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Research Article

Internalization of a thiazole-modified peptide in Sinorhizobium meliloti occurs by BacA-dependent and -independent mechanisms

Silvia Wehmeier, Markus F. F. Arnold, Victoria L. Marlow, Mustapha Aouida, Kamila K. Myka, Vivien Fletcher, Monica Benincasa, Marco Scocchi, Dindial Ramotar, Gail P. Ferguson

  • 1School of Medicine and Dentistry, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
  • 2Maisonneuve-Rosemont Hospital Research Centre, University of Montreal, 5415, Boul. de l'Assomption, Montreal, QC H1T 2M4, Canada
  • 3Department of Life Sciences, University of Trieste, Via Giorgieri 1, 34127 Trieste, Italy
  • Correspondence
    Gail P. Ferguson
    g.ferguson{at}abdn.ac.uk

    • Microbiology 2010; 156(9):2702–2713 · https://doi.org/10.1099/mic.0.039909-0

    View at publisher PubMed

    Abstract

    BacA proteins play key roles in the chronic intracellular infections of Sinorhizobium meliloti, Brucella abortus and Mycobacterium tuberculosis within their respective hosts. S. meliloti, B. abortus and M. tuberculosis BacA-deficient mutants have increased resistance to the thiazole-modified peptide bleomycin. BacA has been previously hypothesized, but not experimentally verified, to be involved in bleomycin uptake. In this paper, we show that a BacA-dependent mechanism is the major route of bleomycin internalization in S. meliloti. We also determined that the B. abortus and S. meliloti BacA proteins are functional homologues and that the B. abortus BacA protein is involved in the uptake of both bleomycin and proline-rich peptides. Our findings also provide evidence that there is a second, BacA-independent minor mechanism for bleomycin internalization in S. meliloti. We determined that the BacA-dependent and -independent mechanisms of bleomycin uptake are energy-dependent, consistent with both mechanisms of bleomycin uptake involving transport systems.

    • ↵†These authors contributed equally to this work.

    Edited by: M. F. Hynes

    INTRODUCTION

    Sinorhizobium meliloti can be found either free-living in the soil or in a symbiotic relationship with leguminous plants such as alfalfa (for recent reviews refer to Gibson et al., 2008; Jones et al., 2007). After entry into alfalfa cells, S. meliloti differentiates into a nitrogen-fixing bacteroid, which persists for extensive periods. Despite being beneficial for the host, this interaction can be viewed as a chronic intracellular infection (Gibson et al., 2008). The BacA protein is essential for the chronic intracellular infection of S. meliloti within alfalfa (Glazebrook et al., 1993), as in the absence of BacA, an S. meliloti mutant can enter into the plant cell but lyses and dies shortly after entry. The chronic mammalian pathogen Brucella abortus also has a BacA protein, which shares 68 % identity with the S. meliloti BacA protein and is essential for chronic infections of BALB/c mice (LeVier et al., 2000). Recently, the Mycobacterium tuberculosis BacA protein (Rv1819c), which is 39 % similar to the B. abortus BacA protein, was also determined to be involved in the maintenance of chronic B6D2/F1 mice infections (Domenech et al., 2009). Since the mechanism of chronic bacterial infections is poorly understood, elucidation of the function of different BacA proteins could reveal important insights into this process.

    To understand more about the function of BacA proteins, S. meliloti, B. abortus and M. tuberculosis bacA mutants have also been characterized in their free-living states relative to their respective parent strains. The lipid A of S. meliloti and B. abortus is unusually modified with very-long-chain fatty acids (VLCFAs), and it was discovered that S. meliloti and B. abortus bacA null mutants have an approximately 50 % reduction in their lipid A VLCFA content (Ferguson et al., 2004). Lipid A is a component of the lipopolysaccharide that forms the outer leaflet of the outer membrane of Gram-negative bacteria (Raetz & Whitfield, 2002). However, by constructing and characterizing S. meliloti acpXL and lpxXL mutants that are deficient in the biosynthesis of the lipid A VLCFA modification, it was found that the unusual modification of the lipid A is important but not essential for the chronic infection of S. meliloti in alfalfa (Ferguson et al., 2005; Haag et al., 2009; Sharypova et al., 2003). Additionally, these studies demonstrated that the effect of BacA on the lipid A VLCFA modification is unlikely solely to account for its essential role in chronic infections. Consistent with this, the BacA protein of M. tuberculosis had no effect on the fatty acid content of membrane lipids (Domenech et al., 2009). Combined, these studies provide support for a model whereby BacA proteins also have another function or effect on bacterial cells that is important for their essential role in chronic bacterial infections.

    The S. meliloti BacA protein also shares 64 % identity with the Escherichia coli SbmA protein (Glazebrook et al., 1993; Ichige & Walker, 1997). The sbmA gene was first identified by its ability to confer sensitivity of E. coli to the antimicrobial peptide microcin B17 (Laviña et al., 1986; Yorgey et al., 1994). Microcin B17 is a glycine-rich, thiazole-modified peptide produced by E. coli strains carrying a naturally occurring plasmid and is an inhibitor of DNA gyrase (Yorgey et al., 1994). Since mutations in sbmA only protected E. coli against exogenous microcin B17 and conferred no growth advantage to the microcin B17-producing strain, this provided indirect evidence that SbmA is involved in peptide uptake (Laviña et al., 1986). It was subsequently shown, using fluorescently labelled peptides, that the SbmA protein is involved in the uptake of truncated Bac7 peptides into E. coli (Mattiuzzo et al., 2007). Full-length Bac7 is produced by bovine leukocytes and is a linear proline/arginine-rich peptide (Frank et al., 1990). At present, it is not known whether SbmA plays a direct or indirect role in peptide uptake in E. coli (Mattiuzzo et al., 2007). However, it has been proposed that SbmA and BacA proteins form the transmembrane domains of an ABC transporter and the M. tuberculosis BacA protein has a fused ATPase domain (Domenech et al., 2009; LeVier & Walker, 2001), suggesting that they could be directly involved in peptide uptake.

