INTRODUCTION

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Bacteriocins are bactericidal compounds, usually proteinaceous, whose activities are often restricted to bacterial strains that are closely related to the producing bacterium (6, 23). Bacteriocins are produced in all major groups of Eubacteria and Archaebacteria (22). Some bacteriocins, the halocins from Halobacteria, have no protein sequence homology to any known bacteriocins (20), whereas others, such as the S-type pyocins of Pseudomonas aeruginosa, some of the colicins of Escherichia coli, and a cloacin of Enterobacter cloacae, reveal protein sequence homologies (16, 17, 19).

Despite their diversity, the bacteriocins share several characteristics (6, 23). They are generally high-molecular-weight proteins that gain entry into target cells by binding to cell surface receptors. Their bactericidal mechanisms vary and include pore formation, degradation of cellular DNA, disruption of specific cleavage of 16S rRNA, and inhibition of peptidoglycan synthesis.

Many phytopathogenic bacteria, including members of the corynebacteria, erwinias, pseudomonads, and xanthomonads, produce proteinaceous bacteriocins (1, 2, 4, 5, 21, 24, 25). Since these bacteriocins are highly specific, can be produced at low cost, and are likely to be safe for both users and the environment, they appear to be excellent candidates for agricultural use in controlling plant pathogens. However, little is known about the chemical compositions, structures, modes of action, and genetics of most bacteriocins. Only a few isolated and/or purified bacteriocins have been reported from Agrobacterium radiobacter (18), Corynebacterium ulcerans (1), Erwinia carotovora subsp. carotovora (4), and Pseudomonas syringae pv. syringae (21).

Thus far, bacteriocin production by xanthomonads has received little attention. Bacteriocin production by Xanthomonas campestris pv. glycines was first reported by Fett et al. (5), who chose the term glycinecin to differentiate the X. campestris bacteriocin from the glycin produced by P. syringae pv. glycinea (21). Previously, X. campestris pv. glycines 8ra was tested for its antimicrobial activity, and it was found to be effective against most tested phytopathogenic Xanthomonas species, such as X. axonopodis, X. campestris pv. campestris, X. campestris pv. citri, X. campestris pv. pruni, X. campestris pv. vesicotoria, and X. oryzae pv. oryzae (24). To identify genes involved in glycinecin production, we screened an X. campestris pv. glycines 8ra cosmid library for bacteriocin production. We found five cosmid clones showing bacteriocin activity against X. campestris pv. vesicatoria Ds94-9. We subcloned a 6.0-kb DNA fragment with antimicrobial activity, which hybridized with only two of the five cosmid clones, suggesting that there was more than one gene encoding the bacteriocin in X. campestris pv. glycines 8ra (2). To distinguish this bacteriocin from the glycinecin discovered by Fett et al. (5), we named it glycinecin A. Testing of 24 X. campestris pv. glycines strains isolated from various geographic regions and soybean cultivars showed that all carried homologous DNA in the region corresponding to glycinecin A production (15).

Here, we report the cloning and characterization of the two structural genes, glyA and glyB, encoding glycinecin A from X. campestris pv. glycines. We also show that glycinecin A exists as a heterodimer and that its stability is improved by chemical cross-linking of the two subunits.

	MATERIALS AND METHODS

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Bacterial strains. X. campestris pv. glycines 8ra was obtained from E. J. Braun of the University of Illinois at Urbana-Champaign. E. coli and Xanthomonas strains were grown in Luria-Bertani (LB) broth or M9 minimal broth supplemented with 0.4% (wt/vol) of appropriate carbon sources. Liquid cultures of E. coli and Xanthomonas were grown in a shaking incubator at 37 and 30°C, respectively.

Bacteriocin activity assay. Bacteriocin activity was determined by examining inhibition zones created on the indicator strain X. campestris pv. vesicatoria Ds94-9 (10). Cell-free bacterial culture supernatants or cell extracts were serially diluted (twofold) in sterile water. A 10-µl aliquot of each sample was then spotted on LB agar plates and allowed to dry for 10 min. The plate was overlaid with 7 ml of soft agar (0.7%, wt/vol) containing 0.1 ml of the indicator strain (optical density at 600 nm, 0.1) and incubated overnight at 30°C. Bacteriocin titer was defined as the reciprocal of the highest dilution factor that showed inhibition of the indicator strain. The activity was calculated as titer¹ × 100 and indicated in arbitrary units per milliliter. When formation of a turbid zone followed formation of a clear inhibition zone, the critical dilution was taken to be the average of the final two dilutions.

