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Applied and Environmental Microbiology, December 2008, p. 7490-7496, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.00767-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Random Mutagenesis Identifies Novel Genes Involved in the Secretion of Antimicrobial, Cell Wall-Lytic Enzymes by Lactococcus lactis

Yu Pei Tan,¹ Philip M. Giffard,² Daniel G. Barry,¹ Wilhelmina M. Huston,¹ and Mark S. Turner^3*

Infectious Diseases Program, Cells and Tissue Domain, Institute of Health and Biomedical Innovation, Queensland University of Technology,¹ School of Land, Crop and Food Sciences, University of Queensland, Brisbane, Queensland,³ Menzies School of Health Research, Darwin, Northern Territory, Australia²

Received 3 April 2008/ Accepted 7 October 2008

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Lactococcus lactis is a gram-positive bacterium that is widely used in the food industry and is therefore desirable as a candidate for the production and secretion of recombinant proteins. Previously, we generated a L. lactis strain that expressed and secreted the antimicrobial cell wall-lytic enzyme lysostaphin. To identify lactococcal gene products that affect the production of lysostaphin, we isolated and characterized mutants generated by random transposon mutagenesis that had altered lysostaphin activity. Out of 35,000 mutants screened, only one with no lysostaphin activity was identified, and it was found to contain an insertion in the lysostaphin expression cassette. Ten mutants with higher lysostaphin activity contained insertions in only four different genes, which encode an uncharacterized putative transmembrane protein (llmg_0609) (three mutants), an enzyme catalyzing the first step in peptidoglycan biosynthesis (murA2) (five mutants), a putative regulator of peptidoglycan modification (trmA) (one mutant), and an uncharacterized enzyme possibly involved in ubiquinone biosynthesis (llmg_2148) (one mutant). These mutants were found to secrete larger amounts of lysostaphin than the control strain (MG1363[lss]), and the greatest increase in secretion was 9.8- to 16.1-fold, for the llmg_0609 mutants. The lysostaphin-oversecreting llmg_0609, murA2, and trmA mutants were also found to secrete larger amounts of another cell wall-lytic enzyme (the Listeria monocytogenes bacteriophage endolysin Ply511) than the control strain, indicating that the phenotype is not limited to lysostaphin.

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Lactococcus lactis is a gram-positive bacterium widely used in the dairy manufacturing industry. Due to its GRAS (generally regarded as safe) status for certain applications, it is often regarded as a promising candidate host for the production of recombinant proteins of therapeutic interest. One such protein is lysostaphin, an endopeptidase that is naturally produced by Staphylococcus simulans biovar staphylolyticus ATCC 1362 (34). Lysostaphin specifically cleaves the pentaglycine cross bridges of Staphylococcus aureus peptidoglycan and is able to cause cell wall breakdown and consequent cell death. Interest in this antimicrobial enzyme has increased in recent years due to the worsening problem of methicillin (meticillin)-resistant S. aureus, and several studies have demonstrated its usefulness in the treatment of infections (3, 4, 17, 27, 28). Recombinant lysostaphin was first produced in Escherichia coli in 1987 (32), and more recently, it has been expressed in L. lactis using the nisin-controlled gene expression system (21, 22). Recent work in our group has demonstrated the expression and secretion of active lysostaphin in several lactic acid bacteria, including L. lactis (39).

Levels of heterologous proteins secreted by L. lactis and other lactic acid bacteria are generally low, and efforts have been made to improve the secretion efficiency by modifying secretion signal sequences (5, 31), inactivating proteases (2, 23, 29), or supplying heterologous secretion machinery (26). Insights into protein secretion in L. lactis were gained when Nouaille et al. identified 13 genes that affect the secretion efficiency of a staphylococcal nuclease reporter enzyme (NucT) by using random mutagenesis (25). The inactivation of these genes resulted in either increased or decreased levels of secreted NucT. One gene of particular interest was dltA, whose product catalyzes the incorporation of D-alanine residues into lipoteichoic acids. The inactivation of dltA reduced NucT secretion efficiency due to modifications of the cell wall leading to negatively charged lipoteichoic acid interacting with the positively charged Nuc and physical entrapment of NucT in the peptidoglycan network.

