Supplementary Information for " Lrs14 transcriptional regulators influence biofilm
formation and cell motility of Crenarchaea"
by Orell et al.
Protein sequence analysis
Similarity searches were conducted using BLASTP at the National Center for Biotechnology
Information (NCBI). Multiple alignment of archaeal Lrs14-like and Lrp/AsnC protein sequences
was constructed using the CLUSTALW program (Thompson et al. 1994), followed by manual
adjustment. Domain analysis on protein sequences was performed using SMART 4.0 (Letunic
et al. 2004). Jpred3 was used for secondary structure prediction (Cole et al. 2008). Phylogenetic
analyses were conducted using MEGA4 (Tamura et al. 2007). The evolutionary history was
inferred using the Neighbor-Joining method (Saitou & Nei 1987). The bootstrap consensus tree
inferred from 1000 replicates was taken to represent the evolutionary history of the analyzed
taxa. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates
were collapsed. The evolutionary distances were computed using the Poisson correction
method (Zuckerkandl & Pauling 1965). All positions containing gaps and missing data were
eliminated from the dataset (complete deletion option).
Construction of plasmids for in-frame gene deletion and in-trans-complementation
For the construction of the deletion mutant plasmids, the respective up- and down-stream
flanking regions of saci0102, saci0446, and saci1242, respectively, were PCR amplified from
S. acidocaldarius genomic DNA using primer pairs as listed in Table S1. The up- and downstream flanking DNA regions were joined by means of overlap extension PCR using the outward
bound primer of the respective primer pair. The overlap extension PCR products were restricted
with PstI and BamHI and subsequently ligated into the plasmid pSVA406, containing the pyrEF
1
cassette from S. solfataricus (Wagner et al. 2009). This ligation yielded deletion plasmids
pSVA452, pSVA453 and pSVA2004 (see Table 1 for details).
For overexpression of Saci0446 in S. acidocaldarius MW001, the saci0446 gene was
cloned into the S. acidocaldarius expression vector pSVA1450, (Wagner and Albers,
unpublished) which is based on pCmalLacS (Berkner et al. 2010) and allows for maltose
inducible expression of proteins. saci0446 was first cloned into pMZ1 yielding pSVA2022,
thereby adding 6 histidine residues to the N-terminus of saci0446, which originate from the
multiple cloning site of pMZ1. pMZ1 contains an expression cassette for Sulfolobus species
including a terminator region (Zolghadr et al. 2007). The saci0446 gene was then excised with
the terminator region from pSVA2022 using NcoI/ EagI and ligated with pSVA1450. This
construct was termed pSVA2026.
A plasmid for the complementation of Saci0446 including its promoter DNA region was
constructed in the expression plasmid pSVA1450. The saci0446 gene including its promoter
region was, therefore, amplified using primer pair 4069/4070. The PCR product was restricted
with SacII/EagI and ligated into pSVA1450 yielding plasmid pSVA2024.
Plasmids pSVA2024 and pSVA2026 were methylated using the E. coli strain ER1821 as
described
by
Wagner
et
al.
(2012).
Methylated
plasmids
were
transformed
into
S. acidocaldarius cells as described previously (Wagner et al. 2012). Plasmid containing
colonies were selected on gellan gum solidified Brock medium plates without uracil. Obtained
colonies were grown in liquid Brock medium and used for the inoculation of biofilms. All
constructs were sequenced to confirm their identity. The primer sequences are given in Table
S1.
Construction of chromosomal deletion mutants
In frame marker-less deletion mutants were generated for genes saci0102, saci0446, and
saci1242. To this end, methylated deletion mutant plasmids pSVA452, pSVA453 and
2
pSVA2004 were electroporated into MW001 as described by Wagner et al. (2012). Integrants
were selected on uracil selective gelrite plates after 5 days of incubation at 75°C and
subsequently subjected to 5-FOA (100 µg/ml) gelrite plates to allow the excision of the DNA
region containing the target gene. In frame marker-less deletion mutants were confirmed by
sequencing of PCR products that were obtained using primers binding at least 100 bp up and
downstream of the respective primers used for the construction of the flanking regions for the
deletion mutant plasmids.
