Cell wall metabolism
Teichoic acids (TA) are molecules found in the bacterial cell envelope of Gram positive bacteria that appear to extend to the surface of the peptidoglycan layer, provide rigidity to the cell wall by attracting cations such as magnesium and sodium and can elicit immunomodulating effects (23)(24)(25)(26)(27). TA also assist in regulation of cell growth by limiting the ability of autolysins to break the β(1-4) bond between the N-acetyl glucosamine and the N-acetylmuramic acid (28). They can be covalently linked to N-acetylmuramic acid of the peptidoglycan layer, to the lipids of the cytoplasmic membrane, or to a terminal D-alanine in the tetrapeptide crosslinkage between N-acetylmuramic acid units. TA that remain anchored to lipids are referred to as lipoteichoic acids (LTA), while TA that are covalently bound to peptidoglycan are referred to as wall teichoic acids (WTA) (29)(30).
The biosynthesis of LTA is conserved among LAB, but some LAB species, such as L. fermentum, L. casei, L. rhamnosus and L. reuteri, cannot biosynthesize WTA (31). Most LAB that produce WTA use a poly(glycerol-3-phosphate[poly(Gro-P)] backbone for the WTA (32). However, in L. plantarum some strains produce a WTA with a poly(ribitol-3-phosphate[poly(Rbo-P)] backbone (33). Responsible for the formation of poly(Rbo-P) is the tar gene cluster (genes tarIJKL – OGs 1679, 1682, 1684, 1685). This gene cluster is universally conserved between all the 54 L. plantarum strains (Table 1). The tag gene cluster (genes tagD1-F1-F2 – OGs 2846, 2914, 3622) is responsible for the formation of poly(Gro-P), but this gene cluster is not present in all strains. Although the strains which produce poly(Gro-P) are able to produce poly(Rbo-P), there have been no reports of strains who produce only poly(Rbo-P) of both TAs, indicating that de presence of the tag-locus exclusively dictates WTA production(33).
A recent study showed that when the tag-locus is disabled, L. plantarum is able to switch to poly(Rbo-P), but variations in growth conditions, such as variation is temperature, pH, concentrations of amino acids, oxygen and NaCl, did not trigger a switch to poly(Rbo-P) (34). This study also showed that the LTA and WTA biosynthesis pathways are independent of each other and that disabling the WTA biosynthesis, though mutation of the tagO gene, does not impact the LTA production. Furthermore this study suggests that WTA does not play a role in immunomodulation (34).
Wall Teichoic Aid (WTA)
Parts of the WTA biosynthesis pathway are highly conserved between the different strains (Table 1). Only strains 19.1, ER, NAB1 and NAB2 appear to have a frameshifted tagB gene. This contradicts the claim in a previous study that mutations in genes other than the tagO gene lead to the build-up of toxic intermediates or the depletion of components like undecaprenyl phosphate that typically function as a scaffold in cell-wall component biosynthesis (32).
The tag gene cluster is only found in 22 of the 54 strains. In strain 19.1 the tagF2 gene (OG_2914) is frameshifted. In strains NIZO1839, NIZO1840 and NIZO2264 the tagF2 gene is absent, but replaced by a gene encoding a 1675-1690 residue protein. When functional regions of both proteins are compared with Interproscan (35) it is seen that both have a CDP-glycerol glycerophosphotransferase domain. In the tagF2 gene it is located at the start of the protein, while in the other protein is near the end and it is separated into two parts. Also the tagF2 gene has a glycosyl transferase family 1 domain behind the CDP-glycerol glycerol-phosphotransferase domain, while the other protein has and glycosyl transferase family 2 domain in front of the CDP-glycerol glycerophosphotransferase domain. It could be that the 1675-1690 amino acid long protein provides the same function as the tagF2 gene, but it is not certain.
All strains also possess a TA ABC-transporter which is coded by the tagG and tagH genes (OGs 671 and 670).
Besides the tagD gene found in the tag gene cluster all strains, except strains NIZO1839, NIZO1840 and NIZO2264, have a paralog tagD gene found elsewhere in the genome. Besides this gene, several other strains have paralog genes of tagD, tagF and tarK gene.
