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Applied and Environmental Microbiology, December 2007, p. 7763-7766, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01818-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Ubericin A, a Class IIa Bacteriocin Produced by Streptococcus uberis ^,

Nicholas C. K. Heng,^* Grace A. Burtenshaw, Ralph W. Jack, and John R. Tagg

Department of Microbiology and Immunology, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand

Received 5 August 2007/ Accepted 5 October 2007

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 
Streptococcus uberis, a causal agent of bovine mastitis, produces ubericin A, a 5.3-kDa class IIa (pediocin-like) bacteriocin, which was purified and characterized. The uba locus comprises two overlapping genes: ubaA (ubericin A precursor peptide) and ubaI (putative immunity protein). Ubericin A is the first streptococcal class IIa bacteriocin to be characterized.

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 
Streptococcus uberis is the main environmental causative agent of bovine mastitis in New Zealand and a prolific producer of proteinaceous antibacterial substances (bacteriocins) targeting other strains of S. uberis and/or other mastitis-associated species, such as Streptococcus agalactiae and Enterococcus faecalis (2, 11, 18-21). Due to their diversity, the bacteriocins elaborated by S. uberis can also be exploited by being incorporated into formulations designed as a preventive treatment against bovine mastitis, a strategy similar to that achieved with the lantibiotics nisin and lacticin 3147 (3, 15). It is therefore important, both practically and conceptually, to elucidate the complete S. uberis antimicrobial repertoire.

To date, two S. uberis bacteriocins (nisin U and uberolysin) have been biochemically characterized and their genetic loci identified (20, 21). However, during screening of S. uberis strains for novel inhibitory activities using an agar-based deferred-antagonism method, we found that S. uberis strain E produces a heat-stable antibacterial activity with an inhibitory spectrum that was distinguishable from that of nisin U or uberolysin by the absence of activity against Micrococcus luteus and certain streptococcal species, such as Streptococcus pyogenes, Streptococcus salivarius, and Streptococcus agalactiae (Table 1). Moreover, this new inhibitory agent was particularly potent against Listeria spp. and also inhibited S. uberis 42 (Table 1), a strain known to synthesize (and to be immune to) both nisin U and uberolysin (20, 21). The aim of the present study was to determine the biochemical and genetic characteristics of the inhibitory agent produced by S. uberis strain E.

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TABLE 1. Inhibitory spectra of Streptococcus uberis strain E and purified ubericin A

(Parts of this work were presented at the 7th ASM Conference on Streptococcal Genetics, Saint Malo, France [22].)

The bacteriocin elaborated by S. uberis E was purified from 2 liters of supernatant recovered from an 18-h Todd-Hewitt broth culture as follows. First, protein (crude bacteriocin preparation) was precipitated with ammonium sulfate (80% saturation at 4°C), harvested by centrifugation (15,000 x g, 30 min, 4°C), redissolved in 400 ml buffer A (20 mM 2-morpholinoethanesulfonic acid, pH 5.8), and applied to a 5-ml HiTrap CM Fast Flow (GE Healthcare Life Sciences, Little Chalfont, United Kingdom) cation-exchange column (equilibrated with buffer A). The column was then developed with a linear gradient of 0 to 0.5 M NaCl (in buffer A) at a flow rate of 5 ml/min. Biologically active fractions (using Listeria grayi ATCC 19120 as the indicator strain) were lyophilized, redissolved in 600 µl of 10% (vol/vol) aqueous acetonitrile containing 0.1% (vol/vol) trifluoroacetic acid, and applied (200-µl aliquots) to a 50- by 2-mm (5-µm-pore-size) Gemini C₁₈ reversed-phase high-performance liquid chromatography column (Phenomenex Inc., Torrance, CA) at a flow rate of 0.4 ml/min. The column was developed at a flow rate of 0.4 ml/min over 30 min in a linear gradient of 10 to 50% (vol/vol) aqueous acetonitrile (containing 0.1% [vol/vol] trifluoroacetic acid).