Gene disruptions by S. solfataricus pyrEF cassette exchange via homologous
recombination were generated for saci0133, saci1219 and saci1223. To this end, 50 bp of the
up and downstream regions of each target gene were added to the 5’ and 3’ ends of the pyrEF
cassette via PCR. S. acidocaldarius MW001 cells were electroporated with ~300 ng of the
corresponding PCR product. Transformed cells were selected on uracil selective gelrite plates
after 5 days of incubation at 75 °C. Obtained colonies were transferred to liquid Brock medium.
Deletion mutants were confirmed by sequencing of PCR products that were obtained using
primers that bound at least 100 bp up and downstream of the target gene and one reverse and
one forward primer annealing in the pyrEF cassette sequence, respectively.
Western blotting
Cultures for immunological analyses were grown in liquid Brock medium to reach an OD600 0.5
and harvested by centrifugation at 3400 x g. The pellet was resuspended in fresh medium
without nutrient source. After 4 h, 0.001% tryptone was added to each culture and further
incubated at 76 °C overnight. Cells were harvested and the pellet was resuspended in buffer
containing 50 mM HEPES and 150 mM NaCl. Proteins were separated by SDS-PAGE
according to the method of Laemmli (Laemmli, 1970) and transferred to a PVDF membrane
(Roche) by semi-dry blotting. Polyclonal peptide antibodies against S. acidocaldarius anti-FlaB
3
were raised in rabbits (Eurogentec). Binding of the secondary antibody, the alkaline
phosphatase goat anti-rabbit immunoglobulin G (Sigma) was detected by using the CDP-star
chemiluminescent detection kit (Roche) according to the manufacturer’s instructions.
Chemiluminescence was measured using the LAS-4000 Luminescent image analyzer (Fujifilm,
Düsseldorf, Germany).
Heterologous expression of saci0446 and protein purification
To express N-terminal histidine tagged Saci0446 protein, saci0446 was amplified by PCR from
genomic DNA of S. acidocaldarius DSM639 using primer (Table S1) and cloned into the
pETDuet-1 vector system (Novagen), yielding plasmid pSVA2009. The constructed plasmid was
verified by sequencing of both strands. Heterologous expression of recombinant Saci0446 was
performed as reported previously (Ghosh et al. 2011), using E. coli BL21 (DE3)-RIL as
expression host strain. For purification of the recombinant protein, the resulting E. coli crude
extracts were diluted 1:1 with 50 mM HEPES, 300 mM KCl (pH 7.5) and subjected to a heat
precipitation for 10 min at 70°C. After heat precipitation, the samples were cleared by
centrifugation (60,000 x g for 30 min at 4°C). The supernatant was applied to a Ni2+-affinity
column (Native IMAC) on the Profinia TM protein purification system (Bio-Rad Laboratories).
Bound protein was eluted with elution buffer (50 mM HEPES, pH 8.0; 300 mM KCl; 250 mM
imidazole). A final desalting step was incorporated during the Profinia purification protocol to
wash the imidazole out of the protein sample. Fractions containing the recombinant protein
(analyzed by SDS-PAGE and anti-his Western blotting) were pooled and concentrated via
centrifugal concentrators (Amicon Ultra Centrifugal Filter 5000 MWCO, Millipore) using buffer A
(50 mM NaH2PO4, pH 7.5; 100 mM NaCl). Protein samples were used for DNA-protein binding
assays or stored at -20°C in presence of 10% glycerol.
4
DNA-protein binding assays and in gel footprinting experiments
For protein-DNA interaction studies, 5’-end
32
P-labeled DNA fragments were generated by
PCR-amplifying desired fragments with S. acidocaldarius genomic DNA as template and with
two primers, one being labeled with [γ-32P]-ATP with T4 polynucleotide kinase. Following primer
pairs were used: ep092/ep093 (promoter/operator (p/o) saci0446), ep094/ep095 (ORF
saci0446), ep096/ep097 (p/o saci1178), ep098/ep099 (p/o saci1177), ep100/ep101 (p/o
saci2314) and ep102/ep103 (p/o saci1908) (Table S1). Labeled probes were purified on a 6%
acrylamide gel. Electrophoretic mobility shift assays (EMSAs) were conducted as described
before (Peeters et al, 2007). Binding reaction mixtures were incubated at 37°C for 25 minutes in
LrpB buffer and contained, besides different amounts of the protein, 7500 cpm of DNA and 25
mg ml-1 sonicated salmon sperm DNA as non-specific competitor. Gel electrophoresis was
performed with 6% native acrylamide gels. EMSA autoradiographs were scanned and
integrated densities of individual bands were measured with ImageJ (Abramoff et al., 2004).
