BACKGROUND
according to the three domains of life paradigm, archaea are distinct life forms and constitute a domain of their own, the other domains being bacteria and eukarya  <cit> .
recent studies suggest that eukaryotes may, in fact, have originated from ancient archaea  <cit> .
many structural and functional attributes of archaea share a high degree of similarity with the corresponding features present in bacterial or eukaryotic cells.
yet, several aspects of archaeal biochemistry and biology are unique  <cit> .
these include, for example, the ether linkages between glycerol and hydrophobic side chains in archaeal membrane phospholipids  <cit>  and the archaeal cell wall, often composed of a single s-layer glycoprotein  <cit> .
moreover, the ability of many archaea to thrive in extreme environments is biologically intriguing and makes archaea a valuable resource for biotechnology applications.
even though a degree of information is available about some unique archaeal features, the biochemical pathways and genetic basis behind many of them remain poorly characterized or entirely uncharacterized.
haloferax volcanii is a moderately halophilic, mesophilic and aerobic archaeon that can be cultured in defined media, and it serves as an excellent model organism for the archaeal domain of life.
the genome consists of one major chromosome , present in up to thirty copies, and three secondary minichromosomes phv <dig> , phv <dig> , and phv <dig> , with copy numbers up to  <dig>   <dig>  and  <dig>  respectively  <cit> .
in addition, the characterized wild type strains also harbor a plasmid phv <dig>   <cit> .
a number of biochemical, genetic and molecular biology methods have been developed for h. volcanii  <cit> , and their successful employment has enabled the identification of key factors involved in several crucial cellular functions, including the components of the two predominant protein transport systems  <cit>  and the proteasome  <cit> .
while several types of useable genome manipulation techniques and selectable markers are available for h. volcanii  <cit> , its more detailed biological characterization remains critically dependent on the development of a wider selection of advanced methodologies.
biochemical pathways of archaea can be discovered using comparative genome analyses, where homologous bacterial or eukaryotic genes may indicate a functional archaeal counterpart.
however, these types of approaches have only a limited utility in identifying genes behind unique archaeal features.
some archaeal genes may be identified through biochemical means, but the low expression level of many gene products often complicates these analyses.
in general, genetics provides the most effective means to discover novel genes, but sophisticated genetics methodologies are yet to be developed for most archaeal species.
a comprehensive collection of mutant clones with randomly distributed genomic alterations provides a valuable resource for studies aimed at delineating molecular mechanisms behind biological functions.
such libraries have proven their immense usefulness, particularly in microbiological research, but only a limited number of archaeal studies have exploited such resources.
chemical and physical mutagens, such as ethyl methanesulfonate  and ultraviolet light, have been used to introduce random mutations into several archaeal species  <cit> .
although these studies have yielded auxotrophic mutants, mapping of the underlying genetic alterations has been an arduous task.
furthermore, the presence of multiple genome copies within a single cell  <cit> , a common feature of archaea, complicates these types of approaches: mutagenesis produces heterozygous cells with initially only one copy of a mutated allele, and without a selective pressure, newly acquired mutations may be lost or fail to result in detectable phenotypic changes due the dominance of the wild type alleles  <cit> .
insertional mutagenesis typically employs selectable markers to tag random genomic loci, and transposition-based methods offer the most straightforward general means for the purpose.
thus far, genome-wide transposon insertion libraries have only been produced for two archaeal species, anaerobic methanogens methanosarcina acetivorans  <cit>  and methanococcus maripaludis  <cit> .
some progress in transposon mutagenesis has also been made with halophilic haloarcula hispanica  <cit> , but comprehensive insertion mutant libraries have not been produced for any haloarchaeal species.
all transposon-mediated mutagenesis approaches employ efficient transpositional recombination machinery, which catalyzes the joining of the transposon dna into target dna, and the most useful transposition systems integrate into a nearly random selection of target sites  <cit> .
the machinery is normally introduced into target cells by the use of plasmid vehicles  <cit>  or via electroporation of pre-assembled dna transposition complexes, transpososomes  <cit> .
the plasmid vehicle strategy requires a functional transposition system, either native or of heterologous origin.
however, no native transposon has so far been recruited for mutagenesis in h. volcanii, and heterologous systems may not be functional due to the high  intracellular potassium chloride concentration in haloarchaea.
the transpososome delivery strategy is critically dependent on electrotransformation under salt-free conditions, and as h. volcanii cells are sensitive to a low salt concentration, the utilization of such a strategy may be difficult or impossible.
however, alternative approaches may be used, if the organism harbors homologous recombination machinery sufficiently operational for efficient gene targeting.
in principle, insertion mutant libraries can be generated by the combined use of in vitro transposition mutagenesis and simultaneous targeting of a mixture of genomic fragments, as shown with yeast and fungi  <cit> .
previous studies have shown that the h. volcanii homologous recombination machinery functions for gene targeting  <cit> , and that the mua-catalyzed in vitro dna transposition reaction can be used to introduce randomly distributed insertions into any target dna  <cit> .
encouraged by these achievements, we developed here a strategy to generate an insertion mutant library for h. volcanii and generated a broad collection of clones, each individually tagged with transposon dna.
we validated the quality of the library for gene discovery by isolating pigmentation-defective and auxotrophic mutants.
the library represents the first comprehensive insertion mutant collection generated in haloarchaea.
as the applied strategy is characteristically species non-specific, it should readily be transferable to other archaeal species.
