Sequence Analysis of Putative potB, potC, and potD Genes from Serratia Rubidae
I
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O
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S
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ISSN 1978-3477
*Corresponding author, Phone/Fax: +62-22-2511575;
Email: sony@sith.itb.ac.id
Volume 4, Number 2, August 2010
p 90-95
Volume 4, 2010
Microbiol Indones
91
Fig 1 (Left) PCR reaction products. Putative potBCD were sized
approximately 1800 bp; (Right) Line1: Recombinant plasmid uncut. Line 2:
Recombinant plasmid cut by EcoRI. M: marker (1 kb DNAladder).
92
SUHANDONO ET AL.
Microbiol Indones
RERC
PotB
SD
*
PotC
M
SD
*
M
PotD
FERC
Fig 2 Nucleotide sequence of potB, potC and potD genes from S. rubidae. SD denotes putative Shine- Delgarno sequence. Codon start indicated by “M”
and codon stop indicated by “*”. Grey boxes indicate a pair of primers.
Volume 4, 2010
Microbiol Indones
93
TM1
PotB_ S.rubidae
: --------------------------------------- ------------------------------------------------------------------------
PotB_ E.coli
:MIVTIVGWLV LFVFLPNLMIIG TSFLTRDDASFVKMVFTLDNYTRLLDPLYFEVLLHSLN
60
TM3
TM2
PotB_ S.rubidae
: ------------------------------ ---------------PQKVRPLLLFLLIVPFWTNSLIRIYGLKIFLSTKGYL 37
PotB_ E.coli
:MALIATLACLVLGYPFAWFL AKLPHKVRPLLL FLLIVPFWTNSLIRIYGLKIFLSTKGYL 120
* :* ******** *******************************
TM4
PotB_ S.rubidae
:NEFLLWLGVIETPIRIMFTP GAVIIGLVYILLPF MVMPLY SSIEKLDK PLLEAAKDLGAS
PotB_ E.coli
:NEFLLWLGVIDTPIRIMFTP SAVIIGLVYILLPF MVMPLY SSIEKLD KPLLEAARDLGAS 180
97
****************************************************************** *********
TM5
PotB_ S.rubidae
:KLQTFVRIIIPLT MPGIIAGCLLVMLPA MGLFYVSDLMGGAKNLLIGNVIKSQFLNIRDW 157
PotB_ E.coli
:KLQTFI RIIIPLTMPGIIAGCLLVMLPA MGLFYVSDLMGGAKNLLIGNVIKVQFLNIRDW 240
****** : *******************************************************************
TM6
PotB_S.rubidae
:PFGSATSITLTVVMGLMLLVY WRASRLLNKKVELE
192
PotB_ E.coli
:PFGAATSITLTI VMGLMLLVY WRASRLLNKKVELE
275
*** : ****************************************
Fig 3 Sequence alignment of putative PotB protein from S. rubidae and PotB from E. coli Acc Number M 64519.1 which were aligned using the Clustal W
algorithm. Grey boxes indicate the six predicted transmembrane segments (TM1 to TM6). Consensus symbols; identical residues: ''*'', conserved substitutions:
'':'', semi-conserved substitutions: ''.''.
