Question

A microbiologist sequenced the genome of Streptococcus species that can cause bacterial meningitis serine-rich repeat (SRR) (Accession No.DQ174691.1).

Q1: Find the open reading frame encoding the serine-rich repeat protein and to identify several other proteins in this DNA sequence using an Open Reading Frame finder and BLAST.  

Q2: Are the longest open reading frames transcribed in both directions on the DNA (located on the + and - strand) or are they transcribed in only one direction?

Q3: Which ORF encodes the SRR protein? List its nucleotide position from the table?

Q4: List three different organisms in which SRRs have been identified and list three different names that have been given to SRRs (not including "unknown" or "unnamed" protein). What kind of organisms are these?

Q5: Identify four other ORFs in this sequence listing the most frequent name found in the BLAST output for each ORF. Because most of these gene names reveal little about function, use PubMed to find abstracts related to each gene and describe the role of each of these genes in one sentence?

Answer 1

The bacteria Streptococcus causes bacterial meningitis. Using BLAST software and ORF reader we can view the details of the genome of the bacteria.

If you don't know how to use the BLAST tool:

BLAST tool: Google- BLAST, NCBI; then click on BLAST, then as the portal open click on nucleotide/ protein BLAST whatever is required, copy the accession no given in the question, Paste it in the box placed on the left-hand side of the screen, then scroll down and click on BLAST, you will get entire details of the organism in the result viewer.

ORF Finder: Google ORF finder; copy the accession no. in the white box given there, and then enter the SUBMIT button.

Now after searching for the ORF, you will get the result viewer as below:

As you can see, ORF 20 is the corresponding open reading frame which codes for serine-rich repeats that is responsible for causing bacterial meningitis. The FASTA sequence (in amino acid format) is given below on the left-hand side below. And the alternate ORFs in the sequence of the streptococcus genome are given in the image above and also below the right-hand side.

.The ORF 20 in the amino acid format is as follows :

>lcl|ORF20
MLKKQFGNFGEKSRKVRVKMRKSGKHWVKSVMTQIGYVILSRFSGKEKSS
KVQTTSEDLSRTKTSASILTAVAALGAVVGGTTDTTSVSAEETPTATELT
GNEKTLATAETVVVAPEVKTVNSDSSSHSTSESQSMSTSTLQSTSASLSA
SESLMDSTSASLSESSSLSEYSSLSLSSSESVSASESVQSSEAATTARVQ
PRAMRVVSSASDMETLPAALISGEGDVTTVQGQDVTDKLQNLDIKLSGGV
QAKAGVINMDKSESMHMSLKFTIDSVNRGDTFEIKLSDNIDTNGASNYSI
VEPIKSPTGEVYATGIYDSQKKSIVYSFTDFAASKNNINGILDIPLWPDD
TTVQNTKEDVLFSVKIKDQEATIKETVKYDPPVRIDFAGGVSVDSRITNI
DDVGKKMTYISQINVDGKSLYNYNGLYTRIYNYSKESTADLKNSTIKIYK
TTSDNIVESMVQDYSSMEDVTSKFANSYPEKGWYDIYWGQFIASNETYVI
VVETPFTNAVTLNTTLSDYNENNGVEHNHTYSSESGYSDVNAQERKILSE
LVSSSESVSSSESVSNSESISTSESVSNSESISSSESVSSSESISTSESV
STSESISSSESVSSSESVSSSESISSSESVSNSESISSSESVSNSESISS
SESVSSSESISNSESISSSESVSTSESISSSESVSNSESISSSESVSSSE
SISNSESISSSESVSTSESISNSESVSSSESVSTSESISSSESVSNSESI
STSESVSTSESISSSESVSSSESISSSESVSNSESISNSESVSSSESVSN
SESISSSESVSNSESISTSESVSTSESISSSESVSNSESISSSESVSNSE
SISSSESVSNSESISSSESVSNSESISSSESVSSSESVSSSESISTSESV
SNSESISSSESVSNSESISSSESVSNSESISSSESVSNSESISSSESVSS
SESISSSESVSSSESVSNSESISSSESVSNSESISSSESVSSSESISSSE
SVSNSESILSSESVSSSESISSSESISSSESVSMSTTESLSESEVSGDSE
ISSSTESSSQSESMNHTEIKSDSESQHEVKHQVLPETGDNSASALGLLGA
GLLLGATKSRKKKKD

If you click on the blue bar or a red line showing ORF 20 in the image give you other details regarding the ORF.

2. The longest open reading frames of some of the viruses are present in both directions and in positive-strand as well as a negative strand. The positive-strand has the same direction as mRNA, so it can be directly transcribed, but negative-strand has ORF complementary to the mRNA so it requires RNA polymerase to reverse transcribe it to positive-stranded DNA and then is transcribed to mRNA. Thus, it has ORF in both directions and it also transcribes in both directions, but the positive-strand transcribes faster than the negative strand.

3. The ORF 20 is positive-stranded and 1115 amino acids, coded by 3348 nucleotides. The ORF coding for SRR is from 5606-8953 nucleotides in the location of the entire genome.

For the fourth question, you need to go for the BLAST tool.

From the BLAST result, you will get similar sequences, as the BLAST tool is to find similar sequences as the query sequence across the entire NCBI database and its linked databases or compare.

4. So, Clicking on the BLAST and further the Protein BLAST and copying the protein sequence (ORF20) we got earlier (in amino acid form) into the query sequence, scroll down further and click on BLAST.

The window shown above will appear if you scroll down the protein result viewer. Here, You can see on left-hand-side the name of the proteins which has similar/identical sequences to that of the SRR sequence of amino acid. The sequence identity to that of SRR protein is given on the right-hand side of the respective name.

Different names for the protein are cell-wall anchor protein, adhesin protein, fibrinogen binding adhesin Sdr G C-terminal domain-containing protein, YIbF, YmcA competence regulator protein which is present in the same organism, Streptococcus agalactiae.

Some of the other organism which has a similar type of protein are Lactobacillus sp., such as Lactobacillus pros, Lactobacillus jonsonni, Streptococcus vestibularis, Streptococcus mitis. These organisms have the same repeats but slightly different amino acid sequences from the given data.

5. Other ORFs and their function are given below:

Total of 83 ORFs have been found in the given sequence:

1. ORF 37: A unique serine repeat-rich protein and novel surface antigen( epsilon ) associated with a virulent lineage of serotype 3 of the organism.

2. ORF 8: Similar to Sec A, putative ATPase component of the SECY2_A2 secretion locus; probably involved in srr2 .

3. ORF 7 and ORF 23: Accessory secretory protein probably involved in srr-2 secretion.

I hope this helps!