What have we learned from Unicellular Genomes?

1. What have we learned from Unicellular Genomes?

2. Propionibacterium acnes • Responsible for acne, its genome sequenced in 2004. • It lives on human skin in sebaceous follicles; feeds on sebum and this stimulates immune response of inflammation. • Can we understand pimples?

3. Anatomy of acne

4. Propionibacterium acnes genome • Sequenced by three different groups. • 32 190 sequencing reactions • 8.7-fold coverage of 2 560 265 bp genome • Error rate of 0.0001 • Genome contains a single circular chromosome and no additional plasmids. • Annotation of 2333 putative genes, allowed for construction of the metabolism.

5. Propionibacterium acnes genome • 12% encoded RNA products (rRNA and tRNA). • 1578 (68%) is orthologous with other organisms and 20% does not match with anything.

6. GC skewing • A non-uniform distribution of guanine and cytosine bases on the two strands of DNA. • Origin of replication have the lowest GC skew (even distribution) • Terminus of replication have higher GC skewing.

8. Horizontal Transfer • Genes appeared in genome through an unknown mechanism. • To find alien genes, scan the genome with a sliding window for segments that have an abnormal GC content (either higher or lower than the species average) and evaluate the codon bias. • Which codon is used more often than other codons for a particular amino acid.

9. Transcriptional Phase Variation • Variation in the Gs is used to produce transcriptional variation. • Initiation of transcription depends on the number of consecutive guanines on a particular strand at a critical location upstream of the coding region. • Regions of replicating bases are difficult to accurately replicate which will affect the transcriptional efficiency.

11. Which genes cause pimples? • Metabolic reconstruction: • Can grow anaerobically and aerobically. • Has many enzymes to degrade lipids, esters and amino acids. • P. acnes digestive enzymes have LPXTG motif that targets proteins to the extracellular wall; these enzymes chew away on your cells. • cell-wall sorting signal LPXTG responsible for covalently anchoring proteins to the cell-wall peptidoglycan • LPxTG, the target for cleavage and covalent coupling to the peptidoglycan by enzymes called sortases

12. Which genes cause pimples? • Cells exterior is decorated with hyaluronate lyase that destroys the extracellular matrix binding your skin cells together and thus facilitates further tissue invasion and digestion.

13. LPxTG Database: Sortase substrates http://bamics3.cmbi.kun.nl/cgi-bin/jos/sortase_substrates/index.py

14. Stimulation of immune response • Genome encodes five CAMP (Christie, Atkins, Munch-Peterson) factors. CAMP factors are secreted proteins that bind to antibodies (IgG and IgM) and can form pores in eukaryotic cell membranes. • Lysis of our cells trigger an immune response.

15. CAMP factors • Proteins from BACTERIA and FUNGI that are soluble enough to be secreted to target ERYTHROCYTES and insert into the membrane to form beta-barrel pores. Biosynthesis may be regulated by HEMOLYSIN FACTORS

16. Quorum Sensing • Many bacteria have evolved the ability to condition culture medium by secreting low-molecular-weight signaling pheromones in association with growth phase to control expression of specific genes, a process termed quorum sensing • Bioluminescence • antibiotic biosynthesis • Pathogenicity • plasmid conjugal transfer

17. Quorum Sensing • LuxS produces the precursor of autoinducer-2 (AI-2), 4,5,-dihydroxy-2,3-pentanedione (DPD), whilst converting S-ribosylhomocysteine to homocysteine.

19. Are all bacteria Living in Us Bad for Us? • An average adult body is composed of about 10 trillion human cells. • Every milliliter of your large intestine’s content is estimated to contain 10 billion microbes and our intestines contain about 1 L.. • There are about 500 to 1000 different species living in an adult’s intestines.

20. Bacteroides thetaiotaomicron • 31 million bases • Assembly of 867 contigs with many gaps. • Finished assembly by PCR • 67 938 sequencing runs into a single 6 260 361 bp circular contig. • Annotated 4779 predicted ORFs with 58% orthologs of known function, 18% orthologs of proteins with no known function and 24% with no recognizable sequence similarity.

22. COGs • Clusters of orthologous group are functional categories of genes. • They are phylogenetic classiciation of proteins encoded in complete genomes. • Transcription • Energy production, etc.

23. http://www.ncbi.nlm.nih.gov/COG/

24. Eukaryotic Clusters

25. ADH

26. CDH

27. Bacteroides thetaiotamicron • It can metabolize sugars. • 170 genes for polysaccharide metabolism; paralogs of 23 genes. • E. coli has only 8 of them. • It can also import sugars into its own cytoplasm. • Has two genes SusC and SusD represented by 163 paralogs.