TM1
PotC_ S.rubidae
: MIGRLLRGGFMTAIHAYLYIPIIILIV NSFNSSRFGINWQ GFTTKWYGLLMNNDSLLQAA
PotC_ E.coli
: MIGRLLRGGFMTAIYAYLYIPIIILI VNSFNSSRFGINWQ GFTTKWYSLLMNNDSLLQAA
60
60
***************** : ************************************** .****************
TM3
TM2
PotC_ S.rubidae
:QHSLTMAVFFATFAT LIGSLTAVAL YRYRFRGKPFVSGML FVVMMSPDIVMAISLLVLFM 120
PotC_ E.coli
:QHSLTMAVFSATFAT LIGSLTAVAL YRYRFRGKPFVSGML FVVMMSPDIVMAISLLVLFM 120
***********
************************************************ ***************
TM4
PotC_ S.rubidae
:LLGVQLGFWSLLFSHITFCLPFVVVTVYARLKGFDVRMLEAAKDLGASEMTILRKI ILPL 180
PotC_ E.coli
:LLG I QLGFWSLLFSHITFCLPFVVVTVYSRLKGFDVRMLEAAKDLGASEFTILRKIILPL 180
*** : ****************************** * :************************** :*** *********
TM5
PotC_ S.rubidae
:AMPAVAAGWLL SFTLSMDDVVVSSFVTGPSYEILPLKIYSMVKVGVSPEVN ALATILLVL 240
PotC_ E.coli
:AMPAVAAGWVISFTLS MDDVVVSSFVTGPSYEILPLKIYSMVKVGVSPEVNALATILLVL 240
************* : ************************************************************* *
TM6
PotC_ S.rubidae
:SLVLVIASQVI ARDKTK - SQGTQK
PotC_ E.coli
:SLVMVIASQLI ARDKTKGNTGDVK
*** : ***** : *********
.
*
263
264
*
Fig 4 Sequence alignment of putative PotC protein from S. rubidae and PotC from E. coli Acc number M64519.1 using the Clustal W algorithm. Grey
boxes indicate the six predicted transmembrane segments (TM 1 to TM 6). Consensus symbols; identical residues: ''*'', conserved substitutions: '':'', semiconserved substitutions: ''.''.
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SUHANDONO ET AL.
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PotD_S.rubidae
PotD_E.coli
:MKKM - - - LAAAATGARNWAAAHAGDCKTLYFYN WTEYVPPGLLEQFTKETGIKVIYSTYE 57
:MKKWSRHLLAAGALA LGMSAAHADDNNTLYFY N WTEYVPPGLLEQFTKETGIKVIYSTYE 60
***
* ** . : * . : **** . * .* : ******************************************
Signal sequence
βA
α1
βB
PotD_S.rubidae
PotD_E.coli
:SNETMYAKLKTYKDGAYDLVVP STYF VDKMRKEGMIQKIDKSKLTNFSNLDPEMLNKPED 117
:SNETMYAKLKTYKDGAYDLVVP STYYVDKMRKEGMIQKIDKSKLTNFSNLDPDMLNKPED 120
*********** ********************** : ********************************** : *********
α2
βB
α3
PotD_S.rubidae
PotD_E.coli
:PNNDYSIPYIWGATAIGVNSDA I DPKRVSSWADLWKPEYKNSL ---------------------------------- 160
:PNNDYSIPYIWGATAIGVNGDAVDPKRVTSWADLWKPEYKGSLLLTD DAREVFQMALRKL 180
*********** ************ . ** : *** * : **************** . **
βD
βE
βF
α4
PotD_S.rubidae
PotD_E.coli
: --------------------------------------------------------------------------------------- --------------------------------:GYSGNTTDPKEIEAAYNELKKLMPNVAAFNSDNPANPYMEGEVNLGMIWNGSAFVARQAG 240
α5
βG
α6
βH
α7
PotD_S.rubidae
PotD_E.coli
: -----------------------------------------------------------------------------------------------------------------------: TPIDVVWPKEGGIFWMDSLAIPANAKNKEGALKLINFLLRPDVAKQVAETIG YPTPNLAA
βI
βJ
α8
α9
α10
PotD_S.rubidae
PotD_E.coli
: -----------------------------------------------------------------------------------------------: RKLLSPEVANDKTLYPDAETIKNGEW QNDVGAASSIYEEYYQKLKAGR 348
α11
α12
α13
300
Fig 5 Sequence alignment between putative PotD protein from S. rubidae and PotD from E. coli Acc. number M 64519.1 Based on Sugiyama et al. (1996)
the active residue which binds to spermidine are indicated by grey boxes. Consensus symbols; identical residues: ''*'', conserved substitutions: '':'', semiconserved substitutions: ''.''.
