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Growth medium-dependent glycine incorporation into the peptidoglycan of Caulobacter crescentus.

Takacs CN, Hocking J, Cabeen MT, Bui NK, Poggio S, Vollmer W, Jacobs-Wagner C - PLoS ONE (2013)

Bottom Line: The PG of Caulobacter crescentus, unlike that of many other Gram-negative bacteria, has repeatedly been shown to contain significant amounts of glycine.High glycine content in the PG had no obvious effects on growth rates, mode of PG incorporation or cell morphology.Hence, the C. crescentus PG is able to retain its physiological functions in cell growth and morphogenesis despite significant alterations in its composition, in what we deem to be unprecedented plasticity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.

ABSTRACT
The peptidoglycan (PG) is a macromolecular component of the bacterial cell wall that maintains the shape and integrity of the cell. The PG of Caulobacter crescentus, unlike that of many other Gram-negative bacteria, has repeatedly been shown to contain significant amounts of glycine. This compositional peculiarity has been deemed an intrinsic characteristic of this species. By performing a comprehensive qualitative and quantitative analysis of the C. crescentus PG by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), we show here that glycine incorporation into the C. crescentus PG depends on the presence of exogenous glycine in the growth medium. High levels of glycine were detected at the fifth position of the peptide side chains of PG isolated from C. crescentus cells grown in the complex laboratory medium PYE or in defined medium (M2G) supplemented with casamino acids or glycine alone. In contrast, glycine incorporation was undetectable when cells were grown in M2G medium lacking glycine. Remarkably, glycine incorporation into C. crescentus peptidoglycan occurred even in the presence of low millimolar to sub-millimolar concentrations of free glycine. High glycine content in the PG had no obvious effects on growth rates, mode of PG incorporation or cell morphology. Hence, the C. crescentus PG is able to retain its physiological functions in cell growth and morphogenesis despite significant alterations in its composition, in what we deem to be unprecedented plasticity.

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Comparison between the PG composition of C. crescentus cells grown in PYE and M2G media.(A). Overlay of HPLC profiles of muropeptides obtained from C. crescentus cultures grown in PYE (black trace) and M2G (red trace) media. Black arrowheads, Penta(Gly)-containing muropeptide peaks present only in the PYE-derived sample; red arrowhead, peak corresponding to the PentaTri muropeptide species, identified in the M2G-derived sample. (B). Relative representation (molar percentage) of each muropeptide species in PG digests obtained from C. crescentus cultures grown in PYE (black) and M2G (grey) media. Red rectangles denote glycine-containing species. Tri, GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap; Tetra, GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap-d-Ala; Penta, GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap-d-Ala-d-Ala; Penta(Gly), GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap-d-Ala-Gly; Anh, 1,6-anhydro- MurNAc; (D,D), m-Dap-D-Ala crosslink; (L,D), m-Dap-m-Dap crosslink. Bars represent averages ± standard deviation for the three samples analyzed. (C). Summary of the composition of C. crescentus PG digests obtained from cultures grown in the indicated media. Major PG characteristics are shown, namely the total degree of crosslinkage, the relative amounts of differentially crosslinked muropeptide classes (monomers, dimers, trimers and tetramers), side chain types (Tri, l-Ala-d-γ-Glu-m-Dap; Tetra, l-Ala-d-γ-Glu-m-Dap-d-Ala; Penta(Ala), l-Ala-d-γ-Glu-m-Dap-d-Ala-d-Ala; Penta(Gly), l-Ala-d-γ-Glu-m-Dap-d-Ala-Gly), or chain ends (anhydro, 1,6-anhydro-MurNAc ). Bars are as in (B). The red rectangle highlights the relative representation of the Penta(Gly) side chain. (B and C).
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pone-0057579-g001: Comparison between the PG composition of C. crescentus cells grown in PYE and M2G media.(A). Overlay of HPLC profiles of muropeptides obtained from C. crescentus cultures grown in PYE (black trace) and M2G (red trace) media. Black arrowheads, Penta(Gly)-containing muropeptide peaks present only in the PYE-derived sample; red arrowhead, peak corresponding to the PentaTri muropeptide species, identified in the M2G-derived sample. (B). Relative representation (molar percentage) of each muropeptide species in PG digests obtained from C. crescentus cultures grown in PYE (black) and M2G (grey) media. Red rectangles denote glycine-containing species. Tri, GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap; Tetra, GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap-d-Ala; Penta, GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap-d-Ala-d-Ala; Penta(Gly), GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap-d-Ala-Gly; Anh, 1,6-anhydro- MurNAc; (D,D), m-Dap-D-Ala crosslink; (L,D), m-Dap-m-Dap crosslink. Bars represent averages ± standard deviation for the three samples analyzed. (C). Summary of the composition of C. crescentus PG digests obtained from cultures grown in the indicated media. Major PG characteristics are shown, namely the total degree of crosslinkage, the relative amounts of differentially crosslinked muropeptide classes (monomers, dimers, trimers and tetramers), side chain types (Tri, l-Ala-d-γ-Glu-m-Dap; Tetra, l-Ala-d-γ-Glu-m-Dap-d-Ala; Penta(Ala), l-Ala-d-γ-Glu-m-Dap-d-Ala-d-Ala; Penta(Gly), l-Ala-d-γ-Glu-m-Dap-d-Ala-Gly), or chain ends (anhydro, 1,6-anhydro-MurNAc ). Bars are as in (B). The red rectangle highlights the relative representation of the Penta(Gly) side chain. (B and C).

