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Two copies of cellulose synthase gene were identified from the genome databases of B. pseudomallei K96243. Both genes are located on the chromosome II and labeled as open reading frame (ORF) BPSS0735 and BPSS1577 respectively. Sequence comparisons revealed that the two genes share low similarity and identity of 35% and 24% respectively. This suggested that the two cellulose synthase genes are of different origins. A study was conducted to characterize the role of BPSS1577 (also annotated as bcsA) in the cellulose biosynthesis of B. pseudomallei. A bcsA deleted mutant was generated to determine whether bcsA was involved in biofilm formation, adherence to abiotic surfaces and desiccation survival potential in this bacterium. Furthermore, the functionality of BPSS0735 is verified if cellulose production is accomplished in the deletion mutant. A markerless deletion mutant of bcsA was generated using suicidal vector, pDM4 that utilized chloramphenicol resistance gene (catR) as selectable marker and levan sucrose gene (sacB) as counter-selectable marker. In conclusion, ?bcsA01 showed no significant differences in cellulose production compared with wild type cells, thus confirming that BcsA is not the only cellulose synthase responsible for cellulose biosynthesis in B. pseudomallei. The ?bcsA01 mutant demonstrated a reduced ability in adherence to plastic, suggesting that bcsA might play a role in adherence to surfaces.
Cellulose is naturally viewed as a property of the plant kingdom. It is widely understood that plants produce cellulose as the structural component of their cell wall. However, single-cell organisms such as bacteria also share the same property. The gene encoding the catalytic subunit of cellulose synthase responsible for ß-glucan chain polymerization in cellulose biosynthesis was first isolated and identified in G. xylinus (Saxena et al., 1990; Wong et al., 1990) which then laid the basis for subsequent identification of cellulose biosynthesis gene clusters in plants (Pear et al., 1996). Available evidence seemed to support the notion that cellulose synthase enzymes originally belong to bacterial invention which was later acquired by diverse eukaryotes via lateral gene transfer mechanisms (Saxena and Brown, 2007).
To date, cellulose biosynthesis has been established for a diverse origin of bacteria at both phenotypic and genetic levels even though the biological significance of cellulose biogenesis in pathogenic, environmental and commensal bacteria is only partially deciphered. Taking into account the historical presence of cellulose biosynthesis gene clusters in bacterial genome and the successive events of lateral gene transfer between eukaryotes and within prokaryotes (Saxena and Brown, 2007), it is proposed that selection pressure may favor the maintenance of cellulose biosynthesis gene clusters in bacteria that increase their adaptive fitness in an environmental niche. In Acetobacteriaceae and Rhizobiaceae, it has been shown that cellulose molecules confer mechanical and chemical protection by maintaining the buoyancy of the cells at the air-liquid interface (Brown et al., 1976). Likewise, it is also involves in the bacterial attachment to their plant host cells (Matthysee, 1983). In Enterobacteriaceae, cellulose is associated with a multicellular morphotype which is also known as red, dry and rough (rdar) morphotype on Congo red plates (Romling et al., 2000). This coordinated multicellular behavior is said to offer various advantages to a bacterial population as compared to single, planktonic cells (Shapiro, 1998). In addition, cellulose is also coiled with the wrinkled spreader phenotype in the Pseudomonadaceae (Spiers et al., 2002) which was found to play essential role in the colonization of plant surface (Ude et al., 2006).
B. pseudomallei is a soil-dwelling bacterium that opportunistically infect both human and animals. It is the causative agent of melioidosis, a disease which recorded for up to 50% of mortality rate in certain endemic region. Over the years, the molecular mechanism of pathogenesis in melioidosis and the development of vaccine against melioidosis had grabbed the limelight in researches pertaining to this deadly bacterium. Less attention is paid to the environmental behavior of B. pseudomallei despite knowing that this bacterium acquires multifaceted adaptation and survival strategies in natural environments (Stevens and Galyov, 2004). As a result, biological systems which enhance the Burkholderia survival outside their host and thereby aiding in the perpetuation of this pathogen might be overlooked.
Having in mind that cellulose might act as a stabilizer that confers adaptive fitness to B. pseudomallei in the natural environments, we are prompted to understand cellulose biosynthesis system in this bacterium. Intriguingly, the complete genome databases of B. pseudomallei K96243 indicated the presence of more than one copy of cellulose synthase gene on the chromosome, namely, bcsA and BPSS0735. Given the complexity of this biological system, we have decided to refine the research questions to be addressed in this study. An overview of our research objectives are summarized below:
- To provide experimental evidence that B. pseudomallei UKMS-01 is able to produce cellulose. The phenotypic characterization of B. pseudomallei as a cellulose producer is achieved by observation on agar plates containing Congo red and Calcofluor binding dyes. Besides, cellulose production in this bacterium is quantified via cellulase digestion assay.
- To investigate the role of bcsA in cellulose production. Allelic replacement mutagenesis is adopted to construct a markerless bcsA deleted mutant by using a suicidal vector, pDM4.
- To study the resulting effects of bcsA deletion on biofilm formation, adherence and desiccation survivability in B. pseudomallei UKMS-01.
As such, this study is another effort towards understanding the function of cellulose synthase and the characteristic of cellulose with regard to bacterial behavior. It is hoped that the findings will strengthen our basis on this subject which allowed for further studies that ultimately contribute towards the establishment of a theoretical framework of cellulose biosynthesis in Burkholderia.