After subtraction of background densities, values were converted to the fraction of bound DNA.
Subsequently, using the Prism 6 software (GraphPad) these data were plotted and fitted to a
non-linear model with the Hill function, yielding for each fragment the apparent equilibrium
dissociation constant (KD) and the Hill coefficient (n), which represents a measure of the binding
cooperativity.
For in gel footprinting with the 1,10-phenantroline-copper ((OP)2-Cu+) ion (Cu-OP) of the
different DNA populations, an EMSA was performed with approximately 100,000 cpm DNA in
each binding reaction before performing chemical footprinting, exposure to a X-ray sensitive
film, excision and elution of different populations and analysis on a 8% denaturing acrylamide
gel. The procedure was followed as described before (Peeters et al, 2004). Reference ladders
were generated by chemical sequencing (Maxam & Gilbert, 1980).
5
References
Berkner S, Wlodkowski A, Albers S V, Lipps G. (2010). Inducible and constitutive promoters for
genetic systems in Sulfolobus acidocaldarius. Extremophiles 14: 249–259.
Cole C, Barber JD, Barton GJ. (2008). The Jpred 3 secondary structure prediction server.
Nucleic Acids Res 36: W197–W201.
Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. (1956). Colorimetric method for
determination of sugars and related substances. Anal Chem 28: 350–356.
Ghosh A, Hartung S, Van Der Does C, Tainer JA, Albers SV. (2011). Archaeal flagellar ATPase
motor shows ATP-dependent hexameric assembly and activity stimula-tion by specific
lipid binding. Biochem J 437: 43–52.
Letunic I, Copley RR, Schmidt S, Ciccarelli FD, Doerks T, Schultz Jet al.(2004). SMART 4.0:
towards genomic data integration. Nucleic Acids Res 32: D142–D144.
Petersen GL. (1977). A simplification of the protein assay of Lowry which is more generally
applicable. Anal Chem 83: 346–353.
Saitou N, Nei M. (1987). The neighbour-joining method: a new method for reconstructing
phylogenetic trees. Mol Biol Evol 4: 406–425.
Tamura K, Dudley J, Nei M, Kumar S. (2007). MEGA4: Molecular Evolutionary Genetics
Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596–1599.
Thompson JD, Higgins DG, Gibson TJ. (1994). CLUSTALW: improving the sensitivity of of
progressive multiple sequence alignment through sequence weighting, position-specific
gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680.
Wagner M, Berkner S, Ajon M, Driessen AJ, Lipps G, Albers SV. (2009). Expanding and
understanding the genetic toolbox of the hyperthermophilic genus Sulfolobus. Biochem
Soc Trans 37: 97–101.
Zuckerkandl E, Pauling L. (1965). In Bryson V, Vogel HJ(eds). Evolutionary divergence and
convergence. In Evolving Genes and Proteins 97: pp 97–166.
6
7
Figure S1. Sequence analysis of archaeal Lrs14-like proteins. (A) Multiple alignments of
archaeal Lrs14 proteins together with E. coli Lrp and S. solfataricus Ss-LrpB protein sequences.
Residues conserved ≥ 90 % are shown by (:) and (.) represents residues sharing 50% of
conservation. Secondary structure prediction for both Lrs14-like proteins and Lrp-like proteins
are indicted above the alignment (α helixes: yellow cylinders, β sheets: green arrows). The six
S. acidocaldarius homologous Lrs14-like proteins are shown in bold. The location of a putative
HTH motif and the RAM domain are indicated above the alignment. Accession numbers and
ORF numbers are indicated. Species abbreviations: S.so, Sulfolobus solfataricus; S.ac,
Sulfolobus acidocaldarius; S.to, Sulfolobus tokodaii; Hsp, Halobacterium sp. NRC1; E.co,
Escherichia coli. (B) The NJ distance tree was constructed using a subset of each archaeal
Lrs14-like and Lrp-like proteins. The six S. acidocaldarius homologous Lrs14-like proteins are
underlined and the corresponding ORF numbers are depicted. Accession numbers are indicated
in parentheses. Bootstrap values, based on 1000 repetitions, are shown next to the branches.
