Question

More on how electrons move, and some tools you can use to ask your own questions:
You have become interested in the anaerobic respiration pathways of Desulfovibrio vulgaris, and specifically in the question of whether or not it can utilize sulfate as a terminal electron acceptor. To see whether it might use sulfate you have decided first to consult the genome sequence, focusing on D. vulgaris strain Hildenborough. A database of all the D. vulgaris str. Hildenborough genes (and complete genome sequences for many other microbial species) is maintained at the National Center for Biotechnology Information.

A. Navigate to the NCBI website and use the tools you find there to see whether D. vulgaris str. Hildenborough has genes encoding sulfate adenylyltransferase, adenylylsulfate reductase, and sulfite reductase (dissimilatory type). If you find them, provide the ‘locus tag’ given for one or more of the protein subunits (DVU####). If you do not find genes for these enzymes, consult with your T.A. or with a colleague from the class.

B. On discovering how easy it is to address such questions using the tools available at NCBI, you realize you’d also like to know whether or not D. vulgaris str. Hildenborough can use nitrate to support respiration. Key enzymes are nitrate reductase and nitrite reductase. Use the NCBI site to determine whether the D. vulgaris genome encodes genes for nitrate reductase and nitrite reductase. Provide the locus tags if you find the genes; if you do not find them, consult with your T.A. or a colleague from the class.

C. If D. vulgaris uses sulfate as a terminal electron acceptor and conserves energy by the accepted mechanism (section 13.18), then it is expected to have a low-potential periplasmic cytochrome, cytochrome c3. Use the tools available on the NCBI site to search for the cytochrome c3 gene. Restrict your search to strain Hildenborough. You should find several genes that encode various cytochromes c3. One of these is of particular interest because it is likely to interact with a hydrogenase. Click on the link for DVU2524, and on the page that opens, in the ‘Genomic context’ area, mouse over the genes adjacent to DVU2524. What genes do you find adjacent to DVU2524? (Provide the names of enzyme(s) they encode, not just locus tags.)

D. Another cytochrome c is thought to form part of the cytochrome hmc complex—and is named accordingly. Identify this one (provide the DVU number).

E. By using appropriate links on the NCBI website (which you are feeling impelled to explore at length now that you realize how much useful stuff is there), find the amino acid sequence of the cytochrome c protein that is encoded in the hmc operon (hint: on the hmc gene page, scroll down to the “mRNA and protein” part). Paste this sequence into your problem set answers (it should start with MSEDV...). It’s helpful to select “FASTA” at the top of the page, to obtain the sequence in FASTA format, which has a carat and the protein name on the first line.

F. Impressed by the unusual size of this cytochrome c, you want to find out what is known about its structure. Copy its amino acid sequence, then navigate to the “protein BLAST” page of the NCBI website and paste the sequence into the appropriate box, using FASTA format (retaining the line with the carat). Carry out a protein-protein BLAST search, specifying that the search should use the protein structure database, called the pdb (if you don’t, it will search the much larger database of nonredundant protein sequences). The search is initiated by the “BLAST” button lower down on the page. You should find some strong ‘hits’ to protein sequences in the pdb. Make a note of the pdb “accession number” for the top hit (given in the ‘accession’ column). Take just the first four characters (the characters _A are not necessary). Write this accession in your problem set answer.

G. Navigate to the RCSB website. In the search box, enter the four-character code (the accession number) you’ve just found and press return, An image of the protein should come up. Click on the “structure” link next to “3D view” below the image. You can manipulate the structure by moving the cursor on it while holding the mouse button down.

i. How would you describe the overall shape of this protein?

ii. What else do you find remarkable about this protein?

iii. Given its structure and the prosthetic groups present, what sort of function(s) do you think the protein might be suited for?

Note that if you’re used to viewing macromolecular structures in another program, you can also just download the coordinates of the protein structure from the RCSB page.

Answer 1

A) To get the Locus tags,

-Go to the homepage of the NCBI website.

-In the search bar, Choose gene, in the smaller left box, and in the right search bar Type the keywords "sulphate adenosyltransferase" and "Desulfovibrio vulgaris strain Hildenborough " and click on search.

- The appropriate results are chosen, and the locus tag is obtained.

sulphate adenosyl transferase-DVU1295

adenylylsulfate reductase- subunit alpha-DVU0847

subunit beta -DVU0846

sulfite reductase- sub unit alpha-DVU0402

  subunit beta -DVU0403

     subunit gamma -DVU2776

B) The same procedure as (A) is followed and the following results are obtained.

nitrate reductase-sub unit gamma-DVU1290

nitrite reductase-catalytic sub unit- DVU0625

C) The genes that code for enzymes, adjacent to cytochrome c3 are:

To the right:

periplasmic [NiFe] hydrogenase small subunit, isozyme 2 - hynB-2

periplasmic [NiFe] hydrogenase large subunit, isozyme 2- hynA-2

To the left:

Lipoprotein- DVU2523

Shikimate kinase- aroK

D). The protein mentioned was:

hmcA high-molecular-weight cytochrome C [ Desulfovibrio vulgaris str. Hildenborough ]- DVU0536