    S. meliloti is highly resistant to killing by microcin B17 (Ichige & Walker, 1997). However, S. meliloti is sensitive to truncated Bac7 peptides, and the BacA protein has been shown to play an essential role in their uptake (Marlow et al., 2009). In contrast, there appear to have been no previous studies investigating the role of the B. abortus and M. tuberculosis BacA proteins in peptide uptake. Nevertheless, every sbmA/bacA mutant characterized to date shows increased resistance to the glycopeptide antibiotic bleomycin (Domenech et al., 2009; Ichige & Walker, 1997; LeVier et al., 2000). In S. meliloti, it was shown that reductions in the lipid A VLCFA do not confer resistance to bleomycin, providing evidence that the increased resistance of the S. meliloti bacA null mutant to bleomycin is independent of its lipid A alteration (Ferguson et al., 2006). Bleomycin is synthesized by the soil bacterium Streptomyces verticillus (Umezawa et al., 1966) and has been shown previously to damage DNA through a Fe(II)-mediated oxidative mechanism (Kane & Hecht, 1994). It had been proposed previously that BacA-mediated sensitivity to bleomycin could be due to BacA being involved in iron uptake (Ichige & Walker, 1997). However, an M. tuberculosis BacA-deficient mutant was unaffected in iron uptake, suggesting that this was not the case (Domenech et al., 2009). Interestingly, although the peptide sequences of bleomycin and microcin B17 differ, both peptides are modified with thiazole rings (Yorgey et al., 1994). Based on this finding and the increased resistance of bacA/sbmA mutants to bleomycin it was hypothesized that SbmA/BacA proteins could also be involved in the uptake of bleomycin into bacterial cells (Ichige & Walker, 1997; Yorgey et al., 1994). However, this hypothesis had never been verified experimentally. Bleomycin is also known to damage the cell wall and membrane of yeast cells (Lim et al., 1995). Therefore, we could not exclude the possibility that BacA/SbmA were sensitizing bacterial cells to bleomycin through another mechanism rather than uptake.

    Since bleomycin is produced by a soil bacterium and plants produce thiazole-containing compounds (Umezawa et al., 1966; Yorgey et al., 1994), the effect of bleomycin on S. meliloti could have relevance to both its free-living and symbiotic states. Additionally, bleomycin is an antibiotic (Umezawa et al., 1966; Yorgey et al., 1994). Therefore, investigating the factors involved in sensitizing B. abortus to this peptide could provide information relevant to the treatment of chronic bacterial infections. In this study, we investigated the hypothesis that BacA proteins are involved in bleomycin internalization. We found that both the S. meliloti and B. abortus BacA proteins play important roles in bleomycin internalization in S. meliloti. However, we also discovered a second, BacA-independent, energy-dependent mechanism for bleomycin internalization in S. meliloti.

    METHODS

    Bacterial strains and growth conditions.

    All bacterial strains and plasmids used in this work are described in Table 1⇓. The S. meliloti strains used are all derivatives of the Rm1021 sequenced strain (Galibert et al., 2001). For all experiments, S. meliloti strains were grown in either Luria–Bertani broth (LB) (Sambrook et al., 1982) prepared with 10 g NaCl l−1or LB supplemented with 2.5 mM CaCl2 and 2.5 mM MgSO4 (LB/MC) for 48 h at 30 °C. Unless indicated otherwise, antibiotics were used at the following concentrations (μg ml−1): gentamicin (Gm), 50; streptomycin (Sm), 500; spectinomycin (Spc), 100; neomycin (Nm) 200; and tetracycline (Tc), 5.

    Table 1.

    Bacterial strains and plasmids used

    Filter disc and liquid culture viability assays.

    For the filter disc assays, late-exponential-phase cultures (OD600 ∼3.0) were washed in LB, resuspended to OD600 ∼0.2 and then the assays were performed exactly as described previously (Ferguson et al., 2002). The agar plates were incubated at 30 °C (72 h) and then the diameters of growth inhibition were recorded. The growth inhibition zone was measured from at least three plates for each strain and condition and the results were averaged. The liquid culture viability assays, unless stated otherwise, were also performed with late-exponential-phase cultures, which were harvested, washed and diluted to OD600 ∼0.1 in LB medium. After addition of the appropriate form of bleomycin, the cultures were incubated at 30 °C. At defined times, samples were removed, serially diluted in LB medium, then 10 μl aliquots were plated in triplicate on LB agar plates. C.f.u. were determined after 72 h at 30 °C. The mean c.f.u. ml−1 in the three 10 μl aliquots was plotted. All experiments were repeated at least twice and the data shown are a representative dataset.

    For the 2,4-dinitrophenol (DNP) treatment, cultures were grown to mid-exponential phase and then resuspended to OD600 ∼0.8 in LB medium supplemented or not with the defined amount of DNP. After 50 min with shaking (200 r.p.m.) at 30 °C, the cultures were washed twice with LB and the viability determined as described above. To determine whether DNP treatment affected sensitivity to bleomycin, the cultures were diluted to OD600 ∼0.1 in LB medium and the bleomycin sensitivity assay performed as described above.

    Genomic DNA degradation assay.

    Cultures were grown to mid-exponential phase (OD600 ∼0.9) in LB/MC, washed three times with LB and then recovered by centrifugation at 2264 g (10 min). The cell pellet was resuspended in 2 ml LB to OD600 ∼2, split equally between two glass test tubes and then one tube was treated with either bleomycin A5 (20 μg ml−1) or the defined amount (1 or 0.2 % v/v) of methyl methane sulfonate (MMS) (Sigma). The second tube served as an untreated control. All tubes were incubated with shaking at 30 °C for 2 h (bleomycin treated) or the defined time (30 min to 2 h) for MMS; the cells were then washed once, recovered by centrifugation at 2264 g (10 min) and the cell pellets were stored at −80 °C until required. The genomic DNA was extracted as described previously (Wilson, 1987) with the following modifications: in step 4 chloroform only was used, in step 5 phenol/chloroform (1 : 1) was used instead of phenol/chloroform/isoamyl alcohol (25 : 24 : 1) and in step 7 the genomic DNA was dissolved in 25 μl H2O. The genomic DNA was quantified using UV spectroscopy and 2 μg of the DNA was analysed on a 1 % (w/v) agarose gel.

    4′,6′-Diamidino-2-phenylindole (DAPI) fluorescence.

    Cultures were treated with and without bleomycin A5 exactly as for the genomic DNA preparations except that following the centrifugation the cells were resuspended in 100 μl 1 % (v/v) toluene, vortexed and stored at 4 °C. When required, the cells were diluted to OD600 ∼0.08 in dilution buffer (10 mM NaCl, 6.6 mM Na2SO4, 5 mM HEPES pH 7.0). The DAPI assay was performed following a previously described procedure (Johnson, 1994) except that DAPI was added to the cells at a final concentration of 0.1 μg ml−1 in a final volume of 100 μl and the cells were incubated with DAPI overnight at 4 °C. The fluorescence intensity was measured using a FLUOstar optima plate reader (BMG Labtech) spectrophotometer with excitation and emission at 350 and 450 nm, respectively.

