Kruse et al., SOM, p.1
Supplementary Online Material, Kruse et al., 2005, Ms No. 050054
Isolation of A22-resistant mutant
In order to identify the target molecule(s) of A22 in E. coli, resistant mutants were isolated. A22 induced turbid growth inhibitory zones on wild type E. coli cells, which made it difficult to isolate resistant mutants from wild-type strains. We first searched for mutant strains showing higher sensitivity to A22 and thus forming clear growth inhibitory zones upon A22 treatment. As a result, we found that an E. coli mutant strain deficient in penicillin-binding protein 1B (ponB-) (Tamaki et al., 1977) was more sensitive to A22. A parent strain JE1011 formed a turbid growth inhibitory zone (≈12 mm) around a paper disk (≈8 mm) containing 200 μg of A22 after 24 h incubation at 37oC, while its ponB derivative, JST975, formed a clear growth inhibitory zone with a larger size (≈32 mm). Several colonies appeared within the clear growth inhibitory zone after prolonged (9 days) incubation of the assay plates. These colonies were purified on LA plates and their A22-sensitivity was examined. One of them, named JA221, was highly resistant to A22. Minimum inhibitory concentrations(MICs) of A22 were 1.6 μg/ml for JST975 and 100 μg/ml for JA221 (Table S1). In the presence of 10 μg/ml of A22, the resistant mutant JA221 grew as rods while its parent strain JST975 formed spherical cells upon A22 treatment (data not shown) as reported for wild-type (ponB+) strains (Iwai et al., 2002). The A22-resistant mutant JA221 was still sensitive to mecillinam (Table S1) and formed spherical cells upon mecillinam treatment (data not shown), indicating that PBP 2 was normally working in the mutant cells. The mutant strain did not produce anucleate cells frequently (data not shown), indicating that the chromosome segregation was normal in the mutant cells.
A22-sensitive transductants were obtained with a frequency of 67 % when an transposon marker zhc-12::Tn10 located at 71 min, which is co-transducible with the mreB129 mutation with a frequency of 66 %, was transduced into JA221 by the P1-phage mediated transduction. A22-resistance phenotype was also transferable from an A22-resistant transductant (A22rzhc-12::Tn10) into the parent strain JST975, as well as into the wild-type stain W3110 with similar frequencies (Table S1). These results indicate that the A22-resistant mutation is located around 71 min. It was also deduced that the ponB mutation was not required for the A22-resistance. That is, this mutation located around 71 min alone renders the cell A22-resistant.
According to the results of the P1-phage mediated transduction mapping, it was expected that the A22-resistant mutation lies within either mreB, mreC or mreD gene. This region of the JA221 chromosomal DNA was PCR-amplified and the amplified fragments were cloned on a plasmid. Three independently isolated clones of each gene were sequenced by the dideoxy method and the sequences of the mutant clone were compared with that of the wild-type clone. As a result, only one transition, G to A, was found in the mreB gene of the mutant strain, which causes a substitution of the 21st Asn to Asp of the MreB protein. From these results, it is concluded that this mutation is responsible for A22-resistance. The mutant allele, named mreB221, could complement the mreB129 mutation (data not shown), which was originally isolated by mecillinam-resistance and showed a spherical cell shape (Wachi et al., 1987). This means that the mutant MreB221 protein still has the ability to maintain the rod shape of the E. coli cells. This concurs well with the fact that the mreB221 mutant strain JA221 grows as rods.
Bacterial strains used
E. coli K-12 strains JE1011 (F-leuB thr trp his thy ara lac xyl mtl rpsL tonA), JST975 (the same as JE1011 but ponB) (Tamaki et al., 1977), W3110 (wild-type), DV14 (F-metB1 panD2 panF1 zhc-12::Tn10) (Vallari and Rock, 1985) were used in this study. Cells were grown in Lennox (L) medium (1 % Bactopeptone, 0.5 % yeast extracts, 0.5 % NaCl, 0.1 % glucose, pH 7.2, supplemented with 20 μg/ml thymine).
A22 resistant mutant JA221 was isolated from JST975 (ponB-) by spontaneous mutagenesis. JST975 cells fully grown in LB medium was inoculated at 105 cells/ml into melting LA agar medium at 50oC and agar was solidified in plastic plates. A paper disk (8 mm) soaked with 20 μl of A22 solution (10 mg/ml methanol solution) was put on the center of the plate, and the plate was incubated at 37oC. After 24 h incubation, a clear growth inhibitory zone of 32 mm was formed around the paper disk. Several colonies were generated within the growth inhibitory zone after 9 days incubation. These colonies were purified on LA plates and their A22-sensitivity was checked. Among them, one clone, highly resistant to A22 and designated JA221, was used in this study.
