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1.  Genome of the Haloarchaeon Natronomonas moolapensis, a Neutrophilic Member of a Previously Haloalkaliphilic Genus 
Genome Announcements  2013;1(2):e00095-13.
The genus Natronomonas contains two species, one haloalkaliphile (N. pharaonis) and one neutrophile (N. moolapensis). Here, we report the genome sequence of N. moolapensis strain 8.8.11. The overall genome properties are similar for the two species. Only the neutrophile contains bacteriorhodopsin and a membrane glycolipid.
PMCID: PMC3623002  PMID: 23516216
2.  The protein interaction network of a taxis signal transduction system in a Halophilic Archaeon 
BMC Microbiology  2012;12:272.
The taxis signaling system of the extreme halophilic archaeon Halobacterium (Hbt.) salinarum differs in several aspects from its model bacterial counterparts Escherichia coli and Bacillus subtilis. We studied the protein interactions in the Hbt. salinarum taxis signaling system to gain an understanding of its structure, to gain knowledge about its known components and to search for new members.
The interaction analysis revealed that the core signaling proteins are involved in different protein complexes and our data provide evidence for dynamic interchanges between them. Fifteen of the eighteen taxis receptors (halobacterial transducers, Htrs) can be assigned to four different groups depending on their interactions with the core signaling proteins. Only one of these groups, which contains six of the eight Htrs with known signals, shows the composition expected for signaling complexes (receptor, kinase CheA, adaptor CheW, response regulator CheY). From the two Hbt. salinarum CheW proteins, only CheW1 is engaged in signaling complexes with Htrs and CheA, whereas CheW2 interacts with Htrs but not with CheA. CheY connects the core signaling structure to a subnetwork consisting of the two CheF proteins (which build a link to the flagellar apparatus), CheD (the hub of the subnetwork), two CheC complexes and the receptor methylesterase CheB.
Based on our findings, we propose two hypotheses. First, Hbt. salinarum might have the capability to dynamically adjust the impact of certain Htrs or Htr clusters depending on its current needs or environmental conditions. Secondly, we propose a hypothetical feedback loop from the response regulator to Htr methylation made from the CheC proteins, CheD and CheB, which might contribute to adaptation analogous to the CheC/CheD system of B. subtilis.
PMCID: PMC3579733  PMID: 23171228
3.  A small basic protein from the brz-brb operon is involved in regulation of bop transcription in Halobacterium salinarum 
BMC Molecular Biology  2011;12:42.
The halophilic archaeon Halobacterium salinarum expresses bacteriorhodopsin, a retinal-protein that allows photosynthetic growth. Transcription of the bop (bacterioopsin) gene is controlled by two transcription factors, Bat and Brz that induce bop when cells are grown anaerobically and under light.
A new gene was identified that is transcribed together with the brz gene that encodes a small basic protein designated as Brb (bacteriorhodopsin-regulating basic protein). The translation activity of the start codon of the brb gene was confirmed by BgaH reporter assays. In vivo site-directed mutagenesis of the brb gene showed that the Brb protein cooperates with Brz in the regulation of bop expression. Using a GFP reporter assay, it was demonstrated that Brb cooperates with both Brz and Bat proteins to activate bop transcription under phototrophic growth conditions.
The activation of the bop promoter was shown to be dependent not only on two major factors, Bat and Brz, but is also tuned by the small basic protein, Brb.
PMCID: PMC3184054  PMID: 21929791
4.  Haloquadratum walsbyi : Limited Diversity in a Global Pond 
PLoS ONE  2011;6(6):e20968.
Haloquadratum walsbyi commonly dominates the microbial flora of hypersaline waters. Its cells are extremely fragile squares requiring >14%(w/v) salt for growth, properties that should limit its dispersal and promote geographical isolation and divergence. To assess this, the genome sequences of two isolates recovered from sites at near maximum distance on Earth, were compared.