    Cloning of the B. abortus and S. meliloti bacA genes.

    The B. abortus bacA gene, including 150 bp upstream, was amplified by PCR using the primers BabacA+150-F (5′-GCTTGGCTGCAGCCTAACACCCTATG GGCCGT-3′) and BabacA-R (5′-ATGATGGATCCTCAGCCCGCCCCTG-3′). The fragment was then digested with BamHI and PstI, and ligated into pRF771 (Wells & Long, 2002), under control of the trp promoter. The S. meliloti bacA gene was amplified by PCR using the primers Smeliloti_bacA_NsiI_RBS_F (5′-GCTAGAATGCATGAAACGAGAGTGCCGTCCCCCTTGTTCCAATCCTTCTTCCCC-3′) and Smeliloti_bacA_XbaI_R (5′-TTATCGTCTAGAATGCCCGCCGTTACAGA-3′). The PCR fragment was then digested with NsiI and XbaI and ligated into pRF771 under control of ptrp. In both cases, the ligated plasmids were transformed into E. coli DH5α and transformants were selected on LB Tc agar. The insertions were then confirmed by amplification of the inserts by PCR using the plasmid-specific primers pkX-US-F (5′-CCGGCTCGTATGTTGTG TGG-3′) and pkX-DS-R (5′-CGAAAGGGGGATGTGCTGC-3′), followed by sequencing. The correct clones, pBabacA and pSmbacA, were then conjugated into the S. meliloti Rm1021 bacA null mutant (Ferguson et al., 2002) using E. coli MT616 with the helper plasmid pRK600 (Finan et al., 1986), and selected on LB Sm Tc agar.

    S. meliloti–alfalfa interaction experiments.

    To determine the ability of S. meliloti to form a successful symbiosis with alfalfa, 3-day-old seedlings were inoculated with 1 ml S. meliloti culture, resuspended to OD600 ∼0.05 in sterile water, on Jensen's agar as described previously (Leigh et al., 1985). The plates were incubated at 25 °C, and plant growth and nodule morphology were determined after 4 weeks.

    Bac7 sensitivity and Bac71–16-BY uptake assays.

    The N-terminal fragments 1–16 and 1–35 of Bac7 were synthesized and prepared as described previously (Benincasa et al., 2004). The Bac7 sensitivity assays were performed using mid-exponential-phase cultures exactly as described previously (Marlow et al., 2009). A fluorescently labelled version of Bac71–16, Bac71–16-BY, was prepared by linkage of the thiol-reactive dye BODIPY-FL N-(2-aminorthyl)maleimide (BY) (Invitrogen) to an additional C-terminal cysteine residue as reported previously (Scocchi et al., 2008). The uptake of Bac71–16-BY was performed as described previously (Marlow et al., 2009), with modifications. Mid-exponential-phase cultures of OD600 ∼1.0 were harvested, washed and resuspended in fresh LB medium. After the addition of 0.5 μM Bac71–16-BY, the cultures were incubated at 30 °C for 1 h. To account for extracellular binding of Bac71–16-BY, the cultures were then treated with the extracellular quencher of fluorescence, Trypan Blue (TB; 1 mg ml−1) for 10 min at room temperature. The fluorescence of cells was determined on poly-l-lysine-coated slides using a Carl Zeiss Axioskop microscope (100× objective magnification with UV light and FITC filter). Images were taken with a Canon Powershot camera and using the AxioVision version 2.0 software.

    Statistical analysis.

    Where shown, the significance of differences among bacterial strains was assessed by the Student's unpaired t-test using Microsoft Excel.

    RESULTS

    BacA sensitizes S. meliloti to different forms of bleomycin

    The R-group of bleomycin differs depending upon the form (Fig. 1a⇓). Previous studies investigating the bleomycin-resistance phenotypes of S. meliloti Rm1021 bacA mutants have used the A2 form (Ferguson et al., 2006; Ichige & Walker, 1997; LeVier & Walker, 2001). To investigate whether the nature of the R-group affected BacA-mediated sensitivity of S. meliloti to bleomycin, we performed disc diffusion assays using different forms of bleomycin and compared the sensitivity of an S. meliloti Rm1021 bacA null mutant and the parent strain (Fig. 1b⇓). We found that the S. meliloti parent strain showed an increased sensitivity to all the forms of bleomycin tested relative to the S. meliloti bacA null mutant. We also determined that bleomycin sulphate and bleomycin A5 reduced the viability of S. meliloti Rm1021 in liquid culture and that deletion of bacA conferred protection against both forms (Fig. 1c, d⇓). These findings show that BacA sensitizes S. meliloti to different forms of bleomycin, irrespective of the nature of the R-group.

    Figure image not available in archive
    Fig. 1.

    BacA confers sensitivity of S. meliloti to different forms of bleomycin independent of the nature of the R-group. (a) Structure of bleomycin A5, prepared using ChemDraw Std 10.0. The dashed line indicates the boundary of the R-group. The A2 and B2 R-group structures are also shown. Bleomycin sulphate is a composed of a mixture of the A2 (55–70 %) and B2 (25–32 %) forms. (b) Cultures of the indicated strains were exposed to different forms of bleomycin, and growth inhibition was determined using the disc diffusion assay. For bleomycin A2, B2 and the sulphate forms, cultures were exposed to 5 μl of a 5 mg ml–1 aqueous stock solution, and for bleomycin A5 cultures were exposed to 5 μl of a 2 mg ml−1 aqueous stock solution. (c) Cultures of the indicated strains were exposed to bleomycin sulphate (15 μg ml−1) and viability was assessed at the times indicated. (d) Same as (c) except that bleomycin A5 (3 μg ml−1) was used. The significance values shown (**P<0.01, ***P<0.001) represent comparisons of the Rm1021 ΔbacA mutant with the Rm1021 parent strain. All datasets shown are representative of trends observed in two independent experiments. The error bars represent sd (n=3) for one experiment. The arrow indicates that no viable cells were detected at the next time point.

