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March 2-4th, 2015.

Objective: gather articles to form research topic/question

 

Project: Examine sigma factors and how they relate to early/middle/late gene expression. 

collect known early/middle/late sigma factors and run blasts on other known sigma factors to determine if they match with early/middle/late. Run a phylogenic tree to see if there is some form of grouping. 

 

March 14-16, 2015.

Objective: Complete annotated bibliography with 5 sources for research project.

Sources/Annotated Bibliography

Sigma Factor Research Project Annotated Bibiliography.doc

The Sigma Factors of Bacillus subtilis: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC239352/pdf/590001.pdf 

 

HALDENWANG, W. G. (1995). The Sigma Factors of Bacillus subtilis. 
     MICROBIOLOGICAL REVIEWS59(1), 1-30. 
       This article discusses the sigma factors in B. Subtilis, and how 
       different s-factors regulate gene expression for sporulation and 
       vegetative –cell states. The technique used to isolate and characterize 
       sigma factors are standard chromatographic techniques. Each sigma factor 
       whose sequence was identified was cloned using oligonucleotide sequence 
       or anti-sB polyclonal antibodies to identify the encoding region. The 
       article briefly compares the RNAP’s of E. coli and B. Subtilis, and how 
       biochemical studies done on E. coli are directly applicable to B. 
       subtilis. Of the ten sigma factors identified in B. Subtilis, nine were 
       cloned, sequenced and mapped to sites on the chromosome; with no rational 
       behind their locations. Only two s-factor genes were linked on the 
       chromosome, sG and s29; these two sigma factors are involved in 
       sporulation-specific gene expression, which could indicate a rational for 
       their locations. s43 and s55 functions were identified as early 
       sporulation and housekeeping gene expression. s32 and s30 control 
       postexponential gene expression. sL controls degradative enzyme gene 
       expression.  sA (s43 and s55) is much like s70 in E. coli; it is the 
       principal sigma factor.

 

Bacteriophage T4 Genome Microbiol. Mol. Biol. Rev.-2003-Miller-86-156(1)(1).pdf

Miller, E. S., Kutter, E., Gisela, M., Arisaka, F., Kunisawa, T., & Ruger, W. 
     (1995). Bacteriophage T4 Genome. Microbiological Review, 87-128. 
     http://dx.doi.org/10.1128/MMBR.67.1.86-156.2003
       The article presented a total analysis of the bacteriophage T4. The T4 
       genome was annotated using GeneMark. A few sigma factors of phage T4 
       was identified for early/middle/late gene expression. Sigma 70 was 
       identified in both early and middle gene expression while sigma 55 was 
       identified for late gene expression. It was noted that although sigma 55 
       is required to initiate late transcription, gp33 of T4 is also needed as 
       a co-activator mediating interactions between sigma 55 and the sliding 
       clamp. This article could potentially help to identify other 
       early/middle/late sigma factors through running blast/clustal omega of 
       sigma 70 and sigma55 on other known sigma factors.  


2009 Stewart SPO1 phage supplementary file.pdf

Stewart, C. R. (2009). The Genome of Bacillus subtilis Bacteriophage SPO1. 
     Journal of Molecular Biology388, 48-70. http://dx.doi.org/10.1016/ 
     j.jmb.2009.03.009 
       This article characterizes B. subtilis infecting phage SPO1. Glimmer, 
       GeneMark and DNA Strider were used to predict ORF’s. ABI377 and ABI3100 
       instruments were used to sequence the phage DNA, and assembled with 
       CONSED. Gp28 is identified as a sigma factor which is involved in 
       middle-to-late gene expression of 8/16 genes with middle promoter 
       regions; the continuation of these genes into the late period of gene 
       expression is still unknown. Gp2.21 was identified as a sigma factor 
       involved in late gene expression; this gene should be expressed well from 
       middle sigma K promoter and therefore should be present for late 
       transcription. Gp2.21 has a predicted amino acid sequence 24% similar to 
       that of B. subtilis sK. Gp 34 is identified as a sigma facor required for 
       late gene expression.

  

 

The Secret Life of the Anthrax Agent Bacillus anthracis: Bacteriophage-Mediated Ecological Adaptations. 2009 Schuch Bacillus anthracis sigma factors.pdf


Schuch, R., & Fischetti, V. A. (2009). The Secret Life of the Anthrax Agent 
     Bacillus anthracis: Bacteriophage-Mediated Ecological Adaptations. 
     PlosOne4(8), 1-23. 
       The study examined the life-cycle of bacillus anthracis with hopes to 
       discover phage-mediated adaptations. The study collected eight novel 
       bacteriophages isolated from Bacillus anthracis. In order to examine the 
       relationship between sigma factors and phenotypic alterations in the 
       host, researchers examined the genomic expression libraries of phages 
       Bcp1 and Wip4. Phage genomic fragments were generated by Tsp509I 
       digestion and cloned into a self-replicating B. anthracis. It was found 
       that gp25/26 of Bcp1 and gp38/39 of Wip4 were responsible for the 
       phenotypic alteration, and their sequences were also closely related to 
       that of known bacterial sigma factors F and G. It was also found that 
       Bcp1- and Wip4-encoded sigma factors act alone  to block sporulation 
       through an experiment involving RT-PCR analysis to determine if the bcp1 
       and wip4 loci could still be expressed when cloned into plasmids as 
       fragments with their relative upstream promotors. This article provides 
       some known phage sigma factors that we could potentially compare with 
       known early/middle/late sigma factors to where they stand.  


