Báo cáo khoa học: AcmA of Lactococcus lactis is an N-acetylglucosaminidase with an optimal number of LysM domains for proper functioning

AcmA of Lactococcus lactis is an N-acetylglucosaminidase with an optimal number of LysM domains for proper functioning

Tác giả: Anton Steen, Girbe Buist, Gavin J. Horsburgh, Gerard Venema, Oscar P. Kuipers, Simon J. Foster, and Jan Kok

Lĩnh vực: Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, the Netherlands; Department of Molecular Biology and Biotechnology, University of Sheffield, UK

Nội dung tài liệu: Nghiên cứu này khám phá cấu trúc mô-đun của enzyme AcmA từ Lactococcus lactis, một autolysin chính chịu trách nhiệm cho sự tách tế bào và ly giải trong pha tĩnh. AcmA bao gồm một miền hoạt động ở đầu N và một miền liên kết peptidoglycan ở đầu C, với ba vùng lặp lại giống nhau thuộc họ miền LysM. Các thí nghiệm với các dẫn xuất AcmA đã loại bỏ hoặc bổ sung các miền LysM cho thấy rằng mặc dù hoạt động thủy phân vẫn còn, nhưng hoạt động tối ưu trong việc liên kết peptidoglycan và chức năng sinh học yêu cầu ba miền LysM. Nghiên cứu cũng xác định AcmA là một N-acetylglucosaminidase, chứ không phải là muramidase như suy đoán ban đầu.

Mục lục chi tiết:

• Keywords
• Correspondence
• Note
• AcmA, the major autolysin of Lactococcus lactis is an N-acetylglucosaminidase with an optimal number of LysM domains for proper functioning
• In order to be able to grow and divide, bacteria produce several types of enzymes that can hydrolyze bonds in the peptidoglycan of the cell wall [1]. Two types of enzymes known as glycosidases hydrolyze the β(1,4) bonds between the alternating N-acetylmuramic acid and N-acetylglucosamine residues of the glycan chains in peptidoglycan. A lysozyme-like enzyme, β-N-acetylmuramidase (muramidase), hydrolyses N-acetylmuramyl, 1,4-β-N-acetylglucosamine bonds, whereas the other, a β-N-acetylglucosaminidase (glucosaminidase), liberates free reducing groups of N-acetylglucosamine. In addition to these glycosidases, bacteria produce amidases, hydrolyzing the bond between the glycan chain and the peptide side chain, and peptidases of varying specificity.
• AcmA is the major autolysin of the Gram-positive bacterium Lactococcus lactis ssp. cremoris MG1363. AcmA is required for cell separation and is responsible for lysis in the stationary phase [2,3]. The 40.3 kDa secreted mature AcmA is subject to proteolytic degradation resulting in a number of activity bands in a zymogram of the supernatant of a lactococcal culture [4,5]. Bands as small as that corresponding to a protein of 29 kDa have been detected [3]. As no activity bands are produced by an L. lactis acmA deletion mutant, all bands represent products of AcmA [3]. Poquet et al.
• Abbreviations
• Results
• Two of the three repeats in AcmA are sufficient for cell separation and autolysis of cells
• Halo formation
• Cell separation and sedimentation
• Enzyme activity
• Binding properties of the AcmA derivatives
• Localization of AcmA and its derivatives on the cell surface
• Isolation of mature AcmA and determination of its specificity
• Discussion
• Experimental procedures
• Bacterial strains, plasmids and growth conditions
• General DNA techniques and transformation
• Primer synthesis, PCR and DNA sequencing
• Construction of AcmA derivatives
• Overexpression and isolation of the AcmA active site domain
• Binding of AcmA and its derivatives to lactococcal cells
• SDS/PAGE and detection of AcmA activity
• Western blotting and immunodetection
• Enzyme assays and absorbance measurements
• Immunofluorescence microscopy
• Isolation of AcmA from the supernatant of L. lactis cultures
• Determination of the hydrolytic activity of AcmA using RP-HPLC analysis
• Acknowledgements
• References