    BacA is involved in the internalization of bleomycin in S. meliloti

    It has been shown previously by transmission electron microscopy (TEM) that bleomycin, in addition to damaging the DNA, also damages the cell wall and membrane of yeast cells (Lim et al., 1995). However, using TEM, we did not detect any bleomycin-induced lesions in the cell envelope components of S. meliloti Rm1021 or the bacA null mutant (data not shown). Additionally, unlike polymyxin B, which damages both the inner and outer membranes (Benincasa et al., 2009), bleomycin A5 treatment did not increase the amount of propidium iodide fluorescence of either the S. meliloti mutant lacking BacA or the parent strain (data not shown). To investigate a potential role for BacA in bleomycin uptake, we prepared fluorescently labelled bleomycin A5 (fluoro-BLM-A5) by conjugating it with FITC (Aouida et al., 2004). The resulting fluoro-BLM-A5 contained a 1 : 1 ratio of FITC and bleomycin A5 (data not shown). However, unlike with yeast cells (Aouida et al., 2004), we found that the conjugation of FITC to bleomycin A5 substantially reduced its toxicity and prevented uptake into S. meliloti (data not shown). For this reason, we used bleomycin-induced DNA degradation in the absence of membrane-damaging effects as an assay to monitor bleomycin A5 internalization in S. meliloti. We found that treatment of an S. meliloti Rm1021 culture with bleomycin A5 induced substantial degradation of the genomic DNA relative to the DNA from the untreated control culture (Fig. 2a⇓), confirming the intracellular presence of bleomycin in S. meliloti. In contrast, we did not observe any significant loss of the genomic DNA after bleomycin A5 treatment of the S. meliloti Rm1021 bacA null mutant relative to the DNA from the untreated control culture (Fig. 2a⇓). Additionally, the presence of pJG51A (encoding the S. meliloti wild-type bacA gene in pRK404) but not the control plasmid (pRK404) in the S. meliloti Rm1021 bacA null mutant strain increased the amount of bleomycin-induced DNA degradation relative to untreated control cultures (Fig. 2a⇓). Taken together, these findings are consistent with a role for BacA in the internalization of bleomycin, which then leads to DNA damage and the subsequent degradation of the genomic DNA.

    Figure image not available in archive
    Fig. 2.

    BacA sensitizes S. meliloti to bleomycin-induced DNA damage. (a) Cultures of the defined strains in LB medium were treated or not with bleomycin A5 (20 μg ml−1) for 2 h as indicated. The genomic DNA was extracted and 2 μg analysed on a 1 % (w/v) agarose gel. (b) Cultures of the indicated strains were treated or not with bleomycin A5 as in (a) and then the DNA was stained with DAPI and the fluorescence intensity was determined. The fluorescence intensity for each bleomycin-treated strain is shown as a percentage relative to that of the untreated control. The significance values (**P<0.01) represent comparisons of the Rm1021ΔbacA mutant with the parent strain and the Rm1021 ΔbacA mutant carrying pJG51A with the Rm1021ΔbacA mutant carrying pRK404. The error bars represent sd (n=3) for one experiment. (c) Same as (a) except that cultures were treated or not with MMS as indicated.

    We also quantified the effect of bleomycin A5 treatment on the S. meliloti parent strain and bacA null mutant in situ by permeabilizing cells and measuring fluorescence of DAPI, a fluorescent dye that binds DNA (Johnson, 1994). We found a substantial decrease in the amount of DAPI fluorescence after treatment of either S. meliloti Rm1021 or the S. meliloti Rm1021 bacA null mutant with pJG51A with bleomycin A5 relative to the untreated control cultures (Fig. 2b⇑). In contrast, treatment of either the S. meliloti Rm1021 bacA null mutant with or without pRK404 (control vector) with bleomycin A5 resulted in a much smaller decrease in the amount of DAPI fluorescence relative to their untreated control cultures (Fig. 2b⇑). These data confirm that BacA-dependent uptake is the major route of bleomycin internalization in S. meliloti. However, the fact that we observed a reduction in DAPI fluorescence after bleomycin treatment of the S. meliloti bacA null mutant shows that there is also a BacA-independent mechanism for bleomycin internalization in S. meliloti.

    BacA does not affect the sensitivity of S. meliloti to other DNA-damaging agents

    Although BacA is predicted to be in the inner membrane (Glazebrook et al., 1993), it has been demonstrated previously that the activation of inner-membrane transport systems can alter the susceptibility of bacterial DNA to damaging agents by altering conditions in the cytoplasm (Ferguson et al., 2000). To rule this out, we analysed the sensitivity of the S. meliloti Rm1021 bacA null mutant and parent strain to methylglyoxal, mitomycin C and MMS, which are also known to induce DNA damage in bacteria (Ferguson et al., 2000; Grzesiuk & Janion, 1996; Otsuji & Murayama, 1972). We found that S. meliloti Rm1021 was sensitized to all three DNA-damaging agents, but in contrast to bleomycin A5, deletion of bacA did not increase the resistance of S. meliloti to these agents (data not shown). Additionally, although we found that treatment of S. meliloti Rm1021 with MMS resulted in DNA degradation, the presence of BacA did not affect the degree of MMS-induced DNA degradation (Fig. 2c⇑). These findings show that BacA does not increase the susceptibility of S. meliloti to other DNA-damaging agents.

    B. abortus BacA complements the peptide uptake defects of an S. meliloti bacA null mutant

    The BacA protein is also responsible for conferring bleomycin sensitivity in B. abortus (LeVier et al., 2000). B. abortus is a highly infectious, containment level 3 pathogen (Franz et al., 1997). Therefore, to investigate the function of its BacA protein, we cloned the B. abortus bacA gene into the broad-host-range vector pRF771 (Wells & Long, 2002), under control of a constitutively active trp promoter, to create pBabacA. We found that the B. abortus BacA protein sensitized the S. meliloti bacA null mutant to bleomycin A5 relative to the mutant strain with the control plasmid (Fig. 3a⇓). Additionally, the B. abortus BacA protein substantially reduced the amount of DAPI fluorescence in the S. meliloti bacA null mutant relative to the mutant strain with the control plasmid (Fig. 3b⇓), demonstrating that the B. abortus BacA protein is functionally homologous to S. meliloti in the internalization of bleomycin.

    Figure image not available in archive
    Fig. 3.

    BacA of B. abortus is also involved in peptide uptake. (a) Cultures, containing approximately 1×109 c.f.u. ml−1, were treated with bleomycin A5 (20 μg ml−1) for 2 h and then the viability was determined. (b) Same as (a) except that cultures were treated or not with bleomycin and then the DNA was quantified by DAPI fluorescence. The fluorescence intensity for each bleomycin-treated strain is shown as a percentage relative to that of the untreated control. (c) Cultures of the indicated strains were incubated with and without Bac71–16 and Bac71–35 (1 μM) in LB medium for 1 h and then the viability was assessed. The arrows indicate that no viable cells were detected. The datasets shown are representative of the trends observed in two independent experiments and the error bars represent sd (n=3) for one experiment. The significance values (***P<0.001) represent comparison of the Rm1021 ΔbacA mutant carrying pRF771 (control) compared with pBabacA. (d) Cultures of the indicated strains were incubated with Bac71–16 labelled with the fluorescent dye BODIPY (Bac71–16-BY), treated with Trypan Blue and then analysed by microscopy as indicated. The images shown are representative fields of view from at least two independent experiments (bars, 20 μm).