RESULTS
plasmid library with h. volcanii inserts tagged with transposon dna
initially, we wanted to generate a plasmid library, which would contain transposon-tagged h. volcanii genomic dna segments as inserts, altogether covering the entire genome as overlapping fragments .
for the tagging we made a shuttle transposon, trpa-cat-mu, which includes two marker genes, cat and trpa, allowing selection in escherichia coli and h. volcanii, respectively .
initially, h. volcanii genomic dna was partially digested, in separate reactions, with three restriction enzymes , each recognizing a 4-bp sequence and generating a protruding 5′-gc.
trpa-cat-mu was then integrated into the generated fragments using an in vitro transposition reaction, and the fragments were pooled.
following chromatographic size-selection,  <dig> to 6 kb fragments were cloned into a unique clai site of psuperscript sk+, ultimately yielding a plasmid library with  <dig>  independently generated member clones .
as h. volcanii harbors approximately  <dig>  genes  <cit> , on the average, each transposon-tagged gene should be present roughly seven times in this plasmid library.figure  <dig>
transposon trpa-cat-mu and overview of the transposon mutagenesis strategy.
the transposon was made from cat-mu  <cit>  by the addition of a gene cassette containing trpa and pfdx.
orange rectangles indicate 50 bp of mu r-end dna.
the trpa gene  under the control of pfdx promoter  was used for the selection in h. volcanii; the cat gene  under the cat promoter  was used for the selection in e. coli.
the transposon was released from its carrier plasmid by bglii digestion.
h. volcanii h <dig> dna was partially digested with hpaii, acii, and taqi and used as a target in an in vitro mu transposition reaction with trpa-cat-mu as a donor dna.
from transposition products,  <dig> to 6 kb fragments were isolated and cloned into the clai site of pbluesript sk+ to yield a plasmid library.
inserts were released by digesting with xhoi + hindiii or kpni + ecorv, gel-purified, and used to transform h. volcanii h <dig> cells to generate a transposon insertion mutant library.
to validate the plasmid library, we isolated plasmid dna from three randomly selected clones.
the h. volcanii dna segment present in each of these plasmids  was identified by sequencing.
plasmid pskt <dig> harbored transposon-tagged dna from the minor chromosome phv <dig>  whereas pskt <dig> and pskt <dig> contained tagged dna from the main chromosome .
these data and the applied cloning strategy with strong biochemical and biological selections imply that each clone in the library includes transposon-tagged h. volcanii dna.table  <dig>
plasmids used in the work
plasmid
relevant feature
reference/source
e. coli cloning vector; ampr
e. coli cloning vector; ampr
e. coli – h. volcanii shuttle vector; ampr, trp+
e. coli – h. volcanii shuttle vector; ampr, trp+
anumbering according to h. volcanii d <dig> phv <dig>  and genomic sequence , respectively.
amp: ampicillin; cam: chloramphenicol.
validation of gene targeting procedure
our initial targeting attempts with circular plasmid library dna were problematic, yielding h. volcanii clones with no genomic transposon insertions but harboring replicative plasmids .
therefore, we decided to use linear dna fragments for targeting en masse .
prior to the actual experiments, we wanted to validate the targeting procedure using known individual fragments.
accordingly, we isolated and gel-purified the inserts from the abovementioned plasmids, pskt <dig>  pskt <dig>  and pskt <dig>  using xhoi + hindiii double digestion.
these fragments, named iskt <dig>  iskt <dig>  and iskt <dig>  were then used to transform h. volcanii h <dig>  for gene targeting.
this strain was chosen, as it portrays an elevated level of homologous recombination activity  <cit> .
as dam methylation potentially influenced the outcome, we used both methylated and unmethylated dna .
the replicative plasmid pta <dig> served as a control for the transformation of circular dna .
while dam-methylation reduced the transformation efficiency of circular dna by tenfold, the methylation status, surprisingly, did not influence the number of produced colonies in transformations with linear dna.
the efficiency of transformation varied among fragments, being  <dig> to  <dig> colony-forming units /μg dna with iskt <dig> and iskt <dig> but only 102 cfu/μg dna with iskt <dig>  these results likely directly reflect the differences in the length of the homology regions flanking the transposon dna in these fragments .
