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The genomes of many prokaryotes contain substantial fractions of gene pairs with overlapping stop and start codons (ATGA or TGATG). A potential benefit of overlapping gene pairs is translational coupling. In 720 genomes of archaea and bacteria representing all major phyla, we identify substantial, albeit highly variable, fractions of co-directed overlapping gene pairs. Various patterns are observed for the utilization of the SD motif for de novo initiation at upstream genes versus reinitiation at overlapping gene pairs. We experimentally test the predicted coupling in 9 gene pairs from the archaeon Haloferax volcanii and 5 gene pairs from the bacterium Escherichia coli. In 13 of 14 cases, translation of both genes is strictly coupled. Mutational analysis of SD motifs located upstream of the downstream genes indicate that the contribution of the SD to translational coupling widely varies from gene to gene. The nearly universal, abundant occurrence of overlapping gene pairs suggests that tight translational coupling is widespread in archaea and bacteria.
Background 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. Results 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 uM, 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. Conclusions 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.
Each lifecycle of the Hepatitis C virus (HCV) produces structural and non-structural (NS) proteins in equimolar. Structural proteins were either assembled or degraded by host proteolysis systems, while NS proteins remain inside the host cells and don’t accumulate. Therefore, they must be degraded. Here, NS3 and NS5A half-lives were quantified in the presence of autolysosome and proteasome different modulators. Inhibitors of both systems increased the half-life, while inducers decreased the half-life. Furthermore, polyubiquitination of NS3 and NS5A was observed. Additionally, their intracellular co-localization with autolysosome (LAMP2) and proteasome (PSMB5) was observed, and inhibitors of both systems increased the degree of co-localization. A better understanding of NS protein degradation might help to improve medical interventions during HCV infections in the future.
Each lifecycle of the Hepatitis C virus (HCV) produces structural and non-structural (NS) proteins in equimolar. Structural proteins were either assembled or degraded by host proteolysis systems, while NS proteins remain inside the host cells and don’t accumulate. Therefore, they must be degraded. Here, NS3 and NS5A half-lives were quantified in the presence of autolysosome and proteasome different modulators. Inhibitors of both systems increased the half-life, while inducers decreased the half-life. Furthermore, polyubiquitination of NS3 and NS5A was observed. Additionally, their intracellular co-localization with autolysosome (LAMP2) and proteasome (PSMB5) was observed, and inhibitors of both systems increased the degree of co-localization. A better understanding of NS protein degradation might help to improve medical interventions during HCV infections in the future.
Zinc finger domains are highly structured and can mediate interactions to DNA, RNA, proteins, lipids, and small molecules. Accordingly, zinc finger proteins are very versatile and involved in many biological functions. Eukaryotes contain a wealth of zinc finger proteins, but zinc finger proteins have also been found in archaea and bacteria. Large zinc finger proteins have been well studied, however, in stark contrast, single domain zinc finger µ-proteins of less than 70 amino acids have not been studied at all, with one single exception. Therefore, 16 zinc finger µ-proteins of the haloarchaeon Haloferax volcanii were chosen and in frame deletion mutants of the cognate genes were generated. The phenotypes of mutants and wild-type were compared under eight different conditions, which were chosen to represent various pathways and involve many genes. None of the mutants differed from the wild-type under optimal or near-optimal conditions. However, 12 of the 16 mutants exhibited a phenotypic difference under at least one of the four following conditions: Growth in synthetic medium with glycerol, growth in the presence of bile acids, biofilm formation, and swarming. In total, 16 loss of function and 11 gain of function phenotypes were observed. Five mutants indicated counter-regulation of a sessile versus a motile life style in H. volcanii. In conclusion, the generation and analysis of a set of deletion mutants demonstrated the high importance of zinc finger µ-proteins for various biological functions, and it will be the basis for future mechanistic insight.
