What will Bacterial Genomics give us?• Bacterial genomics can give us a broader understanding of how a bacteria functions, a bacterias origins, and what bacteria live in our world that we cant study by other means (i.e through obtaining their DNA from the environment and studying it).• Of medical interest, bacterial genomics is also anticipated to play a significant role in speeding up the development of better therapies and vaccines for controlling disease-causing bacteria.• It will also be the cornerstone of anticipated DNA- based diagnostic tools that will hopefully enable doctors to make quicker, more accurate diagnoses of infectious disease.
Size of Bacterial Genome• The size of Bacterial chromosomes ranges from 0.6 Mbp to over 10 Mbp• The smallest Bacterial genome identified thus far is from Mycoplasma genitalium, an obligate intracellular pathogen with a genome size of 0.58 Mbp (580 Kbp). M. genitalium is restricted to the intracellular niche because it lacks genes encoding enzymes required for amino acid biosynthesis and the peptidoglycan cell wall, genes encoding TCA cycle enzymes, and many other biosynthetic genes.• In contrast to such obligate intracellular bacteria, free-living bacteria must dedicate many genes toward the biosynthesis and transport of nutrients and building blocks. The smallest free-living organisms have a genome size over 1 Mbp.• Currently largest sequenced prokaryotic genome is streptomyces coelicolor, 8.7 Mb.
Gene Content• Gene portion of bacterial genome is around 85 – 95 %• Bacteria posess few genes such as in case of Mycobacterium genitalium (480 genes)• The highest gene content is present in Bradyrhizobium japonicum (8317 genes)• The average gene content is 3,100 genes per genome.
COG• Genes shared among prokaryote genome by virtue of common evolutionary descent are frequently evaluated descent are frequently evaluated in terms of “Clustered Orthologous Groups”It is critical to infer orthologous relationships between genes from differentspecies.Orthologs are direct evolutionary counterparts related by vertical descent asopposed to paralogs which are genes within the same genome related byduplication.Typically, orthologous proteins have the samedomain architecture and the samefunctioncin prokaryotes
Base pair composition• 22.4 % GC – Wigglesworthia sp• 72.1 % GC – Streptomyces coelicolor Average – 46.8 % GC• GC Skew: Used to indicate biases in the relative contribution of G or C in an individual DNA strand (G – C) (G + C)
• In most bacterial genomes a difference in base composition between the strands (which could mean leading vs lagging strand of replication or coding vs non-coding strand of genes) is observed, which means that there are different mutation/substitution processes affecting the two strands.• Typically, the leading strand is enriched in G and T, while the lagging strand is enriched in A and C. Deviations from the base frequencies A=T and G=C are called AT- and GC-skews
Analysis of GC Skew• Analysis of GC skew was ﬁrst utilized for the computational prediction of ori and ter positions by examining available genome sequences.• As GC-skew is positive in the leading strand and negative in the lagging strand, the GC-skew changes sign at the origin and terminus where the leading strand becomes the lagging strand and vice versa.• This makes GC-skew analysis a useful tool to identify the origin and the terminus in circular chromosomes. Local changes, which are visible as diagram distortions, can mark recent rearrangements such as sequence inversions or integration of foreign DNA. The loss of DNA would not change the basic shape of the GC-skew curve, whereas recent incorporation of external DNA would probably result in a local deviation.
General features of Bacterial Chromosomes• Not all bacteria have a single circular chromosome.• some bacteria have multiple circular chromosomes.• Many bacteria have linear chromosomes and linear plasmids.• Linear chromosomes and plasmids were not discovered in bacteria until relatively recently.• In 1989, pulsed field gel electrophoresis had been developed, and this new technique provided convincing evidence that the chromosome of Borrelia burgdoferi was linear
• Agrobacterium tumefacians:• One linear (2.1 Mb) +• One circular (3.0 Mb) +• Two circular plasmids (450 kb + 200 Kb)• Vibrio cholerae: twocircular chromosomes. One of these chromosomes contains the genes involved inmetabolism and virulence, while the other contains the remaining essentialgenes• Bacillus thuringiensis:• One circular (5.7 Mb) +• Six plasmids (Each > 50 Kb)
Genome Packaging in Prokaryotes: the Circular Chromosome of E. coli• Prokaryotic cells donot contain nuclei or other membrane-bound organelles.• In fact, the word "prokaryote"literally means "before the nucleus." The nucleoid is simply the area of a prokaryotic cell in which the chromosomal DNA is located.