In this study, we have used random transposon mutagenesis to identify genes that affect the extracellular activity of lysostaphin expressed by L. lactis. Four uncharacterized genes whose inactivation caused increases in the amounts of lysostaphin secreted by L. lactis were identified in this study. One gene encodes a predicted membrane-embedded protein; two genes are likely to affect cell wall peptidoglycan structure; and the fourth gene may be involved in ubiquinone biosynthesis.

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Bacterial strains, plasmids, media, and growth conditions.
The strains, plasmids, and oligonucleotides used are listed in Table 1. Lactococcus lactis subsp. cremoris MG1363 was grown using M17 (Oxoid, Basingstoke, United Kingdom) supplemented with 0.5% glucose (GM17) or 1% glucose (2GM17) and was incubated at 30°C or 37°C as indicated for the experiments described below. Listeria monocytogenes ATCC 19112 and S. aureus ATCC 49476 were grown in brain heart infusion medium (Oxoid) and incubated at 37°C. Escherichia coli JM109 (Promega, Madison, WI) was used in cloning experiments and was grown in either Luria-Bertani medium or brain heart infusion medium (both from Oxoid). Antibiotics were used at the following concentrations: ampicillin, 100 µg/ml for E. coli; erythromycin, 300 µg/ml for E. coli and 5 and 2 µg/ml for plasmid- and chromosome-encoded resistance in L. lactis, respectively.

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TABLE 1. Strains, plasmids, and oligonucleotides used in this study

Construction of the L. lactis MG1363[lss] strain used for random insertional mutagenesis.
We constructed an MG1363 strain carrying the lysostaphin expression cassette (lss) on the chromosome (Fig. 1). Previously, this cassette (containing the Sep promoter, P_Sep; the Sep secretion signal, ss_Sep; a six-histidine tag; and the gene encoding the mature lysostaphin protein) has been used to produce and secrete lysostaphin in MG1363 (39). The lss cassette was integrated into the same region of the inactive histidine biosynthesis (his) operon as previously described (25) (Fig. 1). The strategy used to produce the MG1363[lss] strain was as follows. The lss cassette was isolated as an XbaI/XhoI fragment from pSep-6 x His-Lss (39) (Table 1). This fragment was ligated to two his operon fragments in the same orientation (the upstream his fragment from pBS-his1 digested using SalI/XbaI and the downstream his fragment from pGT-his2 digested using XhoI/EcoRI) and PCR amplified using primers His1Sal5 and His2Eco3 (Table 1). The resulting 3.9-kb PCR fragment was first cloned into the pGEM-T Easy vector (Promega) for sequencing purposes before being cloned into pGhost9:ISS1. Both plasmids were transformed into E. coli JM109 before the pG9-his1-lss-his2 (Lss⁺ Em^r) plasmid was transformed into wild-type MG1363. Stable integration of lss was performed in two steps. First, strain MG1363 with plasmid pG9-his1-lss-his2 was grown at 37°C and selected on a GM17 agar plate with 2 µg/ml erythromycin. This initial temperature-sensitive integrant, MG1363[pG9-his1-lss-his2], was confirmed by PCR using primers lss-Pst and His2-DS (Table 1; Fig. 1) to ensure that the lss cassette was inserted in the his operon. Primer His2-DS binds to the chromosome 26 bp downstream of the cloned his2 fragment. It was also confirmed to have lysostaphin activity on GM17+SaB agar plates. These are GM17 agar plates containing 100 µl of autoclaved 100-fold-concentrated S. aureus ATCC 49476 cells, which are buffered with 0.2 M potassium phosphate buffer (pH 7.0) (39). The integrant was then made temperature stable by excision of pGhost9:ISS1 at 30°C. This temperature-stable integrant, MG1363[lss] (Fig. 1), was then selected for lysostaphin activity and erythromycin sensitivity.

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FIG. 1. Structure of strain MG1363 carrying the lss expression cassette. The mature lysostaphin gene was fused to the Sep promoter (PSep) and secretion signal (ssSep) (hatched box) with a six-histidine tag (filled box) at the N terminus of the lysostaphin gene. The asterisk indicates the lysostaphin stop codon. The lss expression cassette was integrated by single-crossover recombination into the middle of the his operon in MG1363. PCR primers lss-Pst and His2-DS (dashed arrows) were used to confirm that the lss expression cassette was integrated. The pGhost9:ISS1 plasmid was excised from the chromosome by growing the single-crossover integrant at 30°C without erythromycin and selecting for clones with lysostaphin activity and erythromycin sensitivity.

