Lipolytic enzymes in Myxococcus xanthus

Abstract

The genome of Myxococcus xanthus encodes lipolytic enzymes in three different families: patatin lipases, alpha/beta hydrolases, and GDSL lipases. One member of each family was characterized. The protein encoded by MXAN_3852 contains motifs characteristic of patatins. MXAN_5522 encodes a protein with the G-X-S-X-G motif characteristic of the lipase subfamily of alpha/beta hydrolases. MXAN_4569 encodes a member of the GDSL family of lipolytic enzymes. Strains with deletions of MXAN_5522 and MXAN_4569 undergo faster development and earlier myxospore formation than the wild-type strain. The MXAN_5522 mutation results in spore yields substantially higher than those seen for wild-type cells. Gene expression analysis using translational lacZ fusions indicates that while all three genes are expressed during development, only MXAN_5522 and MXAN_4569 are expressed during vegetative growth. The proteins encoded by these genes were overexpressed using a T7 RNA polymerase transcription (pET102/D-TOPO) system in Escherichia coli BL21 Star (DE3) cells. The substrate specificities of the purified enzymes were investigated using p-nitrophenyl esters with chain lengths from C(2) to C(16). These enzymes preferentially hydrolyzed esters of short-chain fatty acids, yielding the highest activity with p-nitrophenyl acetate.

FIG. 1.

Protein sequence alignments of conserved blocks of Myxococcus xanthus enzymes. (A) Alignment of the M. xanthus MXAN_3852 protein with conserved patatin domains. (B) Comparison of the MXAN_5522 protein with characterized α/β hydrolases. (C) Alignment of the MXAN_4569 protein with GDSL enzymes. Shown on gray and black backgrounds are identical amino acids, and functionally similar amino acids are shown in black letters. Residues necessary for catalytic activity are shown on a black background. The numbers between blocks indicate the numbers of amino acids preceding or following the conserved blocks.

FIG. 2.

Comparison of the colony edges of Myxococcus xanthus DK1622 and two motility mutants with the in-frame deletion mutations (LS2500, ΔMXAN_3852; LS2501, ΔMXAN_5522; LS2502, ΔMXAN_4569). Cells were grown in liquid CYE to approximately 5 × 10⁸ cells ml⁻¹ and concentrated to 5 × 10⁹ cells ml⁻¹ in CYE. Ten microliters was spotted in CYE agar. Pictures were taken after 96 h of incubation at 32°C. All the pictures in the same row were taken at the same magnification. All three mutants exhibited normal motility. A, adventurous motility; S, social motility.

FIG. 3.

Morphologies of Myxococcus xanthus DK1622 and deletion mutant fruiting bodies on TPM agar (LS2500, ΔMXAN_3852; LS2501, ΔMXAN_5522; LS2502, ΔMXAN_4569). Cells were grown in liquid CYE to approximately 5 × 10⁸ cells ml⁻¹ and concentrated to 8 × 10⁹ cells ml⁻¹ in CYE. For analysis of aggregation, 10 μl was spotted on TPM agar and incubated at 32°C. Pictures were taken at indicated times. Bar, 1 mm.

FIG. 4.

Morphologies of Myxococcus xanthus DK1622 and deletion mutant fruiting bodies on CF agar (LS2500, ΔMXAN_3852; LS2501, ΔMXAN_5522; LS2502, ΔMXAN_4569). Cells were grown in CYE to approximately 5 × 10⁸ cells ml⁻¹ and concentrated to 8 × 10⁹ cells ml⁻¹ in CYE. Ten microliters was spotted on CF agar and incubated at 32°C. Pictures were taken at indicated times. Bar, 1 mm.

FIG. 5.

Spore formation of Myxococcus xanthus DK1622 (diamonds) and of LS2500 (ΔMXAN_3852) (circles), LS2501 (ΔMXAN_5522) (triangles), and LS2502 (ΔMXAN_4569) (squares) deletion mutants on TPM (A) and CF (B) agars. The results shown are the averages of three different experiments. The error bars indicate standard deviations. Note differences in the scales.

FIG. 6.

Expression of Myxococcus xanthus lipolytic enzyme translational fusions with β-galactosidase. β-Galactosidase-specific activity (nmol of o-nitrophenol produced per min per mg of protein) of LS2530 (MXAN_3852-lacZ) (circles), LS2531 (MXAN_5522-lacZ) (triangles), and LS2532 (MXAN_4569-lacZ) (squares) strains during vegetative growth in CYE agar (A) and development on TPM agar (B) and CF agar (C). The results are the averages of three different experiments. The error bars indicate standard deviations. Note differences in the scales.

FIG. 7.

Relative activities of MXAN_3852 (black bars), MXAN_5522 (gray bars), and MXAN_4569 (white bars) enzymes towards pNP esters with different carbon chain lengths: C₂, pNP acetate; C₄, pNP butyrate; C₆, pNP caproate; C₈, pNP caprylate; C₁₀, pNP caprate; C₁₂, pNP laurate; C₁₄, pNP myristate; and C₁₆, pNP palmitate. Activities on each substrate are expressed as the percentages of activity relative to that with pNPC₂, which was taken as 100%. Values are the averages of triplicate determinations, with standard deviations indicated.