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ABSTRACT: A recently developed real-time PCR method for the determination of genome copy numbers was optimized for the application to cyanobacteria. Three species were chosen to represent a fresh water species, a salt water species, and two strains of a widely used laboratory species. Synechococcus PCC 7942 and Synechococcus WH7803 were found to contain 3-4 genome copies per cell and are thus oligoploid, confirming earlier publications. In contrast, Synechocystis PCC 6803 is highly polyploid. The motile wild-type strain contains 218 genome copies in exponential phase and 58 genome copies in linear and in stationary growth phase. The GT wild-type strain contains 142 genome copies in exponential phase and 42 genome copies in linear and stationary growth phase. These are the highest numbers found for any cyanobacterial species. Notably these values are much higher than the value of 12 genome copies published for the 'Kazusa' strain more than 20 years ago. The results reveal that for Synechocystis PCC 6803 strain differences exist and that the ploidy level is highly growth phase-regulated. A compilation of the ploidy levels of all investigated cyanobacterial species gives an overview of the genome copy number distribution and shows that monoploid, oligoploid, and polyploid cyanobacteria exist.
FEMS Microbiology Letters 10/2011; 323(2):124-31. · 2.04 Impact Factor
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ABSTRACT: Previous studies revealed that one species of methanogenic archaea, Methanocaldococcus jannaschii, is polyploid, while a second species, Methanothermobacter thermoautotrophicus, is diploid. To further investigate the distribution of ploidy in methanogenic archaea, species of two additional genera-Methanosarcina acetivorans and Methanococcus maripaludis-were investigated. M. acetivorans was found to be polyploid during fast growth (t(D) = 6 h; 17 genome copies) and oligoploid during slow growth (doubling time = 49 h; 3 genome copies). M. maripaludis has the highest ploidy level found for any archaeal species, with up to 55 genome copies in exponential phase and ca. 30 in stationary phase. A compilation of archaeal species with quantified ploidy levels reveals a clear dichotomy between Euryarchaeota and Crenarchaeota: none of seven euryarchaeal species of six genera is monoploid (haploid), while, in contrast, all six crenarchaeal species of four genera are monoploid, indicating significant genetic differences between these two kingdoms. Polyploidy in asexual species should lead to accumulation of inactivating mutations until the number of intact chromosomes per cell drops to zero (called "Muller's ratchet"). A mechanism to equalize the genome copies, such as gene conversion, would counteract this phenomenon. Making use of a previously constructed heterozygous mutant strain of the polyploid M. maripaludis we could show that in the absence of selection very fast equalization of genomes in M. maripaludis took place probably via a gene conversion mechanism. In addition, it was shown that the velocity of this phenomenon is inversely correlated to the strength of selection.
Journal of bacteriology 02/2011; 193(3):734-43. · 3.94 Impact Factor
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ABSTRACT: Haloferax volcanii is highly polyploid and contains about 20 copies of the major chromosome. A heterozygous strain was constructed that contained two different types of genomes: the leuB locus contained either the wild-type leuB gene or a leuB:trpA gene introduced by gene replacement. As the trpA locus is devoid of the wild-type trpA gene, growth in the absence of both amino acids is only possible when both types of genomes are simultaneously present, exemplifying gene redundancy and the potential to form heterozygous cells as one possible evolutionary advantage of polyploidy. The heterozygous strain was grown (i) in the presence of tryptophan, selecting for the presence of leuB, (ii) in the presence of leucine selecting for leuB:trpA and (iii) in the absence of selection. Both types of genomes were quantified with real-time PCR. The first condition led to a complete loss of leuB:trpA-containing genomes, while under the second condition leuB-containing genomes were lost. Also in the absence of selection gene conversion led to a fast equalization of genomes and resulted in homozygous leuB-containing cells. Gene conversion leading to genome equalization can explain the escape from 'Muller's ratchet' as well as the ease of mutant construction using polyploid haloarchaea.
Molecular Microbiology 02/2011; 80(3):666-77. · 5.01 Impact Factor
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ABSTRACT: Bacteria are generally assumed to be monoploid (haploid). This assumption is mainly based on generalization of the results obtained with the most intensely studied model bacterium, Escherichia coli (a gamma-proteobacterium), which is monoploid during very slow growth. However, several species of proteobacteria are oligo- or polyploid, respectively. To get a better overview of the distribution of ploidy levels, genome copy numbers were quantified in four species of three different groups of proteobacteria. A recently developed Real Time PCR approach, which had been used to determine the ploidy levels of halophilic archaea, was optimized for the quantification of genome copy numbers of bacteria. Slow-growing (doubling time 103 minutes) and fast-growing (doubling time 25 minutes) E. coli cultures were used as a positive control. The copy numbers of the origin and terminus region of the chromosome were determined and the results were in excellent agreement with published data. The approach was also used to determine the ploidy levels of Caulobacter crescentus (an alpha-proteobacterium) and Wolinella succinogenes (an epsilon-proteobacterium), both of which are monoploid. In contrast, Pseudomonas putida (a gamma-proteobacterium) contains 20 genome copies and is thus polyploid. A survey of the proteobacteria with experimentally-determined genome copy numbers revealed that only three to four of 11 species are monoploid and thus monoploidy is not typical for proteobacteria. The ploidy level is not conserved within the groups of proteobacteria, and there are no obvious correlations between the ploidy levels with other parameters like genome size, optimal growth temperature or mode of life.