Principal Findings
Both chromosomes are 3.1 MB in size, and 84% of each sequence was highly similar to the other (98.6% identity), comprising the core sequence. ORFs of this shared sequence were completely synteneic (conserved in genomic orientation and order), without inversion or rearrangement. Strain-specific insertions/deletions could be precisely mapped, often allowing the genetic events to be inferred. Many inferred deletions were associated with short direct repeats (4–20 bp). Deletion-coupled insertions are frequent, producing different sequences at identical positions. In cases where the inserted and deleted sequences are homologous, this leads to variant genes in a common synteneic background (as already described by others). Cas/CRISPR systems are present in C23T but have been lost in HBSQ001 except for a few spacer remnants. Numerous types of mobile genetic elements occur in both strains, most of which appear to be active, and with some specifically targetting others. Strain C23T carries two ∼6 kb plasmids that show similarity to halovirus His1 and to sequences nearby halovirus/plasmid gene clusters commonly found in haloarchaea.
Deletion-coupled insertions show that Hqr. walsbyi evolves by uptake and precise integration of foreign DNA, probably originating from close relatives. Change is also driven by mobile genetic elements but these do not by themselves explain the atypically low gene coding density found in this species. The remarkable genome conservation despite the presence of active systems for genome rearrangement implies both an efficient global dispersal system, and a high selective fitness for this species.
PMCID: PMC3119063  PMID: 21701686
5.  Characterization of Growth and Metabolism of the Haloalkaliphile Natronomonas pharaonis 
PLoS Computational Biology  2010;6(6):e1000799.
Natronomonas pharaonis is an archaeon adapted to two extreme conditions: high salt concentration and alkaline pH. It has become one of the model organisms for the study of extremophilic life. Here, we present a genome-scale, manually curated metabolic reconstruction for the microorganism. The reconstruction itself represents a knowledge base of the haloalkaliphile's metabolism and, as such, would greatly assist further investigations on archaeal pathways. In addition, we experimentally determined several parameters relevant to growth, including a characterization of the biomass composition and a quantification of carbon and oxygen consumption. Using the metabolic reconstruction and the experimental data, we formulated a constraints-based model which we used to analyze the behavior of the archaeon when grown on a single carbon source. Results of the analysis include the finding that Natronomonas pharaonis, when grown aerobically on acetate, uses a carbon to oxygen consumption ratio that is theoretically near-optimal with respect to growth and energy production. This supports the hypothesis that, under simple conditions, the microorganism optimizes its metabolism with respect to the two objectives. We also found that the archaeon has a very low carbon efficiency of only about 35%. This inefficiency is probably due to a very low P/O ratio as well as to the other difficulties posed by its extreme environment.
Author Summary
Extremophiles are organisms that thrive in physically or geochemically extreme conditions that are detrimental, even lethal, to the majority of life on Earth. Natronomonas pharaonis is one that has been able to adapt to both high salt concentration and an alkaline pH. In this study, we investigate the chemical reactions that occur within the microorganism, collectively referred to as its metabolic network, that allow it to convert the nutrients in its environment to biomass and energy. Specifically, we reconstructed the network by collecting evidence for the existence of reactions from the literature, and then supplemented them with computational approaches, for example by searching the genome of Natronomonas pharaonis for genes that could potentially encode analogs of known enzymes from other organisms. Finally, with the network in hand, we developed a computational model which we used to simulate growth. Among other results, we found indications that Natronomonas pharaonis regulates its metabolism such that energy production and growth are maximized. Despite this however, we also found that Natronomonas pharaonis is only able to incorporate a very small fraction of the total carbon that it consumes (approximately 35%), likely in no small part due to the difficulties posed by its environment.
PMCID: PMC2881530  PMID: 20543878
6.  Transcriptional control by two leucine-responsive regulatory proteins in Halobacterium salinarum R1 
BMC Molecular Biology  2010;11:40.
