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The Streptococcus agalactiae bacteriophage B30 endolysin contains cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), Acm glycosidase and the SH3b cell wall binding domain. Truncations and point mutations revealed that the Acm domain needs the SH3b domain for activity, whereas the CHAP domain is responsible for almost all cell lysis activity.

Peptidoglycan hydrolases are an effective new source of antimicrobials. A chimeric fusion protein of the Ply187 endopeptidase domain and Lys K SH3b cell wall-binding domain is a potent agent against Staphylococcus aureus in four functional assays. [ABSTRACT FROM AUTHOR]

The article discusses the two conflicting primary nucleotide sequences of the Staphylococcus aureus. Details related to the nucleotide differences and alternative translational start site assignments that result in three non-identical protein sequence predictions for the phi11 amidase. It is suggested that it is wise to verify the correct phi11 amidase protein sequence since various versions of the amidase gene have been subcloned, performed deletion analysis including the discussion of the experimental use.

The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan hydrolases is a promising approach to combat bacterial infections. In our study, we constructed a 2 kb-triple-acting fusion gene ( TF ) encoding the N-terminal amidase-5 domain of streptococcal LambdaSA2 prophage endolysin (D-glutamine-L-lysin endopeptidase), a mid-protein amidase-2 domain derived from the staphylococcal phage 2638A endolysin (N-acetylmuramoyl-L-alanine amidase) and the mature version (246 residues) of the Staphylococcus simulans Lysostaphin bacteriocin (glycyl-glycine endopeptidase) at the C-terminus. The TF gene was expressed in Nicotiana benthamiana plants using the non-replicating Cowpea mosaic virus (CPMV)-based vector pEAQ-HT and the replicating Alternanthera mosaic virus ( AltMV)-based pGD5TGB1 L88 23-MCS-CP3 vector, and in Escherichia coli using pET expression vectors pET26b+ and pET28a+. The resulting poor expression of this fusion protein in plants prompted the construction of a TF gene codon-optimized for expression in tobacco plants, resulting in an improved codon adaptation index (CAI) from 0.79 ( TF gene) to 0.93 ( TFnt gene). Incorporation of the TF nt gene into the pEAQ-HT vector, followed by transient expression in N. benthamiana , led to accumulation of TFnt to an approximate level of 0.12 mg/g of fresh leaf weight. Antimicrobial activity of purified plant- and bacterial-produced TFnt proteins was assessed against two strains of Gram-positive Staphylococcus aureus 305 and Newman. The results showed that plant-produced TFnt protein was preferentially active against S. aureus 305, showing 14% of growth inhibition, while the bacterial-produced TFnt revealed significant antimicrobial activity against both strains, showing 68 (IC 50 25 µg/ml) and 60% (IC 50 71 µg/ml) growth inhibition against S. aureus 305 and Newman, respectively. Although the combination of codon optimization and transient expression using the non-replicating pEAQ-HT expression vector facilitated production of the TFnt protein in plants, the most functionally active antimicrobial protein was obtained using the prokaryotic expression system., Competing Interests: Conflict of interest: All authors declare no conflicts of interest in this paper.

There are two conflicting primary nucleotide sequences of the Staphylococcus aureus bacteriophage phi 11 amidase gene in Genbank. Nucleotide sequence differences as well as alternative translational start site assignments result in three non-identical protein sequence predictions for this amidase. Therefore, it is prudent to verify the correct phi11 amidase protein sequence, especially since multiple versions of the amidase gene have been subcloned, deletion analysis performed, and their experimental use described. There is also a resurgence of interest in the expression and use of bacteriophage lytic proteins as bactericidal agents and the phi 11 amidase has a high antimicrobial potential. The correct amidase sequence is identified through a combination of DNA sequence analysis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis of the recombinant purified phi11 amidase protein., ((c) 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)