Bar, 10 % estimated divergence.
8
Figure S2. Expression profile of S. acidocaldarius MW001 lrs14 genes of biofilmassociated cell population versus planktonic cell population. After 3 day biofilm growth the
supernatant of the Petri dishes containing the planktonic cells was carefully removed. The
biofilm was washed with 50 mL of Brock media and consecutively harvested with a cell scraper.
Total RNA was isolated from both cell samples. qRT-PCR analysis were performed using
specific primers for each Lrs14 encoding ORF (shown underneath the plot). Relative transcript
expression levels of each gene were normalized to the internal control gene secY. The values
reflect the fold change in gene expression compared with cDNA prepared from biofilmassociated cell population versus planktonic cell population. The means and standard
deviations of 3 biological replicates are shown.
9
Figure S3. Construction of Lrs14 deletion mutants. (A) PCRs were performed with reference
strain MW001 and the respective in-frame lrs14 deletion mutant genomic DNA revealing a
downshift of the corresponding DNA band of the mutant DNA. (B) PCR of the deletion mutant
generated by insertion of the pyrEF selection cassette. One forward primer annealing up-stream
of the target gene and one primer annealing within the pyrEF cassette sequence were used.
This primer combination allows amplifications only when mutant DNA is used as a template.
10
Figure S4. Planktonic growth of S. acidocaldarius Lrs14 deletion mutant strains. Shaking
cultured mutant strains were sampled at various time points to measure cell density atOD600.
Reference strain MW001 (-◊-) and marker-less deletion mutants Δsaci1242 (-Δ-), Δsaci0446 (-○), Δsaci0102 (-□-). Wild type of pyrEF disrupted deletion mutants MW001+pyrEF(-♦-) and disrupted
deletion mutants Δsaci0133 (-■-), Δsaci1219 (-▲-), Δsaci1223 (-●-). Each pointrepresents the
mean of 3 biological replicas.
11
Figure S5. CLSM analysis of biofilm formed by S. acidocaldarius MW001pyrEF+ strain.
Three days old biofilms were subjeted to CLSM. The blue channel is the DAPI-staining. The
green channel represents the fluorescently labeld lectin ConA that binds to glucose and
mannose residues. The lectin IB4 able to bind to α-galactosyl-residues is shown in yellow. The
overlay image of all three channels is shown (left panel). DIC pictures (midle panel) were taken
from the bottom layer of biofilms and converted into black/white (right panel) to calculate the
surface coverage. Numbers represent the percentage of surface coverage. Scale bar = 20 µm.
Figure S6. His-tagged Saci0446 protein purification. The elution fraction obtained from a
Ni2+-column chromatography was analyzed on a SDS-PAGE and stained with Coomassie. Bars
to the left side indicate molecular weights in kilo Daltons. Expected size of His-tagged saci0446
protein is 14.97 kDa.
12
Figure S7. Footprinting assays of Saci0446 binding to its own promoter. (A) In-gel’ Cu-OP
footprinting analysis of binding of saci0446 to a promoter fragment of its own gene with the top
strand labeled. In the upper part, the preceding EMSA is shown with indicating of the different
free (F) and bound (B) populations as they were excised for further analysis. Protein
concentrations are indicated in µM. In the lower part, the footprint autoradiograph is shown with
indication of the C+T sequencing ladder, aligned to the footprint lanes, and the different
populations. Protected and hyperreactive regions in the higher-order complex are indicated to
the right of the autoradiograph with open and grey rectangles, respectively. (B) In gel Cu-OP
footprinting analysis of binding of saci0446 to a promoter fragment of its own gene with the
bottom strand labeled. Notations are the same as in panel B, except that an A+G ladder has
been included and that ball-and-stick symbols indicate hyperreactivity positions observed in
lower-order complexes. (C) saci0446 promoter sequence with summary of footprint results. The
translational stop and start codon of saci0445 (CAA on top strand) and of saci0446 (ATG on top
strand) have been highlighted in bold. The notation of protected and hyperreactive regions is the
same as in panels B and C.
