Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae

Abstract

The recent availability of bacterial genome sequence information permits the identification of conserved genes that are potential targets for novel antibiotic drug discovery. Using a coupled bioinformatic/experimental approach, a list of candidate conserved genes was generated using a Microbial Concordance bioinformatics tool followed by a targeted disruption campaign. Pneumococcal sequence data allowed for the design of precise PCR primers to clone the desired gene target fragments into the pEVP3 'suicide vector'. An insertion-duplication approach was employed that used the pEVP3 constructs and resulted in the introduction of a selectable chloramphenicol resistance marker into the chromosome. In the case of non-essential genes, cells can survive the disruption and form chloramphenicol-resistant colonies. A total of 347 candidate reading frames were subjected to disruption analysis, with 113 presumed to be essential due to lack of recovery of antibiotic-resistant colonies. In addition to essentiality determination, the same high-throughput methodology was used to overexpress gene products and to examine possible polarity effects for all essential genes.

Biotin uptake in prokaryotes by solute transporters with an optional ATP-binding cassette-containing module

“…Because streptococci have limited biosynthetic capacities, the products of these operons may be involved in the uptake of essential nutrients. Indeed, it has been shown that one of the cbiO orthologs (ybaE) is an essential gene in Streptococcus pneumoniae (16). It was tempting to speculate that single bioY genes encode functional biotin transporters.…”

Functional analysis of 11 putative essential genes in Bacillus subtilis

“…Several genome-wide inactivation studies have been carried out to identify essential genes in different bacterial species. Targeted gene disruptions have been used in Escherichia coli (Arigoni et al, 1998;Freiberg et al, 2001) and Streptococcus pneumoniae (Thanassi et al, 2002;Zalacain et al, 2003). Transposon mutagenesis in Haemophilus influenzae (Akerley et al, 2002) and Mycoplasma sp.…”

Structural and mechanistic insights into prokaryotic energy-coupling factor transporters

“…For example, Listeria monocytogenes lacks the ability to synthesize thiamin and depends on an ECF transporter for the uptake of this essential micronutrient 15 . In other clinically relevant bacteria, such as Mycoplasma genitalium, Streptococcus pneumoniae and Staphylococcus aureus, the genes that encode ECF transporters are also essential [16][17][18][19][20] , probably because these organisms lack the pathways for folate, biotin and thiamin biosynthesis, respectively (Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database). These examples highlight the importance of ECF transporters and suggest that the proteins involved are potentially useful targets for novel antibiotics.…”