    To determine whether B. abortus BacA is also involved in the uptake of truncated Bac7 peptides, we investigated the viability of the S. meliloti bacA null mutant with either pBabacA or pRF771 (control plasmid) in the presence of truncated Bac7 peptides, relative to the parent strain (Fig. 3c⇑). We determined that the B. abortus BacA protein greatly sensitized the S. meliloti bacA null mutant to Bac71–16 (RRIRPRPPRLPRPRPR) and Bac71–35 (RRIRPRPPRLPRPRPRPLPFPRP GPRPIPRPLPFP), consistent with a role for the B. abortus BacA protein in their uptake. To confirm this, cultures of S. meliloti Rm1021 bacA null mutant with either pBabacA or the control vector were incubated with Bac71–16 labelled with the fluorescent dye BODIPY (Bac71–16-BY) (Marlow et al., 2009; Mattiuzzo et al., 2007) and then analysed by fluorescence microscopy. To prevent the fluorescence of extracellular Bac71–16-BY, cultures were also treated with Trypan Blue, which quenches extracellular fluorescence (Benincasa et al., 2009). We found that cells of the S. meliloti Rm1021 bacA null mutant with pBabacA were highly fluorescent whereas, with the exception of an occasional cell, the cells of the mutant with control vector were non-fluorescent (Fig. 3d⇑, i and ii, respectively). These results show that the B. abortus BacA protein is also involved in the internalization of truncated Bac7 peptides into S. meliloti.

    B. abortus and S. meliloti bacA genes complement the chronic infection defect of the S. meliloti bacA null mutant in alfalfa when expressed under control of the trp promoter

    It was demonstrated previously that the E. coli sbmA gene complemented the alfalfa chronic infection defect of the S. meliloti bacA null mutant, but only when cloned under control of the S. meliloti bacA gene promoter and 168 bp upstream of the bacA coding sequence (Ichige & Walker, 1997). This finding led to the hypothesis that expression of the S. meliloti bacA gene is regulated during the legume infection and that the correct expression is key to the essential role of BacA in the plant infection. However, we found that the B. abortus bacA gene (pBabacA) complemented the chronic infection defect of the S. meliloti Rm1021 bacA null mutant within alfalfa, when expressed from the trp promoter in pFL771 (Table 2⇓). Since we included ∼150 bp upstream of the B. abortus bacA coding sequence in pBabacA, we could not rule out the possibility that this region was playing a regulatory role during legume symbiosis. To determine whether the upstream region and promoter of the S. meliloti bacA gene are needed during legume symbiosis, we cloned the S. meliloti bacA gene with 19 bp upstream under control of ptrp (pSmbacA). We found that the S. meliloti bacA gene cloned under control of ptrp was able to complement the symbiotic defect of the S. meliloti bacA null mutant to a similar extent to S. meliloti bacA cloned under control of its own promoter with an extended upstream region (pJG51A) (Table 2⇓). These findings show that the regulated expression of bacA is not essential for S. meliloti to form the legume symbiosis.

    Table 2.

    B. abortus and S. meliloti bacA genes complement the symbiotic defect of an S. meliloti BacA-deficient mutant

    Spermine protects S. meliloti against different forms of bleomycin

    Our earlier findings showed that there is also a BacA-independent mechanism for bleomycin A5 internalization in S. meliloti. Since the R-group of bleomycin A5 has structural similarities to polyamines, it was proposed that a polyamine uptake system(s) could be involved in its uptake (Aouida et al., 2004, 2005). Consistent with this, pre-treatment of yeast cells with the polyamines spermine or spemidine substantially reduced the uptake of fluorescently labelled bleomycin A5 (fluoro-BLM-A5) (Aouida et al., 2004). To investigate whether there was any relationship between polyamines and bleomycin in S. meliloti, we assessed the sensitivity of the parental strain S. meliloti Rm1021 to different forms of bleomycin in the presence of spermine (Fig. 4a⇓). Interestingly, we found that spermine protected this strain against all forms of bleomycin, independent of the R-group. Spermine also protected the S. meliloti bacA null mutant against different forms of bleomycin (Fig. 4b⇓), showing that spermine-mediated protection is independent of the BacA protein. Bleomycin damages DNA in the presence of iron and oxygen, through the generation of reactive oxygen species (Kane & Hecht, 1994). Although spermine has been shown in vitro to protect DNA against damage induced by reactive oxygen species generated by transition metals such as iron and copper (Pedreno et al., 2005), we found that spermine did not protect S. meliloti Rm1021 against H2O2 and mitomycin C (Fig. 4c⇓). Since H2O2 and mitomycin also damage DNA through redox reactions with transition metals (Imlay & Linn, 1988), these results show that spermine does not have a generalized effect in protection of S. meliloti DNA against damaging agents.

    Figure image not available in archive
    Fig. 4.

    Spermine protects S. meliloti against bleomycin but not H2O2 or mitomycin C. (a , b) Cultures of the indicated strains were exposed to different forms of bleomycin, at the concentrations described for Fig. 1(b), and growth inhibition was determined by the disc diffusion assay in the presence (black bars) or absence (white bars) of 1 mM spermine. (c) Cultures of S. meliloti Rm1021 were exposed to either 5 μl H2O2 (30 %, w/w) or mitomycin C (0.5 mg ml−1), and growth inhibition was determined by the disc diffusion assay in the presence (black bars) or absence (white bars) of 1 mM spermine. All datasets shown are a representative set from at least two independent experiments. The error bars represent sd (n=3) for one experiment. The significance values shown (***P<0.001) represent comparisons of the sensitivities of both strains in LB alone and with spermine added.

    Internalization of bleomycin in S. meliloti is energy-dependent

    The BacA protein is predicted to form the transmembrane domain of an ABC transporter, and polyamine uptake is mediated by ABC transport systems in bacteria (Igarashi & Kashiwagi, 1999; LeVier & Walker, 2001). To gain further insights into the BacA-dependent and -independent mechanisms of bleomycin uptake in S. meliloti, we examined the effect of DNP pre-incubation on the sensitivity of S. meliloti Rm1021 parent strain and bacA null mutant to bleomycin A5. DNP is an uncoupler, which dissipates the proton-motive force and prevents ATP synthesis (McLaughlin, 1972). We found that DNP exposure reduced the viability of both S. meliloti Rm1021 and the bacA null mutant to a similar extent (Fig. 5a⇓). However, pre-incubation of Rm1021 and the bacA null mutant with DNP increased their resistance on subsequent exposure to bleomycin A5 (Fig. 5b, c⇓). These findings are consistent with both the BacA-dependent and -independent mechanisms of bleomycin internalization in S. meliloti involving energy-dependent transport mechanisms.

