Beneath the surface: Evolution of methane activity in the bacterial multicomponent monooxygenases.

The bacterial multicomponent monooxygenase (BMM) family has evolved to oxidise a wide array of hydrocarbon substrates of importance to environmental emissions and biotechnology: foremost amongst these is methane, which requires among the most powerful oxidant in biology to activate. To understand how the BMM evolved methane oxidation activity, we investigated the changes in the enzyme family at different levels: operonic, phylogenetic analysis of the catalytic hydroxylase, subunit or folding factor presence, and sequence-function analysis across the entirety of the BMM phylogeny. Our results show that the BMM evolution of new activities was enabled by incremental increases in oxidative power of the active site, and these occur in multiple branches of the hydroxylase phylogenetic tree. While the hydroxylase primary sequence changes that resulted in increased oxidative power of the enzyme appear to be minor, the principle evolutionary advances enabling methane activity occurred in the other components of the BMM complex and in the recruitment of stability proteins. We propose that enzyme assembly and stabilization factors have independently-evolved multiple times in the BMM family to support enzymes that oxidise increasingly difficult substrates. Herein, we show an important example of evolution of catalytic function where modifications to the active site and substrate accessibility, which are the usual focus of enzyme evolution, are overshadowed by broader scale changes to structural stabilization and non-catalytic unit development. Retracing macroscale changes during enzyme evolution, as demonstrated here, should find ready application to other enzyme systems and in protein design.

Malonate degradation in Acinetobacter baylyi ADP1: operon organization and regulation by MdcR.

Transcriptional regulators in the LysR or GntR families are typically encoded in the genomic neighbourhood of bacterial genes for malonate degradation. While these arrangements have been evaluated using bioinformatics methods, experimental studies demonstrating co-transcription of predicted operons were lacking. Here, transcriptional regulation was characterized for a cluster of mdc genes that enable a soil bacterium, Acinetobacter baylyi ADP1, to use malonate as a carbon source. Despite previous assumptions that the mdc-gene set forms one operon, our studies revealed distinct promoters in two different regions of a nine-gene cluster. Furthermore, a single promoter is insufficient to account for transcription of mdcR, a regulatory gene that is convergent to other mdc genes. MdcR, a LysR-type transcriptional regulator, was shown to bind specifically to a site where it can activate mdc-gene transcription. Although mdcR deletion prevented growth on malonate, a 1 nt substitution in the promoter of mdcA enabled MdcR-independent growth on this carbon source. Regulation was characterized by methods including transcriptional fusions, quantitative reverse transcription PCR, reverse transcription PCR, 5'-rapid amplification of cDNA ends and gel shift assays. Moreover, a new technique was developed for transcriptional characterization of low-copy mRNA by increasing the DNA copy number of specific chromosomal regions. MdcR was shown to respond to malonate, in the absence of its catabolism. These studies contribute to ongoing characterization of the structure and function of a set of 44 LysR-type transcriptional regulators in A. baylyi ADP1.

Strategy of tuning gene expression ratio in prokaryotic cell from perspective of noise and correlation.

Genes are organized into operons in procaryote, and these genes in one operon generally have related functions. However, genes in the same operon are usually not equally expressed, and the ratio needs to be fine-tuned for specific functions. We examine the difference of gene expression noise and correlation when tuning the expression level at the transcriptional or translational level in a bicistronic operon driven by a constitutive or a two-state promoter. We get analytic results for the noise and correlation of gene expression levels, which is confirmed by our stochastic simulations. Both the noise and the correlation of gene expressions in an operon with a two-state promoter are higher than in an operon with a constitutive promoter. Premature termination of mRNA induced by transcription terminator in the intergenic region or changing translation rates can tune the protein ratio at the transcriptional level or at the translational level. We find that gene expression correlation between promoter-proximal and promoter-distal genes at the protein level decreases as termination increases. In contrast, changing translation rates in the normal range almost does not alter the correlation. This explains why the translation rate is a key factor of modulating gene expressions in an operon. Our results can be useful to understand the relationship between the operon structure and the biological function of a gene network, and also may help in synthetic biology design.

Conserved transcriptional unit organization of the cag pathogenicity island among Helicobacter pylori strains.

The Helicobacter pyloricag pathogenicity island (cag PAI) encodes a type IV secretion system that is more commonly found in strains isolated from patients with gastroduodenal disease than from those with asymptomatic gastritis. Genome-wide organization of the transcriptional units in H. pylori strain 26695 was recently established using RNA sequence analysis (Sharma et al., 2010). Here we used quantitative reverse-transcription polymerase chain reaction of open reading frames and intergenic regions to identify putative cag PAI operons in H. pylori; these operons were analyzed further by transcript profiling after deletion of selected promoter regions. Additionally, we used a promoter-trap system to identify functional cag PAI promoters. The results demonstrated that expression of genes on the H. pyloricag PAI varies by nearly five orders of magnitude and that the organization of cag PAI genes into transcriptional units is conserved among several H. pylori strains, including, 26695, J99, G27, and J166. We found evidence for 20 transcripts within the cag PAI, many of which likely overlap. Our data suggests that there are at least 11 operons: cag1-4, cag3-4, cag10-9, cag8-7, cag6-5, cag11-12, cag16-17, cag19-18, cag21-20, cag23-22, and cag25-24, as well as five monocistronic genes (cag4, cag13, cag14, cag15, and cag26). Additionally, the location of four of our functionally identified promoters suggests they are directing expression of, in one case, a truncated version of cag26 and in the other three, transcripts that are antisense to cag7, cag17, and cag23. We verified expression of two of these antisense transcripts, those antisense to cag17 and cag23, by reverse-transcription polymerase chain reaction. Taken together, our results suggest that the cag PAI transcriptional profile is generally conserved among H. pylori strains, 26695, J99, G27, and J166, and is likely complex.