Cloning and assembly strategies in microbial genome projects

Frangeul, Lionel; Nelson, Karen E.; Buchrieser, Carmen; Danchin, Antoine; Glaser, Philippe; Kunst, Frank

doi:10.1099/00221287-145-10-2625

Cited by 54 publications

(29 citation statements)

References 69 publications

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“…Genome sequencing was performed using the whole-genome shotgun strategy 45 , as described previously 46,47 . Two libraries (1-2 kb and 2-3 kb inserts) were generated by random mechanical shearing of genomic DNA and cloning into pcDNA-2.1 (Invitrogen) and a medium-size insert library (5-10 kb) was generated in the low copy number vector pSYX34 48 .…”

Section: Methodsmentioning

confidence: 99%

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens

et al. 2003

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Photorhabdus luminescens is an enterobacterium that is symbiotic with soil entomopathogenic nematodes and pathogenic to a wide range of insects. P. luminescens promotes its own transmission among susceptible insect populations using its nematode host as vector 1 . Its life cycle comprises a symbiotic stage in the nematode's gut and a virulent stage in the insect larvae, which it kills through toxemia and septicemia. After the nematode attacks a prey insect and P. luminescens is released, the bacterium produces a wide variety of virulence factors ensuring rapid insect killing. Bioconversion of the insect cadaver by exoenzymes produced by the bacteria allows the bacteria to multiply and the nematode to reproduce. During this process P. luminescens produces antibiotics to prevent invasion of the insect cadaver by bacterial or fungal competitors. Finally, elimination of competitors allows P. luminescens and the nematode to reassociate specifically before leaving the insect cadaver 2,3 .To better understand this complex life style, we determined the genome sequence of P. luminescens subspecies laumondii strain TT01 4 , a symbiont of the nematode Heterorhabditis bacteriophora isolated on Trinidad and Tobago. RESULTS General featuresStrain TT01 possesses a single circular chromosome of 5,688,987 bp with an average GC content of 42.8%. No plasmid replicon was found.A total of 4,839 protein-coding genes, including 157 pseudogenes, seven complete sets (23S, 5S and 16S) of ribosomal RNA operons and 85 tRNA genes, were predicted ( Fig. 1; Supplementary Table 1 online). Toxins against insectsMore toxin genes were predicted in the P. luminescens genome than in any other bacterial genome sequenced yet. A large number of these toxins may be involved in the killing of a wide variety of insects. Some may act synergistically or use redundancy for 'overkill' 5 , ensuring a quick death of the host. In addition, some may kill insects by interfering with their development. In the TT01 genome, two paralogs, plu4092 and plu4436, encode proteins similar to juvenile hormone esterases (JHEs) of the insect Leptinotarsa decemlineata 6 . Juvenile hormone maintains the insect in a larval state. Its inactivation by JHE allows metamorphosis to proceed. JHEs may be used to trigger the insect endocrine machinery at an inappropriate time and thus represents a promising approach for insect control 7 . These genes are located downstream of highly related orphan genes (plu4093 and plu4437), suggesting a locus duplication.The toxicity of the proteins encoded by these two loci was verified experimentally. Two Escherichia coli clones, containing the recombinant BAC1A02 and BAC8C11, were shown to be toxic toward insects. BAC1A02, which contains the locus plu4093-plu4092, exhibited substantial oral toxicity toward three mosquito species, Aedes aegypti,

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Section: Methodsmentioning

confidence: 99%

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens

et al. 2003

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“…Genome sequencing was performed using the conventional whole-genome shotgun strategy (24,26). Two libraries (1-to 2-kb and 2-to 3-kb inserts) were generated by random mechanical shearing of genomic DNA and cloning into pcDNA-2.1 (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Genome Sequence ofStreptococcus gallolyticus: Insights into Its Adaptation to the Bovine Rumen and Its Ability To Cause Endocarditis

et al. 2010

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Streptococcus gallolyticus (formerly known as Streptococcus bovis biotype I) is an increasing cause of endocarditis among streptococci and frequently associated with colon cancer. S. gallolyticus is part of the rumen flora but also a cause of disease in ruminants as well as in birds. Here we report the complete nucleotide sequence of strain UCN34, responsible for endocarditis in a patient also suffering from colon cancer. Analysis of the 2,239 proteins encoded by its 2,350-kb-long genome revealed unique features among streptococci, probably related to its adaptation to the rumen environment and its capacity to cause endocarditis. S. gallolyticus has the capacity to use a broad range of carbohydrates of plant origin, in particular to degrade polysaccharides derived from the plant cell wall. Its genome encodes a large repertoire of transporters and catalytic activities, like tannase, phenolic compounds decarboxylase, and bile salt hydrolase, that should contribute to the detoxification of the gut environment. Furthermore, S. gallolyticus synthesizes all 20 amino acids and more vitamins than any other sequenced Streptococcus species. Many of the genes encoding these specific functions were likely acquired by lateral gene transfer from other bacterial species present in the rumen. The surface properties of strain UCN34 may also contribute to its virulence. A polysaccharide capsule might be implicated in resistance to innate immunity defenses, and glucan mucopolysaccharides, three types of pili, and collagen binding proteins may play a role in adhesion to tissues in the course of endocarditis.

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“…The pGT200 and pGT220 plasmids, containing 7.4-and 16.3-kbp inserts, respectively, were fragmented by nebulization, and gel-purified fragments in the range of 1 to 2 kbp were cloned in the pcDNA2.1 vector using the nonpalindromic cloning method as described before (11). The inserts of randomly chosen clones were sequenced from both ends using a Perkin-Elmer ABI 3700 automated sequencer.…”

Section: Methodsmentioning

confidence: 99%