Archaea combine bacterial-as well as eukaryotic-like features to regulate cellular processes. Halobacterium salinarum R1 encodes eight leucine-responsive regulatory protein (Lrp)-homologues. The function of two of them, Irp (OE3923F) and lrpA1 (OE2621R), were analyzed by gene deletion and overexpression, including genome scale impacts using microarrays.
It was shown that Lrp affects the transcription of multiple target genes, including those encoding enzymes involved in amino acid synthesis, central metabolism, transport processes and other regulators of transcription. In contrast, LrpA1 regulates transcription in a more specific manner. The aspB3 gene, coding for an aspartate transaminase, was repressed by LrpA1 in the presence of L-aspartate. Analytical DNA-affinity chromatography was adapted to high salt, and demonstrated binding of LrpA1 to its own promoter, as well as L-aspartate dependent binding to the aspB3 promoter.
The gene expression profiles of two archaeal Lrp-homologues report in detail their role in H. salinarum R1. LrpA1 and Lrp show similar functions to those already described in bacteria, but in addition they play a key role in regulatory networks, such as controlling the transcription of other regulators. In a more detailed analysis ligand dependent binding of LrpA1 was demonstrated to its target gene aspB3.
PMCID: PMC2894021  PMID: 20509863
7.  A predictive computational model of the kinetic mechanism of stimulus-induced transducer methylation and feedback regulation through CheY in archaeal phototaxis and chemotaxis 
BMC Systems Biology  2010;4:27.
Photo- and chemotaxis of the archaeon Halobacterium salinarum is based on the control of flagellar motor switching through stimulus-specific methyl-accepting transducer proteins that relay the sensory input signal to a two-component system. Certain members of the transducer family function as receptor proteins by directly sensing specific chemical or physical stimuli. Others interact with specific receptor proteins like the phototaxis photoreceptors sensory rhodopsin I and II, or require specific binding proteins as for example some chemotaxis transducers. Receptor activation by light or a change in receptor occupancy by chemical stimuli results in reversible methylation of glutamate residues of the transducer proteins. Both, methylation and demethylation reactions are involved in sensory adaptation and are modulated by the response regulator CheY.
By mathematical modeling we infer the kinetic mechanisms of stimulus-induced transducer methylation and adaptation. The model (deterministic and in the form of ordinary differential equations) correctly predicts experimentally observed transducer demethylation (as detected by released methanol) in response to attractant and repellent stimuli of wildtype cells, a cheY deletion mutant, and a mutant in which the stimulated transducer species is methylation-deficient.
We provide a kinetic model for signal processing in photo- and chemotaxis in the archaeon H. salinarum suggesting an essential role of receptor cooperativity, antagonistic reversible methylation, and a CheY-dependent feedback on transducer demethylation.
PMCID: PMC2857822  PMID: 20298562
8.  Phosphate-Dependent Behavior of the Archaeon Halobacterium salinarum Strain R1▿ † 
Journal of Bacteriology  2009;191(12):3852-3860.
Phosphate is essential for life on earth, since it is an integral part of important biomolecules. The mechanisms applied by bacteria and eukarya to combat phosphate limitation are fairly well understood. However, it is not known how archaea sense phosphate limitation or which genes are regulated upon limitation. We conducted a microarray analysis to explore the phosphate-dependent gene expression of Halobacterium salinarum strain R1. We identified a set of 17 genes whose transcript levels increased up to several hundredfold upon phosphate limitation. Analysis of deletion mutants showed that this set of genes, the PHO stimulon, is very likely independent of signaling via two-component systems. Our experiments further indicate that PHO stimulon induction might be dependent on the intracellular phosphate concentration, which turned out to be subject to substantial changes. Finally, the study revealed that H. salinarum exhibits a phosphate-directed chemotaxis, which is induced by phosphate starvation.
PMCID: PMC2698381  PMID: 19363117
9.  Systems Analysis of Bioenergetics and Growth of the Extreme Halophile Halobacterium salinarum 
PLoS Computational Biology  2009;5(4):e1000332.
