2) In class, we investigated the problem of horizontal transfer of antibiotic ge

Question

2) In class, we investigated the problem of horizontal transfer of antibiotic genes between bacteria found in animals routinely fed antibiotics and bacteria that might affect human health. We focused on ermB gene that enables bacteria to resist the antibiotic erythromycin (A drug commonly used in both therapeutic and agricultural applications). Investigate the same biological problem by doing BLAST search for another potential gene. You need to mention whether you think that there is horizontal gene transfer between bacteria found in animals and bacteria that might affect human health based on your results.

Some potentially helpful information:

Antibiotic Erythromycin

We will focus on genes enabling bacteria to resist the antibiotic erythromycin

A drug commonly used in both therapeutic and agricultural applications

Query Sequence-ermB

As our query sequence, we use an erythromycin-resistance gene called ermB from Streptococcus agalactiae

ermB gene: accession number DQ355148.1

Identifying genes similar to ermB with BLAST

From the NCBI BLAST home page, paste or upload your S. agalactiae ermB sequence

NCBI BLAST home page

http://blast.ncbi.nlm.nih.gov/Blast.cgi?PRO GRAM=blastn&PAGE_TYPE=BlastSearch&LIN K_LOC=blasthome

Notice the section labeled Choose Search Set, where you can specify the sequences to be searched

Importantly, the default set of sequences is the subset of GenBank containing human DNA sequences

This obviously will not work in our case, where we want to retrieve bacterial sequences

Change the database to nucleotide collection (nr/nt), which will search all the unique (“nonredundant” or nr) sequences in GenBank

Many sequences in GenBank are from bacteria that have been sequenced (using DNA harvested from an environmental sample) but never cultured; these are not useful to us because we do not know what species they come from

So check the box to exclude sequences from uncultured samples

There is also an input box where you can limit your search to a particular organism or group of organisms

you could type “bacteria” here to exclude any nonbacterial sequences that might happen to match

Choose blastn for now to see both very similar and less-similar sequences the program might identify

Click the BLAST button to start the search and compare your ermB sequence with the selected sequences

The top-matching sequences are extremely similar

Such similar sequences found in many different species strongly suggests horizontal gene transfer rather than mutation over a long time of descent from a common ancestor

Retrieving Sequences

We will carry out a multiple alignment of some ermB genes from different species, which requires retrieving their sequences in FASTA format

Sequences

Streptococcus_pneumoniae: A significant human pathogenic bacterium

Staphylococcus_aureus: fish pathogen affecting saltwater fish

Lactococcus_garvieae: frequently found in the nose, respiratory tract, and on the skin

Bacillus_cereus: commonly found in soil and food

The Data

CH04FileofdiverseermBsequencessuitablefort heClustalanalysis.txt

ClustalW

http://www.ebi.ac.uk/Tools/msa/clustalw2/

http://clustalw.ddbj.nig.ac.jp/

Explanation / Answer

To demonstrate that Ampicilin resistance gene of Enterobacter cloacae (ampR) we first downloaded the gene sequence fron NCBI-Genebank as a FASTA file.

>AB016612.1 Enterobacter cloacae ampR gene, complete cds
TAGTCTGTTGGATTTGACTGTGGCTGACAAACGGTTAAATTTAGCATTAGCTGTTAATTTTTCTAACGGA
TGTAATGATGACCCGCAGCTATCTCCCGCTTAATTCGCTTCGCGCTTTCGAAGCTGCCGCCAGACACCTC
AGTTTTACGCATGCCGCGATAGAGCTGAACGTGACTCACTCTGCCATCAGCCAGCACGTTAAAGCGCTGG
AGCAGCATCTGAATTGTCAGCTGTTTGTCCGCGTTTCGCGCGGGCTGATGCTGACCACGGAAGGTGAAAA
CTTGCTGCCGGTGTTGAATGATTCATTCGATCGGATCGCCGGAATGCTGGATCGCTTTGCCAGTCATCGC
GCCCAGGAGAAGCTAAAAGTGGGTGTGGTCGGCACCTTTGCTACCGGTGTTTTATTCTCGCAGCTCGCGG
ACTTTCGTCGATGCTATCCGCATATCGATCTTCATCTTTCGACCCATAACAACCGCGTCGATCCCGCGGC
GGAAGGGCTGGATTATACGATTCGCTACGGTGGCGGGGCGTGGCACGGCACTGAAGCGCAGTTCCTGTGT
TCTGCACCGCTGTCCCCGCTTTGCTCACCCGATATTGCTCTCGGGCTCCAGTCTCCGGCCGACATTCTGA
AGTTTACCCTGCTGCGCTCCTACCGACGCGACGAATGGTCGGCGTGGATGCAGGCGGCAGGTGAACATCC
CCCGTCACCCACGCATCGGGTGATGGTCTTTGATTCGTCCGTGACCATGCTGGAAGCCGCGCAGGCAGGT
GTGGGCATCGCCATAGCACCTGTGGATATGTTTACCCATCTGCTCAATAGCGAGCGTATCGTGCAGCCGT
TTGCCACCCGGATTGACCTGGGGAGTTACTGGCTCACGCGATTACAGTCGAGGGCTGAAACCCCCGCAAT
GCATGAGTTTGCTCAATGGCTGGTGGGAAAAATGCAGAAATAAAAGGCCG