the novel finding that methylation status had no effect on the transformation efficiency of linear fragments demonstrates that there is no need to passage the dna to be transformed via a dam- bacterial strain.
accordingly, methylated dna isolated from e. coli dh5α was used for further experiments.table  <dig>
microbial strains used in the work
strain
genotype
use
reference/source
enda <dig> reca <dig> gale <dig> galk <dig> nupg rpsl Δlacx <dig> 80laczΔm <dig> arad <dig> Δ <dig> mcra
arad <dig> Δ <dig> gale <dig> galk <dig> Δlacx <dig> rpsl  hsdr <dig> mcra mcrb1
enda <dig> supe <dig> thi- <dig> reca <dig> rela <dig> gyra <dig> deor nupg 80laczΔm <dig> Δu <dig> hsdr <dig>
ara- <dig> leub <dig> thi- <dig> fhua <dig> lacy <dig> tsx- <dig> galk <dig> galt <dig> glnv <dig> hisg <dig> rpsl <dig>  xyl- <dig> mtl- <dig> dam13::tn <dig>  dcm- <dig> mcrb <dig> hsdr <dig>  mcra recf143
a,b
dna
right flank
left flank
dam
+
dam
-
aresults from one representative experiment are shown; befficiency in cfu/μg dna; cn.a., not applicable.
pcr was then used to verify proper targeting.
initially, genomic dna was isolated from four clones obtained from each of the three transformations , and dna was amplified from the corresponding loci .
properly targeted clones were expected to generate a pcr fragment with a  <dig>  kb size increase  when compared to the corresponding fragment from the parental strain h <dig>  diagnosing successful targeting, the clones from transformations h295/iskt <dig> and h295/iskt <dig> produced prominent  <dig>  kb and  <dig>  kb fragments, respectively .
however, most clones also portrayed faint parental-size fragments, likely indicating the presence of a small number of non-mutated chromosome copies in the studied clonal cell populations.
the results with clones from h295/iskt <dig> transformation were less uniform , as a prominent diagnostic  <dig>  kb fragment was produced by one of the clones.
the other three clones portrayed a major parental fragment of  <dig>  kb, although a faint diagnostic fragment was also visible with two of them.
thus, it seemed that the distribution of mutated versus parental minichromosomes was not uniform among the clones studied.
this finding was somewhat surprising, as all of these clones grew well without tryptophan, indicating the obligate presence of the trpa selection marker.
we hypothesized that correctly targeted minichromosomes nevertheless were present in all of these clones studied, but with a very low copy number when compared with the copy number of the wild type phv <dig>  to test this hypothesis, we devised a pcr strategy that amplified dna only from a properly targeted minichromosome .
all of the four clones studied exhibited a diagnostic  <dig>  kb fragment, indicating proper targeting.
taken together, the data from these experiments showed that linear selection-marker-containing genomic fragments can be targeted in a single step into specific loci, not only in the main chromosome but also in the minichromosome.
the results also showed that a mixture of mutated and non-mutated chromosomes was present in cells even after the selection regime applied, and this was particularly evident with the minichromosome phv <dig>  we suspect these findings directly reflect the multicopy nature of h. volcanii chromosomes and processes involved in the segregation and homogenotization of its genome.figure  <dig>
pcr analysis of transposon insertions.
linear fragments containing transposon trpa-cat-mu and flanking h. volcanii dna were released from plasmid vectors by restriction digestion and transformed into h <dig> cells.
four clones from three transformations were isolated, their dna was extracted, and pcr was used to detect the presence of the inserted transposon.
h295/iskt <dig> clones were analyzed with primers hsp <dig> and hsp <dig>   h295/iskt <dig> clones were analyzed with primers hsp <dig> and hsp <dig>   h295/iskt <dig> clones were analyzed with primers hsp <dig> and hsp <dig> as well as  with primers hsp <dig> and hsp <dig>  white rectangles indicate the  <dig>  kb transposon.
flanking dna from the integrated fragments is shown with purple.
the primer recognition sites are shown with arrows.
h <dig> indicates control pcr with dna from the parental strain.
crtl indicates template-free control reactions.
a 1 kb dna ladder  was used as a size standard.
h. volcanii mutant libraries with genomic insertions
to generate h. volcanii insertion mutants, a 4-6 kb fragment mixture was isolated from the plasmid library, and the mixture was used to transform h <dig> cells for chromosomal targeting en masse, in which each fragment would target its counterpart genomic locus .
in order to avoid a bias caused by restriction enzyme recognition site distribution, two enzyme pairs were used.
fragments released with xhoi + hindiii and kpni + ecorv double digestions both produced transformants with the efficiency of approximately  <dig> × 104 cfu/μg dna.
