Working hypothesis on Mycobacteriophage Receptors

 

 

If  mycobacteriophages L5, D29, Bxz2 and TM4  are capable to growth on Mycobaterium smegmatis MC2 155 and on Mycobacterium ulcerans then these mycobacteria must have the same receptor  or a different receptor for each of  these mycobacteriophage.


Phage..M.ulcerans Receptor (R1) ...M.smegmatis Receptor(R2)

L5..................... R1L5................................................ R2L5


D29................. R1D29.............................................. R2D29


Bxz2............... R1Bxz2............................................. R2Bxz2


TM4 ...............R1TM4.............................................. R2TM4


Suppositions:

M.ulcerans Receptor(R1) 
 
1)R1L5,R1D29,R1Bxz2,R1TM4 (the receptor is the same)
or
R1L5,R1D29,R1Bxz2,R1TM4 (the receptors are different one another).


M.smegmatis Receptor(R2)
 
2)R2L5,R2D29,R2Bxz2,R2TM4 (the receptor is the same)
or
R2L5,R2D29,R2Bxz2,R2TM4 (the receptors are different one another).

Combination:

3)R1L5,R1D29,R1Bxz2,R1TM4 and R2L5,R2D29,R2Bxz2,R2TM4 (the receptor is the same)
or
R1L5,R1D29,R1Bxz2,R1TM4 and R2L5,R2D29,R2Bxz2,R2TM4 (the receptors are different one another).

D29 phage integrase, Prophage and prophage integrase of M.ulcerans

My working hypotesis

"If before phiMU01 and phiMU02 prophages were active mycobacteriophages probably Phage integrase of  D29 mycobacteri0phage (or  L5) could have some common features with these prophages and with  the prophage integrase inside  the genome  of Mycobacterium ulcerans ".
This analysis could give strength to my conviction about the presence of a new specific and  unknown mycobateriophage usable  in Buruli disease but at moment this phage  has not been isolated in the soil or in fresch water or in other sources  .

Mycobacteriophage D29 

    D29p32

predicted 40.0 kD protein; integrase                      
                                                         

Integrase [DNA replication, recombination, and repair]; Region: XerC; COG0582

Phage integrase family; Region: Phage_integrase; pfam00589

Phage integrase, N-terminal SAM-like domain; Region: Phage_int_SAM_3; pfam14659

Statistics on the protein properties:
                                

Distinguishing mark:  VAL is present 27 times                            



  Prophage integrase sequences from Mycobacterium ulcerans genome.



I have chosen  MUL 0529:

 MUL 0529

Site-specific recombinase XerC [DNA replication, recombination, and repair]; Region: XerC; COG4973

DNA breaking-rejoining enzymes, C-terminal catalytic domain. The DNA breaking-rejoining enzyme superfamily includes type IB topoisomerases and tyrosine recombinases that share the same fold in their catalytic domain containing six conserved active site...; Region: DNA_BRE_C; cl00213

Statistics on the protein properties:

Distinguishing mark:  VAL is present 27 times

Comparison:

MUL 0529 Protein / D29p32 Protein Dot plot

D29p32 and MUL 0529 alignment

In this first part of the  analysis  is not  clear if my working hypotesis is confirmed but if I  use the observation: VAL is present 27 times, I can obtain my goal after 1 h0urs of work .


1-by Jemboss and by Wordcount software I select the word with 3 Aa  but I chose only the words that contain Val Aa.
2- in the protein sequence I write all positions of VAL Aa,the start and the end  of the  main miscellaneous features.
3- in the protein sequence I check the positions of the words with the  Val Aa.

In Position 91 in both the sequences is present VAL Aa .
In position 167 and 166 there is a VAL Aa.
LDV and TVP words are present in both the sequences and my working hypotesis is  in part confirmed.

phiMU01 prophage

In the alignment with phiMU01 prophage  and D29 phage  integrase 
I have the confirmation  of my working hypotesis.