    Figure image not available in archive
    Fig. 5.

    Uptake of bleomycin is energy-dependent. (a) Cultures of the indicated strains were treated with (black bars) and without (white bars) 5 mM DNP for 50 min in LB medium and then the viability was determined. (b, c) Cultures of the indicated strains were treated (black bars) or not (white bars) with DNP as described in (a), diluted to OD600 ∼0.1 in LB, exposed to 3 μg bleomycin A5 ml−1 for the indicated time, and then the viability was determined. The results are expressed as a percentage of the viable cells at time zero. The datasets shown are representative of at least two independent experiments and the error bars represent sd (n=3) for one experiment. The significance values shown (**P<0.01, ***P<0.001) represent comparisons of the sensitivities of both strains with and without DNP pretreatment.

    DISCUSSION

    Our data demonstrate that in the presence of either the S. meliloti or B. abortus BacA protein, bleomycin A5 treatment induces substantial degradation of S. meliloti genomic DNA. Since bleomycin exposure did not affect the integrity of the inner membrane and since BacA did not increase the sensitivity of S. meliloti to other DNA-damaging agents, these findings show that BacA plays a key role in the internalization of bleomycin in S. meliloti. We found that BacA-mediated bleomycin uptake is energy-dependent, but it is not known if BacA is directly or indirectly involved in peptide uptake. BacA is predicted to form the transmembrane domain (TMD) of an ABC transporter (LeVier & Walker, 2001). The TMDs of ABC transporters dimerize (Davidson & Chen, 2004) and, consistent with this, it has been shown previously that site-directed mutants in the S. meliloti bacA gene confer a dominant-negative effect on the function of the wild-type BacA protein with respect to bleomycin (LeVier & Walker, 2001). Bacterial ABC transporters involved in uptake require a solute-binding protein (Davidson & Chen, 2004) Therefore, if BacA is directly involved in peptide uptake, this would suggest that the solute-binding protein can interact with different classes of peptides and possibly other thiazole-containing compounds. However, we cannot rule out the possibility that BacA affects the activity of a number of different peptide-uptake systems, which would account for its role in the uptake of structurally diverse peptides. Further biochemical studies will be necessary to confirm that BacA dimerizes and directly participates as part of an ABC transport system in peptide uptake.

    Combining these results with those of our previous study (Marlow et al., 2009), we found that the S. meliloti BacA protein is involved in the uptake of both proline-rich and thiazole-modified peptides. Therefore, within the soil and during legume symbiosis, BacA-mediated uptake of different classes of peptides and/or thiazole-containing compounds into S. meliloti could be important for environmental survival and/or bacteroid development. Interestingly, plants produce thiazole-containing compounds in response to invading micro-organisms (Yorgey et al., 1994). Therefore, the BacA-mediated uptake of one or more of these compounds could be important during the legume symbiosis. Additionally, hundreds of nodule-specific cysteine-rich (NCR) peptides have been identified in the legume host of S. meliloti (Alunni et al., 2007; Mergaert et al., 2003, 2006). The NCR peptides have recently been shown to play a key role in S. meliloti bacteroid differentiation (Van de Velde et al., 2010). At present, S. meliloti factors involved in the NCR-mediated response have not been identified. However, since it has been shown that NCR peptides can enter S. meliloti (Van de Velde et al., 2010) and since we found that BacA is involved in the uptake of structurally diverse peptides, BacA may also be playing a key role in the uptake of one or more of these NCR peptides.

    We found by functional complementation experiments that the B. abortus BacA protein is also involved in uptake of bleomycin and truncated Bac7 peptides in S. meliloti. These results suggest that BacA-mediated peptide uptake could also be important for B. abortus during host infections. B. abortus causes abortions in cattle, and in humans it results in brucellosis, a severe debilitating disease (Franz et al., 1997). During mammalian infections, B. abortus is expected to encounter a number of different classes of peptides such as the proline-rich Bac7 peptides used in this study, as they were derived from a full-length peptide produced by bovine leukocytes (Frank et al., 1990). Other mammalian hosts are also known to produce proline-rich peptides (Gennaro et al., 2002), and three major families of antimicrobial peptides (defensins, cathelicidins and histatins) are produced as part of the human innate immune response (for a recent review refer to Rivas-Santiago et al., 2009). However, further studies will be necessary to understand the effect of these different classes of peptides on B. abortus and the role of BacA in this response.

    Our findings also demonstrate that there is a BacA-independent mechanism of bleomycin uptake in S. meliloti. Based on work with bleomycin in yeast (Aouida et al., 2004, 2005), we hypothesize that the BacA-independent mechanism of bleomycin uptake in S. meliloti occurs via one or more polyamine uptake system(s). Consistent with this, we showed that spermine protected S. meliloti against bleomycin and that the BacA-independent mechanism of bleomycin uptake is energy-dependent. Since our fluorescently labelled bleomycin was unable to enter S. meliloti, we have not been able to demonstrate conclusively that spermine reduces bleomycin uptake in S. meliloti. However, since spermine did not protect S. meliloti against other agents, which damage DNA via a similar mechanism to bleomycin (Imlay & Linn, 1988), our findings suggest that spermine is not mediating its protective effect against bleomycin at the level of the DNA. Polyamine uptake in bacteria has been most extensively investigated in E. coli (Igarashi & Kashiwagi, 1999), in which spermine uptake occurs via the ATP-binding cassette spermidine-preferential uptake system encoded by the four genes potA–D. Based on sequence similarity to the E. coli potA–D genes, we have identified five different probable ABC transporters (SMc01963–66, SMb20380–83, SMc01652–55, SMb20281–84 and PotF–I) encoded in the S. meliloti Rm1021 genome (Galibert et al., 2001), which are potential polyamine-uptake systems. Like the E. coli spermine uptake system, each of the S. meliloti probable ABC transporters consists of four proteins, which have between 22% and 53 % identity to the corresponding proteins in the E. coli PotA–D system (data not shown). Further studies will be necessary to construct single and combination mutants in these different systems, to determine how they affect polyamine and bleomycin uptake.

