A “Double Adaptor” Method for Improved Shotgun Library Construction

Zomorodipour

et al. 1998

Nature

1,527

1,093

We describe here the complete genome sequence (1,111,523 base pairs) of the obligate intracellular parasite Rickettsia prowazekii, the causative agent of epidemic typhus. This genome contains 834 protein-coding genes. The functional pro®les of these genes show similarities to those of mitochondrial genes: no genes required for anaerobic glycolysis are found in either R. prowazekii or mitochondrial genomes, but a complete set of genes encoding components of the tricarboxylic acid cycle and the respiratory-chain complex is found in R. prowazekii. In effect, ATP production in Rickettsia is the same as that in mitochondria. Many genes involved in the biosynthesis and regulation of biosynthesis of amino acids and nucleosides in free-living bacteria are absent from R. prowazekii and mitochondria. Such genes seem to have been replaced by homologues in the nuclear (host) genome. The R. prowazekii genome contains the highest proportion of non-coding DNA (24%) detected so far in a microbial genome. Such non-coding sequences may be degraded remnants of`neutralized' genes that await elimination from the genome. Phylogenetic analyses indicate that R. prowazekii is more closely related to mitochondria than is any other microbe studied so far.The Rickettsia are a-proteobacteria that multiply in eukaryotic cells only. R. prowazekii is the agent of epidemic, louse-borne typhus in humans. Three features of this endocellular parasite deserve our attention. First, R. prowazekii is estimated to have infected 20±30 million humans in the wake of the First World War and killed another few million following the Second World War (ref. 1). Because it is the descendent of free-living organisms 2±4 , its genome provides insight into adaptations to the obligate intracellular lifestyle, with probable practical value. Second, phylogenetic analyses based on sequences of ribosomal RNA and heat-shock proteins indicate that mitochondria may be derived from the aproteobacteria 5,6 . Indeed, the closest extant relatives of the ancestor to mitochondria seem to be the Rickettsia 7±10 . That modern Rickettsia favour an intracellular lifestyle identi®es these bacteria as the sort of organism that might have initiated the endosymbiotic scenario leading to modern mitochondria 11 . Finally, the genome of R. prowazekii is a small one, containing only 1,111,523 base pairs (bp). Its phylogenetic placement and many other characteristics identify it as a descendant of bacteria with substantially larger genomes 2±4 . Thus Rickettsia, like mitochondria, are good examples of highly derived genomes, the products of several types of reductive evolution.The genome sequence of R. prowazekii indicates that these three features may be related. For example, prokaryotic genomes evolving within a cell dominated by a much larger, eukaryote genome and constrained by bottle-necked population dynamics will tend to lose genetic information 12,13 . Predictable sets of expendable genes will tend to disappear from the prokaryotic genome when they are made redundant by the activit...

Section: Methodsmentioning

confidence: 99%

The genome sequence of Rickettsia prowazekii and the origin of mitochondria

Zomorodipour

et al. 1998

Nature

1,527

1,093

“…We constructed plasmid libraries from randomly sheared DNA in pUC18 or pUC19 with insert sizes of 1-2 kb or 2-4 kb, respectively. We also constructed five libraries with insert sizes of 1-4 kb using the TOPO Shotgun subcloning kit (Invitrogen), from nebulized DNA, and one M13 library with insert sizes of 1-2 kb using the double adaptor method 43 from nebulized DNA. We carried out DNA sequencing and assembly as previously described 44 .…”

Section: Methodsmentioning

confidence: 99%

The complete genome sequence of Francisella tularensis, the causative agent of tularemia

et al. 2005

Francisella tularensis is one of the most infectious human pathogens known. In the past, both the former Soviet Union and the US had programs to develop weapons containing the bacterium. We report the complete genome sequence of a highly virulent isolate of F. tularensis (1,892,819 bp). The sequence uncovers previously uncharacterized genes encoding type IV pili, a surface polysaccharide and iron-acquisition systems. Several virulence-associated genes were located in a putative pathogenicity island, which was duplicated in the genome. More than 10% of the putative coding sequences contained insertion-deletion or substitution mutations and seemed to be deteriorating. The genome is rich in IS elements, including IS630 Tc-1 mariner family transposons, which are not expected in a prokaryote. We used a computational method for predicting metabolic pathways and found an unexpectedly high proportion of disrupted pathways, explaining the fastidious nutritional requirements of the bacterium. The loss of biosynthetic pathways indicates that F. tularensis is an obligate host-dependent bacterium in its natural life cycle. Our results have implications for our understanding of how highly virulent human pathogens evolve and will expedite strategies to combat them.

“…Genomic DNA was extracted from agarose plugs using the QIAEXII gel extraction kit (Qiagen). The DNA was sheared by nebulization and end-repaired with T4 DNA polymerase (Roche Molecular Biochemicals) and Klenow (Roche Molecular Biochemicals) (Andersson et al, 1996). The end-repaired DNA was separated on a 1 % (w\v) SeaPlaque low-melting-point agarose (FMC BioProducts) gel.…”

Section: Methodsmentioning

confidence: 99%

Physical and genetic map of the Clostridium saccharobutylicum (formerly Clostridium acetobutylicum) NCP 262 chromosome

2001

A physical and genetic map of the Clostridium saccharobutylicum NCP 262 chromosome was constructed. The order of macrorestriction fragments was determined by analysing fragments generated after single and double digestion with the restriction enzymes BssHII, I-CeuI, Sse8387I, RsrII and SfiI and separation by PFGE. The I-CeuI backbone of C. saccharobutylicum was constructed by indirect end-labelling with rrs-and 3' rrl-specific probes located on either side of the I-CeuI site in the rrn operon, and reciprocal separation of BssHII and I-CeuI digestion products by two-dimensional PFGE. The positions of BssHII fragments on the physical map were determined using a library of linking clones containing BssHII cleavage sites. The size of the circular genome was estimated to be 53 Mb with a mean resolution of approximately 140 kb. The chromosome of C. saccharobutylicum contains 12 rrn operons, located on 46 % of the chromosome, which are transcribed divergently from the deduced origin of replication. The genetic map was constructed by determining the location of 28 genes involved in house-keeping, heat-shock response, sporulation, electron transfer and acid-and solvent-formation. Comparison of the C. saccharobutylicum genetic map with those of the spore-forming bacteria Bacillus subtilis, Clostridium acetobutylicum, Clostridium perfringens and Clostridium beijerinckii indicated C. saccharobutylicum to be most similar to the latter two Clostridium species, with the order of the genes within the gyrAB and recA loci being conserved.