Mutagenesis and screening conditions.
Random insertional mutagenesis was performed on MG1363[lss] with pGhost9:ISS1 as previously described (19). Approximately 50 CFU was plated onto each GM17+SaB or GM17+SaU (unbuffered GM17+SaB) agar plate with 2 µg/ml erythromycin and was incubated at 37°C for 48 h. The mutants were screened for the absence of halos or for halos significantly larger than that produced by the control strain, MG1363[lss]. The average diameter of the halo of the control strain, MG1363[lss], was 3.4 mm. Larger halos from selected mutants had diameters 0.8 mm to 2.8 mm greater than that produced by the control strain.

Characterization of the pGhost9:ISS1 insertion sites and isolation of stable ISS1 mutants.
The sites where pGhost9:ISS1 integrated into the chromosome were determined as previously described (19). The flanking chromosomal DNA was sequenced using primer ISS1-seq1 for the EcoRI chromosomal junction or ISS1-seq2 for the HindIII junction (Table 1). The mutated genes were identified by sequence comparison with the recently sequenced genome of MG1363 (42). The number of pGhost9:ISS1 integrations into the chromosome was determined by Southern hybridization of EcoRI-digested DNA using a digoxigenin-labeled ISS1 fragment as a probe. For further experiments, we isolated stable ISS1 mutants that grew at 30°C without erythromycin as previously described (19). These mutants were then retested for their lysostaphin activities by streaking a loopful of overnight cultures onto GM17+SaB agar and GM17+SaU agar and then incubating the cultures at 30°C (or 37°C where indicated) for 2 days.

Prediction of operon structures.
To determine if genes containing ISS1 insertions were cotranscribed with downstream genes, two operon prediction methods were used (9, 30). The accuracy of the method of Price et al. (30), which has been estimated based on the prediction of experimentally proven operons and from microarray expression data, is 82% for most genomes. We have identified likely cotranscribed downstream genes that were predicted by at least one of these two methods. The programs are available at http://www.microbesonline.org/operons/ and http://operondb.cbcb.umd.edu/cgi-bin/operondb/operons.cgi.

Prediction of subcellular locations of proteins.
Protein sequences were entered into three different online programs: SOSUI, a predictor of transmembrane regions (http://bp.nuap.nagoya-u.ac.jp/sosui/) (14); TMpred, a predictor of transmembrane regions and protein orientation (http://www.ch.embnet.org/software/TMPRED_form.html) (15); and the PHD transmembrane helix predictor (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_htm.html) (33).

Cell fractionation, protein extraction, and Western blot analysis.
Cell-associated fractions were prepared by boiling the cells from late-exponential- or early-stationary-phase cultures in 2x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. Proteins in the supernatants were concentrated using 5% trichloroacetic acid as described previously (37). Proteins were separated using SDS-PAGE, transferred to nitrocellulose filters (GE Healthcare), and blocked with 1% casein (Roche Applied Science, Mannheim, Germany). The membrane was probed with either an anti-His₅ monoclonal antibody (Qiagen, Hilden, Germany) at a 1:1,000 dilution or an anti-His₆ monoclonal antibody (Sigma-Aldrich, St. Louis, MO) at a 1:3,000 dilution, followed by a horseradish peroxidase-conjugated rabbit anti-mouse antibody (Dako, Glostrup, Denmark) at a 1:1,000 dilution. The bound antibodies were detected using the Lumi-Light chemiluminescence kit (Roche Applied Science). To quantify the amounts of proteins, different nonsaturated film exposures were scanned (CanoScan800F; Canon), and the total amounts of proteins from the supernatant and cell-associated fractions were estimated with GeneTools software (Syngene, Frederick, MD). Statistical analysis was carried out using Student's t test.

Ply511 expression and secretion in [lss] mutant strains.
The L. monocytogenes bacteriophage endolysin Ply511 was transformed into wild-type MG1363; the control strain, MG1363[lss]; and the lom[lss], murA2[lss], and trmA[lss] mutant strains. The Ply511 gene was introduced into these strains using the pGhost9:ISS1 plasmid under the control of the Sep expression system (pSep511sec) (39). The transformed strains were tested for Ply511 activity against L. monocytogenes on GM17+LmB agar plates (GM17 with 5 µg/ml erythromycin and 300 µl of an autoclaved 100-fold concentrate of L. monocytogenes ATCC 19112 cells, buffered with 0.2 M potassium phosphate buffer [pH 7.0]) (39). Ply511 was also detected by Western blotting using an anti-His₆ monoclonal antibody (Sigma-Aldrich) as described above.

