Bacterial strains and plasmids.
The strains and plasmids used for this study are listed in .
Strains and plasmids used in this study
Media and culture conditions. Escherichia coli
strains were grown in LB (26
) at 37°C. Actinomycetes
strains were grown in R5 medium (13
) at 30°C. Media were supplemented with antibiotics when necessary to maintain plasmids.
DNA preparation and manipulation.
The methods used for the isolation and manipulation of DNA for E. coli
and actinomycetes were described by Kieser et al. (13
). PCR fragments were isolated from agarose gels by use of a QIAquick kit (Qiagen). Restriction endonucleases were obtained from various suppliers and were used according to their specifications.
Preparation of A. balhimycina RNA. A. balhimycina
cells were cultivated for different times. The cells were then harvested and shock frozen at −70°C. An aliquot was resuspended in 100 μl of buffer P (31
) that contained 10 mg/ml of lysozyme and was then incubated for 7 min at 37°C. The RNA was extracted by use of an RNeasy minikit (Qiagen) according to the manufacturer's instructions.
Reverse transcription (RT) analysis.
From the isolated RNA, cDNA was synthesized and used as a template for amplification of a fragment containing parts of vanH and vanA by use of primers vanH1 and vanA2 and of a fragment containing parts of vanA and vanX by use of primers vanA3 and vanX4. To test for the vanYb transcript (“b” represents the balhimycin biosynthetic gene cluster), the primer pair vanY_RT_rev-vanY_RT_for was used (). Chromosomal DNA from A. balhimycina was used as a positive control, and total RNA was used to test for contamination with chromosomal DNA.
Amplification of an internal vanHAX fragment.
To identify cosmids carrying vanHAX-like genes, the primer pair p2-p3 () was used to generate a labeled probe. With this probe, the A. balhimycina cosmid library was screened by Southern hybridization.
Construction and analysis of Δorf7 and ΔvnlR mutant strains.
The plasmid pSPΔorf7 was constructed to inactivate orf7 by an in-frame deletion of 1,314 bp. A 1,764-bp upstream fragment (frΔorf7-left) and a 1,711-bp downstream fragment (frΔorf7-right) of orf7 were amplified from cosmid DNA by PCRs using the primer pairs prΔorf7F1.1-prΔorf7F1.2 and prΔorf7F2.1-prΔorf7F2.2 (), respectively. The primers contained restriction sites at the 3′ and 5′ ends (EcoRI/XbaI and XbaI/SphI sites, respectively). Both fragments were cloned into the vector pSP1 to generate pSPΔorf7.
For the in-frame deletion (678 bp) of vnlRb, plasmid pSPΔvnlR was constructed. A 1,369-bp downstream fragment (frΔvnlR-left) and a 1,467-bp upstream fragment (frΔvnlR-right) of vnlRb were amplified from cosmid DNA by PCRs using the primer pairs vnlRinakt1.1-vnlRinakt1.2 and vnlRinakt2.1-vnlRinakt2.2 (), respectively. The primers contained restriction sites at the 5′ and 3′ ends (XbaI/PstI and XbaI/SacI sites, respectively). Both fragments were cloned into the vector pSP1 to generate pSPΔvnlR.
Wild-type A. balhimycina
was transformed with pSPΔorf7 or pSPΔvnlR by use of a direct transformation method as described previously (22
). The integration of the plasmids into the chromosome via homologous recombination was confirmed by PCR screening for the erythromycin resistance cassette, using primers ermEleft and ermEright (). To obtain deletion mutants, a second homologous recombination event was provoked by stressing plasmid-carrying colonies as described elsewhere (23
). Colonies were examined for sensitivity to erythromycin, and the deletions were verified by PCR analysis, using primers prΔorf7.1prove and prΔorf7.2prove () to generate a 2,585-bp fragment from the A. balhimycina
mutant and the primers prΔvnlR.1prove and prΔvnlR.2prove () to generate a 310-bp fragment from the A. balhimycina
Bioassay for detection of active balhimycin.
Balhimycin production was determined through the use of bioassays using Bacillus subtilis ATCC 6633 as a test organism and cell supernatants from Amycolatopsis strains grown in R5 medium.
