Identifying the genetic basis of ecologically and biotechnologically useful functions of the bacterium Burkholderia vietnamiensis

Abstract

Signature-tagged mutagenesis (STM) was used to identify genetic determinants of fitness associated with two key ecological processes mediated by bacteria. Burkholderia vietnamiensis strain G4 was used as a model bacterium to investigate: phenol degradation as a model of bioremediation, and pea rhizosphere colonization as a prerequisite to biological control and phytoremediation. A total of 1900 mutants were screened and 196 putative fitness mutants identified; the genetic basis of 137 of these mutations was determined by correlation to the G4 genome. The phenol-STM screen was more successful at identifying phenol degradation mutations (83 mutants; 4.4% hit rate) than a conventional agar-based phenol screen (49 mutants, 5319 screened, 0.92% hit rate). The combination of both screens completely defined the components of the TOM pathway in strain G4 and also identified novel accessory genes not previously implicated in phenol utilization. The rhizosphere-STM screen identified 113 mutants (5.9% hit rate); 107 had reduced tag signals indicative of poor rhizosphere colonization (Rhiz-), while six mutants produced high hybridization signals suggesting increased rhizosphere competence (Rhiz+). Competition assays confirmed that 69% of Rhiz- mutants tested (24/35) were severely compromised in their rhizosphere fitness. Seventy Rhiz- mutations mapped to genes with the following putative functions: amino acid biosynthesis (25; 36%), general metabolism (18; 26%), hypothetical (9; 13%), regulatory genes (4; 5.7%), transport and stress (2 each; 2.8% respectively). One of the most interesting discoveries mediated by the rhizosphere-STM screen was the identification of three Rhiz+ mutants inactivated within a single virulence-associated autotransporter adhesin gene; this mutation consistently produced a hyper-colonization phenotype suggesting a highly novel role for this surface adhesin during plant interactions. Our study has shown that STM can be successfully applied to ecologically important microbial interactions, defining the underlying genetic systems important for biotechnological fitness of environmental bacteria such those from the Burkholderia cepacia complex.

Culture-Independent Analysis of Bacterial Fuel Contamination Provides Insight into the Level of Concordance with the Standard Industry Practice of Aerobic Cultivation

“…To enable parallel comparisons of the efficacies of different growth media in capturing the broadest range of bacteria, fuel sample JW78, representing a marine gas oil (see Table S1 in the supplemental material), was cultivated on TSA, on the BSM minimal salts agar (glucose carbon source) that has been previously shown to support excellent growth of Pseudomonas (19) and Burkholderia (37), and on BH agar as another minimal salts agar (supplemented with glucose) that is recommended for use with fuel-contaminating bacteria (Fig. 3).…”

Phase variation has a role in Burkholderia ambifaria niche adaptation

“…Rhizosphere colonization Pisum sativum seeds were sterilized as described earlier (O'Sullivan et al, 2007). After 3 days, germinated seeds were sown in tubes containing Perlite as the solid substrate and 8 mM KNO 3 À -supplemented Fahraeus liquid medium (Fahraeus, 1957).…”

Molecular Analysis of Phenol-Degrading Microbial Strains

“…It is known that among the strains belonging to Burkholderia there are some able to utilize phenol by two metabolic pathways Ð ortho and/or meta (Rogers et al, 2000). O'Sullivan et al (2007) report a study defining a strain of Burkholderia vietnamiensis able to degrade a number of toxic aromatic compounds through the meta-cleavage pathway of degradation.…”

“…It has been reported that further degradation 0939Ð5075/2008/0100Ð0133 $ 06.00 " 2008 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D of the phenol hydroxylase reaction product catechol may occur via the ortho-or meta-fission pathway in microorganisms. The both modes of catechol degradation might be found in bacteria (Sauret-Ignazi et al, 1996;Polymenakou and Stephanou, 2005;O'Sullivan et al, 2007). The mechanism of phenol utilization most common for yeast and micelial fungi goes through the orthofission pathway (Spå nning and Neujahr, 1991;Yan et al, 2005).…”