Mycobacteriophage D29

By coderet (Jemboss software).
coderet extracts the coding nucleotide sequence (CDS), messenger RNA nucleotide sequence (mRNA),non- coding nucleotide sequence  and translations specified by the feature tables of the input sequence(s).
By input of D29 genome file.gbk in coderet ( 49136 bp  DNA  linear download from:
ftp://ftp.ncbi.nih.gov/genomes/Viruses/),
the result is:

   CDS     mRNA    non-c    Trans       Total        Sequence
 =====   =====     =====   =====     =====       ========
    79            0            156          79            235          NC_001900


From this work  ( and my contributions):

"D29 was first isolated from soil, and shown to be active against Mycobacterium tuberculosis (Froman et al., 1954).

The plaques produced on the tubercle bacillus were clear, indicating that D29 is a lytic phage (i.e. incapable of lysogeny).

Studies of D29 suggest that it is a member of the "L5-like'' family of mycobacteriophages.

The overall organization of the D29 genome is similar to that of L5, although a 3.6 kb deletion removing the repressor gene accounts for the inability of D29 to form lysogens.

L5 genome

D29 genome

Although lytic, D29 is subject to superinfection immunity by L5; in other words, it is incapable of infecting an L5 lysogen of M. smegmatis.
Only the product of L5 gene 71 is required to prevent infection of M. smegmatis by D29, demonstrating that this is true immunity rather than exclusion (Donnelly-Wu et al., 1993).

L5 gene 71

-The protein  sequence of L5 gene 71 is extracts by SnapGene software:

 -By JEmboss software  the predicting secondary structure of Immunity repressor protein:

a-there is one  helix-turn-helix nucleic acid binding motif:

b-the Aa composition of the protein by L5 gene 71:

c-no presence of transmembrane segments in  this protein
 
d-no site of cleavage between a signal sequence and this protein

e-4 poor PEST motifs as potential proteolytic cleavage sites:

Poor PEST motif with 24 amino acids between position 159 and 183.

   159 HTNLTAEGELLWSWPDDIEELLSEP 183

       PEST score: 3.07 

Poor PEST motif with 23 amino acids between position 117 and 141.

   117 RDDDLVLEFDPSIEPYEGMAGGGFR 141

       PEST score: -3.93 

Poor PEST motif with 11 amino acids between position 62 and 74.

    62 RQIVQQNWPWDTR 74

       PEST score: -16.67 

Poor PEST motif with 11 amino acids between position 16 and 28.

    16 RIPLTLSEIEDLR 28

       PEST score: -10.33 

 
In addition to the shared immunity of L5 and D29, these phages appear to have a common mode of entry in M. smegmatis.
In particular, overexpression of the M. smegmatis mpr gene confers resistance to both L5 and D29, but not to other mycobacteriophages (Barsom & Hatfull, 1996).

The identity of the receptor used for adsorption of these phages is not clear,although pyruvylated, glycosylated acyltrehaloses have been implicated in the infection of M. smegmatis by D29 (Besra et al., 1994).

The two phages have similar host ranges (our unpublished observations), though there are some differences in infection requirements (Fullner & Hatfull,1997), and D29 has been reported to adsorb to Mycobacterium leprae (David et al., 1984).

We therefore assessed the morphologies of L5 and D29 by electron microscopy and found then to be virtually identical (Figure 1).

We have also compared the virion proteins of D29 and L5 by SDS-PAGE of whole particles (Figure 2). The patterns are similar but not identical.

In particular,we note that D29, like L5, has several protein species larger than 150 kDa (bands A, B, and C). In L5, this reflects the presence of extensive covalent crosslinking between subunits of the major capsid protein, gp17 (Hatfull & Sarkis, 1993). Presumably, the major head subunit of D29 is also extensively crosslinked. Indeed, amino-terminal sequencing of the D29 equivalent of band C revealed that it is the product of gene 17 (data notshown).