Mentions: However, all published muropeptide analyses have been conducted on PG isolated from cultures grown in the complex medium PYE. After performing a control experiment for a cell wall hydrolase mutant strain [33] that can grow in the defined medium M2G but not in PYE, we became suspicious that the PG composition may be sensitive to the composition of the growth medium (data not shown). Therefore, we performed a thorough comparative analysis of PG from wild-type CB15N cultures grown in either PYE or M2G (n = 3 for each). As expected, we identified both free and crosslinked forms of Penta(Gly)-containing muropeptides in the samples we obtained from PYE-grown cultures (Fig. 1A, black trace and arrowheads, Fig. 1B, red rectangles, and Table 1). In contrast, these glycine-containing muropeptides were virtually undetectable in samples obtained from M2G-grown cultures (Fig. 1A, red trace, Fig. 1B, and Table 1). Penta(Gly) peptides represented 4.96±0.13% of all side chains and 17% of all pentapeptides within the PG of PYE-grown cells (Fig. 1C and Table 2). Their presence represented the major difference between the muropeptide profiles of C. crescentus PG obtained from PYE and M2G cultures.


Growth medium-dependent glycine incorporation into the peptidoglycan of Caulobacter crescentus.

Takacs CN, Hocking J, Cabeen MT, Bui NK, Poggio S, Vollmer W, Jacobs-Wagner C - PLoS ONE (2013)

