LSM2104 Group Project

Weiwei’s Page (U052166X)

CP000521

1. FASTA format for CP000521

    Data retrieved from NCBI Genome

2. Sample Sequences of Antibiotic Determinants doc.file

    For each sample sequence of each antibiotic determinant, perform blast against the Acinetobacter blastable database at: http://sf01.bic.nus.edu.sg/blast/.

     Note: There are in total 12 sample sequences provide under RND pumps

MexB     BpbB     AdeY     AdeE     AdeJ      AdeB     BpeA     MaxA    

AdeX     AdeD     AdeI       AdeA     OprM     AdeZ     AdeK     AdeC

Acinetobacter baumannii ATCC 17978, complete genome

   4. RND Effe Pump v.s. CP000521

       Eg. Steps:   1. Blast MexB against Acinetobacter database at http://sf01.bic.nus.edu.sg/blast/. (tblastn)

                          2. One sequences on CP000521 was found from 3173131-3176265

                          3. Type in CP000521 in ORF finder and the rough region of 3173000-3177000

                          4. Getting ORF from 3173131-3176307

                          5. Question: Are the blast result and the ORF same sequences (doubt because they are in different frame)?

                          6. Blast two sequence: MexB and ORF. If the blast result and ORF found similar score with MexB they could                               be the same.  (blastp)

                          7. Get the protein sequence and DNA sequence

                                       Note: If want to make sure the DNA sequence is exact same as 3173131-3176307 on CP000521,

                                       perform blast against Acinetobacter database at http://sf01.bic.nus.edu.sg/blast/ (blastn)

 

  5. Results of annotation:

             5.1         MexB    BpbB    AdeY     AdeJ

                          The above 4 genes have the same blast results.

                          ORF protein sequence

                          ORF DNA sequence

                          Pfam :

                               Trusted matches: ACR_tran

                               Matches to Pfam-B: Pfam-B_8122

 

             5.2         AdeE

                          ORF protein sequence

                          ORF DNA sequence

                          Pfam

                               Trusted matches: ACR_tran

                               Matches to Pfam-B: Pfam-B_8122

 

             5.3         AdeB    

                          ORF protein sequence:

                          ORF DNA sequence:

                          Pfam :

                               Trusted matches: ACR_tran

                               Matches to Pfam-B: Pfam-B_8122

 

             5.5         BpeA     MaxA    AdeI      AdeX     AdeA     AdeD

                          The above 6 genes have the same blast results.

                          ORF protein sequence:

                          ORF DNA sequence:

                          Pfam :

                                       Trusted matches HlyD

                                       Potential matches: TPR_MLP1_2, DUF260

 

             5.6         AdeZ     AdeK     AdeC     OprM

                          The above 4 genes have the same blast results.

                          ORF protein sequence:

                          ORF DNA sequence:

                          Pfam

                                       Trusted matches             OEP      OEP

                                       Potential matches           HP_OMP           MSP1_C

 

             Brief summary:

                          RND Efflux pump operon contains 3 structure proteins.

                          3171868-3173118: Periplasmic linker protein       HlyD

                          3173131-3176307: Inner membrane protein         ACR_tran

                          3176307-3177761: Outer membrane protein        OEP OEP

 

6. Check the presence of promoter

    6.1 1000 nucleotides upstream of AdeI

             http://www.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb

       Running results (3170868-3171868)

             > test sequence                                                                

                          Length of sequence-      1001

                          Threshold for promoters -  0.20

                          Number of predicted promoters -      3

                          Promoter Pos:    540 LDF-  6.93

                                       -10 box at pos.    525 TTGTATGTT          Score    51

                                       -35 box at pos.    501 TTGCAC                Score    33

                          Promoter Pos:    240 LDF-  3.14

                                       -10 box at pos.    228 TGTTAAAAA         Score    48

                                       -35 box at pos.    206 TTGTTG                Score    39

                          Promoter Pos:    942 LDF-  2.07

                                       -10 box at pos.    927 GGTTATTAC         Score    38

                                       -35 box at pos.    909 TTGCAC                Score    33

                          Oligonucleotides from known TF binding sites:

                          For promoter at    540:

                                        rpoD17:  ATACTATA at position              483 Score -  11

                                        rpoD17:  TTTTGTAT at position               523 Score -   9

                                        argR:  TTTTTTAT at position                    532 Score -  13

                                       tus:  TAGTATGT at position                      546 Score -  16

                                       cysB:  TGTATATA at position                  551 Score -  12

                          For promoter at    240:

                                        fis:  AAATGTGA at position                      186 Score -  11

                                        rpoD19:  ATTGTTTT at position               214 Score -   7

                                        ihf:  TTTCAAAA at position                       239 Score -   6

                                        glpR:  TTCAAAAT at position                    240 Score -   6

                          For promoter at    942:

                                        ihf:  TTTTATTT at position                        898 Score -  13

 

    6.2  Prediction of potential genes

              Seq name: gi|126385999:3170868-3177761 Acinetobacter baumannii ATCC 17978, complete geno

              3170868-3177761

              Length of sequence - 6894 bp

              Number of predicted genes - 5

              Number of transcription units - 2, operons - 1

 

3.  Relationship of RND Effe Pump

1.AdeZ   AdeK   AdeC       OprM     

(These 4 genes code for outer membrane protein. )

 

3.1.2.1.1   BpeB      MexB      AdeJ      AdeY     

3.1.2.1.2   AdeE     

3.1.2.1.3   AdeB     

 (These 6 genes have the same functional annotation from Pfam. Inner membrane protein.)

 

3.1.2.2.1   AdeI      AdeX     

3.1.2.2.2   MexA      BpeA     

3.1.2.2.3   AdeA     

3.1.2.2.4   AdeD     

 (These 6 genes have the same ORF and functional annotation from Pfam.

   Periplasmic linker protein.)