Halobacterium salinarum is a bioenergetically flexible, halophilic microorganism that can generate energy by respiration, photosynthesis, and the fermentation of arginine. In a previous study, using a genome-scale metabolic model, we have shown that the archaeon unexpectedly degrades essential amino acids under aerobic conditions, a behavior that can lead to the termination of growth earlier than necessary. Here, we further integratively investigate energy generation, nutrient utilization, and biomass production using an extended methodology that accounts for dynamically changing transport patterns, including those that arise from interactions among the supplied metabolites. Moreover, we widen the scope of our analysis to include phototrophic conditions to explore the interplay between different bioenergetic modes. Surprisingly, we found that cells also degrade essential amino acids even during phototropy, when energy should already be abundant. We also found that under both conditions considerable amounts of nutrients that were taken up were neither incorporated into the biomass nor used as respiratory substrates, implying the considerable production and accumulation of several metabolites in the medium. Some of these are likely the products of forms of overflow metabolism. In addition, our results also show that arginine fermentation, contrary to what is typically assumed, occurs simultaneously with respiration and photosynthesis and can contribute energy in levels that are comparable to the primary bioenergetic modes, if not more. These findings portray a picture that the organism takes an approach toward growth that favors the here and now, even at the cost of longer-term concerns. We believe that the seemingly “greedy” behavior exhibited actually consists of adaptations by the organism to its natural environments, where nutrients are not only irregularly available but may altogether be absent for extended periods that may span several years. Such a setting probably predisposed the cells to grow as much as possible when the conditions become favorable.
Author Summary
Living cells can produce usable energy through various means. For example, animals derive energy, through respiration, from nutrients that they consume, and plants from light using photosynthesis. The particular microorganism that we study, Halobacterium salinarum, is a model organism for the archaeal domain of life. It is bioenergetically flexible in that it can perform both respiration and photosynthesis and in addition can also derive energy using fermentation. Accordingly, it is a good model system for investigating the interplay between different energy generating mechanisms. In this study, we investigate these relationships as well as how energy production is linked to the other processes involved in growth, including the consumption of nutrients and the production of cellular material. Because Halobacterium salinarum thrives in salt-saturated solutions, such as those that may be found in salt lakes and solar salterns, our study yields insight on how these cellular processes operate in environments that are lethal to most life on Earth.
PMCID: PMC2674319  PMID: 19401785
10.  Identification of Archaea-specific chemotaxis proteins which interact with the flagellar apparatus 
BMC Microbiology  2009;9:56.
Archaea share with bacteria the ability to bias their movement towards more favorable locations, a process known as taxis. Two molecular systems drive this process: the motility apparatus and the chemotaxis signal transduction system. The first consists of the flagellum, the flagellar motor, and its switch, which allows cells to reverse the rotation of flagella. The second targets the flagellar motor switch in order to modulate the switching frequency in response to external stimuli. While the signal transduction system is conserved throughout archaea and bacteria, the archaeal flagellar apparatus is different from the bacterial one. The proteins constituting the flagellar motor and its switch in archaea have not yet been identified, and the connection between the bacterial-like chemotaxis signal transduction system and the archaeal motility apparatus is unknown.
Using protein-protein interaction analysis, we have identified three proteins in Halobacterium salinarum that interact with the chemotaxis (Che) proteins CheY, CheD, and CheC2, as well as the flagella accessory (Fla) proteins FlaCE and FlaD. Two of the proteins belong to the protein family DUF439, the third is a HEAT_PBS family protein. In-frame deletion strains for all three proteins were generated and analyzed as follows: a) photophobic responses were measured by a computer-based cell tracking system b) flagellar rotational bias was determined by dark-field microscopy, and c) chemotactic behavior was analyzed by a swarm plate assay. Strains deleted for the HEAT_PBS protein or one of the DUF439 proteins proved unable to switch the direction of flagellar rotation. In these mutants, flagella rotate only clockwise, resulting in exclusively forward swimming cells that are unable to respond to tactic signals. Deletion of the second DUF439 protein had only minimal effects. HEAT_PBS proteins could be identified in the chemotaxis gene regions of all motile haloarchaea sequenced so far, but not in those of other archaeal species. Genes coding for DUF439 proteins, however, were found to be integral parts of chemotaxis gene regions across the archaeal domain, and they were not detected in other genomic context.
