Genome-wide identification of the tobacco GDSL family and apical meristem-specific expression conferred by the GDSL promoter

Background GDSL esterases/lipases are a large protein subfamily defined by the distinct GDSL motif, and play important roles in plant development and stress responses. However, few studies have reported on the role of GDSLs in the growth and development of axillary buds. This work aims to identify the GDSL family members in tobacco and explore whether the NtGDSL gene contributes to development of the axillary bud in tobacco. Results One hundred fifty-nine GDSL esterase/lipase genes from cultivated tobacco (Nicotiana tabacum) were identified, and the dynamic changes in the expression levels of 93 of these genes in response to topping, as assessed using transcriptome data of topping-induced axillary shoots, were analysed. In total, 13 GDSL esterase/lipase genes responded with changes in expression level. To identify genes and promoters that drive the tissue-specific expression in tobacco apical and axillary buds, the expression patterns of these 13 genes were verified using qRT-PCR. GUS activity and a lethal gene expression pattern driven by the NtGDSL127 promoter in transgenic tobacco demonstrated that NtGDSL127 is specifically expressed in apical buds, axillary buds, and flowers. Three separate deletions in the NtGDSL127 promoter demonstrated that a minimum upstream segment of 235 bp from the translation start site can drive the tissue-specific expression in the apical meristem. Additionally, NtGDSL127 responded to phytohormones, providing strategies for improving tobacco breeding and growth. Conclusion We propose that in tobacco, the NtGDSL127 promoter directs expression specifically in the apical meristem and that expression is closely correlated with axillary bud development.

A thermostable and alkaline GDSL-motif esterase from Bacillus sp. K91: crystallization and X-ray crystallographic analysis

The esterase Est8 from the thermophilic bacterium Bacillus sp. K91 belongs to the GDSL family and is active on a variety of acetylated compounds, including 7-aminocephalosporanic acid. In contrast to other esterases of the GDSL family, the catalytic residues Asp182 and His185 were more pivotal for the catalytic activity of Est8 than the Ser11 residue. To better understand the biochemical and enzymatic properties of Est8, recombinant Est8 protein was purified and crystallized. Crystals of Est8 were obtained by the hanging-drop vapour-diffusion method using 2.0 M ammonium sulfate, 5%(v/v) 2-propanol as the crystallization solution. X-ray diffraction data were collected to a resolution of 2.30 Å with an R merge of 16.4% from a crystal belonging to space group P41212 or P43212, with unit-cell parameters a = b = 68.50, c = 79.57 Å.

Lipolytic Enzymes in Myxococcus xanthus

ABSTRACT The genome of Myxococcus xanthus encodes lipolytic enzymes in three different families: patatin lipases, α/β 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 α/β 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 C2 to C16. These enzymes preferentially hydrolyzed esters of short-chain fatty acids, yielding the highest activity with p-nitrophenyl acetate.

Identification of a Moraxella catarrhalis Outer Membrane Protein Exhibiting Both Adhesin and Lipolytic Activities

ABSTRACT The UspA1 and Hag proteins have previously been shown to be involved in the ability of the Moraxella catarrhalis wild-type strain O35E to bind to human Chang and A549 cells, respectively. In an effort to identify novel adhesins, we generated a plasmid library of M. catarrhalis DNA fragments, which was introduced into a nonadherent Escherichia coli strain. Recombinant E. coli bacteria were subsequently enriched for clones that gained the ability to bind to Chang and A549 cells, yielding the plasmid pELFOS190. Transposon mutagenesis of this plasmid identified the potential adhesin gene mcaP (M. catarrhalis adherence protein). Sequence analysis revealed that McaP is related to autotransporter proteins and has substantial similarity with the GDSL family of lipolytic enzymes, particularly the Moraxella bovis phospholipase B. Expression of the mcaP gene product by E. coli increased adherence to Chang, A549, and 16HBE14o− polarized human bronchial cells 50- to 100-fold. Spectrophotometric assays with p-nitrophenol derivatives also demonstrated that McaP is an esterase. Furthermore, thin-layer chromatography revealed that McaP cleaves both phosphatidylcholine and lysophosphatidylcholine. McaP releases fatty acids and glycerophosphorylcholine upon cleavage of phosphatidylcholine, thus exhibiting phospholipase B activity. The construction and characterization of isogenic M. catarrhalis O35E mutants demonstrated that the lack of McaP expression abolishes esterase activity and considerably decreases adherence to several human cell lines.