarum species have these homologs located in association with nan cluster genes, and presents 59% identity and 76% similarity to the L. plantarum WCFS1 and JDM1 napA4 gene products. As neither a putative sialidase nor polymerizing genes have been identified in the genome of L. sakei 23K, it is likely that only free NANA can be used as an energy source during growth on this sugar alone.

Fig 1.

Genetic organization of the nan gene cluster of L. sakei 23K. Putative promoters (arrows) and putative terminators (vertical lines with circles) are indicated. The genes are represented by large dark arrows oriented according to their transcriptions. The extended part of the figure is intended for visualizing the sequence corresponding to the putative promoter sequence located between nanT and nanK. Empty rectangles represent the localizations of the potential cre boxes.

In order to demonstrate the function of this cluster of nan genes, several strategies were employed. First, using L. sakei strains that were able to grow on NANA as the sole carbon source and strains that were not, we searched to establish a link between the NANA-positive (NANA⁺) phenotype and the presence of the nan cluster. Second, we used E. coli mutants from the Keio collection bearing deletions in various components of the nan gene cluster and tested the capacity of the L. sakei genes to complement these mutations. Finally, we constructed L. sakei mutants deficient in each gene belonging to this cluster and studied their phenotype.

The presence of the nan cluster in L. sakei strains is associated with the ability to grow on NANA.

The ability to grow on NANA as the sole carbon source was investigated on chemically defined medium supplemented with NANA (MCD_NANA) for different L. sakei strains from our collection. Among 73 strains of L. sakei analyzed for intraspecies genomic diversity using 57 individual genes (31), 53 strains were NANA⁺ and encoded nanA, nanE, and lsa1645. Three NANA⁻ strains (LTH675, LTH677, and 64) and three NANA⁺ strains (23K, LV5, and 300) were selected for further characterization. The presence or absence of each of the above-mentioned nan genes was ascertained in the six strains by PCR. All of the NANA⁺ strains harbored the nanTEAR-nanK genes as well as lsa1644-lsa1645, whereas all the wild-type-mutant strains lacking all the essential genes cannot grow on NANA. Thus, a good correlation was observed between the ability to grow on MCD_NANA and the presence of nan genes, including lsa1644 and lsa1645.

L. sakei nan genes can complement E. coli nan mutants.

As no information on transcriptional regulation was available, we chose to use the complete clusters to test L. sakei gene complementation in E. coli. Plasmids pRV386 and pRV392, encompassing nanTEAR-nanK and nanTEAR-nanK-lsa1644-lsa1645, respectively, were introduced into each of the five E. coli strains deficient in NanA, NanE, NanK, and NanT or NanR (34). The five E. coli deletion mutants were first checked for growth on M63 minimal solid medium supplemented with NANA as the sole carbon source (M63_NANA). As already described (22), only the E. coli ΔnanR mutant strain was able to grow on minimal medium with NANA, whereas each of the nanA, nanE, nanT, and nanK knockout strains was unable to use NANA. Surprisingly, after complementation with pRV386 or pRV392, growth on M63_NANA was restored only for the E. coli ΔnanE and ΔnanK strains (Table 2). The L. sakei NanA protein did not complement the E. coli NanA deficiency when grown on M63_NANA. Similarly, the L. sakei NanT protein did not allow growth of E. coli on M63_NANA medium. Since L. sakei NanT belongs to the sodium solute symporter family, and because there is no sodium source in the M63 medium, we carried out complementation on the M9 minimal medium containing NANA. Under these conditions, growth on NANA was restored in all E. coli mutants by complementation with pRV386 (Table 2). However, when pRV392 containing the two additional genes lsa1644 and lsa1645 was used, NANA growth was not restored in nanA and nanT mutants. As LSA1645 is similar to a Na⁺/H⁺ antiporter found only in Firmicutes, its presence in E. coli might be toxic when complementation of nanT occurs. From these heterocomplementation experiments, we conclude that the L. sakei cluster studied is indeed involved in NANA catabolism. Moreover, the complementation of E. coli deleted for nanE and nanK is observed independently of the presence of sodium. Since there are structural differences in the nan gene cluster between E. coli and L. sakei, we decided to test the role of the nan cluster by inactivating each gene of the L. sakei 23K cluster using in-frame deletions.