Altogether, these results demonstrate that, in the archaeal domain, previously unrecognized archaea-specific Che proteins are essential for relaying taxis signaling to the flagellar apparatus.
PMCID: PMC2666748  PMID: 19291314
11.  Ser/Thr/Tyr Protein Phosphorylation in the Archaeon Halobacterium salinarum—A Representative of the Third Domain of Life 
PLoS ONE  2009;4(3):e4777.
In the quest for the origin and evolution of protein phosphorylation, the major regulatory post-translational modification in eukaryotes, the members of archaea, the “third domain of life”, play a protagonistic role. A plethora of studies have demonstrated that archaeal proteins are subject to post-translational modification by covalent phosphorylation, but little is known concerning the identities of the proteins affected, the impact on their functionality, the physiological roles of archaeal protein phosphorylation/dephosphorylation, and the protein kinases/phosphatases involved. These limited studies led to the initial hypothesis that archaea, similarly to other prokaryotes, use mainly histidine/aspartate phosphorylation, in their two-component systems representing a paradigm of prokaryotic signal transduction, while eukaryotes mostly use Ser/Thr/Tyr phosphorylation for creating highly sophisticated regulatory networks. In antithesis to the above hypothesis, several studies showed that Ser/Thr/Tyr phosphorylation is also common in the bacterial cell, and here we present the first genome-wide phosphoproteomic analysis of the model organism of archaea, Halobacterium salinarum, proving the existence/conservation of Ser/Thr/Tyr phosphorylation in the “third domain” of life, allowing a better understanding of the origin and evolution of the so-called “Nature's premier” mechanism for regulating the functional properties of proteins.
PMCID: PMC2652253  PMID: 19274099
12.  Genome information management and integrated data analysis with HaloLex 
Archives of Microbiology  2008;190(3):281-299.
HaloLex is a software system for the central management, integration, curation, and web-based visualization of genomic and other -omics data for any given microorganism. The system has been employed for the manual curation of three haloarchaeal genomes, namely Halobacterium salinarum (strain R1), Natronomonas pharaonis, and Haloquadratum walsbyi. HaloLex, in particular, enables the integrated analysis of genome-wide proteomic results with the underlying genomic data. This has proven indispensable to generate reliable gene predictions for GC-rich genomes, which, due to their characteristically low abundance of stop codons, are known to be hard targets for standard gene finders, especially concerning start codon assignment. The proteomic identification of more than 600 N-terminal peptides has greatly increased the reliability of the start codon assignment for Halobacterium salinarum. Application of homology-based methods to the published genome of Haloarcula marismortui allowed to detect 47 previously unidentified genes (a problem that is particularly serious for short protein sequences) and to correct more than 300 start codon misassignments.
PMCID: PMC2516542  PMID: 18592220
Halophilic archaea; Genome information system; Genome browser; Proteomics; Biological data curation; Start codon assignment; Dinucleotide bias
13.  Metabolism of halophilic archaea 
Extremophiles   2008;12(2):177-196.
In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. The metabolic diversity of halophilic archaea was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species: Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi, and the haloalkaliphile Natronomonas pharaonis. The comparative study reveals different sets of enzyme genes amongst halophilic archaea, e.g. in glycerol degradation, pentose metabolism, and folate synthesis. The carefully assessed metabolic data represent a reliable resource for future system biology approaches as it also links to current experimental data on (halo)archaea from the literature.