for these two sets of fragments, we performed two transformations each, in total yielding approximately  <dig> ×  <dig>  and approximately  <dig> ×  <dig>  colonies.
to detect the presence and copy number of the transposon dna,  <dig> of these clones were then studied by southern hybridization using a transposon-specific probe .
a single fragment was detected with all of the clones, indicating that at least the majority of the library clones harbored the transposon as a single copy.
these data are fully consistent with proper targeting via homologous recombination.figure  <dig>
southern analysis of transposon insertion mutants.
genomic dna  was digested with ecori + kpni and probed with a transposon-specific probe.
purified trpa-cat-mu transposon  and parental h <dig> dna were used as positive and negative controls, respectively.
lanes  <dig> and  <dig> represent pigmentation mutants, lanes  <dig> and  <dig> auxotrophic mutants, and lanes  <dig> to  <dig> randomly selected uncharacterized mutants.
faint bands in lanes  <dig> and  <dig> are due to a smaller amount of dna in these two samples.
pigmentation-defective mutants
h. volcanii synthesizes a distinct set of carotenoids  <cit> , resulting in pink colonies and providing a convenient phenotype to validate a mutant library for gene discovery.
accordingly, we screened the above two libraries for mutants deficient in color formation .
a total of  <dig>  ×  <dig> colonies were visually inspected, and  <dig> whitish clones were discovered.
to identify the genes affected, we determined by sequencing the transposon integration site in  <dig> of these clones.
in each of them, the transposon was located in the crtb gene .
crtb encodes phytoene synthase, which catalyzes the first specific step in the carotenoid synthesis pathway  <cit> , logically explaining the observed phenotype and verifying that the library represents a potent tool for gene identification.
four different insertion sites in crtb were identified , the number falling within the expectancy range of seven hits per gene on the average.
the recovery of several identical sites was not surprising, as replicated sibling plasmids were necessarily present in the plasmid library prepared originally from  <dig>  e. coli colonies.table  <dig>
pigmentation-deficient mutants
mutant
integration site; orientation
a
gene
protein
athe first change compared to h. volcanii chromosomal sequence  is indicated as the transposon integration site.
the orientation is denoted as trpa direction compared to h. volcanii chromosome : +, same direction; -, opposite direction.
auxotrophic mutants
we screened a total of  <dig>  mutants for auxotrophy by initially testing their ability to grow on hv-min minimal medium plates.
sixteen mutants did not grow, and their growth capacity was further studied on hv-min plates each supplemented with a single amino acid .
all biologically relevant amino acids were tested, and auxotrophic mutants were found for arginine , histidine , proline , tyrosine , isoleucine , and phenylalanine .
four of the sixteen mutants did not grow with any of these single amino acid supplements.table  <dig>
amino acid auxotrophic mutants
mutant
auxotrophy
integration site; orientation
a
gene
protein
athe first change compared to h. volcanii chromosomal sequence  is indicated as the transposon integration site.
the orientation is denoted as trpa direction compared to h. volcanii chromosome : +, same direction; -, opposite direction.
the transposon insertion sites in the growth-defective mutants were then determined .
all the five arginine auxotrophs had the transposon inserted in the genes known to be involved in arginine biosynthesis: argb, argc, argd, and cara, of which argc was targeted at two different sites.
the two histidine auxotrophs were identical with the transposon inserted in the same position in hvo_ <dig> .
one proline auxotroph had the insertion in hvo_ <dig>  and the other in hvo_ <dig> .
in the tyrosine auxotroph the transposon interrupted hvo_ <dig> .
the isoleucine auxotroph harbored the transposon in leua <dig>  proposed to encode either 2-isopropylmalate synthase or highly similar -citramalate synthase.
in the phenylalanine auxotroph the transposon was inserted in phea <dig> .
in summary, the transposon insertion sites pinpointed both genes known to be involved in amino acid biosyntesis and genes with no prior annotation.
of the four mutants not growing with single amino acid supplements, three harbored the transposon in a gene involved in amino acid biosynthesis.
two of them, ilvc  and ilvb <dig> , are needed for valine, leucine, and isoleucine biosynthesis.
indeed, the addition of the mixture of these three supplements restored the growth of these mutants.
the third identified gene, thrc <dig>  is required in glycine, serine, and threonine metabolism, and the addition of all three of these amino acids restored the mutant growth.
the sequence data from the fourth growth-deficient mutant suggested that a larger scale genome rearrangement might have taken place, possibly due to an initial insertion of two transposons .
as the revelation of the exact alteration would have required substantial extra efforts in sequencing, this mutant was not studied further.
discussion
more than  <dig> sequenced archaeal genomes are currently available, and the number is rapidly increasing .
although the sequence data are critically important for gene discovery, such data cannot be used to predict archaeal gene or protein functions if homologous counterparts have not been characterized in other organisms.
