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The haloarchaeon Haloferax volcanii contains nearly 2800 small non-coding RNAs (sRNAs). One intergenic sRNA, sRNA132, was chosen for a detailed characterization. A deletion mutant had a growth defect and thus underscored the importance of sRNA132. A microarray analysis identified the transcript of an operon for a phosphate-specific ABC transporter as a putative target of sRNA132. Both the sRNA132 and the operon transcript accumulated under low phosphate concentrations, indicating a positive regulatory role of sRNA132. A kinetic analysis revealed that sRNA132 is essential shortly after the onset of phosphate starvation, while other regulatory processes take over after several hours. Comparison of the transcriptomes of wild-type and the sRNA132 gene deletion mutant 30 min after the onset of phosphate starvation revealed that sRNA132 controls a regulon of about 40 genes. Remarkably, the regulon included a second operon for a phosphate-specific ABC transporter, which also depended on sRNA132 for rapid induction in the absence of phosphate. Competitive growth experiments of the wild-type and ABC transporter operon deletion mutants underscored the importance of both transporters for growth at low phosphate concentrations. Northern blot analyses of four additional members of the sRNA132 regulon verified that all four transcripts depended on sRNA132 for rapid regulation after the onset of phosphate starvation. Importantly, this is the first example for the transient importance of a sRNA for any archaeal and bacterial species. In addition, this study unraveled the first sRNA regulon for haloarchaea.
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.
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.
Haloferax volcanii is a well-established model species for haloarchaea. Small scale RNomics and bioinformatics predictions were used to identify small non-coding RNAs (sRNAs), and deletion mutants revealed that sRNAs have important regulatory functions. A recent dRNA-Seq study was used to characterize the primary transcriptome. Unexpectedly, it was revealed that, under optimal conditions, H. volcanii contains more non-coding sRNAs than protein-encoding mRNAs. However, the dRNA-Seq approach did not contain any length information. Therefore, a mixed RNA-Seq approach was used to determine transcript length and to identify additional transcripts, which are not present under optimal conditions. In total, 50 million paired end reads of 150 nt length were obtained. 1861 protein-coding RNAs (cdRNAs) were detected, which encoded 3092 proteins. This nearly doubled the coverage of cdRNAs, compared to the previous dRNA-Seq study. About 2/3 of the cdRNAs were monocistronic, and 1/3 covered more than one gene. In addition, 1635 non-coding sRNAs were identified. The highest fraction of non-coding RNAs were cis antisense RNAs (asRNAs). Analysis of the length distribution revealed that sRNAs have a median length of about 150 nt. Based on the RNA-Seq and dRNA-Seq results, genes were chosen to exemplify characteristics of the H. volcanii transcriptome by Northern blot analyses, e.g. 1) the transcript patterns of gene clusters can be straightforward, but also very complex, 2) many transcripts differ in expression level under the four analyzed conditions, 3) some genes are transcribed into RNA isoforms of different length, which can be differentially regulated, 4) transcripts with very long 5’-UTRs and with very long 3’-UTRs exist, and 5) about 30% of all cdRNAs have overlapping 3’-ends, which indicates, together with the asRNAs, that H. volcanii makes ample use of sense-antisense interactions. Taken together, this RNA-Seq study, together with a previous dRNA-Seq study, enabled an unprecedented view on the H. volcanii transcriptome.
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.