2013 Lee Arch Virol.pdf

Lee, J.-H. (2013). Characterization and complete genome sequence of a virulent 

     bacteriophage B4 infecting food-borne pathogenic Bacillus cereus. Arch 
     Virol185, 2101-2108. http://dx.doi.org/10.1007/s00705-013-1719-2 
       The article characterized a novel bacteriophage B4 that infected 
       Bacillus cereus. Genome Sequencer FLX titanium sequencer  GS de novo 
       assembler software (Roche) at Macrogen Inc., South Korea were used to 
       sequence and assemble the DNA. Glimmer and Genemark were used for ORF 
       predictions, and BlastP was used for ORF annotation. From the complete B4 
       genome, putative sigma factors were found including one protein that is 
       similar to sigma factor F located on gp143 and RNA polymerase sigma 
       factor at gp181. Though the sigf-like protein was less than 25% 
       compatible with other known B. cereus host SigF proteins, the RNA 
       polymerase sigma factor was 40% identical to other known phage proteins 
       but shows no similarity to the host sigma factors suggesting its use in 
       expressing phage genes. The article offers to more sigma factors from B4 
       that could be compared to known early/middle/late phage genes.  

 

Sigma Factor Spreadsheet (Autosaved).xlsx - gathered from previous projects. List of known sigma factors. Contains some known early/middle/late genes. We hope to add to this table to add new sigma factors and predict if some are early/middle/late gene expression. 

 

March 18th, 2015

Objective: Finalize research project  

My general topic of interest: Sigma Factors 

My research question: 

The data I will use: Amino Acid Sequences of discovered sigma factors, Genome of novel phages, Collection of known sigma factors collected from last year. 

Where I will find my data: Genbank, phamerator

The approach/method/tool I will use:

My goal- I hope I create a figure/table that looks like this:

Phage_GPhostearly/middle/latepredicted functionnotes
     
     
     
     

 

I will need help in this area:



March 23rd, 2015: 

Objective: start exploring sigma factors on phamerator and clustal omega. 


Phylogenic Tree of Pham2021 which includes sigma factors Nigalna_185, Phrodo_163, and TROLL_170. 


 

March 25, 2015:

Objective: start collecting known sigma factor phams from known early/middle/late genes and unknown early middle late genes with hopes to make a phylogenic tree of all phams and predict early/middle/late transcriptions from unknown sigma factors. 

Accessed complete SPO1 genome from genban

run blast on known SPO1 genes that code for sigma factors(gp2.21, gp28, gp34, gp35) 

Started using phamerator to download sigma factor phamilies. 

 

putative conserved domain obtained from running blastp on gp2.21 from SPO1 

phams downloaded: 

2021,1453, SPO1 sigma factors, 715, 2761

NOTE

On Phamerator: 

SPO1 gp2.21–> SPO1 gp531, pham 715(2): potential late specific gene expression (3-4 supplement of SPO1 paper) (GenBank SPO1_56).

SPO1 gp28 --> SPO1 gp1211, pham 2401(2): middle specific gene expression  (Genbank SPO1_126). 

SPO1 gp34–> SPO1 gp1571 pham 992 (2): Late specific gene expression (Genbank SPO1_159)

March 30th, 2015: 

continue gathering phams and compiling a phylogeny tree of all known sigma factor phams in phamerator as well as individual known sigma factor protein sequences. 

continue looking through literature for known early/middle/late genes and run blast to compare proteins to others.

 

 

 

This is a blast p of Troll gp_170. This gene was annotated as a sigma factor. It has a 64% match to sigma factor sigma-28 in phage Hoody_T; this sigma factor is known from previous literature to be involved with middle gene expression. This would indicate that this sigma factor, along with the other that match with it, could possibly be involved in middle gene regulation. 

 

This blast of Troll gp_170 also revealed a 39% similarity to SPO1 gp_34. Gp34 from SPO1 is known from literature to be involved with late gene expression in SPO1. With this information we could potentially add this gene to our phylogenetic tree and contruct a bigger picture as to which phage encoded sigma factors are responsible for middle/late gene expression. 

 

 

Total Sigma Factor Phams- list of all sigma factor phams on phamerator plus individual sigma factor protein sequences. 

 

This is the compiled phylogenic tree of all the sigma factor phams from phamerator and some individual bacillus phage sigma factor protein sequences. From literature, we knew that SPO1_531 was a potential late gene expression sigma factor, SPO1_1211 was middle specific, and SPO1_1571 was late specific. We had hoped to be able to predict the early/middle/late expression of other sigma factors form this tree, but the SPO1 sigma factors were too distantly related. There is a cluster of proteins between SPO1_531 and SPO1_1211 which could be potential middle specific. Analysis of TROLL 221 confirms that it is potentially involved in late gene regulation. 