28. Transposable Elements • 63 TEs contain ORFs (open reading frames) that help spread tetracycline and erythromycin resistance between individual cells and between species in the microbiota of the gut.

29. Coding Capacity • Gene density for B. thetaiotaomicron is 89%. • Average size of a gene is 1170 bp-largest among bacteria. • M. genitalium 1100 bp • H. pylori 1000 bp • E. coli 950 bp

30. Can Microbial Genomes Become Dependent upon Human Genes? • Second smallest bacterial genome of a self-replicating species (589 070 bp). • A team in TIGR (The Institute for Genomic Research) • 5 people, 8 weeks assembled 8472 high-quality sequencing reactions. • Overall GC content is 32% • GC skew reveals the origin of replication as DnaA and DnaN genes. • Right to the OR transcribed from plus strand • Left to the OR transcribed from minus strand • tRNA and rRNA genes have higher GC content, 52 and 44%.

31. Genome Map • 470 ORFs; 88% coding capacity; average gene is 1040bp. • Retained genes for energy metabolism, fatty acid and PL metabolism, replication, transcription, and protein transport. • Lost DNA when no need for it. • aa synthesis • Cofactors • Cell envelope • Regulatory factors

32. Synteny • When a series of genes are conserved in order and orientation between two or more species, the genes are described as syntenic. • M. genitalium and H. influenzae has similar gene orders with respect to two clusters of ribosomal proteins.

33. Minimum Number of Genes • Synthetic biology: to synthesize de novo (from scratch) a functioning genome with as few genes as possible. • Bacillus subtilis – 190 genes • M. genitalium – 260 genes

34. Bacteria vs. Viruses • Smallest genome is an Archean N. equitans (490 kb) • HIV-9200 nt • SARS-29797 nt • Lambda-48502 nt • Acanthamoeba polyphaga-Mimivirus: infects amoeba • dsDNA-1 181 404 bp with 1262 ORFs linear chromosome

35. Mimivirus Genome • 28%GC rich • 90% coding capacity • Uses biased codons-lacking G or C; uses the least common codon in amoeba the least. • It has proteins used for translation, posttranslation modification, DNA repair-sounds more like a eukaryote. • Encodes topoisomerases • Has a self-splicing intron

37. Is Mimivirus Alive? • Mimivirus is most closely associated with Eurkaryota • Infectious after 1 year of incubation at 4 C. • Survived 48 hours of desiccation and 1% survived 55 C. • Mimivirus can participate in all major steps of translation. • A life form • Highly modified virus?

40. Malaria • 3 billion people in the world in tropical and subtropical climates affected. • Malaria causing ekaryotic parasite genus Plasmodium • 2.7 million people die each year.

41. Plasmodium • Plasmodium falciparum is the most lethal form transmitted to humans by Anopheles mosquito. • Infected mosquito bites, parasite leaves salivary glands move to liver and infects hepatocytes. They mature in hepatocytes and hatch out into RBCs. • A new parasite emerges from RBCs by bursting it, release progeny and metabolic waste causing fever followed by chills. • A few cells differentiate into gametes move through blood can be ingested by new mosquito and gatmetes form zygotes and meiosis and to salivary glands.

43. Infection of RBCs • RBC 6 micron; plasmodium 1.2 micron • Plasmodium enters RBC by evading immune system by sticking to RBCs. • Apicoblast organnelle that is made up of a remnant internalized alga retaining its small genome needed for plasmodium survival.

44. Plasmodium Genome • Three genomes • Nuclear: chromosomes separated through pulse-field gel electrophoresis before random fragmentation and cloning; 22 853 764 with 5268 ORFs; 19.4% GC; 52.6% coding capacity; average gene length 2283 bp. • Mitochondrial: 5967 bp encodes 3 proteins • Apicoplastic: 29 422 bp encodes 30 proteins

45. Plasmodium is a eukaryote • 54% of its genes contains one or more intron with an average 13.5%GC (exons have higher GC%). • 60% of ORFs have no known function

46. rRNA genes • In many species rRNA genes appear in linear clusters • In Plasmodium, rRNA gene distribution var, their expression is host specific; some are expressed in human; the other set is active in mosquito

47. Centromeres and telomeres • Centromeres are AT rich (97%) and contain short tandem repeats. • Telomeres have repeated sequences that vary in length; some genes located nearby telomeres are replicated many times therefore genes have paralogs. • Highly variable gene families, var, rif and stevor (polymorphic) and may add variation to the extracellular surface of the Plasmodium.

48. Hydropathy plot http://expasy.org/cgi-bin/protscale.pl

49. Hydropathy plot

50. Plasmodium • 31% of the encoded polypeptides are predicted to be integral proteins. • 1% cell-to-cell adhesion • 4% evasion of immune system