An active fraction corresponding to a single absorbance peak containing the purified bacteriocin was further characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry and N-terminal amino acid sequencing (Protein Microchemistry Facility, Department of Biochemistry, University of Otago). Mass spectrometry conducted using a Finnigan LaserMAT 2000 (Thermo BioAnalysis) mass analyzer yielded a single peak with an average mass of 5,270.5 Da (Fig. 1A). Automated Edman degradation using a Procise Model 492 pulsed liquid/gas-phase microsequencer (Applied Biosystems, Foster City, CA) disclosed the following N-terminal amino acid sequence: KTVNYGNGLYXNQKKXWVNWSETATTIVNNSIMNGLT GGN, where the unidentified residue, X, could represent cysteine. Homology searches using the BLASTP algorithm (http://www.ncbi.nlm.nih.gov/BLAST) revealed that the inhibitory agent produced by S. uberis E, now designated ubericin A, is most similar (64% identity, 78% similarity) to leucocin C, a class IIa bacteriocin produced by Leuconostoc mesenteroides (GenBank accession no. P81053). The class IIa (or pediocin-like) bacteriocins are a large group of antibacterial peptides produced by lactic acid bacteria typified by their potent antilisteria activity, the presence of a highly conserved pentapeptide motif (YGNG[V/L]) or "pediocin box," and at least one disulfide bond that is essential for biological activity (4, 6). Indeed, ubericin A possesses all these characteristics, including the YGNGL motif (underlined above) and an essential disulfide bond (Fig. 1B), thus validating ubericin A as a new member of the pediocin-like family of gram-positive peptide bacteriocins.

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FIG. 1. (A) Matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis of purified ubericin A. Only the primary ubericin A (5,270.5 Da) and internal mass standard (insulin; 5,777.6 Da) peaks are labeled. (B) Ubericin A contains an essential disulfide bond. Purified ubericin A (ca. 40 pmol) was subjected to the reductive alkylation protocol of Jack et al. (10). +, untreated ubericin A (positive control); –, reagent-only control; a, ubericin A plus reaction buffer; b, ubericin A plus 4-vinylpyridine; c, ubericin A plus 2-mercaptoethanol; d, ubericin A plus 2-mercaptoethanol plus 4-vinylpyridine. The indicator strain is L. monocytogenes L45.

The inhibitory spectrum of purified ubericin A accounts for the activity produced by S. uberis strain E in deferred-antagonism assays (Table 1). It is noteworthy that growth of small numbers of colonies within the zones of inhibition was observed with strains of Listeria monocytogenes, Listeria grayi, E. faecalis, and Streptococcus bovis, especially when assayed by the deferred-antagonism method (Table 1). Upon subculture and retesting of a selection of these colonies, they were found to be completely resistant to the purified peptide (Table 1). Whereas the development of resistance to ubericin A in Listeria spp. and E. faecalis is consistent with previous findings for other class IIa bacteriocins (7, 8), our discovery of ubericin A resistance in S. bovis was unexpected. In L. monocytogenes and E. faecalis, development of resistance to class IIa peptides is attributed to the loss of a functional mannose-specific phosphotransferase system (EII_t^Man) presumed to be the bacteriocin receptor (6-8). It would therefore be of interest to ascertain if a similar resistance mechanism applies for S. bovis.