PLoS ONE 01/2011; 6(1):e16392. · 4.09 Impact Factor
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ABSTRACT: A system where archaeal gene expression could be controlled by simple manipulation of growth conditions would enable the construction of conditional lethal mutants in essential genes, and permit the controlled overproduction of proteins in their native host. As tools for the genetic manipulation of Haloferax volcanii are well developed, we set out to identify promoters with a wide dynamic range of expression in this organism. Tryptophan is the most costly amino acid for the cell to make, so we reasoned that tryptophan-regulated promoters might be good candidates. Microarray analysis of H. volcanii gene expression in the presence and absence of tryptophan identified a tryptophanase gene (tna) that showed strong induction in the presence of tryptophan. qRT-PCR revealed a very fast response and an up to 100-fold induction after tryptophan addition. This result has been confirmed using three independent reporter genes (cct1, pyrE2 and bgaH). Vectors containing this promoter will be very useful for investigating gene function in H. volcanii and potentially in other halophilic archaea. To demonstrate this, we used the promoter to follow the consequences of depletion of the essential chaperonin protein CCT1, and to determine the ability of heterologous CCT proteins to function in H. volcanii.
Molecular Microbiology 01/2008; 66(5):1092-106. · 5.01 Impact Factor
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ABSTRACT: The basal transcription apparatus of archaea is well characterized. However, much less is known about the mechanisms of transcription termination and translation initation. Recently, experimental determination of the 5'-ends of ten transcripts from Pyrobaculum aerophilum revealed that these are devoid of a 5'-UTR. Bioinformatic analysis indicated that many transcripts of other archaeal species might also be leaderless. The 5'-ends and 3'-ends of 40 transcripts of two haloarchaeal species, Halobacterium salinarum and Haloferax volcanii, have been determined. They were used to characterize the lengths of 5'-UTRs and 3'-UTRs and to deduce consensus sequence-elements for transcription and translation. The experimental approach was complemented with a bioinformatics analysis of the H. salinarum genome sequence. Furthermore, the influence of selected 5'-UTRs and 3'-UTRs on transcript stability and translational efficiency in vivo was characterized using a newly established reporter gene system, gene fusions, and real-time PCR. Consensus sequences for basal promoter elements could be refined and a novel element was discovered. A consensus motif probably important for transcriptional termination was established. All 40 haloarchaeal transcripts analyzed had a 3'-UTR (average size 57 nt), and their 3'-ends were not posttranscriptionally modified. Experimental data and genome analyses revealed that the majority of haloarchaeal transcripts are leaderless, indicating that this is the predominant mode for translation initiation in haloarchaea. Surprisingly, the 5'-UTRs of most leadered transcripts did not contain a Shine-Dalgarno (SD) sequence. A genome analysis indicated that less than 10% of all genes are preceded by a SD sequence and even most proximal genes in operons lack a SD sequence. Seven different leadered transcripts devoid of a SD sequence were efficiently translated in vivo, including artificial 5'-UTRs of random sequences. Thus, an interaction of the 5'-UTRs of these leadered transcripts with the 16S rRNA could be excluded. Taken together, either a scanning mechanism similar to the mechanism of translation initiation operating in eukaryotes or a novel mechanism must operate on most leadered haloarchaeal transcripts.
PLoS Genetics 01/2008; 3(12):e229. · 8.69 Impact Factor
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ABSTRACT: RNA degradation is an important factor in the regulation of gene expression. It allows organisms to quickly respond to changing environmental conditions by adapting the expression of individual genes. The stability of individual mRNAs within an organism varies considerably, contributing to differential amounts of proteins expressed. In this study we used DNA microarrays to analyze mRNA degradation in exponentially growing cultures of the extremely halophilic euryarchaeon Halobacterium salinarum NRC-1 on a global level. We determined mRNA half-lives for 1,717 open reading frames, 620 of which are part of known or predicted operons. Under the tested conditions transcript stabilities ranged from 5 min to more than 18 min, with 79% of the evaluated mRNAs showing half-lives between 8 and 12 min. The overall mean half-life was 10 min, which is considerably longer than the ones found in the other prokaryotes investigated thus far. As previously observed in Escherichia coli and Saccharomyces cerevisiae, we could not detect a significant correlation between transcript length and transcript stability, but there was a relationship between gene function and transcript stability. Genes that are known or predicted to be transcribed in operons exhibited similar mRNA half-lives. These results provide initial insights into mRNA turnover in a euryarchaeon. Moreover, our model organism, H. salinarum NRC-1, is one of just two archaea sequenced to date that are missing the core subunits of the archaeal exosome. This complex orthologous to the RNA degrading exosome of eukarya is found in all other archaeal genomes sequenced thus far.