Lysozyme resistance analysis.
The lysozyme resistance test was performed as described previously (40). Briefly, solutions of chicken egg white lysozyme (Sigma Aldrich) in GM17 were freshly prepared and diluted 10-fold into molten GM17 agar at 45°C. Then 5 µl of overnight cultures diluted 10-fold was spotted onto GM17 agar with various concentrations of lysozyme. The plates were then incubated at 30°C or 37°C as indicated.

Transmission electron microscopy.
Bacterial cultures grown for 3, 6, or 20 h were fixed in 0.4% glutaraldehyde (ProSciTech)-0.1 M cacodylate buffer (pH 7.3). After overnight fixation, the cells were pelleted and washed in 0.1 M cacodylate buffer. Cells were postfixed in 1% osmium tetroxide and embedded in Spurr epoxy resin. Ultrathin sections (thickness, 50 to 100 nm) were cut and stained with uracyl acetate and lead citrate stains prior to examination and photography using the JEOL 1200 EX transmission electron microscope.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Isolation and identification of mutants with altered lysostaphin activities.
The purpose of this study was to identify host factors in MG1363 that affect the secretion efficiency of the peptidoglycan hydrolase lysostaphin, which has specific activity against S. aureus. We adapted a previously described method whereby random mutagenesis (using the ISS1 element in plasmid pGhost9:ISS1) was used to identify host factors in the secretion of the staphylococcal nuclease reporter (NucT) in MG1363 (25). We screened more than 35,000 clones for significantly increased or decreased lysostaphin activity on GM17+SaB and GM17+SaU agar plates containing 2 µg/ml erythromycin. On that basis, 124 mutants were selected, and their lysostaphin activities were confirmed using this agar plate method by comparison to the control strain (MG1363[lss]). Ten of the 124 initial mutants were confirmed to have halo sizes significantly greater than that of MG1363[lss], while 1 mutant was confirmed as having no halo. The locations of inserted pGhost9:ISS1 were identified by comparison of the flanking sequences with the MG1363 genome sequence (42) (Table 2; Fig. 2). The mutant with no lysostaphin activity resulted from an insertion of pGhost9:ISS1 in the lss expression cassette. This was expected, because ISS1 insertion is a random event (19). Three mutants had independent insertions in a gene encoding an uncharacterized putative transmembrane protein (llmg_0609); five mutants had independent insertions in the murA2 gene; one mutant contained an insertion just upstream of trmA; and one mutant contained an insertion in a gene encoding an uncharacterized putative enzyme involved in ubiquinone biosynthesis (llmg_2148).

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TABLE 2. Characteristics of mutants with lysostaphin activity greater than that of the wild type

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FIG. 2. Identification of the chromosomal loci of the nine lysostaphin-oversecreting mutants. (A) Locations of the three mutants with mutations in the llmg_0609 gene at nucleotide positions 818 (mutant 24185), 1354 (mutant 11135), and 1408 (mutant 2190) from the start of the gene. The predicted single transmembrane helix is located from amino acids 177 to 195 (dotted box). (B) Representative Western blot of lysostaphin in supernatant fractions from MG1363[lss] and mutant 11135[lss] grown at 30°C. (C) The five independent insertions in the murA2 gene are located at nucleotide positions 116 (mutant 28801), 753 (mutant 24189), 766 (mutant 31397), 1050 (mutant 31394), and 1220 (mutant 18662). (D) Representative Western blot of lysostaphin in supernatant fractions from MG1363[lss] and mutant 28801[lss] grown at 37°C. (E) The single mutation in the trmA gene (mutant 16270) is located 34 bp upstream of the start of the gene. (F) Representative Western blot of lysostaphin in supernatant fractions from MG1363[lss] and mutant 16270[lss] grown at 37°C. The variable quantities of supernatants loaded in the Western blots are given in the tables above the blots (B, D, and F).