By Swiss-Model there is  also the confirmation:

The  reference Model :

My contribution by Discovery Studio 4.0:

D29p03

CDS :   2106-2357, 252 bp,  : predicted 9.0 kD protein (83 Aa)
misc_feature: 2142-2294, 153 bp, SdiA-regulated; Region: SdiA-regulated; cl19046

By Snapgen software:

By Artemis software:

Aa sequence (83 Aa):

 

misc_feature Aa sequence:

Region: SdiA-regulated; cl19046

Statistics on the protein properties:

-No helix-turn-helix nucleic acid binding motifs
 
-No prediction of transmembrane segments in protein 

- No PEST motif as potential proteolytic cleavage site 

- N0 site of cleavage between a signal sequence and the "mature exported protein"



Virtual 2D-gel:

Open reading frames of Mycobacteriophage D29

The genetic code is the correspondence between triplets in DNA (or RNA) and amino acids in protein.

A codon is a triplet of nucleotides that represents an amino acid or a termination signal.

The sequence of a coding strand of DNA, read in the direction from 5'>to 3' , consists of nucleotide triplets (codons) corresponding to the amino acid sequence of a protein read from N-terminus to C-terminus.

The coding strand (Sense strand) of DNA has the same sequence as the mRNA(except for possessing T instead of U) and is related by the genetic code to the protein sequence that it represents.

The antisense strand (Template strand) of DNA is complementary to the sense strand, and is the one that acts as the template for synthesis of mRNA.


Gene expression occurs by a two-stage process:

1-Transcription generates a single-stranded RNA identical in sequence with one of the strands of the duplex DNA.

2-Translation converts the nucleotide sequence of mRNA into the sequence of amino acids comprising a protein. The entire length of an mRNA is not translated, but each mRNA contains at least one coding region that is related to a protein sequence by the genetic code: each nucleotide triplet (codon) of the coding region represents one amino acid.

The genetic code is read in non-overlapping triplets from a fixed starting point:
Non-overlapping implies that each codon consists of three nucleotides and that successive codons are represented by successive trinucleotides.

The use of a fixed starting point means that assembly of a protein must start at one end and work to the other, so that different parts of the coding sequence cannot be read independently.There are six potential reading frames in every DNA sequence, with three possible frames on one strand, and another three on the complementary strand.
All ORFs read in the 5' to 3' direction on a given sequence.

A reading frame is one of the three possible ways of reading a nucleotide sequence. Each reading frame divides the sequence into a series of successive triplets. There are three possible reading frames in any sequence, depending on the starting point.

If the first frame starts at position 1, the second frame starts at position 2, and the third frame starts at position 3.

An open reading frame (ORF) is a sequence of DNA consisting of triplets that can be translated into amino acids starting with an initiation codon and ending with a termination codon.

The initiation codon is a special codon (usually AUG) used to start synthesis of a protein.

A stop codon (Termination codon) is one of three triplets (UAG, UAA, UGA) that causes protein synthesis to terminate. They are also known historically as nonsense codons. The UAA codon is called ochre, and the UAG codon is called amber, after the names of the nonsense mutations by which they were originally identified.

A blocked reading frame cannot be translated into protein because of the occurrence of termination codons.

If the genetic code is read in non-overlapping triplets, there are three possible ways of translating any nucleotide sequence into protein, depending on the starting point. These called reading frames.

For the sequence:

A C G A C G A C G A C G A C G A C G the three possible reading frames are:

ACG ACG ACG ACG ACG ACG ACG
-CGA CGA CGA CGA CGA CGA CGA
--GAC GAC GAC GAC GAC GAC GAC

A reading frame that consists exclusively of triplets representing amino acids is called an open reading frame or ORF. A sequence that is translated into protein has a reading frame that starts with a special initiation codon (AUG) and that extends through a series of triplets representing amino acids until it ends at one of three types of termination codon.

A reading frame that cannot be read into protein because termination codons occur frequently is said to be blocked.

If a sequence is blocked in all three reading frames, it cannot have the function of coding for protein.

When the sequence of a DNA region of unknown function is obtained, each possible reading frame ( 6 for each double strand) is analyzed to determine whether it is open or blocked. Usually no more than one of the three possible frames of reading is open in any single stretch of DNA.

