Figure legends
Fig. S1: (A) Zinc binding site prediction by PredZinc version 1.4 (Shu et al., 2008) of Zur Aj (GenBank:
AIG78644). Predicted zinc binding residues are highlighted in red. Cys, His, Asp and Glu are bolded.
Residues are predicted as zinc binding if the score is ≥ 0.450. (B) Structure-based multiple sequence
alignment of Zur from M. tuberculosis (Lucarelli et al ., 2007) and S. coelicolor (Shin et al., 2011)
with its putative ortholog from A. japonicum. Sequences were aligned by using ClustalW2. Zinc
binding sites: ▪ site 1 (structural) , ▪ site 2, ▪ site 3. Protein domains are indicated on the top. Green
bar indicates the N-terminal DNA-binding domain, red bar indicates the amino acids involved in
forming the inter-domain hinge loop and the blue bar indicates the C-terminal dimerization
domain. Amino acid similarity is indicated at the bottom (identical (*), highly similar (:) and similar
(.), respectively).
Fig. S2. Construction of the zur in-frame deletion strain A. japonicum Δzur using the plasmid
st
nd
pGusA21Δzur (Table S1) via homologous recombination. 1 and 2 homologous recombinations
are exemplary illustrated as succession of first, downstream recombination and second, upstream
recombination but could also occur in opposite succession. aac(3)-IV, apramycin resistance gene;
gusA, β-glucuronidase (GUS) gene. Red: upstream flanking region of zur amplified by using the
primer pair pΔzur-US-F and pΔzur-US-R (Table S2). Yellow: downstream flanking region of zur
amplified by using the primer pair pΔzur-DS-F and pΔzur-DS-R (Table S2). Hatched red and yellow
bars illustrate the by PCR generated upstream and downstream fragments which were integrated
into pGusA21. pGusA21Δzur-zur is depicted in dashed lines to illustrate that this plasmid is not
replicating in A. japonicum. The proposed gene products of ajap_2935 – 60 are a conserved
putative secreted protein, a HTH-type transcriptional repressor, a Fur family transcriptional
regulator, a hypothetical protein, a membrane permease and a second membrane permease,
respectively. The deletions of aesA-C and aesH-E were performed accordingly.
Fig. S3. HPLC analysis of the zinc dependent [S,S]-EDDS production.. Strains were grown in
deionized flasks in SM either with or without supplementation of 6 µM ZnSO 4. Commercial [S,S]EDDS from Sigma Aldrich was used as a standard.
Fig. S4. Zinc dependent transcriptional pattern of the putative [S,S]-EDDS biosynthesis genes aesAD of A. japonicum. Cultures were grown in SM in absence of any trace element (-) or supplemented
with (+) 6 μM Zn2+ respectively and samples were taken after 25 h of incubation to isolate RNA.
sigB was used as housekeeping gene to normalize the RNA. Left panel shows transcription pattern
in WT background, right panel in the ∆zur background.
Fig. S5. HPLC chromatograms of culture supernatants of A. japonicum strains grown in deionized
flasks in SM in absence of zinc. Commercial [S,S]-EDDS from Sigma Aldrich was used as a standard.
Fig. S6. 16S rRNA gene phylogenetic tree for 16 members of the genus Amycolatopsis chosen by
criteria of available genome sequence data. The tree was constructed using neighbor-joining with
CLUSTAL W and MEGA4 software. Classification into the phylogenetic clades A-F occured
accordingly to Everest and Meyers 2009. The percentage bootstrap values of 1,000 replications are
shown at each node (only values above 40% are shown). The scale bar indicates 1 nucleotide
substitution per 100 nucleotides. S. scabies was used as an outgroup.
Fig. S7. HPLC chromatograms of culture supernatants of several Amycolatopsis strains grown in
deionized flasks in SM either with or without supplementation of 25 µM ZnSO4. Commercial [S,S]EDDS from Sigma Aldrich was used as a standard.
Fig. S1: (A) Zinc binding site prediction by PredZinc version 1.4 (Shu et al., 2008) of Zur Aj (GenBank:
AIG78644). Predicted zinc binding residues are highlighted in red. Cys, His, Asp and Glu are bolded.