Expression and purification of glycinecin A. For separate expression of each gene product of glyA and glyB, E. coli strain HB101 was transformed with plasmids pKEYH1 and pSGEV1, respectively. Cells were incubated at 37°C overnight and harvested through centrifugation, and cell lysates and culture supernatants were assayed for bacteriocin activity as described above.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Restriction enzyme map and associated bacteriocin activity of plasmid pBL5 and related subclones. (A) Plasmid pBL5 was digested with several restriction enzymes, and each fragment was ligated into pBluescript or pRK415. E. coli was transformed with each construct, and the bacteriocin activities of the cell lysates were determined. Locations of glyA and glyB are indicated. (B) Plasmids containing glyA or glyB were cotransformed into E. coli. The plasmid pRK415 containing an RK2 replicon was used for the construction of pKYEH1, and the plasmid pBluescript was used for pSGEV1 and pKYS26B. Resultant cell lysates were tested for bacteriocin activity. Enzyme abbreviations: E, EcoRI; B, BamHI; H, HindIII; PI, PstI; Hc, HincII; S1, SalI; Sm, SmaI; RV, EcoRV; Xb, XbaI.

N-terminal amino acid sequencing. The subunits of purified glycinecin A were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transblotted onto a polyvinyl difluoride membrane. The transferred polypeptides were visualized with Coomassie brilliant blue, and the glycinecin A bands were cut for N-terminal amino acid sequencing, which was performed using Edman degradation on an Applied Biosystems 477A automatic sequencer.

Molecular weight determination. The subunit composition of glycinecin A was examined by size exclusion chromatography and by SDS-PAGE analysis of chemically cross-linked glycinecin A. Purified glycinecin A was analyzed via size exclusion using a Superdex-75 HR 10/30 fast-performance liquid chromatography column (Pharmacia). The column was equilibrated with 20 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, and the separation was carried out isocratically at a flow rate of 0.4 ml/min. Both A₂₈₀ and glycinecin A activities were determined for all fractions. For cross-linking of glycinecin A, disuccinimidyl suberate (DSS) was used according to the manufacturer's instructions. Cross-linked glycinecin A was analyzed by SDS-PAGE (8) and visualized by silver staining.

Localization of recombinant glycinecin A. E. coli cells expressing glycinecin A were incubated in a 15-ml test tube containing 5 ml of LB medium at 29°C for 21 h with aeration. Cells were harvested by centrifugation at 4,000 × g for 15 min, and the supernatant was analyzed for secreted bacteriocin activity. The cell pellet was resuspended in lysis buffer (phosphate-buffered saline containing 1% Triton X-100), sonicated (5 cycles of 20-s bursts at 300 W at 1-min intervals), and then centrifuged at 10,000 × g for 20 min. The resulting supernatant was used as the cytoplasmic fraction (14).

Subcloning of the genes for glycinecin A. Plasmid pBL5, which contains a 6.0-kb DNA fragment that confers bacteriocin activity, was further digested with various restriction endonucleases for restriction enzyme mapping. Fragments of different sizes were subcloned into pBluescript using various restriction enzymes. Each construct was transformed into E. coli HB101 and cultured in an LB medium containing ampicillin (50 µg/ml) overnight at 37°C.

Nucleotide sequence accession number. The nucleotide sequence reported here has been submitted to the GenBank database under accession no. AF281069.

	RESULTS

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Cloning and nucleotide sequencing of the genes encoding glycinecin A. In a previous work (2), it was shown that plasmid pBL5 carrying a 6.0-kb DNA fragment isolated from X. campestris pv. glycines 8ra conferred bacteriocin activity against X. campestris pv. vesicatoria Ds94-9. We termed this activity glycinecin A. Here, we subcloned different parts of the 6-kb DNA insert from pBL5 into pBluescript. Each subclone was expressed in E. coli HB101, and its ability to produce bacteriocin was tested on nutrient agar media using X. campestris pv. vesicatoria Ds94-9 as an indicator (Fig. 1). Since the 1.9-kb pBL5 DNA fragment carried on plasmid pSGEB1 was the smallest fragment that conferred bacteriocin activity, we sequenced this fragment in both directions using the dideoxy-chain termination method. The nucleotide sequence revealed two complete open reading frames (ORFs) encoding polypeptides of 356 and 151 amino acids.

Purification of glycinecin A. Production of glycinecin A in pSGEB1-transformed E. coli was maximal at pH 7, and a minimum of 10⁷ CFU/ml was required for the detection of bacteriocin activity on the indicator strain. The recombinant glycinecin A was found predominantly in the E. coli cell extracts rather than in the culture medium, whereas glycinecin A from X. campestris pv. glycines 8ra cultures was found predominantly in the culture media (Table 1).

View larger version (36K): [in this window] [in a new window]   FIG. 2.   SDS-PAGE analysis of glycinecin A. (A) SDS-PAGE analysis of glycinecin A purification. Samples were collected from each step of the purification and analyzed by SDS-PAGE and Coomassie blue staining. Lane 1, total cell lysate; lane 2, ammonium sulfate fractionation (3 to 60%); lane 3, active fraction after Q-Sepharose anion-exchange chromatography; lane 4, active fraction after Mono Q anion-exchange chromatography. Arrow, larger subunit of glycinecin A encoded by ORF1. The smaller subunit cannot be observed due to low concentration of the sample. (B) Subunit composition of purified glycinecin A. The subunits of purified glycinecin A (2 µg) were separated by SDS-PAGE and transferred to a polyvinyl difluoride membrane for N-terminal amino acid sequencing.