for example, only about 66% of the predicted genes in seven sequenced haloarchaeal genomes was assigned with a hypothetical protein function in a recent study  <cit> , leaving a significant portion of the genomes non-annotated.
as simple sequence comparisons cannot reveal genes behind unique archaeal features, experimental approaches are needed to advance our knowledge about the biology of these intriguing organisms.
various types of mutant libraries constitute an important resource for genetics/genomics studies, and those containing randomly distributed genomic insertions have been particularly useful for the identification of gene functions.
transposon-mediated mutagenesis provides a straightforward means to produce insertion libraries and has been widely applied in bacteria and eukaryotes  <cit> .
however, the use of insertion mutagenesis has been very limited with archaea  <cit> .
this is presumably due to the difficulties in methods development caused by the challenging biology of most archaeal species.
furthermore, inadequately characterized native transposon systems  <cit>  and potential incompatibility problems with heterologous systems may have hindered progress towards the efficient utilization of transposition-based techniques in archaea.
the aim of this study was to develop a robust insertion mutagenesis methodology for h. volcanii.
we chose to use minimal-component mu in vitro transposition  <cit>  for the distribution of insertions in dna and homologous recombination machinery of h. volcanii for genomic integration  <cit> .
mu in vitro reaction yields transposition products highly efficiently and with a relatively low target site selectivity  <cit> .
in practice, the distribution of integrations is essentially random along any longer segment of dna.
these characteristics have made the mu reaction ideal for a variety of molecular biology  <cit> , protein engineering  <cit> , and genomics  <cit>  applications.
also in this study, the reaction provided a straightforward means to deliver selection cassettes into random positions along the h. volcanii chromosomal dna.
in order to maximize the frequency of gene targeting via homologous recombination into the h. volcanii genome, we chose to use the strain h <dig>  which is devoid of mre11and rad <dig> proteins.
in the wild type strains these proteins form a complex restraining homologous recombination, and in the h <dig> mutant strain this activity is reported to be increased roughly 100-fold  <cit> .
as the targeting frequency with h <dig> in our experiments was approximately  <dig> × 104 cfu/μg dna, it is likely that the generation of sizeable mutant libraries can also be accomplished with strains exhibiting the wild type level of homologous recombination activity.
in order to produce a high quality insertion mutant library for h. volcanii, we paid careful attention to the details of the strategy design and optimized several key steps in the techniques used.
three different restriction endonucleases and partial digestions were used to fragment h. volcanii genomic dna, effectively reducing the bias caused by restriction site distribution and guaranteeing an extensive coverage of the genome with overlapping fragments.
the size-selection following the in vitro transposition reaction prior to ligation ensured that 4-6 kb genomic inserts were enriched in the plasmid library.
accordingly, the effective elimination of short fragments guaranteed that practically all of the cloned inserts were of suitable length for gene targeting.
our preliminary en masse targeting trials with circular plasmids resulted in a large fraction of h. volcanii clones that had not experienced genomic insertions but contained replicative plasmids harboring the selection cassette used and an h. volcanii origin of replication .
these plasmids evidently represented molecules, in which the transposon was originally integrated in the vicinity of an h. volcanii origin of replication, and ultimately resulted in shuttle plasmids capable of replication in both e. coli and h. volcanii.
this prompted us to use linear dna fragments for targeting and to eliminate even trace amounts of dna circles from the targeting preparation.
as alkaline plasmid preparation methods produce a fraction of collapsed supercoiled plasmid forms, which enter the cells effectively and cannot be digested with restriction endonucleases, we removed these plasmid forms by purifying the plasmid library using cesium chloride gradient centrifugation.
in addition, the dna to be transformed was size-selected prior to transformation using a preparative agarose gel, a technique which also selects against circular plasmid forms.
we believe that the circle-elimination procedures were particularly pivotal for the successful generation of a high-quality insertion mutant library.
the plasmid library was made from  <dig>  e. coli colonies.
theoretically, assuming random transposon insertion into h. volcanii dna, the probability  at which every gene is targeted at least once can be calculated using the formula p =  <dig> - n  <cit> , where x is the average gene size, g is the genome size, and n is the number of mutants.
the size of the h. volcanii genome is  <dig>  mb, and the number of genes is  <dig>   <cit> .
thus, the average gene size is 971 bp.
with these numbers, the probability of every gene being tagged in the plasmid library was  <dig> , indicating that all of the h. volcanii genes were represented in this library with a very high likelihood.
note that these calculations do not take into account the known coding density of 86%  <cit> .
it should be emphasized that the plasmid library generated contains a full repertoire of both essential and non-essential h. volcanii genes.
however, the original diversity of the plasmid library could not be maintained in the final h. volcanii genomic insertion mutant library.