Comparison between the PG composition of C. crescentus cells grown in PYE and M2G media.(A). Overlay of HPLC profiles of muropeptides obtained from C. crescentus cultures grown in PYE (black trace) and M2G (red trace) media. Black arrowheads, Penta(Gly)-containing muropeptide peaks present only in the PYE-derived sample; red arrowhead, peak corresponding to the PentaTri muropeptide species, identified in the M2G-derived sample. (B). Relative representation (molar percentage) of each muropeptide species in PG digests obtained from C. crescentus cultures grown in PYE (black) and M2G (grey) media. Red rectangles denote glycine-containing species. Tri, GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap; Tetra, GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap-d-Ala; Penta, GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap-d-Ala-d-Ala; Penta(Gly), GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap-d-Ala-Gly; Anh, 1,6-anhydro- MurNAc; (D,D), m-Dap-D-Ala crosslink; (L,D), m-Dap-m-Dap crosslink. Bars represent averages ± standard deviation for the three samples analyzed. (C). Summary of the composition of C. crescentus PG digests obtained from cultures grown in the indicated media. Major PG characteristics are shown, namely the total degree of crosslinkage, the relative amounts of differentially crosslinked muropeptide classes (monomers, dimers, trimers and tetramers), side chain types (Tri, l-Ala-d-γ-Glu-m-Dap; Tetra, l-Ala-d-γ-Glu-m-Dap-d-Ala; Penta(Ala), l-Ala-d-γ-Glu-m-Dap-d-Ala-d-Ala; Penta(Gly), l-Ala-d-γ-Glu-m-Dap-d-Ala-Gly), or chain ends (anhydro, 1,6-anhydro-MurNAc ). Bars are as in (B). The red rectangle highlights the relative representation of the Penta(Gly) side chain. (B and C).
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pone-0057579-g001: Comparison between the PG composition of C. crescentus cells grown in PYE and M2G media.(A). Overlay of HPLC profiles of muropeptides obtained from C. crescentus cultures grown in PYE (black trace) and M2G (red trace) media. Black arrowheads, Penta(Gly)-containing muropeptide peaks present only in the PYE-derived sample; red arrowhead, peak corresponding to the PentaTri muropeptide species, identified in the M2G-derived sample. (B). Relative representation (molar percentage) of each muropeptide species in PG digests obtained from C. crescentus cultures grown in PYE (black) and M2G (grey) media. Red rectangles denote glycine-containing species. Tri, GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap; Tetra, GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap-d-Ala; Penta, GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap-d-Ala-d-Ala; Penta(Gly), GlcNAc-MurNAc-l-Ala-d-γ-Glu-m-Dap-d-Ala-Gly; Anh, 1,6-anhydro- MurNAc; (D,D), m-Dap-D-Ala crosslink; (L,D), m-Dap-m-Dap crosslink. Bars represent averages ± standard deviation for the three samples analyzed. (C). Summary of the composition of C. crescentus PG digests obtained from cultures grown in the indicated media. Major PG characteristics are shown, namely the total degree of crosslinkage, the relative amounts of differentially crosslinked muropeptide classes (monomers, dimers, trimers and tetramers), side chain types (Tri, l-Ala-d-γ-Glu-m-Dap; Tetra, l-Ala-d-γ-Glu-m-Dap-d-Ala; Penta(Ala), l-Ala-d-γ-Glu-m-Dap-d-Ala-d-Ala; Penta(Gly), l-Ala-d-γ-Glu-m-Dap-d-Ala-Gly), or chain ends (anhydro, 1,6-anhydro-MurNAc ). Bars are as in (B). The red rectangle highlights the relative representation of the Penta(Gly) side chain. (B and C).
Mentions: However, all published muropeptide analyses have been conducted on PG isolated from cultures grown in the complex medium PYE. After performing a control experiment for a cell wall hydrolase mutant strain [33] that can grow in the defined medium M2G but not in PYE, we became suspicious that the PG composition may be sensitive to the composition of the growth medium (data not shown). Therefore, we performed a thorough comparative analysis of PG from wild-type CB15N cultures grown in either PYE or M2G (n = 3 for each). As expected, we identified both free and crosslinked forms of Penta(Gly)-containing muropeptides in the samples we obtained from PYE-grown cultures (Fig. 1A, black trace and arrowheads, Fig. 1B, red rectangles, and Table 1). In contrast, these glycine-containing muropeptides were virtually undetectable in samples obtained from M2G-grown cultures (Fig. 1A, red trace, Fig. 1B, and Table 1). Penta(Gly) peptides represented 4.96±0.13% of all side chains and 17% of all pentapeptides within the PG of PYE-grown cells (Fig. 1C and Table 2). Their presence represented the major difference between the muropeptide profiles of C. crescentus PG obtained from PYE and M2G cultures.

Bottom Line: The PG of Caulobacter crescentus, unlike that of many other Gram-negative bacteria, has repeatedly been shown to contain significant amounts of glycine.High glycine content in the PG had no obvious effects on growth rates, mode of PG incorporation or cell morphology.Hence, the C. crescentus PG is able to retain its physiological functions in cell growth and morphogenesis despite significant alterations in its composition, in what we deem to be unprecedented plasticity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.

ABSTRACT
The peptidoglycan (PG) is a macromolecular component of the bacterial cell wall that maintains the shape and integrity of the cell. The PG of Caulobacter crescentus, unlike that of many other Gram-negative bacteria, has repeatedly been shown to contain significant amounts of glycine. This compositional peculiarity has been deemed an intrinsic characteristic of this species. By performing a comprehensive qualitative and quantitative analysis of the C. crescentus PG by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), we show here that glycine incorporation into the C. crescentus PG depends on the presence of exogenous glycine in the growth medium. High levels of glycine were detected at the fifth position of the peptide side chains of PG isolated from C. crescentus cells grown in the complex laboratory medium PYE or in defined medium (M2G) supplemented with casamino acids or glycine alone. In contrast, glycine incorporation was undetectable when cells were grown in M2G medium lacking glycine. Remarkably, glycine incorporation into C. crescentus peptidoglycan occurred even in the presence of low millimolar to sub-millimolar concentrations of free glycine. High glycine content in the PG had no obvious effects on growth rates, mode of PG incorporation or cell morphology. Hence, the C. crescentus PG is able to retain its physiological functions in cell growth and morphogenesis despite significant alterations in its composition, in what we deem to be unprecedented plasticity.

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