Table 2.

Complementation and growth on M63 or M9 media of E. coli mutants by L. sakei nan genes

E. coli strain	Growtha with indicated carbon source (25 mM) when complimented with plasmid:
	pRV610		pRV386 (pRV610::nanTEARK)			pRV392 (pRV610::nanTEARKlsa1644lsa1645)
			Glucose	NANA		Glucose	NANA
	Glucose	NANA		M63	M9		M63	M9
MG1655 (wild type)	+	+	+	+	+	NT	NT	NT
JW3192 (ΔnanE)	+	−	+	+	+	+	+	+
JW3193 (ΔnanT)	+	−	+	−	+	+	−	−
JW3194 (ΔnanA)	+	−	+	−	+	+	−	−
JW3195 (ΔnanR)	+	+	+	+	+	+	+	+
JW5538 (ΔnanK)	+	−	+	+	+	+	+	+

^a+, growth; −, absence of growth; NT, not tested.

As shown using isogenic mutants, the nan gene cluster of L. sakei 23K plays a role in the utilization of NANA.

In order to determine the role of the nan gene cluster, in-frame deletions of lsa1645, (nanA-nanE), nanK, nanT, and nanR genes were constructed in L. sakei 23K, giving rise to strains RV6030, RV6031, RV6032, RV6033, and RV6034, respectively. The wild-type and knockout strains exhibited similar growth rates on MCD_glucose and MCD_NAG, but while none of the knockout strains were able to metabolize NANA on MCD_NANA, the wild-type strain grew (Table 3). These results indicated that each gene, including lsa1645, is involved in NANA catabolism. On NAM, the two mutant strains RV6031 [Δ(nanA-nanE)] and RV6034 (ΔnanR) were deficient for growth, whereas the other mutant strains grew (Table 3). As the first step in the use of NAM involves the NanE protein, we can conclude that in L. sakei, NanE is the NAM-6P epimerase and that NanR probably regulates nanE gene expression. It is noteworthy that the absence of growth on NANA of the RV6030 (Δlsa1645 mutant) strain suggests that a newly identified protein is involved in catabolism of sialic acid in L. sakei (Table 3). As lsa1645 is the distal gene of the operon nanK-lsa1644-lsa1645, we suggest that LSA1644 may be involved in NANA utilization too.

Table 3.

Growth of L. sakei strains on MCD medium supplemented with various carbon sources (25 mM)

Strain	Carbon source (25 mM)a
	Glucose	NAG	NAM	NANA
23K	+	+	+	+
RV6030 (Δlsa1645)	+	+	+	−
RV6031 [Δ(nanA-nanE)]	+	+	−	−
RV6032 (ΔnanK)	+	+	+	−
RV6034 (ΔnanR)	+	+	−	−
RV6035 (ΔnanT)	+	+	+	−

^aNAG, N-acetylglucosamine; NAM, N-acetylmannosamine; NANA, N-acetylneuraminic acid.

DISCUSSION

The present paper is the first known initial report on the NANA catabolic pathway in L. sakei. These genetic approaches allowed us to establish that L. sakei NanA, NanE, NanT, NanK, NanM, and NanP play a role in the transport and catabolism of sialic acid. Regulation of the expression of this gene cluster is likely to be different from that described for other bacteria since deletion of the putative regulator nanR in L. sakei 23K did not lead to the NANA⁺ phenotype. The L. sakei NanR protein and H. influenzae SiaR belong to the same RpiR family, but their deletion did not lead to the same phenotype. In our case, L. sakei NanR does not seem to be the repressor alone, as its inactivation impaired growth on NANA. Regulation of gene expression for this catabolic pathway should therefore be investigated. A second possibility is that deletion of nanR is toxic in the presence of NANA, as has previously been observed for E. coli (47). Our results suggest that NanR is likely to activate expression of at least the nanE gene, but perhaps also nanA, which seems to be structurally cotranscribed with nanE. Since we only found three putative promoters upstream from nanR, nanT, and nanK, it is possible that the genes spanning nanT to nanA constitute an operon whose expression might be activated by NanR. Between the nanT and nanK start codons, a potential bidirectional catabolic-responsive element (cre) site (TGATAACGCTTACA) is present. This box is also present upstream from the nanR and nanE genes, suggesting potential regulation by the carbon catabolic response. Whether transcription of nanK, lsa1644, and lsa1645 is under the control of NanR and/or carbon catabolite protein A (CcpA) repression, as recently described for their counterparts in the Gram-negative H. influenzae and Vibrio vulnificus species (25, 48), has to be investigated. Moreover, the presence of an additional conserved putative “nan box” upstream from nanR, nanT, and nanK suggests that regulation of this system is complex. Whether NanR and/or CcpA could separately or cooperatively be involved in such a regulation remains to be clarified.

