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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 unsupplemented and glycine-supplemented M2G media.(A). Overlay of HPLC profiles of muropeptides obtained from C. crescentus cultures grown in the indicated media. CAA, casamino acids; empty arrowheads, Penta(Gly)-containing muropeptide peaks; filled arrowheads, peak corresponding to the PentaTri muropeptide species. (B). Relative representation (molar percentage) of each muropeptide species in PG digests obtained from C. crescentus cultures grown in the indicated 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 two (M2G, M2G + 0.2 mM Gly, M2G + 2 mM Gly) or three (M2G + 0.1% CAA) samples analyzed. (C). Summary of the composition of C. crescentus PG digests obtained from C. crescentus 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 ). The red rectangle highlights the relative amounts of the Penta(Gly) side chain. Bars are as in (B).
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pone-0057579-g002: Comparison between the PG composition of C. crescentus cells grown in unsupplemented and glycine-supplemented M2G media.(A). Overlay of HPLC profiles of muropeptides obtained from C. crescentus cultures grown in the indicated media. CAA, casamino acids; empty arrowheads, Penta(Gly)-containing muropeptide peaks; filled arrowheads, peak corresponding to the PentaTri muropeptide species. (B). Relative representation (molar percentage) of each muropeptide species in PG digests obtained from C. crescentus cultures grown in the indicated 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 two (M2G, M2G + 0.2 mM Gly, M2G + 2 mM Gly) or three (M2G + 0.1% CAA) samples analyzed. (C). Summary of the composition of C. crescentus PG digests obtained from C. crescentus 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 ). The red rectangle highlights the relative amounts of the Penta(Gly) side chain. Bars are as in (B).

Mentions: Our finding that the Penta(Gly) side chain was present in the PG of C. crescentus cells grown in the complex medium PYE but not in the minimal medium M2G suggested that the incorporation of glycine may depend on the presence of this amino acid in the growth medium. Furthermore, three studies of C. crescentus PG composition identified different values for the Penta(Gly) levels in the PG of PYE-grown cultures: ∼11%, as calculated from the data reported in a 1983 study [21] and for cultures grown in unsupplemented PYE [22], ∼15%, for cultures grown in PYE supplemented with 20 mM Tris-HCl buffer pH 8.0 [22], and ∼5% for the PYE-grown samples analyzed in this study. As the sources of nutrients in PYE medium are enzymatic digests of animal tissue (peptone) and yeast [25], we expect batch-to-batch variations in the concentrations of the different components of this complex medium, including glycine. Based on an analysis of commercially available peptone and yeast extract [34], we calculated that PYE medium made with Difco nutrients contains approximately 4.6 mM total glycine, including 0.3 mM free glycine. The M2G medium formulation, on the other hand, does not include any amino acids [25]. We thus hypothesized that addition of relatively small amounts of exogenous glycine to the otherwise glycine-free growth medium M2G would cause incorporation of this amino acid into the C. crescentus PG. Consistent with this hypothesis, growing cells in M2G medium supplemented with 2 mM glycine was sufficient to generate high levels of glycine incorporation into the PG, as visualized and quantified from HPLC traces (Fig. 2A and 2B, Table 3). The Penta(Gly) side chains represented 16.5±0.3 % of all the side chains and 45% of all pentapeptides in these samples (Fig. 2C and Table 4). Other differences in C. crescentus PG composition that were observed following growth in glycine-supplemented M2G medium were small. Namely there were slight decreases in the degree of crosslinkage and in the amounts of trimers, tetramers, and tetrapeptides, while monomer and dimer levels and the average length of the glycan chains were only mildly increased (Fig. 2C and Table 4).


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 unsupplemented and glycine-supplemented M2G media.(A). Overlay of HPLC profiles of muropeptides obtained from C. crescentus cultures grown in the indicated media. CAA, casamino acids; empty arrowheads, Penta(Gly)-containing muropeptide peaks; filled arrowheads, peak corresponding to the PentaTri muropeptide species. (B). Relative representation (molar percentage) of each muropeptide species in PG digests obtained from C. crescentus cultures grown in the indicated 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 two (M2G, M2G + 0.2 mM Gly, M2G + 2 mM Gly) or three (M2G + 0.1% CAA) samples analyzed. (C). Summary of the composition of C. crescentus PG digests obtained from C. crescentus 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 ). The red rectangle highlights the relative amounts of the Penta(Gly) side chain. Bars are as in (B).
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pone-0057579-g002: Comparison between the PG composition of C. crescentus cells grown in unsupplemented and glycine-supplemented M2G media.(A). Overlay of HPLC profiles of muropeptides obtained from C. crescentus cultures grown in the indicated media. CAA, casamino acids; empty arrowheads, Penta(Gly)-containing muropeptide peaks; filled arrowheads, peak corresponding to the PentaTri muropeptide species. (B). Relative representation (molar percentage) of each muropeptide species in PG digests obtained from C. crescentus cultures grown in the indicated 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 two (M2G, M2G + 0.2 mM Gly, M2G + 2 mM Gly) or three (M2G + 0.1% CAA) samples analyzed. (C). Summary of the composition of C. crescentus PG digests obtained from C. crescentus 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 ). The red rectangle highlights the relative amounts of the Penta(Gly) side chain. Bars are as in (B).
Mentions: Our finding that the Penta(Gly) side chain was present in the PG of C. crescentus cells grown in the complex medium PYE but not in the minimal medium M2G suggested that the incorporation of glycine may depend on the presence of this amino acid in the growth medium. Furthermore, three studies of C. crescentus PG composition identified different values for the Penta(Gly) levels in the PG of PYE-grown cultures: ∼11%, as calculated from the data reported in a 1983 study [21] and for cultures grown in unsupplemented PYE [22], ∼15%, for cultures grown in PYE supplemented with 20 mM Tris-HCl buffer pH 8.0 [22], and ∼5% for the PYE-grown samples analyzed in this study. As the sources of nutrients in PYE medium are enzymatic digests of animal tissue (peptone) and yeast [25], we expect batch-to-batch variations in the concentrations of the different components of this complex medium, including glycine. Based on an analysis of commercially available peptone and yeast extract [34], we calculated that PYE medium made with Difco nutrients contains approximately 4.6 mM total glycine, including 0.3 mM free glycine. The M2G medium formulation, on the other hand, does not include any amino acids [25]. We thus hypothesized that addition of relatively small amounts of exogenous glycine to the otherwise glycine-free growth medium M2G would cause incorporation of this amino acid into the C. crescentus PG. Consistent with this hypothesis, growing cells in M2G medium supplemented with 2 mM glycine was sufficient to generate high levels of glycine incorporation into the PG, as visualized and quantified from HPLC traces (Fig. 2A and 2B, Table 3). The Penta(Gly) side chains represented 16.5±0.3 % of all the side chains and 45% of all pentapeptides in these samples (Fig. 2C and Table 4). Other differences in C. crescentus PG composition that were observed following growth in glycine-supplemented M2G medium were small. Namely there were slight decreases in the degree of crosslinkage and in the amounts of trimers, tetramers, and tetrapeptides, while monomer and dimer levels and the average length of the glycan chains were only mildly increased (Fig. 2C and Table 4).

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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