Recently a first genome-wide analysis of translational regulation using prokaryotic species had been performed which revealed that regulation of translational efficiency plays an important role in haloarchaea. In fact, the fractions of genes under differential growth phase-dependent translational control in the two species Halobacterium salinarum and Haloferax volcanii were as high as in eukaryotes. However, nothing is known about the mechanisms of translational regulation in archaea. Therefore, two genes exhibiting opposing directions of regulation were selected to unravel the importance of untranslated regions (UTRs) for differential translational control in vivo. Differential translational regulation in exponentially growing versus stationary phase cells was studied by comparing translational efficiencies using a reporter gene system. Translational regulation was not observed when 5'-UTRs or 3'-UTRs alone were fused to the reporter gene. However, their simultaneous presence was sufficient to transfer differential translational control from the native transcript to the reporter transcript. This was true for both directions of translational control. Translational regulation was completely abolished when stem loops in the 5'-UTR were changed by mutagenesis. An “UTR-swap” experiment demonstrated that the direction of translational regulation is encoded in the 3'-UTR, not in the 5'-UTR. While much is known about 5'-UTR-dependent translational control in bacteria, the reported findings provide the first examples that both 5'- and 3'-UTRs are essential and sufficient to drive differential translational regulation in a prokaryote and therefore have to functionally interact in vivo. The current results indicate that 3'-UTR-dependent translational control had already evolved before capping and polyadenylation of transcripts were invented, which are essential for circularization of transcripts in eukaryotes.
Polyploidy is common in higher eukaryotes, especially in plants, but it is generally assumed that most prokaryotes contain a single copy of a circular chromosome and are therefore monoploid. We have used two independent methods to determine the genome copy number in halophilic archaea, 1) cell lysis in agarose blocks and Southern blot analysis, and 2) Real-Time quantitative PCR. Fast growing H. salinarum cells contain on average about 25 copies of the chromosome in exponential phase, and their ploidy is downregulated to 15 copies in early stationary phase. The chromosome copy number is identical in cultures with a twofold lower growth rate, in contrast to the results reported for several other prokaryotic species. Of three additional replicons of H. salinarum, two have a low copy number that is not growth-phase regulated, while one replicon even shows a higher degree of growth phase-dependent regulation than the main replicon. The genome copy number of H. volcanii is similarly high during exponential phase (on average 18 copies/cell), and it is also downregulated (to 10 copies) as the cells enter stationary phase. The variation of genome copy numbers in the population was addressed by fluorescence microscopy and by FACS analysis. These methods allowed us to verify the growth phase-dependent regulation of ploidy in H. salinarum, and they revealed that there is a wide variation in genome copy numbers in individual cells that is much larger in exponential than in stationary phase. Our results indicate that polyploidy might be more widespread in archaea (or even prokaryotes in general) than previously assumed. Moreover, the presence of so many genome copies in a prokaryote raises questions about the evolutionary significance of this strategy.
Iron is part of many redox and other enzymes and, thus, it is essential for all living beings. Many oxic environments have extremely low concentrations of free iron. Therefore, many prokaryotic species evolved siderophores, i.e., small organic molecules that complex Fe3+ with very high affinity. Siderophores of bacteria are intensely studied, in contrast to those of archaea. The haloarchaeon Haloferax volcanii contains a gene cluster that putatively encodes siderophore biosynthesis genes, including four iron uptake chelate (iuc) genes. Underscoring this hypothesis, Northern blot analyses revealed that a hexacistronic transcript is generated that is highly induced under iron starvation. A quadruple iuc deletion mutant was generated, which had a growth defect solely at very low concentrations of Fe3+, not Fe2+. Two experimental approaches showed that the wild type produced and exported an Fe3+-specific siderophore under low iron concentrations, in contrast to the iuc deletion mutant. Bioinformatic analyses revealed that haloarchaea obtained the gene cluster by lateral transfer from bacteria and enabled the prediction of enzymatic functions of all six gene products. Notably, a biosynthetic pathway is proposed that starts with aspartic acid, uses several group donors and citrate, and leads to the hydroxamate siderophore Schizokinen.
Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure–function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.
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.
Proteins can be acetylated at the alpha-amino group of the N-terminal amino acid (methionine or the penultimate amino acid after methionine removal) or at the epsilon-amino group of internal lysines. In eukaryotes the majority of proteins are N-terminally acetylated, while this is extremely rare in bacteria. A variety of studies about N-terminal acetylation in archaea have been reported recently, and it was revealed that a considerable fraction of proteins is N-terminally acetylated in haloarchaea and Sulfolobus, while this does not seem to apply for methanogenic archaea. Many eukaryotic proteins are modified by differential internal acetylation, which is important for a variety of processes. Until very recently, only two bacterial proteins were known to be acetylation targets, but now 125 acetylation sites are known for E. coli. Knowledge about internal acetylation in archaea is extremely limited; only two target proteins are known, only one of which--Alba--was used to study differential acetylation. However, indications accumulate that the degree of internal acetylation of archaeal proteins might be underestimated, and differential acetylation has been shown to be essential for the viability of haloarchaea. Focused proteomic approaches are needed to get an overview of the extent of internal protein acetylation in archaea.