    In summary, our findings show the importance of BacA proteins in the uptake of structurally diverse peptides. Since the BacA protein also sensitizes M. tuberculosis to bleomycin (Domenech et al., 2009), it will be interesting to determine whether this protein is also involved in peptide uptake in this pathogen, and whether BacA-mediated peptide uptake is important during the host infection.

    Acknowledgments

    An MRC New Investigator (G0501107) grant and a BBSRC (BB/D000564/1) grant awarded to G. P. F. supported this work. S. W. was funded by the BBSRC and V. F. is funded by the MRC grant. The studentship of V. L. M. was funded by the BBSRC and M. A. and K. M. are SULSA-funded PhD students. The Canadian Institute of Health (MPO-67196) funds D. R. Thanks to Graham Walker and Andreas Haag for helpful discussion and Marty Roop II for provision of the B. abortus DNA. Thanks also to Hazel Phillips for preparing the bleomycin structure figure and Xiaming Yang for technical assistance.

    References

    1. Alunni, B., Kevei, Z., Redondo-Nieto, M., Kondorosi, A., Mergaert, P. & Kondorosi, E. (2007). Genomic organisation and evolutionary insights on GRP and NCR genes, two large nodule-specific gene families in Medicago truncatula. Mol Plant Microbe Interact 20, 1138–1148.
    2. Aouida, M., Leduc, A., Wang, H. & Ramotar, D. (2004). Characterization of a transport and detoxification pathway for the antitumour drug bleomycin in Saccharomyces cerevisiae. Biochem J 384, 47–58.
    3. Aouida, M., Leduc, A., Poulin, R. & Ramotar, D. (2005). AGP2 encodes the major permease for high affinity polyamine import in Saccharomyces cerevisiae. J Biol Chem 280, 24267–24276.
    4. Benincasa, M., Scocchi, M., Podda, E., Skerlavaj, B., Dolzani, L. & Gennaro, R. (2004). Antimicrobial activity of Bac7 fragments against drug-resistant clinical isolates. Peptides 25, 2055–2061.
    5. Benincasa, M., Pacor, S., Gennaro, R. & Scocchi, M. (2009). Rapid and reliable detection of antimicrobial peptide penetration into Gram-negative bacteria based on fluorescence quenching. Antimicrob Agents Chemother 53, 3501–3504.
    6. Davidson, A. L. & Chen, J. (2004). ATP-binding cassette transporters in bacteria. Annu Rev Biochem 73, 241–268.
    7. Ditta, G., Schmidhauser, T., Yakobson, E., Lu, P., Liang, X.-W., Finlay, D. R., Guiney, D. & Helinski, D. R. (1985). Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid 13, 149–153.
    8. Domenech, P., Kobayashi, H., Levier, K., Walker, G. C. & Barry, C. E., III (2009). BacA: an ABC transporter involved in maintenance of chronic murine infections with Mycobacterium tuberculosis. J Bacteriol 191, 477–485.
    9. Ferguson, G. P., Battista, J. R., Lee, A. T. & Booth, I. R. (2000). Protection of the DNA during the exposure of Escherichia coli cells to a toxic metabolite: the role of the KefB and KefC potassium channels. Mol Microbiol 35, 113–122.
    10. Ferguson, G. P., Roop, R. M., II & Walker, G. C. (2002). Deficiency of Sinorhizobium meliloti bacA mutant in alfalfa symbiosis correlates with alteration of cell envelope. J Bacteriol 184, 5625–5632.
    11. Ferguson, G. P., Datta, A., Baumgartner, J., Roop, R. M., II, Carlson, R. W. & Walker, G. C. (2004). Similarity to peroxisomal-membrane protein family reveals that Sinorhizobium and Brucella BacA affect lipid-A fatty acids. Proc Natl Acad Sci U S A 101, 5012–5017.
    12. Ferguson, G. P., Datta, A., Carlson, R. W. & Walker, G. C. (2005). Importance of unusually modified lipid A in Sinorhizobium stress resistance and legume symbiosis. Mol Microbiol 56, 68–80.
    13. Ferguson, G. P., Jansen, A., Marlow, V. L. & Walker, G. C. (2006). BacA-mediated bleomycin sensitivity in Sinorhizobium meliloti is independent of the unusual lipid A modification. J Bacteriol 188, 3143–3148.
    14. Finan, T. M., Kunkel, B., de Vos, G. F. & Signer, E. R. (1986). Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol 167, 66–72.
    15. Frank, R. W., Gennaro, R., Schneider, K., Przybylski, M. & Romeo, D. (1990). Amino acid sequences of two proline-rich bactenecins. Antimicrobial peptides of bovine neutrophils. J Biol Chem 265, 18871–18874.
    16. Franz, D. R., Jahrling, P. B., Friedlander, A. M., McClain, D. J., Hoover, D. L., Bryne, W. R., Pavlin, J. A., Christopher, G. W. & Eitzen, E. M., Jr (1997). Clinical recognition and management of patients exposed to biological warfare agents. JAMA 278, 399–411.
    17. Galibert, F., Finan, T. M., Long, S. R., Puhler, A., Abola, P., Ampe, F., Barloy-Hubler, F., Barnett, M. J., Becker, A. & other authors (2001). The composite genome of the legume symbiont Sinorhizobium meliloti. Science 293, 668–672.
    18. Gennaro, R., Zanetti, M., Benincasa, M., Podda, E. & Miani, M. (2002). Pro-rich antimicrobial peptides from animals: structure, biological functions and mechanism of action. Curr Pharm Des 8, 763–778.
    19. Gibson, K. E., Kobayashi, H. & Walker, G. C. (2008). Molecular determinants of a symbiotic chronic infection. Annu Rev Genet 42, 413–441.
    20. Glazebrook, J., Ichige, A. & Walker, G. C. (1993). A Rhizobium meliloti homolog of the Escherichia coli peptide-antibiotic transport protein SbmA is essential for bacteroid development. Genes Dev 7, 1485–1497.
    21. Grzesiuk, E. & Janion, C. (1996). MMS-induced mutagenesis and DNA repair in Escherichia coli dnaQ49: contribution of UmuD′ to DNA repair. Mutat Res 362, 147–154.
    22. Haag, A. F., Wehmeier, S., Beck, S., Marlow, V. L., Fletcher, V., James, E. K. & Ferguson, G. P. (2009). The Sinorhizobium meliloti LpxXL and AcpXL proteins play important roles in bacteroid development within alfalfa. J Bacteriol 191, 4681–4686.
