Material and Methods s1
Supplementary Data
Structural peculiarities of the micrococcal linear megaplasmid pLMA1 interfere with pyrosequencing reads assembly
Martin Wagenknecht, Julián R. Dib, Andrea Thürmer, Rolf Daniel, María E. Farías, Friedhelm Meinhardt*
*Corresponding author:
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstr. 3, D-48149 Münster, Germany
E-mail address:
Journal: Biotechnology Letters
Supplementary Table S1 Primers used in this study
Primer name and sequence (5´-3´) / Purposeop15-revw1
GAGAATCCCGCCGCAGTGAC / Used for the sequencing of the cloned 8.6-kb fragment of pLMA1 (yielding pP86-15) by primer walking.
op15-revw2
GAGTGTGGATCACCTGATCCC
op15-revw3
CGGCCCGTAGAAAGGATCTGC
op15-revw4
ATACCTGGTAGCCACGCCAC
op15-revw5a
CGTGGAAGTCGACGCGAGC
op15-revw6
CGTCGTTGATGTCGGCTGTGG
op15-revw7
GCATCGGTGACCGTTGGTGC
op15-uniw1
GCAGACCGTTGGGCACATCG
op15-uniw2
GCTCGGTTCTTTCCAGACCC
op15-uniw3
CAATGTCTCGAGGTCGTGTCC
op15-uniw3-1a
CTTCTCCGGCTCGCAGATCC
op15-uniw4
CCATCCTCGCGCTCATCTCC
op34-revw1
CGACCAGATGGTGATGGATGC / Used for the sequencing of the cloned 6.2-kb fragment of pLMA1 (yielding pP62-34) by primer walking.
op34-revw2
TGGAGAATCACCCAGCTGCAC
op34-revw3
GTGCTGATCACCGGCGACC
op34-revw3-1a
ACTCCAAGGGTCACCGGATCG
op34-uniw1
AATCTGCTGGCCGTGGATGG
op34-uniw2
CCACCGAGAACTGGATGGC
op34-uniw3
GTCGGCAGCTGGACTACAAG
op34-uniw4a
CTAGACGCCAGCCAGTGCTCG
op34-uniw5
GATCAGGCGTCCAGGATGGTG
op54-revw1
TCGTTGACGGAGTAGTAGAAGG / Used for the sequencing of the cloned 4.5-kb fragment of pLMA1 (yielding pP45-54) by primer walking.
op54-revw2a
AGATGGTGCGCGCCCAGAC
op54-uniw1
AGGGCCGGTGGAGGTCGC
op54-uniw2a
CAGCAGTCCCTGCGCACC
op54-uniw2-1
CAGTTCTGATGGCCACCAGC
op62-revw1
GCCGCTCAGCAGGCCTCC / Used for the sequencing of the cloned 3.7-kb chromosomal fragment (yielding pP37-62) by primer walking.
op62-revw2a
GGTCACGCTCGCCAGTGTG
op62-uniw1
GGCCGGCGATCACGTGGTCC
op62-uniw2a
CCGAACACGCCGTCGTAGC
op74-revw1
GGTGAAGGCCATGCTCCACAG / Used for the sequencing of the cloned 3.7-kb fragment of pLMA1 (yielding pP37-74) by primer walking.
op74-revw2a
CGCCACCGCTTGATCGTCG
op74-revw3
CTATCGGCCGCAGACCAACG
op74-uniw1a
TCGGACACGAGCGGGTACATC
op74-uniw2
CACATCCTTCCCTGCGGTGTG
op15-1
CATGTAGCCGGTGTTCTGAC / Used for gap closure during primer walking.