Bioassay for determination of balhimycin resistance.
Amycolatopsis and Streptomyces strains were grown for 3 days at 30°C on R5/HA plates containing balhimycin at different concentrations.
Isolation of the cell wall. Actinomycetes
cells were grown at 30°C with shaking and were then cooled rapidly and harvested by centrifugation at 10,000 × g
for 10 min. The pellet was resuspended in 50 mM Tris-HCl (pH 7.0), and this solution was added dropwise to a boiling SDS solution (5%). The solution was stirred the entire time, and boiling was continued for 15 min before cooling of the mixture to room temperature. The crude cell wall material was collected, and the pellet was washed free of SDS by a continuous resuspending and washing procedure consisting of two washes with 20 ml of 1 M NaCl and then one wash with water. The washing procedure was continued until no SDS was detectable by use of a published assay (8
). Glass beads (diameter, 0.1 to 0.5 mm) and 1 ml of the cell wall suspension were mixed, and the walls were broken mechanically in a FastPrep instrument (Precellys; 6 passages for 20 to 30 s at 6,500 × g
) on ice. After removal of the glass beads and the unbroken cells (by centrifugation at 2,000 × g
for 5 min), the broken cell walls were sedimented by centrifugation at 25,000 × g
for 15 min at room temperature. The pellet was then resuspended in 3 ml of 100 mM Tris-HCl (pH 7.5) with 20 mM MgSO4
. DNase (10 μg/ml) and RNase (50 μg/ml) were added, and the sample was incubated for 2 h with stirring. Afterwards, 10 mM CaCl2
and 100 μg/ml trypsin were added, and the sample was incubated for 18 h with stirring. SDS was added to a final concentration of 1%, and the sample was incubated at 80°C for 15 min. The volume was adjusted with water to 20 ml, and the cell wall was sedimented by centrifugation at 25,000 × g
for 30 min. The pellet was resuspended in 10 ml 8 M LiCl and incubated at 37°C for 15 min. After harvesting, the pellet was resuspended in 10 ml of 100 mM EDTA (pH 7.0) and incubated at 37°C for 15 min. The pellet was then washed successively with water, acetone, and water. After the last step, the pellet was resuspended in a small amount of water, and the material obtained was lyophilized.
Isolation of peptidoglycan from cell walls (peptidoglycan-teichoic acid complexes).
For cell wall composition analyses, it was essential to cleave the peptidoglycan into smaller fragments (muropeptides). Our initial attempt to release muropeptides from the cell wall, i.e., the peptidoglycan-teichoic acid complex, by use of cellosyl failed, presumably because the teichoic acids inhibited the enzyme. Therefore, teichoic acid was removed with hydrofluoric acid (HF) to isolate the peptidoglycan. The purified cell wall (5 mg) was resuspended in ice-cold 48% HF. The sample was stirred for 48 h at 4°C. The sample was centrifuged at 100,000 × g for 45 min at 4°C and then washed twice with ice-cold 100 mM Tris-HCl (pH 7) and twice with ice-cold water. It was then resuspended in 750 μl of water, and potassium azide was added (final concentration, 0.05%).
Preparation and reduction of muropeptides for high-performance liquid chromatography (HPLC) analysis.
One hundred microliters of 25 mM potassium phosphate (pH 5.5) was added to 100 μl of the peptidoglycan suspension (10 to 15 mg/ml). Ten microliters of 10 mg/ml mutanolysin (Sigma-Aldrich) was added, and the sample was incubated at 37°C for 18 h. The sample was then boiled for 10 min and centrifuged. The supernatant was mixed with the same volume of potassium borate (0.5 M). Sodium borohydride (1 to 2 mg) was added, and the sample was incubated at room temperature for 30 min. Excess borohydride was destroyed by the successive addition of phosphoric acid until a pH of 1 to 2 was reached.
Peptidoglycan precursor extraction.