    23. Ichige, A. & Walker, G. C. (1997). Genetic analysis of the Rhizobium meliloti bacA gene: functional interchangeability with the Escherichia coli sbmA gene and phenotypes of mutants. J Bacteriol 179, 209–216.
    24. Igarashi, K. & Kashiwagi, K. (1999). Polyamine transport in bacteria and yeast. Biochem J 344, 633–642.
    25. Imlay, J. A. & Linn, S. (1988). DNA damage and oxygen radical toxicity. Science 240, 1302–1309.
    26. Johnson, J. L. (1994). Similarity analysis of DNAs. In Methods for General and Molecular Bacteriology, pp. 655–682. Edited by Gerhardt, P., Murray, R. G. E., Wood, W. A. & Kreig, N. R.. Washington, DC. : American Society of Microbiology.
    27. Jones, K. M., Kobayashi, H., Davies, B. W., Taga, M. E. & Walker, G. C. (2007). How rhizobial symbionts invade plants: the Sinorhizobium–Medicago model. Nat Rev Microbiol 5, 619–633.
    28. Kane, S. A. & Hecht, S. M. (1994). Polynucleotide recognition and degradation by bleomycin. Prog Nucleic Acid Res Mol Biol 49, 313–352.
    29. Laviña, M., Pugsley, A. P. & Moreno, F. (1986). Identification, mapping, cloning and characterization of a gene (sbmA) required for microcin B17 action on Escherichia coli K12. J Gen Microbiol 132, 1685–1693.
    30. Leigh, J. A., Signer, E. R. & Walker, G. C. (1985). Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules. Proc Natl Acad Sci U S A 82, 6231–6235.
    31. LeVier, K. & Walker, G. C. (2001). Genetic analysis of the Sinorhizobium meliloti BacA protein: differential effects of mutations on phenotypes. J Bacteriol 183, 6444–6453.
    32. LeVier, K., Phillips, R. W., Grippe, V. K., Roop, R. M., II & Walker, G. C. (2000). Similar requirements of a plant symbiont and a mammalian pathogen for prolonged intracellular survival. Science 287, 2492–2493.
    33. Lim, S. T., Jue, C. K., Moore, C. W. & Lipke, P. N. (1995). Oxidative cell wall damage mediated by bleomycin-Fe(II) in Saccharomyces cerevisiae. J Bacteriol 177, 3534–3539.
    34. Marlow, V. L., Haag, A. F., Kobayashi, H., Fletcher, V., Scocchi, M., Walker, G. C. & Ferguson, G. P. (2009). Essential role for the BacA protein in the uptake of a truncated eukaryotic peptide in Sinorhizobium meliloti. J Bacteriol 191, 1519–1527.
    35. Mattiuzzo, M., Bandiera, A., Gennaro, R., Benincasa, M., Pacor, S., Antcheva, N. & Scocchi, M. (2007). Role of the Escherichia coli SbmA in the antimicrobial activity of proline-rich peptides. Mol Microbiol 66, 151–163.
    36. McLaughlin, S. (1972). The mechanism of action of DNP on phospholipid bilayer membranes. J Membr Biol 9, 361–372.
    37. Meade, H. M., Long, S. R., Ruvkun, G. B., Brown, S. E. & Ausubel, F. M. (1982). Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149, 114–122.
    38. Mergaert, P., Nikovics, K., Keleman, Z., Maunoury, N., Vaubert, D., Kondorosi, A. & Kondorosi, E. (2003). A novel family of Medicago truncatula consisting of more than 300 nodule-specific genes coding for small, secreted polypeptides with conserved cysteine motifs. Plant Physiol 132, 161–173.
    39. Mergaert, P., Uchiumi, T., Alunni, B., Evanno, G., Cheron, A., Catrice, O., Mausett, A. E., Barloy-Hubler, F., Galibert, F. & other authors (2006). Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium–legume symbiosis. Proc Natl Acad Sci U S A 103, 5230–5235.
    40. Otsuji, N. & Murayama, I. (1972). Deoxyribonucleic acid damage by monofunctional mitomycins and its repair in Escherichia coli. J Bacteriol 109, 475–483.
    41. Pedreno, E., Lopez-Contreras, A. J., Cremades, A. & Penafiel, R. (2005). Protecting or promoting effects of spermine on DNA strand breakage induced by iron or copper ions as a function of metal concentration. J Inorg Biochem 99, 2074–2080.
    42. Raetz, C. R. & Whitfield, C. (2002). Lipopolysaccharide endotoxins. Annu Rev Biochem 71, 635–700.
    43. Rivas-Santiago, B., Serrano, C. J. & Enciso-Moreno, J. A. (2009). Susceptibility to infectious diseases based on antimicrobial peptide production. Infect Immun 77, 4690–4695.
    44. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1982). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY. : Cold Spring Harbor Laboratory.
    45. Scocchi, M., Mattiuzzo, M., Benincasa, M., Antcheva, N., Tossi, A. & Gennaro, R. (2008). Investigating the mode of action of proline-rich antimicrobial peptides using a genetic approach: a tool to identify new bacterial targets amenable to the design of novel antibiotics. Methods Mol Biol 494, 161–176.
    46. Sharypova, L. A., Niehaus, K., Scheidle, H., Holst, O. & Becker, A. (2003). Sinorhizobium meliloti acpXL mutant lacks the C28 hydroxylated fatty acid moiety of lipid A and does not express a slow migrating form of lipopolysaccharide. J Biol Chem 278, 12946–12954.
    47. Umezawa, H., Maeda, K., Takeuchi, T. & Okami, Y. (1966). New antibiotics, bleomycin A and B. J Antibiot (Tokyo) 19, 200–209.
    48. Van de Velde, W., Zehirov, G., Szatmari, A., Debreczeny, M., Ishihara, H., Kevei, Z., Farkas, A., Mikulass, K., Nagy, A. & other authors (2010). Plant peptides govern terminal differentiation of bacteria in symbiosis. Science 327, 1122–1126.
    49. Wells, D. H. & Long, S. R. (2002). The Sinorhizobium meliloti stringent response affects multiple aspects of symbiosis. Mol Microbiol 43, 1115–1127.
    50. Wilson, K. (1987). Preparation of genomic DNA from bacteria. In Current Protocols in Molecular Biology, pp. 2.4.1–2.4.5. Edited by Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K.. New York. : Green Publishing & Wiley-Interscience.
    51. Yorgey, P., Lee, J., Kordel, J., Vivas, E., Warner, P., Jebaratnam, D. & Kolter, R. (1994). Posttranslational modifications in microcin B17 define an additional class of DNA gyrase inhibitor. Proc Natl Acad Sci U S A 91, 4519–4523.