DNA Supercoiling• One way prokaryotes compress their DNA into smaller spaces is through supercoiling.• Genomes can be negatively supercoiled, meaning that the DNA is twisted in the opposite direction of the double helix, or positively supercoiled, meaning that the DNA is twisted in the same direction as the double helix. Most bacterial genomes are negatively supercoiled during normal growth.
Proteins Involved in Supercoiling• Multiple proteins act together to fold and condense prokaryotic DNA.• In particular, one protein called HU, which is themost abundant protein in the nucleoid, works with an enzyme called topoisomerase I to bind DNA and introduce sharp bends in the chromosome, generating the tension necessary for negative supercoiling.• Integration host factor (IHF), can bind to specific sequences within the genome and introduce additional bends.• The folded DNA is then organized into a variety of conformations that are supercoiled and wound around tetramers of the HU protein, much like eukaryotic chromosomes are wrapped around histones.• Once the prokaryoticgenome has been condensed, DNA topoisomerase I, DNA gyrase, and other proteins help maintain the supercoils.• One of these maintenance proteins, H-NS, plays anactive role in transcription by modulating the expression of the genes involved in the response to environmental stimuli.• Another maintenance protein, factor for inversion stimulation (FIS), is abundant during exponential growth and regulates the expression of more than 231 genes, including DNA topoisomerase I .
Accessing Supercoiled Genes• It has been determined that prokaryotic DNA replication occurs at arate of 1,000 nucleotides per second, and prokaryotic transcription occurs at arate of about 40 nucleotides per second .• During transcription, small regions of the chromosome can be seen to project from the nucleoid into the cytoplasm• Because there is nonuclear membrane to separate prokaryotic DNA from the ribosomes within the cytoplasm, transcription and translation occur simultaneously in these organisms
Operons• When different genes are to be expressed in exactly the same amount because they are part of a complex, transcription of all genes in a single transcript diminishes gene expression noise and ensures more precise stoichiometry.• Pairs of divergently oriented operons show correlated expression levels this is because sometimes they share bidirectional regulatory regions that allow coregulation of the two operons.
Replication-Associated Gene Dosage Effects• The possibility of starting a new round of replication before the previous round ﬁnishes, i.e., of having simultaneous replication rounds, allows cells of E. coli to double every 20 min, whereas the chromosome takes three times longer to replicate. The estimated number of simultaneous replication rounds (R) is the ratio between the time required to replicate the chromosome and the time between two successive cell divisions.• If R is close to zero, then chromosome replication rarely takes place in the cell.• IfRis 1, one replication round starts when the previous ends.• When R > 1, cells experience multiple simultaneous replication rounds.
Problems faced by linear DNA• 1. Free double-stranded DNA ends are very sensitive to degradation by intracellular nucleases, there must be a mechanism to protect the ends.• 2. Ends of linear DNA molecules must have a special mechanism for DNA replication.
• Protected by both types of telomeres: palindromic hairpin loops are protected by the lack of free double-stranded ends, and invertron telomeres are protected by proteins that bind to the 5-ends.
Table : Prokaryotic versusEukaryotic Chromosomes Prokaryotic Chromosomes Eukaryotic Chromosomes•Many prokaryotes contain a single circular •Eukaryotes contain multiple linearchromosome. chromosomes.•Prokaryotic chromosomes are condensed in •Eukaryotic chromosomes are condensed in athe nucleoid via DNA supercoiling and the membrane-bound nucleus via histones.binding of various architectural proteins. •In eukaryotes, transcription occurs in the•Because prokaryotic DNA can interact with the nucleus, and translation occurs in thecytoplasm, transcription and translation occur cytoplasm.simultaneously. •Most eukaryotes contain two copies of each•Most prokaryotes contain only one copy of gene (i.e., they are diploid).each gene (i.e., they are haploid). •Some eukaryotic genomes are organized into•Nonessential prokaryotic genes are commonly operons, but most are not.encoded on extrachromosomal plasmids. •Extrachromosomal plasmids are not•Prokaryotic genomes are efficient and commonly present in eukaryotes.compact, containing little repetitive DNA. •Eukaryotes contain large amounts of noncoding and repetitive DNA.