13
Explanation belonging to the footprinting assays:
To identify potential recognition motifs, we performed in gel Cu-OP footprinting with the p/o
saci0446 probe, which is bound by Saci0446 with one of the highest affinities (Fig. 7B). For the
nucleoprotein complexes with the highest electrophoretic mobility and lowest binding
stoichiometries, no obvious protection zones were observed. This result suggested that complex
populations consists of complexes bound at different locations and that Saci0446 binds with a
low sequence specificity, a conclusion that is corroborated by the fact that the number of
electrophoretically distinct complexes is proportional to DNA fragment length (data not shown).
The protected regions observed in the complexes with higher stoichiometry are not well
delineated and did not lead to the identification of a recognition binding motif (Fig. 6B).
Interestingly, a negative correlation exists between the AT level of the tested DNA sequence
and the apparent KD (Pearson’s correlation coefficient r −0.8522 and R2 0.73), demonstrating
that Saci0446 interacts with AT-rich sequences with a higher affinity.
In the footprint experiment with the bottom strand labeled, two positions exhibited
hyperreactivity in the lower-order complexes, demonstrating protein-induced deformations at
these specific locations (Fig. 7B/D). In the higher-order complexes extensive hyperreactivity
occurs, leading to higher cleavage efficiencies of longer DNA molecules. Based on these
cleavage patterns, it is likely that considerable conformational changes occurred in the DNA
upon cooperative binding of multiple Saci0446 molecules. A similar footprinting pattern was
observed for p/o Saci1908 (data not shown).
14
Table S1. Oligonucleotides used in this study
Primer
no.
1004
sequence
description
5‘-GGGCCATGGAACTCAGGGTGAAAACCTAC
Forward primer for upstream region Δsaci0102
with ApaI restriction site
1007
5‘CAAGGGAATTACTGGGACATTTATCTCACAAATA
AAGTTC
5‘GTGAGATAAATGTCCCAGTAATTCCCTTGACTTT
TCCCC
5‘-GCGGGATCCGGTTTGCGTGCTATATTCAG
2413
2414
5‘-TTGGGCTACAGAGGGACTTC
5‘-TTTGTCCACGAGGACTAACG
1012
1019
5‘-GGGCCATGGTTCCGTCGGAAGTGTCAAC
5’GCATAATTCCTCTTCAATACTCATTTTAATCTCG
CCTTTG
5’GAGTATTGAAGAGGAATTATGCAAAGAATTAAA
CCAAG
1073
5’-GATGGATCCGTAGGCTCAGTGGCTTTAAC
2415
2416
5‘-GACGATACGCCTGTAGTTTG
5‘-ACTGAAGGGCGGTTTCTATC
2417
5‘-GTAGGGCCCCAGGCATGAGACCCAATACG
2418
5‘-GAACCAATGAGTTAATATATTCAATTTTTAAC
2419
5‘-ATTGAATATATTAACTCATTGGTTCTTGGCTG
2420
2421
2422
5‘-GATGGATCCCCTCTAGCAGGAAGTCTTTG
5‘-AGGGTATCTCGTTTCACCAG
5‘-TGCAGTTAAGGCAACTGTGG
5‘CACTTTTTTTAGTTAGCAAAAACAAGTAATATTC
GGAGTGATACAAAATGTTTGAGCAGTTCTAG
5‘CTATAGTAATAAGAGGAGATAATGTTATCTTAGT
GTCTCCTGTTTAAGACGACCGGCTATTTTTTCAC
5‘TTTACTTTTTAATGAAAAGATTTAAATATGAGTAT
TTAAAATGAATTAATTTTGAGCAGTTCTAG
5‘ATTTGATGAAATAAATCCTAAACCTGTAATTAAT