The investigated haloarchaeal species, Halobacterium salinarum, Haloferax mediterranei, and H. volcanii, have all been shown to be polyploid. They contain several replicons that have independent copy number regulation, and most have a higher copy number during exponential growth phase than in stationary phase. The possible evolutionary advantages of polyploidy for haloarchaea, most of which have experimental support for at least one species, are discussed. These advantages include a low mutation rate and high resistance toward X-ray irradiation and desiccation, which depend on homologous recombination. For H. volcanii, it has been shown that gene conversion operates in the absence of selection, which leads to the equalization of genome copies. On the other hand, selective forces might lead to heterozygous cells, which have been verified in the laboratory. Additional advantages of polyploidy are survival over geological times in halite deposits as well as at extreme conditions on earth and at simulated Mars conditions. Recently, it was found that H. volcanii uses genomic DNA as genetic material and as a storage polymer for phosphate. In the absence of phosphate, H. volcanii dramatically decreases its genome copy number, thereby enabling cell multiplication, but diminishing the genetic advantages of polyploidy. Stable storage of phosphate is proposed as an alternative driving force for the emergence of DNA in early evolution. Several additional potential advantages of polyploidy are discussed that have not been addressed experimentally for haloarchaea. An outlook summarizes selected current trends and possible future developments.
Gene homologs of GlnK PII regulators and AmtB-type ammonium transporters are often paired on prokaryotic genomes, suggesting these proteins share an ancient functional relationship. Here, we demonstrate for the first time in Archaea that GlnK associates with AmtB in membrane fractions after ammonium shock, thus, providing a further insight into GlnK-AmtB as an ancient nitrogen sensor pair. For this work, Haloferax mediterranei was advanced for study through the generation of a pyrE2-based counterselection system that was used for targeted gene deletion and expression of Flag-tagged proteins from their native promoters. AmtB1-Flag was detected in membrane fractions of cells grown on nitrate and was found to coimmunoprecipitate with GlnK after ammonium shock. Thus, in analogy to bacteria, the archaeal GlnK PII may block the AmtB1 ammonium transporter under nitrogen-rich conditions. In addition to this regulated protein–protein interaction, the archaeal amtB-glnK gene pairs were found to be highly regulated by nitrogen availability with transcript levels high under conditions of nitrogen limitation and low during nitrogen excess. While transcript levels of glnK-amtB are similarly regulated by nitrogen availability in bacteria, transcriptional regulators of the bacterial glnK promoter including activation by the two-component signal transduction proteins NtrC (GlnG, NRI) and NtrB (GlnL, NRII) and sigma factor σN (σ54) are not conserved in archaea suggesting a novel mechanism of transcriptional control.
Background: Phototrophy of the extremely halophilic archaeon Halobacterium salinarum was explored for decades. The research was mainly focused on the expression of bacteriorhodopsin and its functional properties. In contrast, less is known about genome wide transcriptional changes and their impact on the physiological adaptation to phototrophy. The tool of choice to record transcriptional profiles is the DNA microarray technique. However, the technique is still rarely used for transcriptome analysis in archaea. Methodology/Principal Findings: We developed a whole-genome DNA microarray based on our sequence data of the Hbt. salinarum strain R1 genome. The potential of our tool is exemplified by the comparison of cells growing under aerobic and phototrophic conditions, respectively. We processed the raw fluorescence data by several stringent filtering steps and a subsequent MAANOVA analysis. The study revealed a lot of transcriptional differences between the two cell states. We found that the transcriptional changes were relatively weak, though significant. Finally, the DNA microarray data were independently verified by a real-time PCR analysis. Conclusion/Significance: This is the first DNA microarray analysis of Hbt. salinarum cells that were actually grown under phototrophic conditions. By comparing the transcriptomics data with current knowledge we could show that our DNA microarray tool is well applicable for transcriptome analysis in the extremely halophilic archaeon Hbt. salinarum. The reliability of our tool is based on both the high-quality array of DNA probes and the stringent data handling including MAANOVA analysis. Among the regulated genes more than 50% had unknown functions. This underlines the fact that haloarchaeal phototrophy is still far away from being completely understood. Hence, the data recorded in this study will be subject to future systems biology analysis.