Volume 4, 2010
Microbiol Indones
95
N
D
O
N
E
S
I
A
ISSN 1978-3477
*Corresponding author, Phone/Fax: +62-22-2511575;
Email: sony@sith.itb.ac.id
Volume 4, Number 2, August 2010
p 90-95
Volume 4, 2010
Microbiol Indones
91
Fig 1 (Left) PCR reaction products. Putative potBCD were sized
approximately 1800 bp; (Right) Line1: Recombinant plasmid uncut. Line 2:
Recombinant plasmid cut by EcoRI. M: marker (1 kb DNAladder).
92
SUHANDONO ET AL.
Microbiol Indones
RERC
PotB
SD
*
PotC
M
SD
*
M
PotD
FERC
Fig 2 Nucleotide sequence of potB, potC and potD genes from S. rubidae. SD denotes putative Shine- Delgarno sequence. Codon start indicated by “M”
and codon stop indicated by “*”. Grey boxes indicate a pair of primers.
Volume 4, 2010
Microbiol Indones
93
TM1
PotB_ S.rubidae
: --------------------------------------- ------------------------------------------------------------------------
PotB_ E.coli
:MIVTIVGWLV LFVFLPNLMIIG TSFLTRDDASFVKMVFTLDNYTRLLDPLYFEVLLHSLN
60
TM3
TM2
PotB_ S.rubidae
: ------------------------------ ---------------PQKVRPLLLFLLIVPFWTNSLIRIYGLKIFLSTKGYL 37
PotB_ E.coli
:MALIATLACLVLGYPFAWFL AKLPHKVRPLLL FLLIVPFWTNSLIRIYGLKIFLSTKGYL 120
* :* ******** *******************************
TM4
PotB_ S.rubidae
:NEFLLWLGVIETPIRIMFTP GAVIIGLVYILLPF MVMPLY SSIEKLDK PLLEAAKDLGAS
PotB_ E.coli
:NEFLLWLGVIDTPIRIMFTP SAVIIGLVYILLPF MVMPLY SSIEKLD KPLLEAARDLGAS 180
97
****************************************************************** *********
TM5
PotB_ S.rubidae
:KLQTFVRIIIPLT MPGIIAGCLLVMLPA MGLFYVSDLMGGAKNLLIGNVIKSQFLNIRDW 157
PotB_ E.coli
:KLQTFI RIIIPLTMPGIIAGCLLVMLPA MGLFYVSDLMGGAKNLLIGNVIKVQFLNIRDW 240
****** : *******************************************************************
TM6
PotB_S.rubidae
:PFGSATSITLTVVMGLMLLVY WRASRLLNKKVELE
192
PotB_ E.coli
:PFGAATSITLTI VMGLMLLVY WRASRLLNKKVELE
275
*** : ****************************************
Fig 3 Sequence alignment of putative PotB protein from S. rubidae and PotB from E. coli Acc Number M 64519.1 which were aligned using the Clustal W
algorithm. Grey boxes indicate the six predicted transmembrane segments (TM1 to TM6). Consensus symbols; identical residues: ''*'', conserved substitutions:
'':'', semi-conserved substitutions: ''.''.