Examination of the MG1363 genome showed that it is possible that the llmg_0609, murA2, and llmg_2148 genes could be part of different operons. Downstream genes predicted to be located in these operons by at least one of two computational prediction programs (9, 30) are shown in Table 2. Transcript analysis by Dupont et al. (7) has shown that, due to the presence of a promoter in the ISS1 element, ISS1 (and pGhost9:ISS1) insertion does not lead to polar effects on downstream genes. Such effects are observed, however, when the ISS1 sequence, which encodes a putative transposase, is orientated in the same direction as the interrupted genes. In our study, ISS1 elements were orientated in the same direction as the interrupted genes in two llmg_0609 mutants (2190 and 24185) and two murA2 mutants (18662 and 31397). In these mutants, it would be expected that the genes downstream would be transcribed and that the phenotypes would not be due to polar effects. In the llmg_2148 mutant, the ISS1 element is in the opposite orientation to the interrupted gene and therefore may affect the transcription of the downstream fmt gene (Table 2). It should also be noted that the ISS1 element in mutant 16270 has inserted 34 bp upstream of the trmA gene and in the opposite orientation and is therefore expected to prevent its transcription.

Plasmid pGhost9:ISS1 was excised from all mutants to create temperature-stable mutants for further investigation. These temperature-stable mutants were streaked onto GM17+SaB agar (without erythromycin) to reconfirm the lysostaphin activity phenotype. Southern hybridization analysis showed that for each temperature-stable mutant, ISS1 transposition occurred only at one site (data not shown). The llmg_0609 and llmg_2148 mutants retained their increased lysostaphin activities at 30°C, while large zones of activity were observed only at 37°C for the murA2 and trmA mutants. The amount of lysostaphin secreted, the generation time, and the final culture pH were measured for all mutants (Table 2). The increases in the lysostaphin activities of the 10 mutants were determined to be due to increases in the secretion of lysostaphin over that by the control strain, MG1363[lss], as determined by Western blot analysis (Fig. 2; Table 2). The levels were determined by direct comparison to the amount secreted by MG1363[lss], which was assigned a value of 1.0 (Table 2). The average increases (± standard deviations) in the lysostaphin levels in the cell extracts and supernatants of the three llmg_0609 mutants were 3.0-fold (±0.2-fold) and 12.0-fold (±3.6-fold), respectively; both levels were significantly higher than those of the control strain (P < 0.01). The average increase (± standard deviation) in the lysostaphin level in the supernatants of the five murA2 mutants grown at 37°C was 6.2-fold (±0.7-fold), and the level was significantly higher than that of the control strain (P < 0.01).

Characterization of genes that affect lysostaphin secretion.
The llmg_0609 gene has been annotated in the MG1363 genome as encoding PabC (42) but is unlikely to function as a 4-amino-4-deoxychorismate lyase like the PabC proteins of E. coli and Bacillus subtilis (11, 35). It has recently been proposed that the open reading frame of the true pabC gene in MG1363 is contained within the llmg_1154 open reading frame as a fusion to pabB (41). Therefore, we propose to rename llmg_0609 as lom (lysostaphin-oversecreting mutant), in order to avoid confusion. GenBank searches revealed that Lom has significant similarity only to hypothetical proteins or proteins with putative functions not yet experimentally tested. Protein sequence analysis of Lom revealed that a single transmembrane helix was predicted to lie in the central region (amino acids 177 to 195) (Fig. 2) and that the C-terminal region extends outside of the membrane.

MurA2 is a putative UDP-N-acetylglucosamine enopyruvyl transferase that catalyzes the first step in the biosynthesis of peptidoglycan (20). As with other low-G+C gram-positive bacteria (6), MG1363 has two MurA enzymes (MurA1 and MurA2) (42). MurA2 in MG1363 is more closely related to enzymes in related species that are annotated as the primary MurA enzyme. For example, MG1363 MurA2 is 61% and 40% identical to the B. subtilis MurAA and MurAB proteins, respectively. Similarly, MG1363 MurA2 is 70% and 44% identical to the Streptococcus pneumoniae MurA1 and MurA2 proteins, respectively. Interestingly, MG1363 MurA2, B. subtilis MurAA, and S. pneumoniae MurA1 are more similar to the MurA in E. coli and other species with just one MurA than are the MurA paralogs in the gram-positive species. The simplest explanation of this is that MG1363 MurA2, B. subtilis MurAA, and S. pneumoniae MurA1 are orthologs of E. coli MurA and may represent the primary MurA. Du et al. (6) demonstrated that MurA1 and MurA2 were both enzymatically functional and could substitute for each other in S. pneumoniae. In contrast, MurAB could not substitute for MurAA in B. subtilis (16). The lack of a growth defect in the MG1363 murA2 mutants (Table 2) would suggest that the MG1363 murA1 paralog is able to substitute functionally for murA2. The hypothesis that the role of MurA2 is distinct from that of MurA1 is supported by previous studies that identified MurA2 proteins in the cytoplasm of wild-type Lactococcus lactis subsp. lactis IL1403 (12) and, in particular, showed that MurA2 proteins were upregulated in response to acid stress in two MG1363 mutants (1). Since the lysostaphin oversecretion phenotype was observed primarily under heat stress conditions at 37°C, it is possible that MurA1 is not fully functional at this higher temperature and that this allowed lysostaphin to be readily exported through a defective cell wall. However, both examination of cell morphology using light microscopy and determination of the thickness of cell walls using transmission electron microscopy failed to reveal any differences between the murA2 mutants and the MG1363[lss] control strain grown at 30°C or 37°C (data not shown). SDS-PAGE analysis of Coomassie-stained proteins from supernatant fractions showed that the murA2 mutants did not release greater amounts of intracellular proteins than the control strain (MG1363[lss]) (data not shown), suggesting that the former are not leaky.