The five knockout mutants constructed in L. sakei 23K allowed us to demonstrate that the cluster of nan genes is functional and is involved in NANA catabolism. To our knowledge, two newly identified proteins not yet described for such a metabolism were first identified in this work, and their functions still remained to be identified. The protein LSA1644 is similar to a putative sugar isomerase/epimerase found in several lactobacilli. We suggest that this protein could play a role in determining the conformation of the NANA molecule that is taken up and propose to name it NanM. In solution, the NANA molecule is mostly found in the α-anomer form whereas the physiological form is a β-anomer (49). Recently, it has been shown in E. coli that a periplasmic protein called YjhT, which is well conserved among pathogens, is a mutarotase which facilitates the NANA uptake in vivo (49). However, genome analysis seeking such a function in L. sakei 23K did not find evidence of any potential gene encoding an eventual NANA-specific mutarotase, and further studies must be carried out in order to define the function of NanM protein. The LSA1645 protein resembles a sodium/H⁺ antiporter. We postulate that this protein is involved in the regulation of the intracellular pH or the sodium gradient in L. sakei. In fact, internalization and metabolism of sialic acid as a carbon source led to acidification of the medium. Specialized machinery must be present for excreting protons, and such a function is of utmost importance in lactic acid bacteria. Indeed, fermentation of NANA, which has a pK_a = 2.6, by lactic acid bacteria could be stressful due to the high rate of lactic acid production during glycolysis. These fermentative bacteria may therefore require highly tuned intracellular pH. In LAB, the observation that similar Na⁺/H⁺ antiporters (LSA1645 and the L. plantarum LP3565, NapA4) are found at a position close to the cluster containing the nan genes must be of great importance. The LSA1645 protein might have just such a regulatory role in maintaining internal pH, and we propose to name it NanP. It has recently been reported that in Vibrio cholerae, the NhaP1 described to be an K⁺(Na⁺)/H⁺ antiporter is required for growth and internal pH homeostasis (50). Although LSA1645 presents no homology with NhaP1, they could play similar roles.

The NANA catabolic pathway, as proposed in Fig. 2, is likely to be similar to the pathway described in E. coli (22) and other bacteria (28), although the regulation and the transport system are specific to L. sakei. This means that when grown on NANA, L. sakei is able to use it only as an energy source. We suggest that in L. sakei 23K, transport of NANA into the cytoplasm by NanT would be concomitant with transport of a sodium cation as previously described (51). The catabolism of NANA into fructose-6P is identical to that described for E. coli (22), and NanP would be involved in exporting protons and Na⁺ excess from the cytoplasm (Fig. 2) (50).

Fig 2.

Model proposed for the metabolism of the NANA in L. sakei. NANA is transported through NanT, a sodium/proton symporter. Intracellular NANA molecule is cleaved by NanA into pyruvate and NAM. Nam is phosphorylated by NanK into NAM-6-P. NanE epimerase transforms NAM-6-P into NAG-6-P and is deacetylated by NagA into GlcN-6-P which enters the glycolysis pathway. NanP (LSA1645) is supposed to export protons and sodium excess. NAM and NAG pathways are convergent to the NANA metabolism, respectively, transported by ManXYZ and NagE.

Taken all together, these data demonstrate that the L. sakei 23K cluster of nan genes is involved in NANA catabolism and that regulation of the expression of the genes involved this catabolism is unlike that in other bacteria so far described. Transcriptional analysis is under way. This is the first known study describing the NANA catabolic pathway in a Gram-positive lactic acid bacterium.