NAD is a coenzyme central to metabolism that was also found to serve as a 5’-terminal cap of bacterial and eukaryotic RNA species. The presence and functionality of NAD-capped RNAs (NAD-RNAs) in the archaeal domain remain to be characterized in detail. Here, by combining LC-MS and NAD captureSeq methodology, we quantified the total levels of NAD-RNAs and determined the identity of NAD-RNAs in the two model archaea, Sulfolobus acidocaldarius and Haloferax volcanii. A complementary differential RNA-Seq (dRNA-Seq) analysis revealed that NAD transcription start sites (NAD-TSS) correlate with well-defined promoter regions and often overlap with primary transcription start sites (pTSS). The population of NAD-RNAs in the two archaeal organisms shows clear differences, with S. acidocaldarius possessing more capped small non-coding RNAs (sncRNAs) and leader sequences. The NAD-cap did not prevent 5’→3’ exonucleolytic activity by the RNase Saci-aCPSF2. To investigate enzymes that facilitate the removal of the NAD-cap, four Nudix proteins of S. acidocaldarius were screened. None of the recombinant proteins showed NAD decapping activity. Instead, the Nudix protein Saci_NudT5 showed activity after incubating NAD-RNAs at elevated temperatures. Hyperthermophilic environments promote the thermal degradation of NAD into the toxic product ADPR. Incorporating NAD into RNAs and the regulation of ADPR-RNA decapping by Saci_NudT5 is proposed to provide additional layers of maintaining stable NAD levels in archaeal cells.
Importance: This study reports the first characterization of 5’-terminally modified RNA molecules in Archaea and establishes that NAD-RNA modifications, previously only identified in the other two domains of life, are also prevalent in the archaeal model organisms Sulfolobus acidocaldarius and Haloferax volcanii. We screened for NUDIX hydrolases that could remove the NAD-RNA cap and showed that none of these enzymes removed NAD modifications, but we discovered an enzyme that hydrolyzes ADPR-RNA. We propose that these activities influence the stabilization of NAD and its thermal degradation to potentially toxic ADPR products at elevated growth temperatures.
Halophilic archaea cultivated from surface sterilized middle-late Eocene rock salt are polyploid
(2014)
Live bacteria and archaea have been isolated from several rock salt deposits of up to hundreds of millions of years of age from all around the world. A key factor affecting their longevity is the ability to keep their genomic DNA intact, for which efficient repair mechanisms are needed. Polyploid microbes are known to have an increased resistance towards mutations and DNA damage, and it has been suggested that microbes from deeply buried rock salt would carry several copies of their genomes. Here, cultivable halophilic microbes were isolated from a surface sterilized middle-late Eocene (38–41 million years ago) rock salt sample, drilled from the depth of 800 m at Yunying salt mine, China. Eight unique isolates were obtained, which represented two haloarchaeal genera, Halobacterium and Halolamina. We used real-time PCR to show that our isolates are polyploid, with genome copy numbers of 11–14 genomes per cell in exponential growth phase. The ploidy level was slightly downregulated in stationary growth phase, but the cells still had an average genome copy number of 6–8. The polyploidy of halophilic archaea living in ancient rock salt might be a factor explaining how these organisms are able to overcome the challenge of prolonged survival during their entombment.