TrmA has homology to Spx, an oxidative stress regulator in B. subtilis (24), and seven genes in the trmA family are encoded by the MG1363 genome (42). The inactivation of this gene has been reported to confer various stress-resistant phenotypes. Various random mutagenesis studies have identified trmA mutants as able to relieve temperature sensitivity in recA (8) and clpP (13) mutant strains and to confer resistance to tellurite and oxidative stress on the wild type (38). In addition, a trmA mutant has increased resistance against the hydrolytic activity of lysozyme over the wild-type strain (40), due to a modified cell wall. Veiga et al. hypothesized that TrmA competes with SpxB, one of its paralogs, for the binding of RpoA, the subunit of RNA polymerase (40). The inactivation of trmA may allow SpxB to better bind RpoA, and this interaction activates the expression of oatA, encoding the lactococcal peptidoglycan O-acetylase, thus modifying the cell wall so that it becomes lysozyme resistant. The reason for the increased lysostaphin secretion levels of the trmA mutant is not clear, but as with the murA2 mutant, they may be due to an altered cell wall structure.

Finally, one of the mutants selected from the screening had an inactivation in the gene annotated as llmg_2148. The basis for the increased lysostaphin secretion of this mutant remains unclear. This gene is annotated as a putative enzyme, 3-demethylubiquinone-9 3-methyltransferase (UbiG) (42), which in E. coli is involved in ubiquinone biosynthesis (36). However, llmg_2148 has no homology to functionally characterized UbiG enzymes in databases.

The lom, murA2, and trmA mutants oversecrete another hydrolytic enzyme, Ply511.
To examine whether this increase in lysostaphin secretion is specific to lysostaphin, we compared the abilities of the wild-type MG1363, the control strain (MG1363[lss]), and the lysostaphin-secreting mutant strains (lom[lss], murA2[lss], and trmA[lss]) to express another heterologous protein, Ply511. Ply511 is a peptidoglycan-hydrolytic endolysin (N-acetylmuramoyl-L-alanine amidase) from an L. monocytogenes bacteriophage (18). All the strains transformed with the Ply511 expression cassette pSep511sec (Table 1) demonstrated activity against both L. monocytogenes and S. aureus, as evidenced by zones of clearing on agar plates containing autoclaved cells. We compared the zones of activity of Ply511 on GM17+LmB agar plates but observed no differences in the activity zones between the control strain and the mutant strains (data not shown). Western blot analysis, however, conclusively demonstrated that the amounts of Ply511 secreted into the cell-associated and supernatant fractions by the lom[lss][511], murA2[lss][511], and trmA[lss][511] mutant strains were significantly greater than those for the control strain, MG1363[lss][511] (Fig. 3). The lack of correlation between Western blot analysis and clearing zone size for Ply511 may be because the zone size for wild-type Lactococcus expressing Ply511 (MG1363[lss][511]) is already very large (compared to clearing zones from lysostaphin activity) and therefore may have saturated the assay, despite the fact that only very low levels of enzyme were produced. The large clearing zones generated by Ply511 could be due to its ability to diffuse through the bacterial-cell-containing agar more efficiently than lysostaphin.

