Chromosomal position shift of a regulatory gene alters the bacterial phenotype

Recent studies strongly suggest that in bacterial cells the order of genes along the chromosomal origin-to-terminus axis is determinative for regulation of the growth phase-dependent gene expression. The prediction from this observation is that positional displacement of pleiotropic genes will affect the genetic regulation and hence, the cellular phenotype. To test this prediction we inserted the origin-proximal dusB-fis operon encoding the global regulator FIS in the vicinity of replication terminus on both arms of the Escherichia coli chromosome. We found that the lower fis gene dosage in the strains with terminus-proximal dusB-fis operons was compensated by increased fis expression such that the intracellular concentration of FIS was homeostatically adjusted. Nevertheless, despite unchanged FIS levels the positional displacement of dusB-fis impaired the competitive growth fitness of cells and altered the state of the overarching network regulating DNA topology, as well as the cellular response to environmental stress, hazardous substances and antibiotics. Our finding that the chromosomal repositioning of a regulatory gene can determine the cellular phenotype unveils an important yet unexplored facet of the genetic control mechanisms and paves the way for novel approaches to manipulate bacterial physiology.     

Isolation and properties of a plasmid which expresses the E. coli Su+7 amber suppressor tRNA gene.

Summary The gene of the amber suppressor tRNA derived from tRNA^Try, Su⁺7, has been inserted into a col E₁-derived vehicle by selecting for its expression. Despite selection for a suppressor phenotype, and the plasmid's stable presence at ca. 180 copies/cell during balanced growth, the level of mature tRNA maintained by the gene is less than that of the normal haploid tRNA^Try locus in the bacterial chromosome. Transfer RNA genes, both the plasmid Su⁺7 gene and chromosomal tRNA's are expressed during inhibition of protein synthesis. During, e.g. chloramphenicol inhibition, Su^-7 and Su⁺7 tRNA can be elevated similarly in the plasmid-containing cell; Su⁺7 reaches levels of molecules/cell which ordinarily characterize a major tRNA.The recombinant plasmid, but not the cloning vehicle alone, has a more general effect on tRNA levels; accumulation of tRNA from three chromosomal tRNA loci including tRNA^Try, continues during extensive isoleucine limitation. The plasmid therefore contains a locus which probably alters the relaxedstringent circuit, whose effects is disseminated to at least 3 widely separated loci.     

Evolution of multiple phosphodiesterase isoforms in stickleback involved in cAMP signal transduction pathway

Background  

Duplicate genes are considered to have evolved through the partitioning of ancestral functions among duplicates (subfunctionalization) and/or the acquisition of novel functions from a beneficial mutation (neofunctionalization). Additionally, an increase in gene dosage resulting from duplication may also confer an advantageous effect, as has been suggested for histone, tRNA, and rRNA genes. Currently, there is little understanding of the effect of increased gene dosage on subcellular networks like signal transduction pathways. Addressing this issue may provide further insights into the evolution by gene duplication.     

Multiple nutritional phenotypes of fission yeast mutants defective in genes encoding essential mitochondrial proteins

Mitochondria are essential for regulation of cellular respiration, energy production, small molecule metabolism, anti-oxidation and cell ageing, among other things. While the mitochondrial genome contains a small number of protein-coding genes, the great majority of mitochondrial proteins are encoded by chromosomal genes. In the fission yeast Schizosaccharomyces pombe, 770 proteins encoded by chromosomal genes are located in mitochondria. Of these, 195 proteins, many of which are implicated in translation and transport, are absolutely essential for viability. We isolated and characterized eight temperature-sensitive (ts) strains with mutations in essential mitochondrial proteins. Interestingly, they are also sensitive to limited nutrition (glucose and/or nitrogen), producing low-glucose-sensitive and ‘super-housekeeping'' phenotypes. They fail to produce colonies under low-glucose conditions at the permissive temperature or lose cell viability under nitrogen starvation at the restrictive temperature. The majority of these ts mitochondrial mutations may cause defects of gene expression in the mitochondrial genome. mrp4 and mrp17 are defective in mitochondrial ribosomal proteins. ppr3 is defective in rRNA expression, and trz2 and vrs2 are defective in tRNA maturation. This study promises potentially large dividends because mitochondrial quiescent functions are vital for human brain and muscle, and also for longevity.     

