Cloning and Expression Analysis of Genes Encoding Lytic Endopeptidases L1 and L5 from Lysobacter sp. Strain XL1

Abstract

c Lytic enzymes are the group of hydrolases that break down structural polymers of the cell walls of various microorganisms. In this work, we determined the nucleotide sequences of the Lysobacter sp. strain XL1 alpA and alpB genes, which code for, respectively, secreted lytic endopeptidases L1 (AlpA) and L5 (AlpB). In silico analysis of their amino acid sequences showed these endopeptidases to be homologous proteins synthesized as precursors similar in structural organization: the mature enzyme sequence is preceded by an N-terminal signal peptide and a pro region. On the basis of phylogenetic analysis, endopeptidases AlpA and AlpB were assigned to the S1E family [clan PA(S)] of serine peptidases. Expression of the alpA and alpB open reading frames (ORFs) in Escherichia coli confirmed that they code for functionally active lytic enzymes. Each ORF was predicted to have the Shine-Dalgarno sequence located at a canonical distance from the start codon and a potential Rho-independent transcription terminator immediately after the stop codon. The alpA and alpB mRNAs were experimentally found to be monocistronic; transcription start points were determined for both mRNAs. The synthesis of the alpA and alpB mRNAs was shown to occur predominantly in the late logarithmic growth phase. The amount of alpA mRNA in cells of Lysobacter sp. strain XL1 was much higher, which correlates with greater production of endopeptidase L1 than of L5.

Bacteriophage of the Skin Microbiome in Patients with Psoriasis and Healthy Family Controls

“…The phage-related COGs can be further described as follows. Firstly, AlpA is a hydrolase, and its function is to break down the bacterial cell (Lapteva et al, 2012). Secondly, phage endonuclease and primase are essential enzymes for phage DNA replication (Duerkop et al, 2012;Ilyina et al, 1992) and the former can induce the degradation of bacterial genomic DNA (Panayotatos and Fontaine, 1985).…”

Biofilm-degrading enzymes fromLysobacter gummosus

“…XL1, with 78% and 58% sequence identity to the L. enzymogenes preproenzyme. 28,29 Another α-lytic protease was purified from the culture medium of Achromobacter lyticus, and N-terminal sequencing/ amino acid compositional analysis of the mature enzyme suggested that large segments are identical to the L. enzymogenes prototype. 30 The A. lyticus α-lytic protease was shown to cleave the N-acetylmuramoyl-l-alanine amide bond and the d-Ala-Gly and Gly-Gly bonds of S. aureus peptidoglycan.…”

The Role of Membrane Vesicles in Secretion of <b><i>Lysobacter</i></b> sp. Bacteriolytic Enzymes

“…Enzymes L1 and L5 are synthesized in the bacterial cytosol as pre-pro-proteins, which suggests secretion through the cytoplasmic membrane via a Sec-export mechanism [Granovsky et al, 2010[Granovsky et al, , 2011Muranova et al, 2004;Lapteva et al, 2012]. For Lysobacter , there has been no data defining the existence of a similar mechanism.…”

“…The most investigated among these enzymes are lytic proteases L1 and L5. The gene structures of these proteins ( alp A and alp B) suggest them to be synthesized as pre-pro-proteins, as has been shown for the alpha-lytic protease of Lysobacter enzymogenes , which is homologous to them [Granovsky et al, 2010[Granovsky et al, , 2011Lapteva et al, 2012;Muranova et al, 2004]. In accordance with this, it could be assumed that secretion of enzymes L1 and L5 from the cytosol into the environment should proceed by the same protocol, in two stages.…”