Article

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

Laboratory of Molecular Microbiology, G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
Applied and environmental microbiology (impact factor: 3.69). 08/2012; 78(19):7082-9. DOI:10.1128/AEM.01621-12 pp.7082-9
Source: PubMed

ABSTRACT 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.

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Keywords

alpA mRNA
 
amino acid sequences
 
endopeptidases AlpA
 
Escherichia coli
 
functionally active lytic enzymes
 
homologous proteins synthesized
 
logarithmic growth phase
 
Lysobacter sp
 
Lytic enzymes
 
mature enzyme sequence
 
N-terminal signal peptide
 
potential Rho-independent transcription terminator
 
precursors similar
 
S1E family [clan PA(S)]
 
secreted lytic endopeptidases L1
 
serine peptidases
 
start codon
 
stop codon
 
strain XL1 alpA
 
transcription start points
 

Yu S Lapteva