Tit-Yee Wong

Role of Premature Stop Codons in Bacterial Evolution

When the stop codons TGA, TAA, and TAG are found in the second and third reading frames of a protein-encoding gene, they are considered premature stop codons (PSC). Deinococcus radiodurans disproportionately favored TGA more than the other two triplets as a PSC. The TGA triplet was also found more often in noncoding regions and as a stop codon, though the bias was less pronounced. We investigated this phenomenon in 72 bacterial species with widely differing chromosomal GC contents. Although TGA and TAG were compositionally similar, we found a great variation in use of TGA but a very limited range of use of TAG. The frequency of use of TGA in the gene sequences generally increased with the GC content of the chromosome, while the frequency of use of TAG, like that of TAA, was inversely proportional to the GC content of the chromosome. The patterns of use of TAA, TGA and TAG as real stop codons were less biased and less influenced by the GC content of the chromosome. Bacteria with higher chromosomal GC contents often contained fewer PSC trimers in their genes. Phylogenetically related bacteria often exhibited similar PSC ratios. In addition, metabolically versatile bacteria have significantly fewer PSC trimers in their genes. The bias toward TGA but against TAG as a PSC could not be explained either by the preferential usage of specific codons or by the GC contents of individual chromosomes. We proposed that the quantity and the quality of the PSC in the genome might be important in bacterial evolution.

Analysis of the coral associated bacterial community structures in healthy and diseased corals from off-shore of southern Taiwan

The methods of denaturing gradient gel electrophoresis (DGGE) and DNA sequencing were used to analyze the ribotypes of microbial communities associated with corals. Both healthy and diseased coral of different species were collected at three locations off the southern coast of Taiwan. Ribotyping results suggested that the microbial communities were diverse. The microbial community profiles, even among the same species of corals from different geographical locations, differ significantly. The coral-associated bacterial communities contain many bacteria common to the habitants of various invertebrates. However, some bacteria were unexpected. The presence of some unusual species, such as Staphylococcus, Clostridium and Legionella, associated with corals that were likely the results of human activities. Human activities, such as thermal pollution from the nearby nuclear plant, active fishing and tourism industries in the region might have all contributed to the change in bacterial communities and the death of coral colonies around the region.

Analysis of Codon Usage Patterns in Toxic Dinoflagellate Alexandrium tamarense through Expressed Sequence Tag Data

We have analyzed synonymous codon usage in the genome of A. tamarense CCMP 1598 for protein-coding sequences from 10865 expressed sequence tags (ESTs). We reconstructed a total of 4284 unigenes, including 74 ribosomal protein and 40 plastid-related genes, from ESTs using FrameDP, an open reading frame (ORF) prediction program. Correspondence analysis of A. tamarense genes based on codon usage showed that the GC content at the third base of synonymous codons (GC3s) was strongly correlated with the first axis (r = 0.93 with P < .001). On the other hand, the second axis discriminated between presumed highly and low expressed genes, with expression levels being confirmed by the analysis of EST frequencies (r = −0.89 with P < .001). Our results suggest that mutational bias is the major factor in shaping codon usage in A. tamarense genome, but other factors, namely, translational selection, hydropathy, and aromaticity, also appear to influence the selection of codon usage in this species.

Bacterial phylogenetic tree construction based on genomic translation stop signals