Open reading frames of Mycobacterium D29

1 2 3 4 5 6 10 11 12 13 14 15 16 17 18 19 19.1 20 21 22 23 24 25 26 27 28 29 30 31 32 32.1 33 34.1 36 36.1 38 39 41 41.1 42 43.1 44 44.1 46 NP_597858.1 48 49 50 51 52 53 53.1 54 55 56 57 58 59 59.1 59.2 61 62 63 64 65 66 66.1 68 68.1 69 70 orf_2 82.1 82.2 84 86 87 88 89

Mycobacterial Cell Wall

From Internet by Google: mycobacterial cell wall images





Schematic diagram of Mycobacterial cell wall.

1. outer lipids
2. mycolic acid
3. polysaccharides (arabinogalactan)
4. peptidoglycan
5. plasma membrane
6. lipoarabinomannan (LAM)
7. phosphatidylinositol mannoside
8. cell wall skeleton

New ( Year 2007)

Why Mycobacteriophage DS6A?

About eighteen years ago I was working on Mycobacterium tuberculosis and I was studying a specific Mycobacteriophage: DS6A. Before picking out this phage I had spent a lot of time for reading documents and scientific journals.
I selected this phage for its lytic power expressed on Mycobacterium tuberculosis complex (M.tuberculosis, M.bovis, M.africanum, M.microti).
The lytic activity of phage DS6A is always expressed on all strains collected from any part of the world. In the meanwhile C.R.Merril and colleagues wrote a scientific paper:
“ Long-circulating bacteriophage as antibacterial agents” Proc. Natl. Acad. Sci. USA, published in 1996.
After this reading I thought a potential application of phage DS6A for a peculiar pathological situation in the Man:Multidrug-resistant (MDR) tuberculous meningitis and AIDS.
This crazy idea was originated by one of features of Mycobacteria : the intracellular location. Mycobacteria reside in monocytes, reticuloendothelial cells and giant cells but when cause Meningitis they remain out off the cells and are available for a phage attack by intrathecal administration.
On that occasion I wrote a letter to C.R. Merril and intentionally I did not write to him my idea.
Today Antimicrobial (Drug) Resistance is a big problem and Phage Therapy if properly works might be a valid and cheap solution to the antibiotics.

Here

Mycobacterium phage L5 repressor

From :

Transcriptional silencing by the mycobacteriophage L5 repressor

I have verified if the L5 gp71 protein is a protein (183 Aa) with a binding capacity to the DNA.

See:

Helix-turn-helix

Basic-helix-loop-helix

DNA-binding protein

I have used mEMBOSS software (open source)for finding the part of the protein with a binding capacity:

"FNQTEIAELYGVTRQAVSWHKK"





















This is the primary and secondary structure of L5 gp 71 repressor:












L5 is a lysogenic mycobacteriophage growing in mycobacteria and for that reason also in M.smegmatis and in M.ulcerans .

The lysogenic cycle is maintained by production of gp71 repressor.


D29 mycobacteriophage does not synthesizes this gp71 repressor

Dot plots

This a short explanation of Dot plots  an easy and powerful means of sequence analysis, useful for searching out regions of similarity in two sequences and repeats within a single sequence.

This the principle:


These are some examples of reading:





By Gepard ("GEnome PAir - Rapid Dotter" is an open source from Helmholtz Zentrum München ) we have two variable parameters:

· Word length - minimum word length for identical subsequences which create a hit in the dotplot.

· Window size - If word length =0 "normal" dotplot mode will be activated where all characters of both sequences are compared against each other.

This parameter specifies the window size over which an average dot value will be calculated.

· Word length is number of bases in a sliding window that is moved along each sequence and compared to generate a single data point on the plot. Word lenght must be an number.

· Mismatch Limit ( is the window size) determines how similar the two sequences in a word lenght must be to "match". For example, if word lenght size is 9 and mismatch limit is 2, then up to 2 mismatches in a 9 base word lenght will still be classified as a match.


If we compare the genomes of My. smegmatis and My. ulcerans by Gepard (two FASTA format sequence files ) and changing the parameters for increasing the sensibility,we have:





This is the best: Here it is possible to observe common regions, some repeats and palindromic sequences. By the co-ordinate axes it is possible to select a single gene for further evidence.

The next image it doesn't make any difference.



Control: My.smegmatis versus My.smegmatis
 


Control: My.ulcerans versus My.ulcerans