The genetic locus responsible for ubericin A production (designated uba) was identified by PCR-based methods including "vectorette" PCR (16). The erythromycin resistance gene ermAM (1) served as the "vectorette" component. A complete list of oligonucleotide primers and their sequences is listed in Table S1 in the supplemental material. The uba locus contains two genes, ubaA and ubaI, which overlap by one base pair, i.e., the last nucleotide of the ubaA stop codon is the first nucleotide of the ubaI start codon. ubaA encodes the 70-amino-acid ubericin A prepeptide, which comprises the mature ubericin A propeptide (49 amino acids) preceded by a 21-amino-acid secretion signal peptide containing a "double-glycine" (GG) motif, a distinctive characteristic of many peptide bacteriocins produced by gram-positive bacteria (5, 14). The deduced amino acid sequence of ubericin A not only has a calculated molecular mass (5,271.5 Da) consistent with that obtained by mass spectrometry but also confirms the residues (Cys¹¹ and Cys¹⁶) that form the essential disulfide bond (Fig. 2). ubaI specifies UbaI, a 99-amino-acid polypeptide displaying 52% identity (71% similarity) to MunC, the protein which confers immunity to the class IIa peptide mundticin ERL35 (GenBank accession no. AAQ95743), indicating that the probable function of UbaI is to protect the ubericin A producer strain from the lethal effects of its own bacteriocin.

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FIG. 2. Genetic organization of the ubericin A (uba) locus in S. uberis E and comparison with the corresponding region of the S. uberis genome sequence reference strain 0140J. For simplicity, the ORFs are not drawn to scale. The incompletely sequenced DNA region and gene (CHP) in strain E are indicated by the dotted line and unfilled arrow, respectively. The deduced amino acid sequence of the ubericin A prepeptide (UbaA) is shown with the residues obtained by N-terminal sequencing underlined. The inverted arrow highlights the cleavage site of the signal peptide, and the disulfide bond (SS) formed between Cys11 and Cys16 is also indicated. ORF1, putative ABC transporter; ORF2(HK) and ORF3(RR), putative histidine kinase and response regulator, respectively; fpg, formamidopyrimidine glycosylase; DPCK, dephosphocoenzyme A kinase; ORF(MF), putative membrane fusion protein; ORF(CHP), conserved hypothetical protein of unknown function.

The S. uberis genome sequence reference strain 0140J does exhibit antilisteria activity but differs from strain E in not inhibiting enterococci or S. bovis (19; G. A. Burtenshaw, unpublished results), leading us to speculate that 0140J may produce a variant of ubericin A having a somewhat different inhibitory spectrum. However, when a BLAST search of the S. uberis 0140J genome sequence (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/s_uberis) was conducted using the uba locus as the query sequence, we found that ubaA is completely absent (Fig. 2), indicating that strain 0140J produces another as-yet-unidentified antilisteria agent. On the other hand, strain 0140J does contain a ubaI homologue, which encodes a polypeptide with four amino acid differences (Thr¹Ser, Ala²Tyr, Glu¹⁶Gly, and Phe⁶⁷Leu) from UbaI in strain E. These substitutions, however, do not appear to compromise the function of the protein, since the growth of strain 0140J is not inhibited upon exposure to purified ubericin A (Table 1).

The majority of class IIa bacteriocins, like the ubericin A prepeptide, contain secretion leader sequences with GG motifs (4, 6). These peptides are usually secreted (with concomitant proteolytic cleavage of the signal peptide) via dedicated export complexes usually composed of an ATP-binding cassette (ABC) transporter and an accessory protein (5, 6). The ABC transporter component characteristically comprises three domains: (i) a N-terminal peptidase, (ii) a membrane-spanning permease, and (iii) a C-terminal ATPase (5). Since most of the genetic loci encoding class IIa bacteriocins described to date are organized such that the transport-associated genes are located adjacent to the bacteriocin structural genes (4, 6), we anticipated that the ubericin A export locus would reside close to ubaAI. However, the gene immediately downstream of ubaAI is fgp, which encodes formamidopyrimidine glycosylase, a conserved "housekeeping" protein (Fig. 2). Moreover, additional bioinformatic analyses and sequencing of the DNA regions downstream of ubaAI revealed a genetic complement and organization identical to those of strain 0140J (Fig. 2).

