High Confidence Prediction of Essential Genes in Burkholderia Cenocepacia

BACKGROUND: Essential genes are absolutely required for the survival of an organism. The identification of essential genes, besides being one of the most fundamental questions in biology, is also of interest for the emerging science of synthetic biology and for the development of novel antimicrobials. New antimicrobial therapies are desperately needed to treat multidrug-resistant pathogens, such as members of the Burkholderia cepacia complex. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesize that essential genes may be highly conserved within a group of evolutionary closely related organisms. Using a bioinformatics approach we determined that the core genome of the order Burkholderiales consists of 649 genes. All but two of these identified genes were located on chromosome 1 of Burkholderia cenocepacia. Although many of the 649 core genes of Burkholderiales have been shown to be essential in other bacteria, we were also able to identify a number of novel essential genes present mainly, or exclusively, within this order. The essentiality of some of the core genes, including the known essential genes infB, gyrB, ubiB, and valS, as well as the so far uncharacterized genes BCAL1882, BCAL2769, BCAL3142 and BCAL3369 has been confirmed experimentally in B. cenocepacia. CONCLUSIONS/SIGNIFICANCE: We report on the identification of essential genes using a novel bioinformatics strategy and provide bioinformatics and experimental evidence that the large majority of the identified genes are indeed essential. The essential genes identified here may represent valuable targets for the development of novel antimicrobials and their detailed study may shed new light on the functions required to support life

Conditional lethal phenotype of the rhamnose-dependent mutants of the <i>B. cenocepacia</i> essential genes.

<p>The constructed rhamnose-inducible mutants H111<i>infB</i>, H111<i>gyrB</i>, H111<i>uniB</i>, H111<i>valS</i>, H111<i>BCAL1882</i>, H111<i>BCAL2769</i>, H111<i>BCAL3142</i> and H111<i>BCAL3369</i> grew on LB plates supplemented with rhamnose but not with glucose as expected for mutants with essential genes under the control of rhamnose promoter. Complementation of mutants H111<i>infB</i>c, H111<i>ubiB</i>c, H111<i>valS</i>c, H111<i>1882</i>c and H111<i>3142</i>c <i>in trans</i> has restored their ability to grow on glucose. Undiluted and 10-fold diluted cultures of mutants (0, 1) usually grew visibly on plates supplemented with either glucose or rhamnose prior to depletion of the existing protein; however, at 100, 1000 and 10000- fold dilutions (2, 3, 4) mutants were unable to grow on plates supplemented with glucose enough to be seen by eye.</p

Chromosome 1 harbours most of the core genome. A)

<p>Schematic view of chromosomes 1–3 of <i>Burkholderia cenocepacia</i> J2315. The 649 genes belonging to our core genome are indicated by blue and red bars (positive or negative gene direction respectively). Core genes with homologues within the core genome are printed in light blue and rose. Other genes, which are less conserved in respect of presence within the <i>Burkholderiales</i> are indicated by grey bars (the height indicates the degree of conservation). The black graph also indicates the degree of conservation. 636 core genes belong to chromosome 1. Out of the remaining 13 genes, only two that are located on chromosome 2 are singletons (do not have other homologues within the genome). B) 454 core genes have homologues in the DEG database and are thus predicted to be essential (violet). Our core genome contains 195 genes without clear orthologues in the DEG database (yellow) 111 of these genes do show weak homology to DEG. The other 84 are potentially novel essential genes. 574 of <i>B. cenocepacia</i> J2315 homologues to the DEG database do not belong to our core genome (green).</p

Microscopy and live-dead staining.

<p>Live-dead staining of the wild type strain H111 and of the rhamnose-inducible mutant H111<i>infB</i> grown in the presence of rhamnose or glucose. Green fluorescence indicates viable cells, while dead bacteria appear fluorescent red. The figure depicts the reduced ability of H111<i>infB</i> to survive in the medium with glucose.</p

Investigated genes are essential for growth and viability of <i>B. cenocepacia</i>.

<p>Growth curves of the wild type H111 (circles), and rhamnose-inducible mutants: H111<i>infB</i>, H111<i>gyrB</i>, H111<i>uniB</i>, H111<i>valS</i>, H111<i>BCAL1882</i>, H111<i>BCAL2769</i>, H111<i>BCAL3142</i> and H111<i>BCAL3369</i> in the presence of rhamnose (squares) or glucose (triangles). Complementation of mutants H111<i>infB</i>c, H111<i>ubiB</i>c, H111<i>valS</i>c, H111<i>1882</i>c and H111<i>3142</i>c <i>in trans</i> has restored their ability to grow in the presence of glucose (stars). Values are the mean and standard deviation of a representative experiment with triplicate values.</p