this is because an insertion into any essential gene would result in cell death upon mutant genome homogenotization, leading to a situation where the cell would not be able to survive following the loss of wild type chromosome copies.
likewise, it should be remembered that not all of the insertions generate a loss-of-function mutation.
as dam-methylation had earlier been shown to reduce transformation efficiency in h. volcanii  <cit> , we used both dam+ and dam- dna in our experiments.
the transformation efficiency with plasmid dna was shown to be dependent on its methylation status, being ten times higher with unmethylated than methylated dna.
this difference is smaller than the 100- or  <dig> -fold difference described earlier for h. volcanii  <cit> .
the reason for this apparent discrepancy is not known but may plausibly relate to the use of different h. volcanii strains.
intriguingly, the methylation status had no effect on the transformation efficiency with linear fragments.
this is a novel finding, as  comparative transformation experiments with linear dna fragments have not previously been conducted with h. volcanii.
these data might indicate that targeting is a relatively rapid process and/or that the homologous recombination machinery of h. volcanii protects the incoming dna from degradation.
this conclusion is in line with earlier studies with h. volcanii, showing that integration into the chromosome can rescue methylated foreign dna from being destroyed  <cit> .
an important practical consequence of our findings is that the linear dna used for transformation can be purified from a variety of sources, and it does not need to be passaged through a dam- microbial strain.
we performed important quality measures to control the library generation process.
initially, we studied the accuracy of the targeting procedure by introducing three separate linear transposon-tagged h. volcanii dna fragments into cells.
with each fragment, the transposon was found in the expected locus in the genome, confirming that targeting proceeded accurately via homologous recombination.
thus, linear tagged dna fragments can be used effectively in a fast, one-step procedure to generate site-specific insertions into the h. volcanii genome.
however, in theory there exists a possibility that the integration of incoming dna could also follow a non-homologous recombination pathway.
on the basis of our data, we believe that such pathways, if operational, do not nevertheless prevail in h. volcanii.
this is demonstrated by our results from the gene discovery experiments: sixteen out of eighteen phenotypically screened insertion sites  identified a known gene on a logical biochemical pathway, a result incompatible with a dominating integration via non-homologous recombination machinery.
as the second quality measure, we determined the number of transposon copies present in the genome of twelve insertion mutant library clones.
in each case southern analysis revealed only one resident copy.
as the stoichiometry in the transformation of linear fragments should favor single-copy genomic integrations , the result was not surprising.
thus, each mutant clone should harbor only one tagged gene, establishing the genomic insertion library as a powerful tool for genetic screens.
to validate the insertion mutant library for gene identification, we initially searched for mutants defective in pigment formation.
the crtb gene, identified in the search, encodes phytoene synthase .
this enzyme, which has been characterized in several bacterial, archaeal, and plant species, catalyzes the first specific step in the carotenoid biosynthesis pathway, that is, the formation of phytoene from two molecules of geranylgeranyl pyrophosphate   <cit> .
the loss of pigmentation was thus an expected outcome upon the interruption of crtb.
we were not able to identify insertion mutations in the genes required for the synthesis of ggpp.
this molecule is required not only for the carotenoid biosynthesis, but it is also a precursor of isoprenoid lipids, essential components of the archaeal cell membrane  <cit> .
thus, it is likely that insertions into any of the genes needed for ggpp biosynthesis would result in cell death, and our data are consistent with this supposition.
in archaea, the colorless phytoene is converted into the red-colored lycopene by phytoene dehydrogenase  <cit> .
in h. volcanii, the crti gene  has been annotated to encode this enzyme  <cit> .
if this gene is solely responsible for the phytoene hydrogenase activity in h. volcanii, we should have pinpointed it with our screen.
a closer look at the h. volcanii genome annotation revealed hvo_ <dig>  annotated to encode a flavin-containing amine-oxidoreductase.
this enzyme family contains various amine oxidases, also including phytoene dehydrogenases and related enzymes.
homology searches with other halophilic archaea indicated that the deduced protein sequence of hvo_ <dig> shares a high degree of similarity with haloferax mediterranei and halobacterium salinarum phytoene dehydrogenases:  <dig> % and  <dig> % identical amino acids with the products of crti  and crti <dig> , respectively .
thus, h. volcanii hvo_ <dig> most likely encodes a phytoene dehydrogenase.
considering that the theoretical likelihood for an average gene not being tagged in our study was as low as  <dig> , the fact that we were unable to pinpoint either hvo_ <dig> or hvo_ <dig> strongly suggests that these two genes encode overlapping functions and thus complement each other.
as similar redundant phytoene dehydrogenase genes are present also in h. mediterranei and h. salinarum , such a gene arrangement may be a common feature among haloarchaea.figure  <dig>
carotenoid biosyntehesis pathway in haloarchaea.
the pathway from geranylgeranyl pyrophosphate to lycopene is shown.