 

Next: 

We will continue to examine the literature to find other known early/middle/late specific sigma factors and attempt to develop predictions. 

 

April 9th update by AJ:

I looked at phage Bcp1 from the Schuch paper because Bcp1 is a myovirus, should be virulent, and have similar gene order to Phrodo. I don't understand why they say these phage can form lysogens with the host bacteria because there is no integrase. I think they are wrong but will try to figure that out. The genbank file is here.

gp 143 is predicted to be a sigma factor:

MPRNVEKEQEQIWNGNRFMVSTEETTGVFIRDVDKLFHQYRNLR
MSIYNQYKGYLSSDPVTQDELRSYIDEQFIRLVKEYDIGSDVDFPGYIKIKLHSRVKN
SFIKSVFRDKQRVFVTRNSFDVSNLIEQNPCNDEELDYYATLEYAIGGFKLTQMEKEV
LFYLLQELTDPQIERAIKDDHPRERISSATIRETIKDMSELLRTKLLESLEN

gp 199 is predicted to be a sigma factor sigma-70 like, sigma-B/F/G subfamily:

MNKKRAGSTYTFAKKVLTKEETYELIERSQAGEEEATESLVEHN
ARLVTYVVRRKNNPYHEYDDLFQLGMIGLITAIAKFDTSKGLQFSTYAVRWIDAEIGN
YLKNRTSILKVPREIGAIVNKILAVKLKNEEPAIIMEKLKLDASQLDNVTIALEIIHN
EVISLDKQTGEEKDDSLSSIVGQDVNQDWFSGLAFYDIIRFLDDKEQSVLTLKYVHDM
SSNKIATLFGTYANKISRLEKVALDKLRERYTYEELIN

gp200 is predicted to be a sigma factor sigma-70 like, sigma-B/F/G subfamily also:

MILWKVRGIELTREETIELVKEARAGSEEAKEKIILGYKGMVYT
LAKRFSRSKQHEFEDMFQEGLTILLEVVDKFDIDSGYAFSTYAYPFVFGKMNNVRKRY
NPIKISAHITDIISRIRKYKLTDRSEKEIYEFLNKEYELKWVRAALEYMRRGKVLSLE
KTFAEDEESDWAATLKEVVSKDANGDWELMMDIKGCVxPSLTSYEQYAFCEHILKDRMQ
SDIAEELGVKPQTVSKHVNKACRKIKLELGGV

In the Schuchs paper, Bcp 25,26 are sigma factors that block sporulation. They numbered them 25,26 with a partially sequenced genome, and have since submitted the whole genome sequence including a change in gp numbering. I suspect 25,26 are 199, 200 since they're sequential.

Can you use these to find Phrodo homologs? If one but not both match, or something like that, we might be able to have some information about Phrodo's ability to impact sporulation process in host bacteria.

 

4/13/15

objective: 

attempt to find homologs with VCU phages based on known sigma factors from BCP sigma factors and attempt to propose early/middle/late specificity based on syntenny

 

blasted DIGNKC genbank file with gp143 from Bcp and found a match with gp180. possible middle specific. 

Phrodo 163 possible middle specific? 

blast of phrodo 199 reveals match with troll 209 not troll 221—> possibly middle/late 

 

April 15, 2015: 

continue finding more sigma factors and if they are early/middle/late transcription. 

start to compile Excel sheet of gathered sigma factors

clean up phylogeny tree to include only VCU phage sigma factors and their phamilies + known early/middle/late sigma factors  

 

 

Ran blast of Phrodo 163/199 and 199/200 with BCP1 sigma factors—> phrodo 163 had a 26% identity with BCP1 199 and BcP1 200:

BLAST RESULTS FROM BCP1_199 and Phrodo_199. 

Blast Results from BCP1_200 and Phrodo_199


 

Blast P of Troll_209 revealed a SigF-like sigma factor in Troll, Big Bertha, Spock and B4. 

 

 

 

Blast p of SPO1 gene 34. 

revealed

 

Query of gp34 SPO1 compared to Troll GenBank File. Match with Troll_170. 

 

Blastp of SPO1 gp2.21; from literature it is a known sigma factor. 

Blastp of SPO1 gp2.21 revealed similarity to 3 phage sigma factor genes, and then all other hits are bacteria sigma factor genes. 

 

Blastp of phrodo gen bank file with SPO1_159. match with phrodo_163.

Phylogenic tree of Phrodo_163 and its phamily (1885) along with SPO1_34 and BCP1_143 which was found to match with phrodo_163 and can be predicted as late transcription regulators. 

 

Phylogenic tree of Phrodo_199 and its phamily (1481) along with BCP1_199 and BCP1_200 which matched with phrodo that potentially block sporulation. 

 

 

Bcp1 199 vs. 200 blastp result, they are 32% identical.

blast of each protein vs. NR database shows sigma70 r2 conserved domain is in both, but right domain might be different.

Bcp1 199:

Bcp2 200:

Can you construct a rationale for why Phrodo 199 matches both (with fairly low protein identity of 26%)?

final Phrodo Genbank file which includes gp163 and 199 

Sequence file used for phylogeny tree 

 

 

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