The complete genomic sequence of Mycoplasma penetrans,an intracellular bacterial pathogen in humans

The complete genomic sequence of an intracellular bacterial pathogen, Mycoplasma penetrans HF-2 strain, was determined. The HF-2 genome consists of a 1 358 633 bp single circular chromosome containing 1038 predicted coding sequences (CDSs), one set of rRNA genes and 30 tRNA genes. Among the 1038 CDSs, 264 predicted proteins are common to the Mycoplasmataceae sequenced thus far and 463 are M.penetrans specific. The genome contains the two-component system but lacks the essential cellular gene, uridine kinase. The relatively large genome of M.penetrans HF-2 among mycoplasma species may be accounted for by both its rich core proteome and the presence of a number of paralog families corresponding to 25.4% of all CDSs. The largest paralog family is the p35 family, which encodes surface lipoproteins including the major antigen, P35. A total of 44 genes for p35 and p35 homologs were identified and 30 of them form one large cluster in the chromosome. The genetic tree of p35 paralogs suggests the occurrence of dynamic chromosomal rearrangement in paralog formation during evolution. Thus, M.penetrans HF-2 may have acquired diverse repertoires of antigenic variation-related genes to allow its persistent infection in humans.     

Construction of the physical map of the chloroplast DNA of Phaseolus vulgaris and localization of ribosomal and transfer RNA genes

Construction of a physical map of the chloroplast DNA from Phaseolus vulgaris showed that this circular molecule is segmentally organized into four regions. Unlike other chloroplast DNAs which have analogous organization, two single-copy regions that separate two inverted repeats have been demonstrated to exist in both relative orientations, giving rise to two populations of DNA molecules.Hybridization studies using individual rRNA and tRNA species revealed the location of a set of rRNA genes and at least seven tRNA genes in each inverted repeat region, a minimum of 17 tRNA genes in the large single-copy region and one tRNA gene in the small single-copy region. The tRNA genes code for 24 tRNA species corresponding to 16 amino acids. Comparison of this gene map with those of other chloroplast DNAs suggests that DNA sequence rearrangements, involving some tRNA genes, have occurred.     

Control of rRNA synthesis in Escherichia coli at increased rrn gene dosage. Role of guanosine tetraphosphate and ribosome feedback.

The effects of extra, plasmid-borne rRNA genes on the synthesis rate of rRNA in Escherichia coli were examined by measuring the fraction of total RNA synthesis that is rRNA and tRNA (rs/rt), the cytoplasmic concentration of guanosine tetraphosphate (ppGpp), and the absolute rates of RNA and protein synthesis. Experiments were carried out in different growth media and with two different strains of E. coli, B/r and K-12. The results indicated: 1) increased rrn gene dosage from either intact or defective rrn genes reduced bacterial growth rates and ribosome activity (protein synthesis rate/average ribosome), and increased rs/rt. 2) Extra intact, but not extra defective, plasmid-borne rrn genes caused the level of ppGpp to be increased in comparison to the pBR322-carrying control strain. 3) As a function of ppGpp, rs/rt was increased with either intact or defective rrn genes. 4) The rRNA synthesis rate/rrn gene was reduced in the presence of extra rrn genes; this reduction in gene activity was greater with intact than with defective rrn genes. An analysis of these results showed that they are consistent with the ppGpp hypothesis of rRNA control but not with a feedback effector role of translating ribosomes.     