Peptidoglycan precursors were extracted by a method previously described for Bacillus cereus
), with some modifications. Briefly, mycelium was grown in the appropriate medium for 2 days before bacitracin (Sigma) was added and growth was continued for 1 h. Bacitracin binds the C55
-isoprenyl pyrophosphate and prevents the transport of cell wall precursors outside the cell, resulting in the accumulation of cytoplasmic UDP-linked precursors (30
). Cells were harvested, and the pellet was resuspended in 25 ml water. The solution was boiled for 20 min and then centrifuged. The supernatant was collected and lyophilized. The lyophilized supernatant was resuspended in 1 ml water and extracted with 200 μl chloroform. After vortexing and centrifugation (13,000 rpm for 2 min), the suspension was used for HPLC and HPLC-mass spectrometry (HPLC-MS).
HPLC-MS analysis of peptidoglycan precursors.
Five microliters of the sample was injected by use of an HPLC-MS instrument (XCT 6330 LC/MSD Ultra Trap system; Agilent Technologies) and a Nucleosil 100 C18 column (3 μm by 100 mm by 2 mm internal diameter). The HPLC parameters were as follows: the gradient was linear from 0% eluent A (H2O, 0.1% HCOOH) to 10% eluent B (acetonitrile, 0.06% HCOOH), at a flow rate of 400 μl/min, and took 25 min. The MS parameters were as follows: ionization alternated between positive and negative, the capillary voltage was 3.5 kV, and the temperature was 350°C. Tandem MS (MS/MS) was performed in the negative mode with the corresponding target mass.
VanY assays. (i) Fluorometric OPTA method.
The VanY activity was determined using a previously described method (33
). Briefly, a calibration curve was initially made. The concentration of released d
-Ala was determined by using a calibration curve in which the relative fluorescence was plotted over the free d
-Ala concentration. Several concentrations of free d
-Ala were measured thrice. From a stock solution of 50 mM d
-Ala, different concentrations were set up in a zinc acetate (ZnAc)-NaHCO3
solution (5 mM ZnAc and 50 mM NaHCO3
) in a final volume of 20 μl. The reaction was quenched by the addition of 5 μl HCl (250 mM) followed by the addition of 75 μl H2
-Ala was monitored by the addition of 100 μl fluoraldehyde (o
-phthaldialdehyde [OPTA]) solution followed by incubation at room temperature for 5 min. After the addition of 800 μl H2
O, the solution was diluted 100-fold and measured in a fluorescence reader (Infinite 200; Tecan) with a λex
value of 340 nm and a λem
value of 455 nm. The background value for a probe without d
-Ala was subtracted.
To test the activity with the artificial substrate Nα,Nε-diacetyl-Lys-d-Ala-d-Ala, the reaction mixture was set up with 5 mM ZnAc, 50 mM NaHCO3, and 10 mM substrate. To 10 μl of this mixture, crude cell extract with VanYb (from the induced E. coli strain BL21 at a final protein concentration of 50 μg/ml) was added in a final volume of 20 μl and incubated at 37°C for 1 h. The sample was further prepared in a similar manner to that for the calibration curve before the measurement was taken in a fluorescence reader. As a blank value, crude cell extract lacking VanYb was used.
(ii) HPLC-DAD analysis.
Using HPLC, we measured the carboxypeptidase substrate Nα,Nε-diacetyl-Lys-d-Ala-d-Ala and the carboxyesterase substrate Nα,Nε-diacetyl-Lys-d-Ala-d-Lac and traced the differences in the substrate concentrations. The samples were prepared as follows. Five microliters of a 10 mM substrate solution was added to 20 μl of crude cell extract and incubated at 37°C for 30 min. Crude cell extract lacking VanYb was used as a blank. To stop the reaction, the sample was boiled. Twenty-five microliters of H2O was added, and after centrifugation, the sample was injected by use of an HPLC-DAD (diode array detector) instrument (HP 1090M chromatograph with autosampler, DAD, and HP Kayak XM 600 Chem station; Agilent Technologies) and a Nucleosil 100 C18 column. The gradient was linear from 0% eluent A (H2O, 0.1% H3PO4) to 100% eluent B (acetonitrile) at a flow rate of 2 ml/min. The substrate peaks were monitored at 210 nm and quantified by integration. The threshold of this method was 0.03 mM substrate. Therefore, values below this threshold were not detectable.