Determination of MIC
MICs were determined by using a standard 2-fold serial dilution format on L agar plates. Fully grown cultures were diluted with 0.85 % NaCl at 106 cells /ml and 10 μl of cell suspensions were spotted on the plates. The plates were incubated at 37oC for 24 h. The lowest concentration that caused > 99.9 % inhibition of colony formation was denoted to be MIC. MICs were also determined on Mueller Hinton agar plates (0.2 % beef extract, 1.75 % acid digest of casein, 0.15 % soluble starch, 1.5 % agar, pH 7.3).
Identification of the mutation point
The mreB regions of JST975 and JA221 were PCR-amplified by using a set of primers, 5'-ATGGGAGTGTGCTTGTCGACTCGCC-3' and 5'-ACCAGGCAGGGTCGACAGACACTTC-3'. The amplified DNA fragments were digested with SalI and cloned into the SalI site of pMW218 (Takara Bio Inc., Ohtsu, Japan). Independently isolated three clones for JST975 and JA221, respectively, were sequenced by the dideoxy method using an automated fluorescence DNA sequencer (Long-ReadTower, Amersham Bioscience, NJ, USA) and the Thermo Sequenase Dye Terminator (Cy5.5 and Cy5) sequencing kit (Amersham Bioscience). Similarly, the mreC and mreD regions were sequenced by using a set of primers, 5'-GGAGAACCGGAAGCTTAATACAGAG-3' and 5'-TTTAGCATGCCTGGTCTGATACGAG-3'.
Table S1. MICs of mecillinam and A22
Strains / Alleles / MICs (μg/ml) a)ponB / mreB / mecillinam / A22
JE1011 / + / + / 0.25 / 6.25
JST975 / - / + / 0.125 / 3.125
JA221 / - / mreB221 / 0.125 / 100
JSTA221 b) / - / mreB221 / 0.125 / 100
JSTA220 b) / - / + / 0.125 / 3.125
WA221 c) / + / mreB221 / 0.25 / >100
WA220 c) / + / + / 0.25 / 6.25
a) MICs were determined by 2-fold serial dilutions.
b) JSTA221 and JSTA220 were constructed by the P1-phage mediated transduction using JA221 zhc-12::Tn10 (mreB221 zhc-12::Tn10) as a donor and JST975 (ponB) as a recipient.
c) WA221 and WA220 were constructed by the P1-phage mediated transduction using JA221 zhc-12::Tn10 (mreB221 zhc-12::Tn10) as a donor and W3110 (wild-type) as a recipient.
References
Iwai,N., K.Nagai, and M.Wachi. 2002. Novel S-benzylisothiourea compound that induces spherical cells in Escherichia coli probably by acting on a rod-shape-determining protein(s) other than penicillin-binding protein 2. Biosci. Biotechnol. Biochem.66: 2658-2662.
Tamaki,S., S.Nakajima, and M.Matsuhashi. 1977. Thermosensitive mutation in Escherichia coli simultaneously causing defects in penicillin-binding protein-1Bs and in enzyme activity for peptidoglycan synthesis in vitro. Proc. Natl. Acad. Sci. U. S. A74: 5472-5476.
Vallari,D.S. and C.O.Rock. 1985. Isolation and characterization of Escherichia coli pantothenate permease (panF) mutants. J. Bacteriol.164: 136-142.
Wachi,M., M.Doi, S.Tamaki, W.Park, S.Nakajima-Iijima, and M.Matsuhashi. 1987. Mutant isolation and molecular cloning of mre genes, which determine cell shape, sensitivity to mecillinam, and amount of penicillin-binding proteins in Escherichia coli. J. Bacteriol.169: 4935-4940.
Legend to Figure S1. Mass spectrometric identification of MreB-interacting proteins. Several peptide sequences of RNA polymerase beta and beta prime chains were obtained from gel bands I and II (see Figure 2) using LC-MS/MS. (A) The figure shows the fragmentation pattern of one of the doubly-charged tryptic peptides derived from RNA polymerase beta chain at m/z value of 532.33 (indicated with *). (B) Fragmentation pattern of one of the doubly charged tryptic peptides from RNA polymerase beta prime chain at m/z value of 460.76 (indicated with *). The sequences of the peptides derived from these spectra are shown in the top left corners. The y series of ions (C-terminus containing fragments) that are produced due to fragmentation are labelled, as well as those from the a and b ion series (N-terminus containing fragments).