TM1
PotC_ S.rubidae
: MIGRLLRGGFMTAIHAYLYIPIIILIV NSFNSSRFGINWQ GFTTKWYGLLMNNDSLLQAA
PotC_ E.coli
: MIGRLLRGGFMTAIYAYLYIPIIILI VNSFNSSRFGINWQ GFTTKWYSLLMNNDSLLQAA
60
60
***************** : ************************************** .****************
TM3
TM2
PotC_ S.rubidae
:QHSLTMAVFFATFAT LIGSLTAVAL YRYRFRGKPFVSGML FVVMMSPDIVMAISLLVLFM 120
PotC_ E.coli
:QHSLTMAVFSATFAT LIGSLTAVAL YRYRFRGKPFVSGML FVVMMSPDIVMAISLLVLFM 120
***********
************************************************ ***************
TM4
PotC_ S.rubidae
:LLGVQLGFWSLLFSHITFCLPFVVVTVYARLKGFDVRMLEAAKDLGASEMTILRKI ILPL 180
PotC_ E.coli
:LLG I QLGFWSLLFSHITFCLPFVVVTVYSRLKGFDVRMLEAAKDLGASEFTILRKIILPL 180
*** : ****************************** * :************************** :*** *********
TM5
PotC_ S.rubidae
:AMPAVAAGWLL SFTLSMDDVVVSSFVTGPSYEILPLKIYSMVKVGVSPEVN ALATILLVL 240
PotC_ E.coli
:AMPAVAAGWVISFTLS MDDVVVSSFVTGPSYEILPLKIYSMVKVGVSPEVNALATILLVL 240
************* : ************************************************************* *
TM6
PotC_ S.rubidae
:SLVLVIASQVI ARDKTK - SQGTQK
PotC_ E.coli
:SLVMVIASQLI ARDKTKGNTGDVK
*** : ***** : *********
.
*
263
264
*
Fig 4 Sequence alignment of putative PotC protein from S. rubidae and PotC from E. coli Acc number M64519.1 using the Clustal W algorithm. Grey
boxes indicate the six predicted transmembrane segments (TM 1 to TM 6). Consensus symbols; identical residues: ''*'', conserved substitutions: '':'', semiconserved substitutions: ''.''.
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SUHANDONO ET AL.
Microbiol Indones
PotD_S.rubidae
PotD_E.coli
:MKKM - - - LAAAATGARNWAAAHAGDCKTLYFYN WTEYVPPGLLEQFTKETGIKVIYSTYE 57
:MKKWSRHLLAAGALA LGMSAAHADDNNTLYFY N WTEYVPPGLLEQFTKETGIKVIYSTYE 60
***
* ** . : * . : **** . * .* : ******************************************
Signal sequence
βA
α1
βB
PotD_S.rubidae
PotD_E.coli
:SNETMYAKLKTYKDGAYDLVVP STYF VDKMRKEGMIQKIDKSKLTNFSNLDPEMLNKPED 117
:SNETMYAKLKTYKDGAYDLVVP STYYVDKMRKEGMIQKIDKSKLTNFSNLDPDMLNKPED 120
*********** ********************** : ********************************** : *********
α2
βB
α3
PotD_S.rubidae
PotD_E.coli
:PNNDYSIPYIWGATAIGVNSDA I DPKRVSSWADLWKPEYKNSL ---------------------------------- 160
:PNNDYSIPYIWGATAIGVNGDAVDPKRVTSWADLWKPEYKGSLLLTD DAREVFQMALRKL 180
*********** ************ . ** : *** * : **************** . **
βD
βE
βF
α4
PotD_S.rubidae
PotD_E.coli
: --------------------------------------------------------------------------------------- --------------------------------:GYSGNTTDPKEIEAAYNELKKLMPNVAAFNSDNPANPYMEGEVNLGMIWNGSAFVARQAG 240
α5
βG
α6
βH
α7
PotD_S.rubidae
PotD_E.coli
: -----------------------------------------------------------------------------------------------------------------------: TPIDVVWPKEGGIFWMDSLAIPANAKNKEGALKLINFLLRPDVAKQVAETIG YPTPNLAA
βI
βJ
α8
α9
α10
PotD_S.rubidae
PotD_E.coli
: -----------------------------------------------------------------------------------------------: RKLLSPEVANDKTLYPDAETIKNGEW QNDVGAASSIYEEYYQKLKAGR 348
α11
α12
α13
300
Fig 5 Sequence alignment between putative PotD protein from S. rubidae and PotD from E. coli Acc. number M 64519.1 Based on Sugiyama et al. (1996)
the active residue which binds to spermidine are indicated by grey boxes. Consensus symbols; identical residues: ''*'', conserved substitutions: '':'', semiconserved substitutions: ''.''.
Volume 4, 2010
Microbiol Indones
95