Then we opened the FASTA file in Word software and copied the gene sequence of ampR of Enterobacter cloacae pasted it in the seaech box of NCBI-BLASTn webpage.

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch

choose set boxes were unfilled as we wanted to BLASTn search for whole sequence. We can specify the region of gene sequence we want to do BLASTn searcgh by using these boxes

In the Data base box "nucleotide collection nr/nt" was selected and "human" option was unselected.

In the organism box "bacteria taxid:2" was selected.

Then the sequence was subjected for BLASTn search.

https://blast.ncbi.nlm.nih.gov/Blast.cgi

Enterobacter cloacae-ampR gene sequence showed most similaritity with sequence of Enterobacter asburiae ( causes necrotizing fasciitis disease in humans), Enterobacter ludwigi (causes human nosocomial bloodstream infections) and Klebsiella pneumoniae (causes pneumonia ).

The aligned sequences of the above mentioned bacteria are downloaded

>CP011863.1 Enterobacter asburiae strain ATCC 35953, complete sequence
GGCCTTTTATTTCTGCATCTTCCCCACCAGCCACTGGGCAAACTCGCGCATTGCGGGCGTTTCTGTCCGTGACTGTAGTC
TTGTGAGCCAGTAGCTGCCGAGGTCAATCTGCGTTGCAAACGGCTGCACGATGCGCTCGCTGCTGAGTAAATGGGTAAAC
ATGTCGACAGGCGCAATCGCAATCCCTACGCCTGCCTGCGCGGCTTCCAGCATGGTCACGGACGAATCAAACACCATCAC
CCGGTGCGTCGGCGAGGGAGGATGTTCACCCGCTGCCTGCATCCACGCAGCCCACTCGTCGCGCCGGTAGGAGCGCAGAA
GGGTAAACTTCAGGATATCTGCCGGGCTTTTGAGAACTGAGGCTATGTCAGGTGTACAGAGCGGAGACAGCGGCGCAGGG
CAAAGGAATTCAGCATCGGTTCCATGCCACGCGCCGCCACCGTAGCGAATAGTGTAATCAAGCCCTTCGGCCGCCGGATC
GACGCGATTGTTATGGGTGGAAAGCTGAAGATCAATATGCGGATAGCAGCGGCGAAAGTCCGCGAGCAGCGAGAATAAAA
CACCCGTGGCAAAAGTCCCTACCACACCTACTTTAAGCTTCTCCTGAGCGCGCTGGCTGGCAAAACGATCCAGCATTCCG
GCGATGCGATCGAACGAGTCATTCAGCACCGGCAGCAAATTCTCGCCTTCGGTGGTCAGCATCAGCCCTCGCGAAACGCG
GACAAACAGCTGGCAATTAAGGTGCTGCTCCAGCGCCTTGACGTGCTGGCTAATCGCGGAATGGGTGACGTTCAGCTCGA
TGGCGGCATGAGTAAAACTGAGATGCCTGGCAGCCGCTTCAAACGCCCGCAGGGAATTAAGAGGGAGATAGCTGCGCGTC
ATGGTCTCGTCCGTTAGAAAAATTAACAGCTAATGCTAAATTTAACCGTTTGTCAGCCATAGTCAAATCCAACAGACTA