the insert portrays the comparison of the deduced phytoene dehydrogenase sequences of haloferax volcanii  to those of haloferax mediterranei  and halobacterium salinarum .
the numbers indicate the percentage of identical amino acids between the compared proteins.
pairwise analyses were made by using emboss water  <cit> .
the screen for amino acid auxotrophic mutants mostly identified genes known to be involved in amino acid biosynthesis: argb, argc, argd, and cara for arginine, hvo_ <dig>  for tyrosine, ilvc and ilvb <dig> for valine, leucine and isoleucine, and thr <dig> for glycine, serine and threonine.
interestingly, the transposon insertion in the isoleucine auxotroph was in the leua <dig> gene, which has been proposed to encode either a 2-isopropylmalate synthase required for leucine biosynthesis or an –citramalate synthase required for isoleucine biosynthesis  <cit> .
our results thus support the latter hypothesis.
the proline auxotroph  had the transposon inserted in hvo_ <dig>  which encodes glutamate synthase  large chain.
glutamate synthase is a key enzyme in ammonia assimilation, and the interruption of hvo_ <dig> is expected to result in unbalanced glutamate and nitrogen metabolism.
more work would be needed to find out why the growth defect of this mutant is restored by the addition of proline, but not by any other amino acid.
in addition to these known amino acid biosynthesis genes, we obtained two auxotrophic mutants having insertions in putative or unidentified genes: hvo_ <dig>  for histidine and hvo_ <dig>  for proline.
our study thus proposes the involvement of these genes in amino acid biosynthesis pathways, illustrating the huge potential of the library for the discovery of novel genes.
the copy number of the h. volcanii major chromosome is dependent on growth phase and conditions, being as high as  <dig>  while the minichromosome copy numbers are somewhat lower   <cit> .
a study with h. mediterranei minichromosome phm <dig> suggests that the replication of the major and minor chromosomes is controlled independently  <cit> , and we suspect that this may be the case also in h. volcanii.
in transposition mutagenesis, the transposon is integrated into the dna of one chromosomal copy, while others remain wild type.
therefore, during the first few cell generations following mutagenesis, the transposon-containing genome copies are necessarily in a minority.
as the mixture of mutated and non-mutated chromosomes may persist in a cell for some time, the cells thus being heterozygous, it may be intuitively somewhat surprising that loss-of function mutants could be isolated in our study relatively easily.
our results suggest that chromosome homogenotization takes place under the selection pressure applied, and the pcr data on the targeting into the major chromosome supports this conclusion.
the findings are consistent with the results of an earlier h. volcanii study, showing that genome copies will be equalized under selection pressure relatively rapidly  <cit> .
in the paper the authors suggested efficient gene conversion machinery as a primary driving force behind the phenomenon, although mutually non-exclusive uneven segregation could not entirely be ruled out.
our phv <dig> targeting data showed variation in chromosome homogenotization, possibly indicating that this process may be slow or inefficient with minichromosomes.
there are a few important issues that need to be considered when generating and using mutant libraries for microbes as described in this study:  two copies of the transposon may be integrated in vitro into the same target dna fragment, although rarely, as the reaction stoichiometry favors single insertions.
if such a fragment passes the size selection applied, it may experience recombination between the transposon copies.
this can occur either in e. coli or in the target organism, and it could ultimately lead to a deletion or inversion, both relatively uninformative with regard to gene discovery.
the mutagenesis strategy produces siblings in the plasmid library.
thus, following transformation into the target organism, the recovery of identical genomic insertion clones is expected, which was particularly well illustrated with our crtb mutants.
therefore, a large number of mutants with a desired phenotype should be collected in order to guarantee the finding of all possible insertion sites.
the target genome may contain regions that cannot be cloned into a plasmid vector, and such regions will not be recovered as transposon-tagged clones in the final insertion library.
the possibility of polar effects should be remembered when working with insertion mutant libraries of microbes.
although rarely, spontaneous mutations may generate false positive results, in which case the transposon tag will identify a gene not involved in the observed phenotype.
the genetic screens should always be regarded as a first step towards gene discovery.
in order to verify the genotype-phenotype connection, complementation or site-directed mutagenesis studies should be executed with each potential candidate gene.
CONCLUSIONS
to conclude, we present here the generation of the first genome-wide insertion mutant library for haloarchaea using the model archaeon h. volcanii.
the library includes clones that harbor a single transposon insertion in their genome, and it can be used for gene identification in h. volcanii.
the strategy used provides a straightforward general means to generate comprehensive mutant libraries, is characteristically species non-specific and should readily be transferable to other archaeal species.
thus, the library represents a significant step towards advanced genetics studies in archaea.