op15-2
ACGTCTCCGGTAAGTACACC
op34-1
CTCAACGAGGAGGGCAAG
op34-2
GTATAACCATACACGCGCAAG
op74-1
GAAGATCGAACGCTTCCAC
op74-2
CACCACAGATCGTATGTCACC
a These primers were also used for the amplification of probe fragments
Supplementary Table S2 Predicted ORFs on cloned PstI fragments of pLMA1
(bp) / Protein sizeb
(aa) / Function of closest relative, source, (no. of amino acids) / Accession number / No. of identical amino acids/length of aligned region (%) / E value
pP86-15:
1 / 192–617 c / 141 / Transposase, Micrococcus luteus NCTC 2665, (127) / YP_002957448 / 96/98 (97) / 1e–43
2 / 878–1216 c / 112 / Lsr2-like protein, Streptomyces hygroscopicus ATCC 53653, (111) / ZP_05516738 / 50/109 (45) / 5e–14
3 / 1269–1634 / 121 / HNH endonuclease, Lactobacillus ruminis ATCC 25644, (111) / ZP_03957443 / 38/103 (36) / 3e–08
4 / 1866–2192 c / 108 / No significant similarities
5 / 2608–2853 / 81 / Hypothetical protein, Mycobacterium avium, (102) / NP_961697 / 28/53 (52) / 5e–04
6 / 3424–4290 c / 288 / Conserved hypothetical protein, Corynebacterium jeikeium ATCC 43734, (228) / ZP_05847000 / 119/228 (52) / 2e–59
7 / 4458–5231 / 257 / Methyltransferase type 11, Bacillus coagulans 36D1, (258) / ZP_04430662 / 110/256 (45) / 7e–56
8 / 5721–6206 / 161 / ADP-ribose pyrophosphatase, Micrococcus luteus NCTC 2665, (154) / YP_002958126 / 115/152 (75) / 3e–60
9 / 6334–6765 / 143 / Cytidine deaminase, Kocuria rhizophila DC2201, (145) / YP_001854367 / 75/145 (51) / 6e–32
10 / 6791–7474 c / 227 / Methylase, Micrococcus
luteus NCTC 2665, (200) / YP_002956146 / 96/194 (49) / 1e–44
11 / 7506–8075 c / 189 / Acetyltransferase of GNAT family, Kytococcus sedentarius
DSM 20547, (186) / YP_003147978 / 98/187 (52) / 7e–43
12 / 8238–8621 c / >127 / Hypothetical protein, Bacillus sp. B14905, (177) / ZP_01723109 / 45/120 (37) / 2e–17
pP62-34:
1 / 27–764 / 245 / Hypothetical protein, Arthrobacter chlorophenolicus A6, (244) / YP_002477823 / 104/241 (43) / 3e–49
2 / 847–2508 / 553 / Transposase, Streptomyces sp. SPB74, (504) / ZP_04991734 / 205/506 (40) / 2e–86
3 / 2604–2801 c / 65 / Conserved hypothetical protein, Actinomyces urogenitalis DSM 15434, (171) / ZP_03927583 / 26/61 (42) / 0.13
4 / 2932–3483 c / 183 / Hypothetical protein, Micrococcus luteus SK58, (118) / ZP_06502621 / 40/112 (35) / 1e–08
5 / 3512–3781 c / 89 / Conserved hypothetical protein, Perkinsus marinus ATCC 50983, (588) / EER00427 / 26/75 (34) / 3.0
6 / 4291–4620 c / 109 / Hypothetical protein, Arthrobacter chlorophenolicus A6, (122) / YP_002477854 / 26/78 (33) / 8e–04
7 / 5224–6195 / >324 / Transposase, Micrococcus luteus NCTC 2665, (335) / YP_002956624 / 264/299 (88) / 4e–151
pP45-54:
1 / 31–774 / 247 / No significant similarities
2 / 1631–2500 c / 289 / Hypothetical protein, Streptomyces griseus subsp. griseus NBRC 13350, (375) / YP_001825356 / 58/240 (24) / 2e–07
3 / 2568–3353 c / 261 / DNA replication protein, Micrococcus luteus NCTC 2665, (261) / YP_002957278 / 260/261 (100) / 5e–148
4 / 3350–3790 c / 146 / Transposase, Micrococcus luteus NCTC 2665, (488) / YP_002957277 / 145/146 (99) / 2e–75
5 / 3848–4498 / >217 / No significant similarities
pP37-74:
1 / 1–1161 / >386 / Putative transposase, Micrococcus sp. 28, (415) / NP_862387 / 361/385 (93) / 0.0
2 / 1167–1418 / 83 / Hypothetical protein, putative NADH-flavin reductase, Corynebacterium jeikeium K411, (217) / YP_251537 / 64/71 (90) / 1e–29
3 / 1505–2491 / 328 / Transposase for Tn3595, Corynebacterium jeikeium K411, (328) / YP_250595 / 288/327 (88) / 2e–170
4 / 2669–2971 / 100 / Putative transposase subunit, Micrococcus sp. 28, (100) / NP_862412 / 95/100 (96) / 1e–49
5 / 2968–3681 / >238 / Putative transposase, Micrococcus sp. 28, (294) / NP_862413 / 213/238 (89) / 3e–122
a c, complementary strand
b The size of putative proteins starting with ‘>’ refers to annotated genes whose nucleotide
sequence is incomplete as they are located at the ends of the cloned PstI fragments. aa,
amino acids
Fig. S1 PstI restriction fragments of pLMA1 selected for cloning. Plasmid pLMA1, isolated by electroelution from a preparative PF gel, was digested using restriction endonuclease PstI and separated on a 1.0% agarose gel. Arrows indicate restriction fragments that where selected for cloning in pUC18. M, DNA size standard
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