Electronic supplementary material
The online version of this article (doi:10.1007/s00792-008-0138-x) contains supplementary material, which is available to authorized users.
PMCID: PMC2262144  PMID: 18278431
Metabolism; Archaea; Haloarchaea; Halobacterium salinarum; Pathway database; Metabolic pathways; Enzymes; Comparative genomics
14.  A small protein from the bop–brp intergenic region of Halobacterium salinarum contains a zinc finger motif and regulates bop and crtB1 transcription 
Molecular Microbiology  2008;67(4):772-780.
Bacteriorhodopsin, the photosynthetic protein of Halobacterium salinarum, is optimally expressed under anaerobic growth conditions. We identified Brz (OE3104F, bacteriorhodopsin-regulating zinc finger protein), a new regulator of the bop gene. It is a small protein with a zinc finger motif, encoded directly upstream of the bop gene in the same orientation. Deletion of the brz gene caused a large decrease of bop mRNA levels as shown by Northern blot and microarray analysis. A similar effect was obtained by site-directed mutagenesis of Cys and His residues in the zinc finger motif, indicating the importance of this motif for the function of the protein. In silico analysis of the genomes from H. salinarum and other archaea revealed a large family of similar small zinc finger motif proteins, some of which may also be involved in transcription regulation of their adjacent genes.
PMCID: PMC2253796  PMID: 18179416
15.  Genome-wide analysis of growth phase-dependent translational and transcriptional regulation in halophilic archaea 
BMC Genomics  2007;8:415.
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.
PMCID: PMC3225822  PMID: 17997854
16.  Microarray Analysis in the Archaeon Halobacterium salinarum Strain R1 
PLoS ONE  2007;2(10):e1064.
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.
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.
PMCID: PMC2020435  PMID: 17957248
17.  Autoinducer-2-Producing Protein LuxS, a Novel Salt- and Chloride-Induced Protein in the Moderately Halophilic Bacterium Halobacillus halophilus▿  
The moderately halophilic bacterium Halobacillus halophilus carries a homologue of LuxS, a protein involved in the activated methyl cycle and the production of autoinducer-2, which mediates quorum sensing between certain species. luxS of H. halophilus is part of an operon that encodes an S-adenosylmethionine-dependent methyltransferase, a cysteine synthase, and a β-cystathionine lyase. Expression of luxS was growth phase dependent, with maximal expression in the mid-exponential growth phase. In addition, transcription of luxS was strictly salt dependent; maximal mRNA concentrations were observed with 2.0 M NaCl in the growth medium. Chloride ions stimulated luxS transcription by a factor of three. Western blot analyses demonstrated a growth phase- and salinity-dependent production of LuxS. Moreover, cellular LuxS levels were strictly chloride dependent. Maximal accumulation of LuxS was observed at 0.5 to 1.0 M Cl− and depended on the salinity.
PMCID: PMC1796989  PMID: 17085700
18.  Biochemical and Molecular Characterization of the Biosynthesis of Glutamine and Glutamate, Two Major Compatible Solutes in the Moderately Halophilic Bacterium Halobacillus halophilus†  
Journal of Bacteriology  2006;188(19):6808-6815.
The moderately halophilic, chloride-dependent bacterium Halobacillus halophilus produces glutamate and glutamine as main compatible solutes at external salinities of 1.0 to 1.5 M NaCl. The routes for the biosynthesis of these solutes and their regulation were examined. The genome contains two genes potentially encoding glutamate dehydrogenases and two genes for the small subunit of a glutamate synthase, but only one gene for the large subunit. However, the expression of these genes was not salt dependent, nor were the corresponding enzymatic activities detectable in cell extracts of cells grown at different salinities. In contrast, glutamine synthetase activity was readily detectable in H. halophilus. Induction of glutamine synthetase activity was strictly salt dependent and reached a maximum at 3.0 M NaCl; chloride stimulated the production of active enzyme by about 300%. Two potential genes encoding a glutamine synthetase, glnA1 and glnA2, were identified. The expression of glnA2 but not of glnA1 was increased up to fourfold in cells adapted to high salt, indicating that GlnA2 is the glutamine synthetase involved in the synthesis of the solutes glutamate and glutamine. Furthermore, expression of glnA2 was stimulated twofold by the presence of chloride ions. Chloride exerted an even more pronounced effect on the enzymatic activity of preformed enzyme: in the absence of chloride in the assay buffer, glutamine synthetase activity was decreased by as much as 90%. These data demonstrate for the first time a regulatory role of a component of common salt, chloride, in the biosynthesis of compatible solutes.