>CP017279.1 Enterobacter ludwigii strain EN-119, complete genome
AGTCTGTTGGATTTGACTGTGGCTGACAAACGGTTAAATTTAGCATTAGCTGTTAATTTTTCTAACGGATGGGTCGATGA
CACGCAGCTATTTACCGCTTAATTCGCTTCGTGCCTTCGAAGCCGCTGCAAGGCACCTCAGTTTTACTAATGCTGCCATT
GAGCTGAATGTCACCCATTCAGCCATCAGTCAGCACGTTAAGACGCTGGAACAGCATCTGAACTGTCAGCTGTTTGTCCG
GGTTTCGCGCGGATTGATGCTGACGACAGAGGGCGAGAATCTGCTGCCGGTGCTGAACGATTCGTTTGATCGTATCGCCG
GCATGCTGGATCGCTTTGCCAGTCATCGCGCGCTGGAGAAGCTTAAAATTGGCGTGGTGGGGACTTTTGCGACCGGGGTG
TTGTTCGCGCAGCTGGATGATTTCCGTCGCCGCTATCCGCATATCGATCTGCATCTTTCGACCCATAATAACCGAGTCGA
TCCGGCCGCCGAAGGGCTTGATTACACGATCCGCTACGGTGGAGGGGCGTGGCATGGCACTGAAGCGACGTTTCTGTGTT
CTGCGCCGCTGGCTCCGCTGTGCACGCCTGAAATTGCCGTCGGGCTACAAGCACCGGCGGACATACTAAAATTTACGCTG
CTGCGCTCCTACCGACGCGACGAATGGTCGGCGTGGATGCAGGCCGCGGGCGAACTCCCGCCGTCACCAACGCATCGGGT
GATGGTGTTTGATTCATCCGTTACCATGCTGGAAGCCGCGCAGGCCGGCGTGGGAATTGCTATCGCGCCTGTGAGTATGT
TCACTCATCTGCTGAACAGCGAACGTATCATTCAGCCGTTTGCGACACAGATTGAGCTGGGCAGTTACTGGTTAACCCGA
TTGCAGTCACGGACAGAAACCCCTGCAATGCGTGAGTTTGCACAGTGGCTGGTGGGGAAAATGCAGAAATGACAGGCC

>AF362955.1 Klebsiella pneumoniae AmpR (ampR) gene, complete cds; and Act-1 beta-lactamase (act-1) gene, partial cds
GGCCTTTTATTTCTGCATCTTCCCCACCAGCCACTGGGCAAATTCGCGCATTGCGGGCGTTTCTGACCGTGACCGTGACT
GTAGCCTTGTCAGCCAGTAGCTGCCGAGATCAATCTGCGTTGCAAACGGCTGCACGATGCGCTCGCTGCTGAGTAAATGG
GTAAACATGTCGACAGGCGCAATGGCAATCCCTACGCCTGCCTGCGCGGCTTCCAGCATGGTCACGGACCAATCAAACAC
CATCACCCGGTGCGTCGGCGAAGGAGAATGTTCGCCCGCTGCCTGCATCCACGCAGCCCACTCGTCGCGCCGGTAGGAGC
GCAGCAGGGTAAACTTCAGGATATCTGCCGGGCTTTTGAAAACTGAGGCAATCTCGGGTGTACAGAACGGAAACAGCGGC
GCAGGGCAAAGGAATTCAGCATCGGTTCCATGCCACGCGCCGCCACCGTAGCGAATAATGTAATCAAGCCCTTCGGCCGC
TGGATCGACGCGATTGTTATGGGTGGAAAGCTGAAGATCAATATGCGGATAGCAGCGGCGAAAGTCCGCGAGCAGCGAGA
ATAAAACACCCGTGGCAAAAGTCCCTACCACACCTATTTTGAGCTTCTCCTGAGCGCGCTGGCTGGCAAAACGATCCAGC
ATTCCGGCGATGCGATCGAACGAGTCATTCAGCACCGGCAGCAAATTCTCGCCTTCGGTGGTCAACATCAGCCCTCGCGA
AACGCGGACAAACAGCTGGCAATTAAGGTGCTGTTCCAGCGCCTTGACGTGCTGGCTAATCGCGGAATGGGTGACATTCA
ACTCGATAGCGGCATGAGTAAAACTGAGATGCCTGGCGGCTGCTTCAAACGCCCGCAGGGAATTAAGAGGGAGATAGCTG
CGCGTCATGGTCTCGTCCGTTAGAAAAATTAACAGCTAATGCTAAATTTAACCGTTTGTCAGCCATAGTCAAATCCAATA
GACTA
Thus the similarities in the sequence of ampR gene indifferent bacterial species causing digestive system diseases in human, strongly suggest occurance of horizontal gene transfer among these bacteria species.

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