BackgroundThe efficiencies of the stop codons TAA, TAG, and TGA in protein synthesis termination are not the same. These variations could allow many genes to be regulated. There are many similar nucleotide trimers found on the second and third reading-frames of a gene. They are called premature stop codons (PSC). Like stop codons, the PSC in bacterial genomes are also highly bias in terms of their quantities and qualities on the genes. Phylogenetically related species often share a similar PSC profile. We want to know whether the selective forces that influence the stop codons and the PSC usage biases in a genome are related. We also wish to know how strong these trimers in a genome are related to the natural history of the bacterium. Knowing these relations may provide better knowledge in the phylogeny of bacteriaResultsA 16SrRNA-alignment tree of 19 well-studied α-, β- and γ-Proteobacteria Type species is used as standard reference for bacterial phylogeny. The genomes of sixty-one bacteria, belonging to the α-, β- and γ-Proteobacteria subphyla, are used for this study. The stop codons and PSC are collectively termed “Translation Stop Signals” (TSS). A gene is represented by nine scalars corresponding to the numbers of counts of TAA, TAG, and TGA on each of the three reading-frames of that gene. “Translation Stop Signals Ratio” (TSSR) is the ratio between the TSS counts. Four types of TSSR are investigated. The TSSR-1, TSSR-2 and TSSR-3 are each a 3-scalar series corresponding respectively to the average ratio of TAA: TAG: TGA on the first, second, and third reading-frames of all genes in a genome. The Genomic-TSSR is a 9-scalar series representing the ratio of distribution of all TSS on the three reading-frames of all genes in a genome. Results show that bacteria grouped by their similarities based on TSSR-1, TSSR-2, or TSSR-3 values could only partially resolve the phylogeny of the species. However, grouping bacteria based on thier Genomic-TSSR values resulted in clusters of bacteria identical to those bacterial clusters of the reference tree. Unlike the 16SrRNA method, the Genomic-TSSR tree is also able to separate closely related species/strains at high resolution. Species and strains separated by the Genomic-TSSR grouping method are often in good agreement with those classified by other taxonomic methods. Correspondence analysis of individual genes shows that most genes in a bacterial genome share a similar TSSR value. However, within a chromosome, the Genic-TSSR values of genes near the replication origin region (Ori) are more similar to each other than those genes near the terminus region (Ter).ConclusionThe translation stop signals on the three reading-frames of the genes on a bacterial genome are interrelated, possibly due to frequent off-frame recombination facilitated by translational-associated recombination (TSR). However, TSR may not occur randomly in a bacterial chromosome. Genes near the Ori region are often highly expressed and a bacterium always maintains multiple copies of Ori. Frequent collisions between DNA- polymerase and RNA-polymerase would create many DNA strand-breaks on the genes; whereas DNA strand-break induced homologues-recombination is more likely to take place between genes with similar sequence. Thus, localized recombination could explain why the TSSR of genes near the Ori region are more similar to each other. The quantity and quality of these TSS in a genome strongly reflect the natural history of a bacterium. We propose that the Genomic- TSSR can be used as a subjective biomarker to represent the phyletic status of a bacterium.

ATP-dependent fructose uptake system in Deinococcus radiodurans

The bacterial phosphoenolpyruvate (PEP)-dependent group translocation system (PTS) requires the presence of both membrane-bound and cytoplasmic components to phosphorylate and translocate sugar. Deinococcus radiodurans has a functional fruA gene coding for the membrane-bound components of the fructose-specific PTS. However, fruB gene coding for the fructose-specific cytosolic components of PTS is a pseudogene. Yet, this bacterium metabolized fructose readily. In vitro studies showed that both cell membranes and cytoplasmic fractions of the cells were needed for fructose phosphorylation. Further studies showed that fructose phosphorylation required ATP, not PEP, as the phosphate donor. Unlike most PEP-dependent PTS systems, fructose phosphorylation is sensitive to sodium fluoride, a kinase inhibitor. Fructose phosphorylation was also inhibited in the presence of antiserum against a kinase phosphorylation site. Rhodobacter capsulatus has a functional fruA–fruB system. Complementation assays by reconstituting the membrane fraction of D. radiodurans to the cytoplasmic fraction of R. capsulatus resulted in a PEP-dependent fructose phosphorylation, whereas mixing the membranes of R. capsulatus and the deinococcal cytosol in vitro resulted in an ATP-dependent fructose phosphorylation.

The effect of Mn(II) on the autoinducing growth inhibition factor in Deinococcus radiodurans.