PMCID: PMC1595520  PMID: 16980483
19.  The genome of the square archaeon Haloquadratum walsbyi : life at the limits of water activity 
BMC Genomics  2006;7:169.
The square halophilic archaeon Haloquadratum walsbyi dominates NaCl-saturated and MgCl2 enriched aquatic ecosystems, which imposes a serious desiccation stress, caused by the extremely low water activity. The genome sequence was analyzed and physiological and physical experiments were carried out in order to reveal how H. walsbyi has specialized into its narrow and hostile ecological niche and found ways to cope with the desiccation stress.
A rich repertoire of proteins involved in phosphate metabolism, phototrophic growth and extracellular protective polymers, including the largest archaeal protein (9159 amino acids), a homolog to eukaryotic mucins, are amongst the most outstanding features. A relatively low GC content (47.9%), 15–20% less than in other halophilic archaea, and one of the lowest coding densities (76.5%) known for prokaryotes might be an indication for the specialization in its unique environment
Although no direct genetic indication was found that can explain how this peculiar organism retains its square shape, the genome revealed several unique adaptive traits that allow this organism to thrive in its specific and extreme niche.
PMCID: PMC1544339  PMID: 16820047
20.  Amino Acid Biosynthesis in the Halophilic Archaeon Haloarcula hispanica 
Journal of Bacteriology  1999;181(10):3226-3237.
Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13C labeling with a mixture of 90% unlabeled and 10% uniformly 13C-labeled glycerol. The resulting 13C-labeling patterns in the amino acids were analyzed by two-dimensional 13C,1H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya.
PMCID: PMC93780  PMID: 10322026
21.  Regulation of Switching Frequency and Bias of the Bacterial Flagellar Motor by CheY and Fumarate 
Journal of Bacteriology  1998;180(13):3375-3380.
The effect of CheY and fumarate on switching frequency and rotational bias of the bacterial flagellar motor was analyzed by computer-aided tracking of tethered Escherichia coli. Plots of cells overexpressing CheY in a gutted background showed a bell-shaped correlation curve of switching frequency and bias centering at about 50% clockwise rotation. Gutted cells (i.e., with cheA to cheZ deleted) with a low CheY level but a high cytoplasmic fumarate concentration displayed the same correlation of switching frequency and bias as cells overexpressing CheY at the wild-type fumarate level. Hence, a high fumarate level can phenotypically mimic CheY overexpression by simultaneously changing the switching frequency and the bias. A linear correlation of cytoplasmic fumarate concentration and clockwise rotation bias was found and predicts exclusively counterclockwise rotation without switching when fumarate is absent. This suggests that (i) fumarate is essential for clockwise rotation in vivo and (ii) any metabolically induced fluctuation of its cytoplasmic concentration will result in a transient change in bias and switching probability. A high fumarate level resulted in a dose-response curve linking bias and cytoplasmic CheY concentration that was offset but with a slope similar to that for a low fumarate level. It is concluded that fumarate and CheY act additively presumably at different reaction steps in the conformational transition of the switch complex from counterclockwise to clockwise motor rotation.
