|Genes and Operons Organization|
We are interested in genes and operons organization related to the production of violacein, particularly the vio operon in Chromobacterium violaceum, the bcs operon in cellulose producing bacteria, and the trp operon in several bacterial strains.
Genes Involved in Matrix Formation in Chromobacterium violaceum Biofilm
n the majority of natural environments a structure known as biofilm is associated to prevailing microbial style of life. Bacterial biofilm consists of bacteria colonies inserted in their own extracellular polymeric matrix. That structure constitutes a kind of self-protection that allows the microorganism to survive in adverse environments. From the extracellular matrix components the exopolysaccharides (EPS) have generated great interest in industrial and medical applications. They have also been associated with immunological mechanisms that fight bacterial infections. Cellulose has been identified as an exopolysaccharide (EPS) in the extracellular matrix produced by several bacterial species during biofilm formation. Cellulose production has been well established for a-proteobacteria Acetobacter xylinum and Agrobacterium tumefaciens. Cellulose production has also been recently observed for g-proteobacteria such as Salmonella typhimurium, Escherichia coli, Pseudomonas fluorescens SBW25, among others.
Chromobacterium violaceum is a very interesting Gram-negative β-proteobacterium, common in soil and water. This bacterium is responsible for the production of a diversity of secondary metabolites with great biotechnological potential. The Brazilian National Genome Project Consortium has sequenced the C. violaceum strain ATCC 12472 genome (www.brgene.lncc.br; www.ncbi.nlm.nih.gov, access number NC_005085). Genome analysis of C. violaceum showed the presence of three genes related to cellulose biosynthesis: bcsC, bcsB and bcsA. Genes involved in bacterial cellulose biosynthesis are usually organized as an operon.
We have analyzed the structure and organization of those genes directly involved in cellulose biosynthesis, and looked for experimental evidences of the presence of cellulose in the extracellular matrix of biofilms produced by C. violaceum. The applied methodology made use of bioinformatics tools in order to exploit genomic information, such as gene structure, as well as conserved sequences related to regulatory and domain motifs.
Sequence alignment of S. typhimurium, E. coli and C. violaceum genomes revealed four genes involved in C. violaceum cellulose production: bcsA, bcsB, bcsZ and bcsC. Moreover, local alignment identified ORF CV2679 – previously annotated as a conserved hypothetical protein – as being similar to the yhjQ gene. In S. typhimurium and E. coli this gene is part of the bcs operon.
It is interesting to notice that our computational analysis revealed that the bcs operon comprises five structural genes: yhjQ (corresponding to ORF CV2679), bcsA, bcsB, bcsZ and bcsC, respectively encoding, in the reverse strand, for five cellulose synthase complex proteins: YhjQ, BcsA, BcsB, BcsZ, and BcsC.
Tryptophan Genes Structure in Chromobacterium violaceum
In Chromobacterium violaceum ATCC 12472 tryptophan is also a precursor for the building of secondary metabolites such as violacein and other compounds of pharmacological interest. In its genome the genes trpA-F and pabA-B that encode the tryptophan biosynthesis pathway enzymes have been identified. The pabA-B genes also encode the folate pathway enzymes. All genes are dispersed throughout the reverse strand, except trpC. The trpF, trpB and trpA genes form a sequence, with some residues between them. According to the annotation, trpE, pabA and pabB gene products are anthranilate synthase (AS) subunits; however, ORF CV0568 is a probable AS. The trpD gene encodes anthranilate phosphoribosyl transferase; trpF, phosphoribosyl-anthranilate isomerase; trpC, indoleglycerol phosphate synthase; trpA, tryptophan synthase, a-subunit; and trpB, tryptophan syntase, b-subunit.
The tryptophan biosynthesis pathway in Escherichia coli and C. violaceum was analyzed by using the Pathway-Genome Databases EcoCyc and CvioCyc, both available at http://cviocyc.intelab.ufsc.br. CvioCyc was built from Pathway-Tools, a software suite developed by SRI International. Additionally, the gene sequences that encode tryptophan pathway enzymes have been located and identified in other microorganisms (C. violaceum, Salmonella typhimurium, Pseudomonas aeruginosa, E. coli and Bacillus subtilis) by using bioinformatics tools available on the Web, including GenBank resources (www.ncbi.nih.gov), Artemis (www.sanger.ac.uk) and BioEdit (www.mbio.ncsu.edu).
A common feature of almost every bacterium is the order of the genes trpF, trpB and trpA. Generally they appear in that order in a cluster. So, this characteristic is independent of possessing or not a trp operon, or having or not transcriptional regulation. In C. violaceum, despite the genes are dispersed throughout its genome, this gene order is conserved: trpF before trpB, following by trpA. That suggests that the three genes could be under the same regulation, organized a single operon.
We have shown by calculating molecular weights that AS in C. violaceum is composed by a (trpE) and b (pabA) subunits. This is in agreement with values determined experimentally. In addition, AS subunits sequence alignment of several bacteria showed that there are conserved amino acids residues in specific loci (catalytic sites). pabB gene and ORF CV0568 were annotated as being an AS subunit and a probable anthranilate synthase, respectively. It is interesting to notice that cysteine, histidine and glutamate residues that are essential to provide allosteric and catalytic function to the enzyme are not conserved in those genes. However, ORF CV0568 is a chorismate binding enzyme family member. Therefore, we concluded that anthranilate synthase of C. violaceum is composed by single TrpE and PabA subunits. TrpE and PabA subunits have catalytic sites and the TrpE subunit shows an allosteric site.
Alignment among other tryptophan biosynthesis pathway enzymes (trpA-D, F) shows good sequence similarity for the studied bacteria C. violaceum, S. typhimurium, P. aeruginosa, E. coli and B. subtilis. That is computational evidence that these annotated trpA-D and trpF genes belong to the tryptophan biosynthesis pathway in C. violaceum.
Quorum sensing in Chromobacterium violaceum and the role of CviR response regulator
Analysis of the bcsZ Gene of Chromobacterium violaceum
Biotechnological applications of cellulases are being subject of investigation, such as bioconversion of cellulosic residues to ethanol production and textile applications.
In our laboratory we are using Chromobacterium violaceum, a Gram-negative β-proteobacterium sequenced in Brazil (www.brgene.lncc.br), as a model to study gene structures and potential biotech applications.
Annotation of the strain ATCC12472 showed the presence of genes related to cellulose biosynthesis. We have found that those genes are organized as a bcs operon, comprising ORF CV2679, bcsA, bcsB, bcsZ and bcsC genes. The bcsZ gene is found in the AAQ60346 locus and codifies for a protein of 382 amino acids, an endoglucanase that belongs to the glycosyl hydrolase family 8 (endo-1,4-D-glucanase, EC 126.96.36.199). Contrary to what its name suggests, cellulase function in the bcs operon remains unknown. A possible role for this protein is in the production of a primer that initiates cellulose biosynthesis.We are currently using other bacteria endoglucanase known domains to identify similar amino acid sequences in the C. violaceum BcsZ protein. In silico screening showed that the BcsZ protein does not possess a carbohydrate binding domain (CBD) indicating that it does not have high cellulolytic activity due to lower affinity for cellulose, as demonstrated by previous works. Nevertheless, CBD could be included in cellulases by using recombinant DNA technology.