ATTTTCACAGGCTAAAGACCGGCTATTTTTTCAC
5‘CAGAAGAAGGAGGAAAAGCAAGAAGAAAGTAAA
AGTTAGATACTTTAGTTTTTGAGCAGTTCTAG
5‘ATAAAAATCGAAGGTAAAGTTTTTTAATTTTTAAT
AACTTTATATTGCTTGACCGGCTATTTTTTCAC
5‘-AGTAGCCTATGGTCTTTCTGAATC
5‘-TCAACTAATCCTGCATCTGAAAGC
1005
1006
1018
2497
2498
4043
4044
4055
4056
2488
2489
Reverse primer for upstream region Δsaci0102,
overlapping region
Forward primer for downstream region
Δsaci0102, overlapping region
Reverse primer for downstream region
Δsaci0102 with BamHI
Forward sequencing primer Δsaci0102
Reverse sequencing primer Δsaci0102
Forward primer for upstream region Δsaci0446
with ApaI restriction site
Reverse primer for upstream region Δsaci0446 ,
overlapping region
Forward primer for downstream region
Δsaci0446 , overlapping region
Reverse primer for downstream region
Δsaci0446 with BamHI
Forward sequencing primer Δsaci0446
Reverse sequencing primer Δsaci0446
Forward primer for upstream region Δsaci1242
with ApaI restriction site
Reverse primer for upstream region Δsaci1242,
overlapping region
Forward primer for downstream region
Δsaci1242, overlapping region
Reverse primer for downstream region
Δsaci1242 with BamHI
Forward sequencing primer Δsaci1242
Reverse sequencing primer Δsaci1242
Δsaci1223 Forward for pyrEF exchange
Δsaci1223 Reverse for pyrEF exchange
Δsaci0133 Forward for pyrEF exchange
Δsaci0133 Reverse for pyrEF exchange
Δsaci1219 Forward for pyrEF exchange
Δsaci1219 Reverse for pyrEF exchange
saci0446 forward qPCR primer
saci0446 reverse qPCR primer
15
2490
2491
2492
2493
1112
1113
1114
1115
1116
1117
1424
1425
1426
1427
696
697
3512
3513
2079
2080
2075
2075
1480
1481
4077
4078
4079
4080
4319
4320
4081
4082
4083
4084
4085
4086
4321
4322
4323
4324
4087
4088
5‘-AGCGGTGCTAAAGGCACAGAAG
5‘-GGTCTACCCGCCTTATTTACAG
5‘-GAGGCGTTGAAGTTCTGCTATGAC
5‘-CGCTCCTGTTTATGGAGGCTTTAG
5‘-GGGCCATGGTTCCGTCGGAAGTGTCAAC
5‘-GGAGACAGTACTTCAAATTCCATATC
5‘-GATTAAAATGAGTATAAACCAAG
5‘-GCCATATCCTCACTTATGACTTGG
5‘-CTGAGAGGCTAACGTCTCTAAATC
5‘-GAAGCAGGAGAAGAGAAGAAGAAG
5‘-ACTGCGTCTACTGCGTTATCTTTATC
5’-GGAGATAAGTCTACACTAGATACACCAGAA
5‘-GCAGTTGAAGAGTTAGCCTTATCTGTG
5‘-CCTACTAACTGACTTACGGTACTAATCT
5’-CTCTAATTTTAACGTCTCAGTAACTAGC
5’-CCTACTTGTTCCATAGGATTGTTAGG
5’-CTCCTGACTACCAACTGACTATTTATC
5’-GTTCACCAGTAGAATAGCTCTTTACAC
5’-TAGCCAGGGTATGTTCAGTAATC
5’-ACCTAAGTTCCCGTTATTGAC
5’-GCTAGTAAAGCCAACAAGAGTG
5’-ATATAGTCGCTGCTACCCTATG
5’-CCTGCAACATCTATCCATAACATACCGA
5’-CCTCATAGTGTATATGCTTTAGTAGTAG’
5’-CGTCTATCGCTTTCGTGATCTG
5’-TCTTACCCTACGTACACGAGAG
5’-GCTCCCGAAGTCCATATAAGG
5’-GTTCTAGGTGGACTCGGTAAG
5’-AGTCGGACCATAGACACTAGAG
5’-GACACGCCAGGAGCTTTATATC
5’-ATCCTTATGCTGGTGGCTCTG
5’-TCTCGTTCCTCCCTTCCAATC
5’-CACCAGCCCTCTTCTCTAC
5’-GTCCTGCACTGACCAATACC
5’-GTGTTGTGATACCGGCATAC
5’-GGCGGAGTCGAACCATATAC
5’-TGCCTTCCCGTTATCATCAGTC
5’-TACAGTCGCTCTGAACGGATAC