PMCID: PMC107292  PMID: 9642190
22.  Lysis of Halobacteria in Bacto-Peptone by Bile Acids 
All tested strains of halophilic archaebacteria of the genera Halobacterium, Haloarcula, Haloferax, and Natronobacterium lysed in 1% Bacto-Peptone (Difco) containing 25% NaCl, whereas no lysis was observed with other strains belonging to archaebacteria of the genera Halococcus, Natronococcus, and Sulfolobus, methanogenic bacteria, and moderately halophilic eubacteria. Substances in Bacto-Peptone which caused lysis of halobacteria were purified and identified as taurocholic acid and glycocholic acid. High-performance liquid chromatography analyses of peptones revealed that Bacto-Peptone contained nine different bile acids, with a total content of 9.53 mg/g, whereas much lower amounts were found in Peptone Bacteriological Technical (Difco) and Oxoid Peptone. Different kinds of peptones can be used to distinguish halophilic eubacteria and archaebacteria in mixed cultures from hypersaline environments.
PMCID: PMC202585  PMID: 16347619
23.  Lag1p and Lac1p Are Essential for the Acyl-CoA–dependent Ceramide Synthase Reaction in Saccharomyces cerevisae 
Molecular Biology of the Cell  2001;12(11):3417-3427.
Lag1p and Lac1p are two homologous transmembrane proteins of the endoplasmic reticulum in Saccharomyces cerevisiae. Homologous genes have been found in a wide variety of eukaryotes. In yeast, both genes, LAC1 and LAG1, are required for efficient endoplasmic reticulum-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins. In this study, we show that lag1Δlac1Δ cells have reduced sphingolipid levels due to a block of the fumonisin B1-sensitive and acyl-CoA–dependent ceramide synthase reaction. The sphingolipid synthesis defect in lag1Δlac1Δ cells can be partially corrected by overexpression of YPC1 or YDC1, encoding ceramidases that have been reported to have acyl-CoA–independent ceramide synthesis activity. Quadruple mutant cells (lag1Δlac1Δypc1Δydc1Δ) do not make any sphingolipids, but are still viable probably because they produce novel lipids. Moreover, lag1Δlac1Δ cells are resistant to aureobasidin A, an inhibitor of the inositolphosphorylceramide synthase, suggesting that aureobasidin A may be toxic because it leads to increased ceramide levels. Based on these data, LAG1 and LAC1 are the first genes to be identified that are required for the fumonisin B1-sensitive and acyl-CoA–dependent ceramide synthase reaction.
PMCID: PMC60264  PMID: 11694577
24.  A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T 
Environmental Microbiology  2011;13(8):1973-1994.
The halophilic γ-proteobacterium Halomonas elongata DSM 2581T thrives at high salinity by synthesizing and accumulating the compatible solute ectoine. Ectoine levels are highly regulated according to external salt levels but the overall picture of its metabolism and control is not well understood. Apart from its critical role in cell adaptation to halophilic environments, ectoine can be used as a stabilizer for enzymes and as a cell protectant in skin and health care applications and is thus produced annually on a scale of tons in an industrial process using H. elongata as producer strain. This paper presents the complete genome sequence of H. elongata (4 061 296 bp) and includes experiments and analysis identifying and characterizing the entire ectoine metabolism, including a newly discovered pathway for ectoine degradation and its cyclic connection to ectoine synthesis. The degradation of ectoine (doe) proceeds via hydrolysis of ectoine (DoeA) to Nα-acetyl-l-2,4-diaminobutyric acid, followed by deacetylation to diaminobutyric acid (DoeB). In H. elongata, diaminobutyric acid can either flow off to aspartate or re-enter the ectoine synthesis pathway, forming a cycle of ectoine synthesis and degradation. Genome comparison revealed that the ectoine degradation pathway exists predominantly in non-halophilic bacteria unable to synthesize ectoine. Based on the resulting genetic and biochemical data, a metabolic flux model of ectoine metabolism was derived that can be used to understand the way H. elongata survives under varying salt stresses and that provides a basis for a model-driven improvement of industrial ectoine production.
PMCID: PMC3187862  PMID: 20849449

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