The gene cluster of aureocyclicin 4185: the first cyclic bacteriocin of Staphylococcus aureus

Staphylococcus aureus 4185 was previously shown to produce at least two bacteriocins. One of them is encoded by pRJ101. To detect the bacteriocin-encoding gene cluster, an ~9160 kb region of pRJ101 was sequenced. In silico analyses identified 10 genes (aclX, aclB, aclI, aclT, aclC, aclD, aclA, aclF, aclG and aclH) that might be involved in the production of a novel cyclic bacteriocin named aureocyclicin 4185. The organization of these genes was quite similar to that of the gene cluster responsible for carnocyclin A production and immunity. Four putative proteins encoded by these genes (AclT, AclC, AclD and AclA) also exhibited similarity to proteins encoded by cyclic bacteriocin gene clusters. Mutants derived from insertion of Tn917-lac into aclC, aclF, aclH and aclX were affected in bacteriocin production and growth. AclX is a 205 aa putative protein not encoded by the gene clusters of other cyclic bacteriocins. AclX exhibits 50 % similarity to a permease and has five putative membrane-spanning domains. Transcription analyses suggested that aclX is part of the aureocyclicin 4185 gene cluster, encoding a protein required for bacteriocin production. The aclA gene is the structural gene of aureocyclicin 4185, which shows 65 % similarity to garvicin ML. AclA is proposed to be cleaved off, generating a mature peptide with a predicted M r of 5607 Da (60 aa). By homology modelling, AclA presents four α-helices, like carnocyclin A. AclA could not be found at detectable levels in the culture supernatant of a strain carrying only pRJ101. To our knowledge, this is the first report of a cyclic bacteriocin gene cluster in the genus Staphylococcus.

Stress Response and Adaptation of Listeria monocytogenes 08-5923 Exposed to a Sublethal Dose of Carnocyclin A

ABSTRACTCarnocyclin A (CCLA) is an antimicrobial peptide produced byCarnobacterium maltaromaticumATCC PTA-5313, which can be used to control the growth ofListeria monocytogenesin ready-to-eat meat products. The aim of this research was to elucidate the cellular responses ofL. monocytogenes08-5923 exposed to a sublethal dose of CCLA. Microarray, quantitative reverse transcription-PCR, tandem mass spectrometry, and electron microscopy were used to investigate the alteration in gene expression, protein production, and morphological changes in cells ofListeriafollowing treatment with CCLA. The genes involved in metabolism (baiE,trn, andpykA), cell wall synthesis (murZanddacB2), and cell division (clpEanddivIVA) were upregulated following a 15-min exposure to CCLA as a result of stress responses. Genes involved in cell division, cell wall synthesis, flagellar synthesis, and metabolism were downregulated after 4 h as a result of adaptation. Analysis of total soluble proteins confirmed the downregulation ofpykAandgndafter 4 h of exposure to CCLA. The absence of flagella was observed inL. monocytogenesfollowing 30 h of exposure to CCLA. A sublethal dose of CCLA induced adaptation inL. monocytogenes08-5923 by inhibition of expression of genes and proteins critical for synthesis of cell wall structures and maintaining metabolic functions. Both the mannose- and cellobiose-specific phosphotransferase systems could be targets for CCLA.

Isolation and Characterization of Carnocyclin A, a Novel Circular Bacteriocin Produced by Carnobacterium maltaromaticum UAL307

ABSTRACT Carnobacterium maltaromaticum UAL307, isolated from fresh pork, exhibits potent activity against a number of gram-positive organisms, including numerous Listeria species. Three bacteriocins were isolated from culture supernatant, and using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Edman sequencing, two of these bacteriocins were identified as piscicolin 126 and carnobacteriocin BM1, both of which have previously been described. The remaining bacteriocin, with a molecular mass of 5,862 Da, could not be sequenced by traditional methods, suggesting that the peptide was either cyclic or N-terminally blocked. This bacteriocin showed remarkable stability over a wide temperature and pH range and was unaffected by a variety of proteases. After digestion with trypsin and α-chymotrypsin, the peptide was de novo sequenced by tandem mass spectrometry and a linear sequence deduced, consisting of 60 amino acids. Based on this sequence, the molecular mass was predicted to be 5,880 Da, 18 units higher than the observed molecular mass, which suggested that the peptide has a cyclic structure. Identification of the genetic sequence revealed that this peptide is circular, formed by a covalent linkage between the N and C termini following cleavage of a 4-residue peptide leader sequence. The results of structural studies suggest that the peptide is highly structured in aqueous conditions. This bacteriocin, named carnocyclin A, is the first reported example of a circular bacteriocin produced by Carnobacterium spp.