The Genomic Pattern of tDNA Operon Expression in E. coli

In fast-growing microorganisms, a tRNA concentration profile enriched in major isoacceptors selects for the biased usage of cognate codons. This optimizes translational rate for the least mass invested in the translational apparatus. Such translational streamlining is thought to be growth-regulated, but its genetic basis is poorly understood. First, we found in reanalysis of the E. coli tRNA profile that the degree to which it is translationally streamlined is nearly invariant with growth rate. Then, using least squares multiple regression, we partitioned tRNA isoacceptor pools to predicted tDNA operons from the E. coli K12 genome. Co-expression of tDNAs in operons explains the tRNA profile significantly better than tDNA gene dosage alone. Also, operon expression increases significantly with proximity to the origin of replication, oriC, at all growth rates. Genome location explains about 15% of expression variation in a form, at a given growth rate, that is consistent with replication-dependent gene concentration effects. Yet the change in the tRNA profile with growth rate is less than would be expected from such effects. We estimated per-copy expression rates for all tDNA operons that were consistent with independent estimates for rDNA operons. We also found that tDNA operon location, and the location dependence of expression, were significantly different in the leading and lagging strands. The operonic organization and genomic location of tDNA operons are significant factors influencing their expression. Nonrandom patterns of location and strandedness shown by tDNA operons in E. coli suggest that their genomic architecture may be under selection to satisfy physiological demand for tRNA expression at high growth rates

Vibrionaceae core, shell and cloud genes are non-randomly distributed on Chr 1: An hypothesis that links the genomic location of genes with their intracellular placement

Background - The genome of Vibrionaceae bacteria, which consists of two circular chromosomes, is replicated in a highly ordered fashion. In fast-growing bacteria, multifork replication results in higher gene copy numbers and increased expression of genes located close to the origin of replication of Chr 1 (ori1). This is believed to be a growth optimization strategy to satisfy the high demand of essential growth factors during fast growth. The relationship between ori1-proximate growth-related genes and gene expression during fast growth has been investigated by many researchers. However, it remains unclear which other gene categories that are present close to ori1 and if expression of all ori1-proximate genes is increased during fast growth, or if expression is selectively elevated for certain gene categories. Results - We calculated the pangenome of all complete genomes from the Vibrionaceae family and mapped the four pangene categories, core, softcore, shell and cloud, to their chromosomal positions. This revealed that core and softcore genes were found heavily biased towards ori1, while shell genes were overrepresented at the opposite part of Chr 1 (i.e., close to ter1). RNA-seq of Aliivibrio salmonicida and Vibrio natriegens showed global gene expression patterns that consistently correlated with chromosomal distance to ori1. Despite a biased gene distribution pattern, all pangene categories contributed to a skewed expression pattern at fast-growing conditions, whereas at slow-growing conditions, softcore, shell and cloud genes were responsible for elevated expression. Conclusion - The pangene categories were non-randomly organized on Chr 1, with an overrepresentation of core and softcore genes around ori1, and overrepresentation of shell and cloud genes around ter1. Furthermore, we mapped our gene distribution data on to the intracellular positioning of chromatin described for V. cholerae, and found that core/softcore and shell/cloud genes appear enriched at two spatially separated intracellular regions. Based on these observations, we hypothesize that there is a link between the genomic location of genes and their cellular placement

The Systemic Imprint of Growth and Its Uses in Ecological (Meta)Genomics

Microbial minimal generation times range from a few minutes to several weeks. They are evolutionarily determined by variables such as environment stability, nutrient availability, and community diversity. Selection for fast growth adaptively imprints genomes, resulting in gene amplification, adapted chromosomal organization, and biased codon usage. We found that these growth-related traits in 214 species of bacteria and archaea are highly correlated, suggesting they all result from growth optimization. While modeling their association with maximal growth rates in view of synthetic biology applications, we observed that codon usage biases are better correlates of growth rates than any other trait, including rRNA copy number. Systematic deviations to our model reveal two distinct evolutionary processes. First, genome organization shows more evolutionary inertia than growth rates. This results in over-representation of growth-related traits in fast degrading genomes. Second, selection for these traits depends on optimal growth temperature: for similar generation times purifying selection is stronger in psychrophiles, intermediate in mesophiles, and lower in thermophiles. Using this information, we created a predictor of maximal growth rate adapted to small genome fragments. We applied it to three metagenomic environmental samples to show that a transiently rich environment, as the human gut, selects for fast-growers, that a toxic environment, as the acid mine biofilm, selects for low growth rates, whereas a diverse environment, like the soil, shows all ranges of growth rates. We also demonstrate that microbial colonizers of babies gut grow faster than stabilized human adults gut communities. In conclusion, we show that one can predict maximal growth rates from sequence data alone, and we propose that such information can be used to facilitate the manipulation of generation times. Our predictor allows inferring growth rates in the vast majority of uncultivable prokaryotes and paves the way to the understanding of community dynamics from metagenomic data

Engineering Parageobacillus thermoglucosidans as a robust platform for bioethanol production

Philosophiae Doctor - PhD (Biotechnology)Parageobacillus thermoglucosidans is a promising “platform” organism to use in the production ofa range of useful metabolites with demonstrated ability to produce ethanol, isobutanol and polylactic acid for bio-degradable plastics. Extensive work has been done in engineering the organism for enhanced ethanol production. However, an often used and highly effective alternative pathway (pyruvate decarboxylase mediated) for ethanol production has not yet been demonstrated in P. thermoglucosidans. We first characterize two novel bacterial pyruvate decarboxylase enzymes (PDC’s) then attempt to express the more thermostable of these enzymes from Gluconobacter oxydans in P. thermoglucosidans to improve ethanol yields. Initial expression was unsuccessful. Analysis of the codon usage pattern for the gene revealed that the codon usage was suboptimal in the heterologous host P. thermoglucosidans. After codon harmonization, we could demonstrate successful expression of the enzyme at 45°C, however not at the bacterium’s optimum growth temperature of 60°C. This was concomitant with enhanced ethanol production close to the theoretical yield possible (0.5g/l)