Also the tarIJKL gene cluster (OGs 1679, 1682, 1684, 1685) is conserved between all strains. Only strains NAB1 and NAB2 have one frameshifted gene in this gene cluster: in NAB1 this is the tarL gene and in NAB2 this is the tarK gene. Although the tar gene cluster is present in all strains, the nucleotide sequences of the different genes was found to vary more than for other genes (1). The two sequence variants of tarIJKL genes are also found for the present set of 54 genomes, although a third variant in strains NIZO1839, NIZO1840 and NIZO2264 may be correlated with the alternative tagDF cluster (Figure 26).
This biosynthesis pathway was first studied in Bacillus subtilis and Staphylococcus aureus (36).The biosynthesis of LTA starts with a diacylglycerol (DAG), which serves as an anchor for the LTA. Previously unidentified enzymes (YpfP) couple sugar groups to its glycerol head, after which a flippase (ltaA) flips the glycerol and sugar groups to the exterior of the cell. A primase (ltaP) couples a phosphatidylglycerol (PtdG) to this molecule after which a LTA polymerase (ltaS) elongates the LTA by adding glycerophosphate groups (36). The proposed genes and enzymes of the biosynthesis pathway of LTA in L. plantarum are listed in Table 8. These 4 genes are found to be conserved and complete in all 54 L. plantarum strains. The flippase LtaA has not been identified yet, but it could be one of the flippases in the EPS biosynthesis gene clusters.
Both WTA and LTA can have D-Ala substitutions. The dlt gene cluster (dltXABC1D – OGs 123, 445, 447, 448, 1457) is highly conserved between the different L. plantarum strains. Only strains ER and NIZO2776 have a frameshifted dltA gene (OG_123). Besides the presence or absence conservation, the protein sequences of these proteins are also highly similar. All strains also possess the complete dltC2 gene OG_734, which is found outside the dlt gene cluster.
The peptidoglycan (PG) layer is the protective layer that exists between the bacteria and the environment. In L. plantarum the genes for the biosynthesis of the peptidoglycan layer are highly conserved (Figure 27). Only strains 19.1 and NAB2 have one putative frameshifted murF gene (EC: 188.8.131.52 - OG_1890). For details see Table 1.
There are many cell-wall hydrolases for turnover of the peptidoglycan. In a variety of bacteria the autolytic machinery has been shown to involve several different hydrolytic activities that include N-acetylmuramidases, N-acetylglucosaminidases, N-acetylmuramyl-L-alanine amidases, endopeptidases, lysozymes and transglycosylases. The latter two are also known as N-acetyl--D-muramidases (or muramidases). These enzymes play a role in cell separation, cell wall turnover, sporulation, competence and flagella formation (37). Autolysins can also play a role in biofilm formation. In general, they function outside the cell, and hence must be translocated across the cell membrane, via the sec pathway. Therefore they can be considered as part of the secretome.
Lytic transglycosylases are an important class of bacterial enzymes that act on peptidoglycan with the same substrate specificity as lysozyme. Unlike the latter enzymes, however, the lytic transglycosylases are not hydrolases but instead cleave the glycosidic linkage between N-actetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anydromuramoyl product.
In L. plantarum WCFS1, the analysis of its genome sequence has revealed the presence of 16 putative peptidoglycan hydrolases (PGHs), including 12 candidates displaying similarity with well-characterized PGHs, 3 hypothetical (lytic) transglycosylases containing a WY domain, and a pseudogene (acm3, three fragments) (38)(39).
The PGHs can be classified into five families according to the PFAM accession number of their catalytic domains: Acm1 (lp_1138) and Acm2 (lp_2645) belong to the family of mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (PF01832); lp_1158 and lp_3093, renamed Lys1 and Lys2, respectively, belong to the muramidase family of Glycosyl hydrolases 25 (PF01183); LytH (lp_1982) belongs to the N-acetylmuramoyl-L-alanine amidase family (PF01520); lp_3421, lp_2162, lp_2520, and lp_1242, renamed LytA, LytB, LytC, and LytD, respectively, belong to the γ-D-Glu-mDAP muropeptidase family containing a NLPC/P60 catalytic domain (PF00877); Lp_0302, Lp_3014, and Lp_3015, renamed MltA, MltB, and MltC, respectively, belong to the lytic transglycosylase family (PF06737).
Our present comparison of 54 L. plantarum genomes shows that the 16 PGHs are present and generally complete in all strains, with only a few exceptions (see Table 1for details). Lys2 (OG_2426) is present in 47 strains, MltA (OG_2141) is absent only in NIZO2741, and a transglycosylase (OG_5404) is absent only in IPLA88. Acm3 (OG_2599) is a pseudogene or absent in most strains.
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