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FIG. 3. Western blot detection of MG1363 strains secreting lysostaphin and/or Ply511 in the cell-associated and supernatant fractions. The different strains and the quantities loaded on the Western blots are given in the tables above the blots. Molecular mass standards are expressed in kilodaltons. The amount of cell-associated protein is equivalent to 1 ml of late-exponential-phase culture. The amount of supernatant protein loaded in each lane is equivalent to 900 µl of late-exponential-phase culture for the control strain (MG1363[lss][511]) and 450 µl for mutant strains. The Ply511 protein resolved at approximately 40 kDa. The lysostaphin protein resolved at approximately 31 kDa. The Ply511 protein can be observed in the cell-associated fraction and supernatant of the control strain after longer exposures. wt, wild type.

murA2 and trmA mutants were more resistant to lysozyme hydrolysis.
A recent study has shown that a mutation in trmA results in lysozyme resistance in MG1363 (40). In this study, we found that a trmA mutant that expresses lysostaphin (trmA[lss]) is also more resistant to lysozyme than the wild-type lysostaphin-expressing strain (MG1363[lss]) (Fig. 4). Interestingly, we observed that MG1363[lss] was much more sensitive to lysozyme than the non-lysostaphin-secreting wild-type strain, MG1363 (Fig. 4B). This result suggests that lysostaphin may be cleaving lactococcal peptidoglycan during its passage through the cell wall, thereby heightening the sensitivity to lysozyme. According to Veiga et al. (40), the resultant interactions within a trmA mutant give rise to changes in the acetylation and thickness of peptidoglycan, thereby making it more resistant to lysozyme hydrolysis. Therefore, the trmA mutant may also be more resistant to the nonspecific degradation caused during the secretion of lysostaphin and, as a result, may be able to tolerate higher levels of protein secretion. We also observed that the murA2[lss] mutant strain was moderately more resistant to lysozyme than the control strain (MG1363[lss]) and that the lysozyme resistance of wild-type MG1363 was greater at 30°C than at 37°C (Fig. 4B and C). Therefore, protection from cell wall-lytic enzymes (lysozyme and lysostaphin) is likely to be attributable to modifications in the peptidoglycan structure resulting from mutations in trmA and murA2; however, this has yet to be confirmed.

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FIG. 4. The murA2[lss] and trmA[lss] mutant strains are more resistant to lysozyme hydrolysis than the control strain, MG1363[lss]. The wild-type strain, MG1363, is more resistant than the control strain, which expresses lysostaphin. All strains showed identical growth on GM17 agar plates without lysozyme at 30°C and 37°C. Therefore, only the growth of MG1363 is shown. Strains were grown at 30°C without lysozyme (A) or with 0.25 mg/ml lysozyme (B) or at 37°C with 0.25 mg/ml lysozyme (C).

In conclusion, we have identified four genes involved in the secretion of lysostaphin in MG1363. These genes are different from those previously identified in the NucT study (25), suggesting that lactococcal host factors that affect the secretion of heterologous proteins differ depending on the protein of interest. While the mechanisms remain unknown, the inactivation of these four genes significantly increased the amount of lysostaphin secreted without any detrimental effects to the host cell and the growth rate. We also describe the construction of novel MG1363 strains that are able to secrete two kinds of peptidoglycan hydrolases, lysostaphin and Ply511. The results of this study clearly provide new insights into lactococcal host factors important for the secretion efficiency of heterologous proteins, which may have applications in the food and pharmaceutical industries.

 
We thank Xavier Chan, Terry Walsh, Sarah Mathews, and Shea Carter for invaluable discussions and technical assistance on various aspects of this project. We also acknowledge Aldert L. Zomer (University of Groningen, Groningen, The Netherlands) for the provision of the MG1363 histidine biosynthesis operon sequences ahead of publication and Christina Theodoropoulos for assistance with the transmission electron microscopy work.

This research was funded by the National Health and Medical Research Council (grant 290526). Y.P.T. is supported by scholarships from Dairy Australia (grant QUT11991) and the Queensland University of Technology.

 
* Corresponding author. Mailing address: School of Land, Crop and Food Sciences, University of Queensland, Brisbane, Queensland 4072, Australia. Phone: (61-7) 3365-1171. Fax: (61-7) 3365-1177. E-mail: m.turner2{at}uq.edu.au

Published ahead of print on 17 October 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

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Applied and Environmental Microbiology, December 2008, p. 7490-7496, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.00767-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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