Mitochondrial tRNA gene clusters in Aspergillus nidulans: Organization and nucleotide sequence

The organization of tRNA genes on the circular 32 kb mitochondrial genome of the ascomycete Aspergillus nidulans has been studied by gel transfer hybridization and by DNA sequencing. Most of the tRNA genes are tightly clustered within two regions (1 kb each) flanking the split gene for the large ribosomal subunit RNA. The upstream cluster contains nine genes, the downstream cluster eleven genes. The twenty tRNA genes are on the same strand as the two rRNA genes and are separated from each other by AT-rich spacer sequences, usually consisting of only a few nucleotides. Two tRNA genes (leul and ala) are joined end to end. The occurrence of two tRNA^Gty genes is the first exception to the observation that in mitochondria all four-codon families are read by a single tRNA. Both genes are adjacent and show extensive sequence homology, suggesting relatively recent origin by gene duplication. The product of glyl has a U in the wobble position as do all other tRNA gene products specific for four-codon families, whereas the gly2 product, which has a rare A in the same position, should read only the codon GGU. The products of metl and thr have an A and G in positions 18 and 55, respectively, like the mitochondrial tRNA^fMet and tRNA^Thr of Neurospora crassa. Other unusual features are the replacement of the invariant G-C pair at positions 53 and 61 by A-T in met2, glyl and gly2, the replacement of the invariant T at position 8 by A in phe and G in pro and the deletion of a nucleotide at position 9 in ser2.     

Variation in the strength of selected codon usage bias among bacteria

Among bacteria, many species have synonymous codon usage patterns that have been influenced by natural selection for those codons that are translated more accurately and/or efficiently. However, in other species selection appears to have been ineffective. Here, we introduce a population genetics-based model for quantifying the extent to which selection has been effective. The approach is applied to 80 phylogenetically diverse bacterial species for which whole genome sequences are available. The strength of selected codon usage bias, S, is found to vary substantially among species; in 30% of the genomes examined, there was no significant evidence that selection had been effective. Values of S are highly positively correlated with both the number of rRNA operons and the number of tRNA genes. These results are consistent with the hypothesis that species exposed to selection for rapid growth have more rRNA operons, more tRNA genes and more strongly selected codon usage bias. For example, Clostridium perfringens, the species with the highest value of S, can have a generation time as short as 7 min.     

Mitochondrial Genome Analysis of Wild Rice (Oryza minuta) and Its Comparison with Other Related Species

Oryza minuta (Poaceae family) is a tetraploid wild relative of cultivated rice with a BBCC genome. O. minuta has the potential to resist against various pathogenic diseases such as bacterial blight (BB), white backed planthopper (WBPH) and brown plant hopper (BPH). Here, we sequenced and annotated the complete mitochondrial genome of O. minuta. The mtDNA genome is 515,022 bp, containing 60 protein coding genes, 31 tRNA genes and two rRNA genes. The mitochondrial genome organization and the gene content at the nucleotide level are highly similar (89%) to that of O. rufipogon. Comparison with other related species revealed that most of the genes with known function are conserved among the Poaceae members. Similarly, O. minuta mt genome shared 24 protein-coding genes, 15 tRNA genes and 1 ribosomal RNA gene with other rice species (indica and japonica). The evolutionary relationship and phylogenetic analysis revealed that O. minuta is more closely related to O. rufipogon than to any other related species. Such studies are essential to understand the evolutionary divergence among species and analyze common gene pools to combat risks in the current scenario of a changing environment.     