Residues are predicted as zinc binding if the score is ≥ 0.450. (B) Structure-based multiple sequence
alignment of Zur from M. tuberculosis (Lucarelli et al ., 2007) and S. coelicolor (Shin et al., 2011)
with its putative ortholog from A. japonicum. Sequences were aligned by using ClustalW2. Zinc
binding sites: ▪ site 1 (structural) , ▪ site 2, ▪ site 3. Protein domains are indicated on the top. Green
bar indicates the N-terminal DNA-binding domain, red bar indicates the amino acids involved in
forming the inter-domain hinge loop and the blue bar indicates the C-terminal dimerization
domain. Amino acid similarity is indicated at the bottom (identical (*), highly similar (:) and similar
(.), respectively).
Fig. S2. Construction of the zur in-frame deletion strain A. japonicum Δzur using the plasmid
st
nd
pGusA21Δzur (Table S1) via homologous recombination. 1 and 2 homologous recombinations
are exemplary illustrated as succession of first, downstream recombination and second, upstream
recombination but could also occur in opposite succession. aac(3)-IV, apramycin resistance gene;
gusA, β-glucuronidase (GUS) gene. Red: upstream flanking region of zur amplified by using the
primer pair pΔzur-US-F and pΔzur-US-R (Table S2). Yellow: downstream flanking region of zur
amplified by using the primer pair pΔzur-DS-F and pΔzur-DS-R (Table S2). Hatched red and yellow
bars illustrate the by PCR generated upstream and downstream fragments which were integrated
into pGusA21. pGusA21Δzur-zur is depicted in dashed lines to illustrate that this plasmid is not
replicating in A. japonicum. The proposed gene products of ajap_2935 – 60 are a conserved
putative secreted protein, a HTH-type transcriptional repressor, a Fur family transcriptional
regulator, a hypothetical protein, a membrane permease and a second membrane permease,
respectively. The deletions of aesA-C and aesH-E were performed accordingly.
Fig. S3. HPLC analysis of the zinc dependent [S,S]-EDDS production. Strains were grown in
deionized flasks in SM either with or without supplementation of 6 µM ZnSO 4. Commercial [S,S]EDDS from Sigma Aldrich was used as a standard.
Fig. S4. Zinc dependent transcriptional pattern of the putative [S,S]-EDDS biosynthesis genes aesAD of A. japonicum. Cultures were grown in SM in absence of any trace element (-) or supplemented
with (+) 6 μM Zn2+ respectively and samples were taken after 25 h of incubation to isolate RNA.
sigB was used as housekeeping gene to normalize the RNA. Left panel shows transcription pattern
in WT background, right panel in the ∆zur background.
Fig. S5. HPLC chromatograms of culture supernatants of A. japonicum strains grown in deionized
flasks in SM in absence of zinc. Commercial [S,S]-EDDS from Sigma Aldrich was used as a standard.
Fig. S6. 16S rRNA gene phylogenetic tree for 16 members of the genus Amycolatopsis chosen by
criteria of available genome sequence data. The tree was constructed using neighbor-joining with
CLUSTAL W and MEGA4 software. Classification into the phylogenetic clades A-F occured
accordingly to Everest and Meyers 2009. The percentage bootstrap values of 1,000 replications are
shown at each node (only values above 40% are shown). The scale bar indicates 1 nucleotide
substitution per 100 nucleotides. S. scabies was used as an outgroup.
Fig. S7. HPLC chromatograms of culture supernatants of several Amycolatopsis strains grown in
deionized flasks in SM either with or without supplementation of 25 µM ZnSO4. Commercial [S,S]EDDS from Sigma Aldrich was used as a standard.
Table S1: Bacterial strains and plasmids used in this study
Table S2: Oligonucleotides used in this study
Table S3: Manual BLAST analysis of AesA-H. Comparison of A. japonicum AesA-H to homologs in
other Amycolatopsis species.
Table S4: Zur-boxes 5‘ upstream of aesA homologues