Functional analysis of glycinecin A. Sequencing of the pSGEB1 insert revealed two ORFs, and SDS-PAGE of purified glycinecin A showed two bands. In order to determine which ORF was responsible for the bacteriocin activity, several subclones covering various regions of the insert were tested for bacteriocin production (Fig. 1A). Only subclones that carried both ORFs were shown to produce antimicrobial activity against X. campestris pv. vesicatoria Ds94-9. No bacteriocin activity was detected when either ORF1 or ORF2 was expressed separately (Fig. 1A), but activity was detected when cells were cotransformed with two compatible plasmids, each carrying one of the two genes. Hence, bacteriocin activity did not require cis-acting glyA and glyB genes (Fig. 1B). However, a mixture of cell extracts from cells expressing glyA and glyB individually did not show activity.

View larger version (43K): [in this window] [in a new window]   FIG. 3.   Glycinecin A forms heterodimers in solution. Purified glycinecin A was cross-linked by treatment with DSS as described in Materials and Methods. After cross-linking, each reaction product was analyzed by SDS-PAGE followed by silver staining.

View larger version (54K): [in this window] [in a new window]   FIG. 4.   Cross-linking increases the thermal stability of glycinecin A. Glycinecin A and cross-linked glycinecin A were incubated at various temperatures (40, 60, 75, and 90°C) for 15 min and then assayed for bacteriocin activity.

	DISCUSSION

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The genes responsible for the production of glycinecin A in X. campestris pv. glycines 8ra were identified and cloned. Nucleotide sequence analysis revealed two structural genes, glyA and glyB, whose deduced amino acid sequences correspond to the N-terminal amino acid sequences of the two components of glycinecin A. Recombinant glycinecin A produced in E. coli was found predominantly in cell extracts, whereas native glycinecin A produced in X. campestris pv. glycines was found predominantly in the culture media.

Colonies of E. coli transformed with pBL5 containing glyA and glyB on a 6-kb DNA insert show a wide inhibitory zone, whereas those transformed with pSGEB1 show a very small inhibitory zone, if any (data not shown). These results suggest that the secretion of glycinecin A may require a specific secretory apparatus that is not present in E. coli, and genes for its secretion may also be located on the 6-kb insert of pBL5. Genes related to bacteriocin production are usually organized in an operon comprised of a structural gene, a dedicated immunity gene, and a gene encoding an accessory protein essential for externalization. The above results indicate that the 1.9-kb DNA fragment that we cloned contained only structural genes and not the genes required for glycinecin A transport or immunity. Many bacteriocins from lactic acid bacteria have consensus N-terminal leader sequences for recognition by ABC transporters (11). These sequences are characterized by the presence of glycine residues in positions 2 and 1 relative to the processing site. However, neither glyA nor glyB has a double-glycine residue in its leader sequence, suggesting that glycinecin A requires a specific transporter distinct from those found in lactic acid bacteria.

Our results also indicate that glycinecin A exists as a heterodimer (Fig. 2A and 3), and coexpression of both subunits in the same host is essential for bacteriocin activity (Fig. 1B). Some bacteriocins require the complementary action of two different peptides to achieve biological activity; this two-peptide group includes lactacin F (3), lactococcin G (13), and cytolysin L (7). The complementary action of bacteriocins is a rather common occurrence in gram-positive bacteria, but bacteriocins from gram-negative bacteria are rarely multimeric. Glycinecin A is unique in this respect, since its natural producer, X. campestris pv. glycines, is gram negative and its subunits are relatively large (39 and 14 kDa). Since BLAST searches did not match glycinecin A to any known protein or gene sequence, it is unclear whether both subunits are responsible for bactericidal activity or whether one subunit acts as a receptor and the other acts as the bacteriocin. Although each of the two subunits contains a separate leader peptide, mixtures of extracts from cells transformed with either glyA or glyB did not show bacteriocin activity. This finding suggests that both subunits are required for proper folding in the periplasm after translocation of each subunit through the plasma membrane.

Using the promoter analysis Neural Network Promoter Prediction program (M. G. Reese, Neural Network Promoter Prediction program, 1998 version [http://www.fruitfly.org./seq_tools/promoter.html]), we found typical 35- and 10-type promoter sequences upstream of glyB with high probability (96%), as well as a ribosome-binding site upstream of glyB. However, we found only one feasible promoter sequence in front of the start codon of glyA. This putative promoter gave a low probability score (45%) and had high homology to the 10 but not to the 35 sequence. Several genes related to bacteriocin are commonly organized as operons under the control of a single promoter (4, 9). Since we cloned only the structural genes, the strong promoter that controls the expression of the operon may be located outside the cloned sequence. On the other hand, the high probability score for the putative promoter of glyB is consistent with independent control of each gene.