It was long assumed that translation initiation in prokaryotes generally occurs via the so-called Shine Dalgarno (SD) mechanism. Recently, it became clear that translation initiation in prokaryotes is more heterogeneous. In the haloarchaeon Haloferax volcanii, the majority of transcripts is leaderless and most transcripts with a 5′-UTR lack a SD motif. Nevertheless, a bioinformatic analysis predicted that 20–30% of all genes are preceded by a SD motif in haloarchaea. To analyze the importance of the SD mechanism for translation initiation in haloarchaea experimentally the monocistronic sod gene was chosen, which contains a 5′-UTR with an extensive SD motif of seven nucleotides and a length of 19 nt, the average length of 5′UTRs in this organism. A translational fusion of part of the sod gene with the dhfr reporter gene was constructed. A mutant series was generated that matched the SD motif from zero to eight positions, respectively. Surprisingly, there was no correlation between the base pairing ability between transcripts and 16S rRNA and translational efficiency in vivo under several different growth conditions. Furthermore, complete replacement of the SD motif by three unrelated sequences did not reduce translational efficiency. The results indicate that H. volcanii does not make use of the SD mechanism for translation initiation in 5′-UTRs. A genome analysis revealed that while the number of SD motifs in 5′-UTRs is rare, their fraction within open reading frames is high. Possible biological functions for intragenic SD motifs are discussed, including re-initiation of translation at distal genes in operons.
Background: Differential RNA-Seq (dRNA-Seq) is a recently developed method of performing primary transcriptome analyses that allows for the genome-wide mapping of transcriptional start sites (TSSs) and the identification of novel transcripts. Although the transcriptomes of diverse bacterial species have been characterized by dRNA-Seq, the transcriptome analysis of archaeal species is still rather limited. Therefore, we used dRNA-Seq to characterize the primary transcriptome of the model archaeon Haloferax volcanii.
Results: Three independent cultures of Hfx. volcanii grown under optimal conditions to the mid-exponential growth phase were used to determine the primary transcriptome and map the 5′-ends of the transcripts. In total, 4749 potential TSSs were detected. A position weight matrix (PWM) was derived for the promoter predictions, and the results showed that 64 % of the TSSs were preceded by stringent or relaxed basal promoters. Of the identified TSSs, 1851 belonged to protein-coding genes. Thus, fewer than half (46 %) of the 4040 protein-coding genes were expressed under optimal growth conditions. Seventy-two percent of all protein-coding transcripts were leaderless, which emphasized that this pathway is the major pathway for translation initiation in haloarchaea. A total of 2898 of the TSSs belonged to potential non-coding RNAs, which accounted for an unexpectedly high fraction (61 %) of all transcripts. Most of the non-coding TSSs had not been previously described (2792) and represented novel sequences (59 % of all TSSs). A large fraction of the potential novel non-coding transcripts were cis-antisense RNAs (1244 aTSSs). A strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs was found, which suggested that the negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts corresponded to internal transcripts overlapping with mRNAs (1153 iTSSs) and intergenic small RNA (sRNA) candidates (395 TSSs).
Conclusion: This study provides a comprehensive map of the primary transcriptome of Hfx. volcanii grown under optimal conditions. Fewer than half of all protein-coding genes have been transcribed under these conditions. Unexpectedly, more than half of the detected TSSs belonged to several classes of non-coding RNAs. Thus, RNA-based regulation appears to play a more important role in haloarchaea than previously anticipated.
Background 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. Results 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. Conclusions 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.
The alpha-proteobacterium Zymomonas mobilis is a promising biofuel producer, based on its native metabolism that efficiently converts sugars to ethanol. Therefore, it has a high potential for industrial-scale biofuel production. Two previous studies suggested that Z. mobilis strain Zm4 might not be monoploid. However, a systematic analysis of the genome copy number is still missing, in spite of the high potential importance of Z. mobilis. To get a deep insight into the ploidy level of Z. mobilis and its regulation, the genome copy numbers of three strains were quantified. The analyses revealed that, during anaerobic growth, the lab strain Zm6, the Zm6 type strain obtained from DSMZ (German Collection of Microorganisms), and the lab strain Zm4, have copy numbers of 18.9, 22.3 and 16.2, respectively, of an origin-adjacent region. The copy numbers of a terminus-adjacent region were somewhat lower with 9.3, 15.8, and 12.9, respectively. The values were similar throughout the growth curves, and they were only slightly downregulated in late stationary phase. During aerobic growth, the copy numbers of the lab strain Zm6 were much higher with around 40 origin-adjacent copies and 17 terminus-adjacent copies. However, the cells were larger during aerobic growth, and the copy numbers per μm3 cell volume were rather similar. Taken together, this first systematic analysis revealed that Z. mobilis is polyploid under regular laboratory growth conditions. The copy number is constant during growth, in contrast to many other polyploid bacteria. This knowledge should be considered in further engineering of the strain for industrial applications.