Decreases in cell division at the stationary phase in bacterial cultures are often due to the depletion of nutrients and/or accumulation of toxic waste products. Yet, during the stationary phase, the highly radiation-resistant bacterium Deinococcus radiodurans undergoes new rounds of cell division when Mn(II) is added to the medium in a phenomenon known as manganese-induced cell division (MnCD). When cells were cultured in medium without Mn(II)-enrichment, a heat-resistant, proteinase K-resistant factor (or factors) with a molecular mass less than 10 kD accumulated in the spent medium. Inclusion of the concentrated spent medium in fresh medium could inhibit the growth of D. radiodurans significantly, and the degree of inhibition was dose dependent. However, the relative stimulatory effect of MnCD was also dose dependent—the higher the inhibition, the stronger was the MnCD response. Previous studies have shown that nutrients were not limiting and deinococcal cells would continue metabolizing its nutrients at stationary phase. Cells became more sensitive to radiation when nutrients in the medium eventually became depleted. We speculated that D. radiodurans might produce this factor in the medium to control its population density. The reduction in cell population would conserve the nutrients that in turn might enhance the survival of the species.

A new drug design strategy: Killing drug resistant bacteria by deactivating their hypothetical genes

ABSTRACT Despite that a bacterial genome is complicated by large numbers of horizontally transferred (HT) genes and function unknown hypothetical (FUN) genes, the Genic-Transcriptional-Stop-Signals-Ratio (TSSR) of a genome shows that HT and FUN genes are complementary to all other genes in the genome. When HT or certain FUN genes are omitted from the Escherichia coli K-12 genome, its Genomic-TSSR value becomes totally incomparable to other E. coli strains. The Genic-TSSR correlation tree of a pathogen shows that some FUN genes would form a unique cluster. Removing these genes by site-specific mutation or gene-knockout should lead to the demise of this pathogen.

Protein Mis-Termination Initiates Genetic Diseases, Cancers, and Restricts Bacterial Genome Expansion

Protein termination is an important cellular process. Protein termination relies on the stop-codons in the mRNA interacting properly with the releasing factors on the ribosome. One third of inherited diseases, including cancers, are associated with the mutation of the stop-codons. Many pathogens and viruses are able to manipulate their stop-codons to express their virulence. The influence of stop-codons is not limited to the primary reading frame of the genes. Stop-codons in the second and third reading frames are referred as premature stop signals (PSC). Stop-codons and PSCs together are collectively referred as stop-signals. The ratios of the stop-signals (referred as translation stop-signals ratio or TSSR) of genetically related bacteria, despite their great differences in gene contents, are much alike. This nearly identical Genomic-TSSR value of genetically related bacteria may suggest that bacterial genome expansion is limited by their unique stop-signals bias. We review the protein termination process and the different types of stop-codon mutation in plants, animals, microbes, and viruses, with special emphasis on the role of PSCs in directing bacterial evolution in their natural environments. Knowing the limit of genomic boundary could facilitate the formulation of new strategies in controlling the spread of diseases and combat antibiotic-resistant bacteria.

Synonymous Codon Usage in Marine Pico-Cyanobacterium Synechococcus sp. WH8102

Unicellular cyanobacteria Synechococcus are ubiquitous, abundant, and extremely competitive phytoplankton in the marine ecosystem. It may be expected that selection pressure would be important during the evolution of these cyanobacteria. In this study, synonymous codon usage in the genome of marine picoplankton Synechococcus sp. WH8102 was analyzed. The overall codon usage pattern indicates that G-or C-ending codons are predominant in Synechococcus sp. WH8102 because this organism is a GC rich organism (59.41%). However, highly-and lowly-expressed genes were found to cluster at opposite ends of the first major axis using correspondence analysis of WH8 102 genes based on relative synonymous codon usage (RSCU). Moreover, all of the 21 optimal codons found in highly -expressed genes ended with G or C. The results of this study indicate that both translational selection and mutational bias are crucial factors and hydrophobicity is a minor factor in shaping the codon usage pattern of Synechococcus sp. WH8102.