Journal of Bacteriology 11/2007; 189(19):6936-44. · 3.83 Impact Factor
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ABSTRACT: Differential expression of genes can be regulated on many different levels. Most global studies of gene regulation concentrate on transcript level regulation, and very few global analyses of differential translational efficiencies exist. The studies have revealed that in Saccharomyces cerevisiae, Arabidopsis thaliana, and human cell lines translational regulation plays a significant role. Additional species have not been investigated yet. Particularly, until now no global study of translational control with any prokaryotic species was available.
A global analysis of translational control was performed with two haloarchaeal model species, Halobacterium salinarum and Haloferax volcanii. To identify differentially regulated genes, exponentially growing and stationary phase cells were compared. More than 20% of H. salinarum transcripts are translated with non-average efficiencies. By far the largest group is comprised of genes that are translated with above-average efficiency specifically in exponential phase, including genes for many ribosomal proteins, RNA polymerase subunits, enzymes, and chemotaxis proteins. Translation of 1% of all genes is specifically repressed in either of the two growth phases. For comparison, DNA microarrays were also used to identify differential transcriptional regulation in H. salinarum, and 17% of all genes were found to have non-average transcript levels in exponential versus stationary phase. In H. volcanii, 12% of all genes are translated with non-average efficiencies. The overlap with H. salinarum is negligible. In contrast to H. salinarum, 4.6% of genes have non-average translational efficiency in both growth phases, and thus they might be regulated by other stimuli than growth phase.
For the first time in any prokaryotic species it was shown that a significant fraction of genes is under differential translational control. Groups of genes with different regulatory patterns were discovered. However, neither the fractions nor the identity of regulated genes are conserved between H. salinarum and H. volcanii, indicating that prokaryotes as well as eukaryotes use differential translational control for the regulation of gene expression, but that the identity of regulated genes is not conserved. For 70 H. salinarum genes potentiation of regulation was observed, but for the majority of regulated genes either transcriptional or translational regulation is employed.
BMC Genomics 02/2007; 8:415. · 4.07 Impact Factor
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ABSTRACT: The cell cycle of all organisms includes mass increase by a factor of two, replication of the genetic material, segregation of the genome to different parts of the cell, and cell division into two daughter cells. It is tightly regulated and typically includes cell cycle-specific oscillations of the levels of transcripts, proteins, protein modifications, and signaling molecules. Until now cell cycle-specific transcriptome changes have been described for four eukaryotic species ranging from yeast to human, but only for two prokaryotic species. Similarly, oscillations of small signaling molecules have been identified in very few eukaryotic species, but not in any prokaryote.
A synchronization procedure for the archaeon Halobacterium salinarum was optimized, so that nearly 100% of all cells divide in a time interval that is 1/4th of the generation time of exponentially growing cells. The method was used to characterize cell cycle-dependent transcriptome changes using a genome-wide DNA microarray. The transcript levels of 87 genes were found to be cell cycle-regulated, corresponding to 3% of all genes. They could be clustered into seven groups with different transcript level profiles. Cluster-specific sequence motifs were detected around the start of the genes that are predicted to be involved in cell cycle-specific transcriptional regulation. Notably, many cell cycle genes that have oscillating transcript levels in eukaryotes are not regulated on the transcriptional level in H. salinarum. Synchronized cultures were also used to identify putative small signaling molecules. H. salinarum was found to contain a basal cAMP concentration of 200 microM, considerably higher than that of yeast. The cAMP concentration is shortly induced directly prior to and after cell division, and thus cAMP probably is an important signal for cell cycle progression.
The analysis of cell cycle-specific transcriptome changes of H. salinarum allowed to identify a strategy of transcript level regulation that is different from all previously characterized species. The transcript levels of only 3% of all genes are regulated, a fraction that is considerably lower than has been reported for four eukaryotic species (6%-28%) and for the bacterium C. crescentus (19%). It was shown that cAMP is present in significant concentrations in an archaeon, and the phylogenetic profile of the adenylate cyclase indicates that this signaling molecule is widely distributed in archaea. The occurrence of cell cycle-dependent oscillations of the cAMP concentration in an archaeon and in several eukaryotic species indicates that cAMP level changes might be a phylogenetically old signal for cell cycle progression.
BMC Cell Biology 02/2007; 8:21. · 2.59 Impact Factor