Genomic imbalance determines positive and negative modulation of gene expression in diploid maize

Genomic imbalance caused by changing the dosage of individual chromosomes (aneuploidy) has a more detrimental effect than varying the dosage of complete sets of chromosomes (ploidy). We examined the impact of both increased and decreased dosage of 15 distal and 1 interstitial chromosomal regions via RNA-seq of maize (Zea mays) mature leaf tissue to reveal new aspects of genomic imbalance. The results indicate that significant changes in gene expression in aneuploids occur both on the varied chromosome (cis) and the remainder of the genome (trans), with a wider spread of modulation compared with the whole-ploidy series of haploid to tetraploid. In general, cis genes in aneuploids range from a gene-dosage effect to dosage compensation, whereas for trans genes the most common effect is an inverse correlation in that expression is modulated toward the opposite direction of the varied chromosomal dosage, although positive modulations also occur. Furthermore, this analysis revealed the existence of increased and decreased effects in which the expression of many genes under genome imbalance are modulated toward the same direction regardless of increased or decreased chromosomal dosage, which is predicted from kinetic considerations of multicomponent molecular interactions. The findings provide novel insights into understanding mechanistic aspects of gene regulation.

Genomic imbalance caused by the addition or subtraction of chromosomal segments leads to modulations of gene expression inversely or directly related to chromosomal dosage but also to nonlinear responses.     

Feedback regulation of rRNA synthesis. A mutational alteration in the anti-Shine-Dalgarno region of the 16 S rRNA gene abolishes regulation

It was shown that induction of rRNA (and ribosome) synthesis from the lambda PL promoter/operator by temperature shift-up causes a repression of rRNA and tRNA synthesis from chromosomal genes. We have carried out experiments using a similar conditional rRNA gene expression system in which a mutational alteration was introduced in the anti-Shine-Dalgarno region at the 3'-end of the 16 S rRNA gene. It was found that the repression observed with the wild-type gene was largely abolished by the mutation. It appears that ribosomes inefficient in translational initiation are unable to cause feedback regulation of rRNA synthesis. It is suggested that the cell regulates rRNA (and tRNA) synthesis by monitoring the production of ribosomes, and that this monitoring is apparently carried out through their activity in the initiation (and perhaps subsequent steps) of translation.     

Global tRNA misacylation induced by anaerobiosis and antibiotic exposure broadly increases stress resistance in Escherichia coli

High translational fidelity is commonly considered a requirement for optimal cellular health and protein function. However, recent findings have shown that inducible mistranslation specifically with methionine engendered at the tRNA charging level occurs in mammalian cells, yeast and archaea, yet it was unknown whether bacteria were capable of mounting a similar response. Here, we demonstrate that Escherichia coli misacylates non-methionyl-tRNAs with methionine in response to anaerobiosis and antibiotic exposure via the methionyl–tRNA synthetase (MetRS). Two MetRS succinyl-lysine modifications independently confer high tRNA charging fidelity to the otherwise promiscuous, unmodified enzyme. Strains incapable of tRNA mismethionylation are less adept at growth in the presence of antibiotics and stressors. The presence of tRNA mismethionylation and its potential role in mistranslation within the bacterial domain establishes this response as a pervasive biological mechanism and connects it to diverse cellular functions and modes of fitness.     

A Broad-Host-Range, Generalized Transducing Phage (SN-T) Acquires 16S rRNA Genes from Different Genera of Bacteria

Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance as well as their ability to affect lateral gene transfer and to harbor bacterial 16S rRNA gene sequences. Here, we demonstrate the ability of broad-host-range, generalized transducing phages to acquire 16S rRNA genes and gene sequences. Using PCR and primers specific to conserved regions of the 16S rRNA gene, we have found that generalized transducing phages (D3112, UT1, and SN-T), but not specialized transducing phages (D3), acquired entire bacterial 16S rRNA genes. Furthermore, we show that the broad-host-range, generalized transducing phage SN-T is capable of acquiring the 16S rRNA gene from two different genera: Sphaerotilus natans, the host from which SN-T was originally isolated, and Pseudomonas aeruginosa. In sequential infections, SN-T harbored only 16S rRNA gene sequences of the final host as determined by restriction fragment length polymorphism analysis. The frequency of 16S rRNA gene sequences in SN-T populations was determined to be 1 × 10⁻⁹ transductants/PFU. Our findings further implicate transduction in the horizontal transfer of 16S rRNA genes between different species or genera of bacteria.