In order to identify the ubericin A-associated ABC transporter in S. uberis strain E, we used a pair of degenerate PCR primers that bind to DNA segments (ca. 1.8 kb apart) encoding the conserved N-terminal peptidase and C-terminal ATPase domains. Amplicons of the expected size were cloned into pBluescript II (Stratagene), and five clones were sequenced, all of which corresponded to an internal segment of an open reading frame (ORF), ORF1 (Fig. 2), in strain 0140J, which encodes a 717-amino-acid polypeptide displaying considerable homology (at least 50% identity) to ABC transporters known to secrete peptide bacteriocins. Interestingly, ORF1 is the only gene specifying a three-domain ABC transporter in the strain 0140J genome, and no apparent accessory protein-encoding counterpart could be detected (Fig. 2). Furthermore, two ORFs (ORF2 and ORF3) located immediately downstream of ORF1 could potentially encode a putative two-component signal transduction system (Fig. 2). Sequencing of additional PCR amplicons (obtained with genomic DNA of strain E as a template) and subsequent PCR analyses revealed not only perfect conservation of ORF1 to ORF3 in strain E but also that the intergenic spacing between ORF1 and ubaAI (after subtracting 0.2 kb, the size of ubaA) was comparable to that found in strain 0140J (Fig. 2). Since the biosynthesis of some class IIa bacteriocins, e.g., carnobacteriocin A (12), is induced via two-component signal transduction systems, it is tempting to speculate that ORF2 and ORF3 might be involved in ubericin A biogenesis. Future functional genomic studies will focus on elucidating the roles, in both strains E and 0140J, of all three ORFs in bacteriocin production.

In conclusion, we have characterized ubericin A, which to the best of our knowledge is the first class IIa bacteriocin to be characterized from a member of the genus Streptococcus. Ubericin A bears the distinctive hallmarks of other members of this bacteriocin class, including potent antilisteria activity, the "pediocin box," and an essential disulfide bond. In addition, ubaA and ubaI are organized in an overlapping fashion, a genetic configuration not previously reported for any class IIa bacteriocin. An overlapping bacteriocin gene system has so far been described only for the locus encoding sakacin Q, an unmodified nonpediocin bacteriocin produced by Lactobacillus sakei, in which case the expression of sppQ (bacteriocin) and spiQ (immunity) depends on translational coupling (13). Whether translational coupling plays a role in expression of the uba locus remains to be determined. Finally, this work has further highlighted the diversity of the S. uberis bacteriocin repertoire, which now includes members of three (i.e., class I [lantibiotics], class II [unmodified peptides], and class IV [cyclic peptides]) of the four known classes (9) of bacteriocins produced by gram-positive bacteria. From a practical perspective, any antimastitis formulation incorporating S. uberis bacteriocins will likely be a "bacteriocin cocktail" containing at least ubericin A, nisin U, and uberolysin.

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 
The nucleotide sequences reported in this article have been deposited in GenBank under accession numbers EF203953 (ubaAI locus) and EF203954 (putative bacteriocin-associated ABC transporter).

 
This study was supported in part by a University of Otago Research Award (BLIS I.P.), the University of Otago Research Grants Committee, and the Otago Medical Research Foundation.

We are grateful to Bushan Jayarao (Pennsylvania State University) for provision of S. uberis and S. bovis strains, Megan Inglis for expert technical assistance, and The Wellcome Trust Sanger Institute for making the S. uberis 0140J genome sequence publicly available.

 
* Corresponding author. Present address: Department of Oral Sciences, Faculty of Dentistry, University of Otago, P.O. Box 647, Dunedin 9054, New Zealand. Phone: 64 3 479-5290. Fax: 64 3 479-7078. E-mail: nicholas.heng{at}stonebow.otago.ac.nz

Published ahead of print on 12 October 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 

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Applied and Environmental Microbiology, December 2007, p. 7763-7766, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01818-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Heng, N. C. K. Articles by Tagg, J. R. Search for Related Content PubMed PubMed Citation Articles by Heng, N. C. K. Articles by Tagg, J. R. Agricola Articles by Heng, N. C. K. Articles by Tagg, J. R.