5’-GGTCGATTGAGATCCCAGTTGTTC
5’-CTTTCTCCCTGACCTCCTTAAACC
5’-ATGTACCCGGACCTGGATATG
5’-TCGGATGCTGGCAAATCAC
2453
4067
5‘-CCCCCGAATTCGATGAGTATTGAAATTACTG
5‘CCCCGCGGCCGCTTAAATAAAAGACTTAATAAC
5‘GTACCATGGGTATTGAAATTACTGAAAAATATG
4068
5‘-GATGGATCCAATAAAAGACTTAATAACTTGG
4069
5‘-GATCCGCGGATACCCTGTCTGTTCTCTTC
5‘GTACGGCCGTTAAATAAAAGACTTAATAACTTG
G
5’-ATACCCTGTCTGTTCTCTTC
5'-CAATACTCATTTTAATCTCGCC
5'-CAGTAGCCTATGGTCTTTCTGAATC
5’-CCTTCATGTTATCTCCTTTTTCCT
2454
4070
ep092
ep093
ep094
ep095
saci0102 forward qPCR primer
saci0102 reverse qPCR primer
saci0133 forward qPCR primer
saci0133 reverse qPCR primer
Saci1223 forward qPCR primer
Saci1223 reverse qPCR primer
Saci1242 forward qPCR primer
Saci1242 reverse qPCR primer
Saci1219 forward qPCR primer
Saci1219 reverse qPCR primer
flaB forward qPCR primer
flaB reverse qPCR primer
flaX forward qPCR primer
flaX reverse qPCR primer
aapA forward qPCR primer
aapA reverse qPCR primer
aapF forward qPCR primer
aapF reverse qPCR primer
upsA forward qPCR primer
upsA reverse qPCR primer
upsE forward qPCR primer
upsE reverse qPCR primer
secY forward qPCR primer
secY reverse qPCR primer
saci1904 forward qPCR primer
saci1904 reverse qPCR primer
saci1905 forward qPCR primer
saci1905 reverse qPCR primer
saci1906 forward qPCR primer
saci1906 reverse qPCR primer
saci1907 forward qPCR primer
saci1907 reverse qPCR primer
saci1908 forward qPCR primer
saci1908 reverse qPCR primer
saci1909 forward qPCR primer
saci1909 reverse qPCR primer
saci1910 forward qPCR primer
saci1910 reverse qPCR primer
saci1911 forward qPCR primer
saci1911 reverse qPCR primer
saci1912 forward qPCR primer
saci1912 reverse qPCR primer
Forward primer for cloning saci0446 into
pETduET with EcoRI restriction site
Reverse primer for cloning saci0446 into
pETduET with NotI restriction site
Forward primer for cloning saci0446 into pMZ1
with NcoI restriction site
Reverse primer for cloning saci0446 into pMZ1
with BamHI restriction site
Forward primer for cloning saci0446 into
pSVA1450 with SacII restriction site
Reverse primer for cloning saci0446 into
pSVA1450 with EagI restriction site
Forward EMSA probe (p/o) saci0446
Reverse EMSA probe (p/o) saci0446
Forward EMSA probe (ORF) saci0446
Reverse EMSA probe (ORF) saci0446
16
ep096
ep097
ep098
ep099
ep100
ep101
ep102
ep103
5’-ATTGCCTTCTCATCAGTATCATG
5’-CCTTCTTTTCATGTATATCATGTT
5’-CAGTATATCTATCAGCCTGATGG
5’-CTATAGGTATACCAACTCCTATC
5’-CTAATTACTTGTATACATTTGTAAG
5’-TAACGTGATATCATGGTAATCTTA
5’-CCGAAAACGATTAATGGTAAGGA
5’-GTGCACTCCTTAAAGAAAACACA
Forward EMSA probe (p/o) saci1178
Reverse EMSA probe (p/o) saci1178
Forward EMSA probe (p/o) saci1177
Reverse EMSA probe (p/o) saci1177
Forward EMSA probe (p/o) saci2314
Reverse EMSA probe (p/o) saci2314
Forward EMSA probe (p/o) saci1908
Reverse EMSA probe (p/o) saci1908
17