Many of the L5 proteins (including bands F, H,and J) are represented in similar sizes in D29,although proteins D and E are either absent or in low abundance in D29 (Figure 2). Additionally, a band corresponding to the L5 band I appears to be missing in D29. Instead, D29 has a protein band that migrates only slightly faster than band H. Finally, the intensity of band F is lower in D29 than in L5. In L5, band J corresponds to the major tail subunit protein gp23 (Hatfull & Sarkis, 1993).
Sequencing of the amino-terminal end of the D29 equivalent of band J (data not shown) revealed that the first ten amino acids of this protein are identical to the first ten amino acids of L5 gp23, and also match the predicted N-terminal sequence of D29 gp23. Therefore, gp23 most likely constitutes the major tail subunit of D29.

The G+C content of the D29 genome is 63.6%, a value consistent with that of the mycobacterial hosts (Clark-Curtiss,1990), and virtually identical to the
G+C content of L5 (63.2%; Hatfull & Sarkis, 1993).

 Sequencing directly from D29 DNA we found that the D29 chromosome contains cohesive termini identical to those found in L5 (5'-GGTCGGTTA-3' with a 3' extension; data not shown).

 Although D29 is not a temperate phage, it does contain a phage attachment site (attP) that is very similar to L5's, including the common core (although one position at the extreme end is different such that only 42 bp are common to the M. smegmatis attB sequence; see PenÄa et al., 1997).

 The D29 attachment site (coordinates of the common core are 26,634 to 26,676) is located somewhat to the right of center of the genome and divides the genome into a left and right arm as described for L5 (Figure 3).

While the D29 and L5 genomes are closely related, the level of similarity is
not the same across the genomes. In particular, the left arms can be aligned without introducing substantial gaps and are approximately 80% identical at the nucleic acid level. The right arms are considerably less closely related although the differences are not evenly distributed. Thus, in the right arm, regions of high similarity are punctuated by segments of unrelated DNA (see below).

We identified three database matches for D29 gene products for which there is no homolog in L5. One of these is an internal segment of gp10 
(which is absent from L5) that matches HI1415, a hypothetical protein of
unknown function from  Haemophilus influenzae Rd (Fleischmann et al., 1995).
The remainder (genes 36.1 and 59.2) have reasonable similarities to deoxycytidinylate deaminases and non-heme haloperoxidases, respectively.

 Although D29 is a close relative of temperate L5 and has an attP-integration system, it does not form lysogens. The simplest explanation for D29's exclusively lytic lifestyle is a 3.6 kb deletion relative to L5 at the right end of its genome which removes part of the repressor gene (71) and several adjacent genes (Figure 4).

Alignment of the genome sequences indicates that D29 has lost 3620 bp relative to L5 (between L5 coordinates 44,700 and 48,321; the junction of the flanking sequences in D29 occurs between coordinates 45,706 and 45,707) probably through a simple deletion without substantial additional rearrangements (Figure 4A).

Deletion derivatives of L5 which have lost all or part of the repressor gene (71) and are incapable of forming lysogens have also been isolated (Donnelly-Wu et al., 1993).
By comparing the L5 and D29 coding regions at the point of deletion,
it is apparent that D29 has lost about half of both genes 71 and 82 and
presumably all of the intervening genes (Figure 4B). In L5, gene 71 encodes 
the phage repressor, and is required for the maintenance of lysogeny and is sufficient to confer superinfection immunity (Donnelly-Wu et al., 1993).

Checking by Gepard software and Mauve software 

 
The observation that D29 does not have an intact copy of gene 71 thus completely explains its lytic properties. In addition, because D29 presumably has lost L5 genes 71 to 82, this region is most likely not essential for phage growth, an observation consistent with previous reports that the entire region from 71 to 88 is non-essential (Sarkis et al., 1995). Little is known about the functions of any of the genes in this area of the L5 genome, although a gene in the 72 to 82 interval is potentially involved in lysogenic establishment (Sarkis et al., 1995).