 according to the three domains of life paradigm, archaea are distinct life forms and constitute a domain of their own, the other domains being bacteria and eukarya. recent studies suggest that eukaryotes may, in fact, have originated from ancient archaea. many structural and functional attributes of archaea share a high degree of similarity with the corresponding features present in bacterial or eukaryotic cells. yet, several aspects of archaeal biochemistry and biology are unique. these include, for example, the ether linkages between glycerol and hydrophobic side chains in archaeal membrane phospholipids and the archaeal cell wall, often composed of a single s-layer glycoprotein. moreover, the ability of many archaea to thrive in extreme environments is biologically intriguing and makes archaea a valuable resource for biotechnology applications. even though a degree of information is available about some unique archaeal features, the biochemical pathways and genetic basis behind many of them remain poorly characterized or entirely uncharacterized. haloferax volcanii is a moderately halophilic, mesophilic and aerobic archaeon that can be cultured in defined media, and it serves as an excellent model organism for the archaeal domain of life. the genome consists of one major chromosome, present in up to thirty copies, and three secondary minichromosomes phv, phv, and phv, with copy numbers up to and respectively. in addition, the characterized wild type strains also harbor a plasmid phv. a number of biochemical, genetic and molecular biology methods have been developed for h. volcanii, and their successful employment has enabled the identification of key factors involved in several crucial cellular functions, including the components of the two predominant protein transport systems and the proteasome. while several types of useable genome manipulation techniques and selectable markers are available for h. volcanii, its more detailed biological characterization remains critically dependent on the development of a wider selection of advanced methodologies. biochemical pathways of archaea can be discovered using comparative genome analyses, where homologous bacterial or eukaryotic genes may indicate a functional archaeal counterpart. however, these types of approaches have only a limited utility in identifying genes behind unique archaeal features. some archaeal genes may be identified through biochemical means, but the low expression level of many gene products often complicates these analyses. in general, genetics provides the most effective means to discover novel genes, but sophisticated genetics methodologies are yet to be developed for most archaeal species. a comprehensive collection of mutant clones with randomly distributed genomic alterations provides a valuable resource for studies aimed at delineating molecular mechanisms behind biological functions. such libraries have proven their immense usefulness, particularly in microbiological research, but only a limited number of archaeal studies have exploited such resources. chemical and physical mutagens, such as ethyl methanesulfonate and ultraviolet light, have been used to introduce random mutations into several archaeal species. although these studies have yielded auxotrophic mutants, mapping of the underlying genetic alterations has been an arduous task. furthermore, the presence of multiple genome copies within a single cell, a common feature of archaea, complicates these types of approaches: mutagenesis produces heterozygous cells with initially only one copy of a mutated allele, and without a selective pressure, newly acquired mutations may be lost or fail to result in detectable phenotypic changes due the dominance of the wild type alleles. insertional mutagenesis typically employs selectable markers to tag random genomic loci, and transposition-based methods offer the most straightforward general means for the purpose. thus far, genome-wide transposon insertion libraries have only been produced for two archaeal species, anaerobic methanogens methanosarcina acetivorans and methanococcus maripaludis. some progress in transposon mutagenesis has also been made with halophilic haloarcula hispanica, but comprehensive insertion mutant libraries have not been produced for any haloarchaeal species. all transposon-mediated mutagenesis approaches employ efficient transpositional recombination machinery, which catalyzes the joining of the transposon dna into target dna, and the most useful transposition systems integrate into a nearly random selection of target sites. the machinery is normally introduced into target cells by the use of plasmid vehicles or via electroporation of pre-assembled dna transposition complexes, transpososomes. the plasmid vehicle strategy requires a functional transposition system, either native or of heterologous origin. however, no native transposon has so far been recruited for mutagenesis in h. volcanii, and heterologous systems may not be functional due to the high intracellular potassium chloride concentration in haloarchaea. the transpososome delivery strategy is critically dependent on electrotransformation under salt-free conditions, and as h. volcanii cells are sensitive to a low salt concentration, the utilization of such a strategy may be difficult or impossible. however, alternative approaches may be used, if the organism harbors homologous recombination machinery sufficiently operational for efficient gene targeting. in principle, insertion mutant libraries can be generated by the combined use of in vitro transposition mutagenesis and simultaneous targeting of a mixture of genomic fragments, as shown with yeast and fungi. previous studies have shown that the h. volcanii homologous recombination machinery functions for gene targeting, and that the mua-catalyzed in vitro dna transposition reaction can be used to introduce randomly distributed insertions into any target dna. encouraged by these achievements, we developed here a strategy to generate an insertion mutant library for h. volcanii and generated a broad collection of clones, each individually tagged with transposon dna. we validated the quality of the library for gene discovery by isolating pigmentation-defective and auxotrophic mutants. the library represents the first comprehensive insertion mutant collection generated in haloarchaea. as the applied strategy is characteristically species non-specific, it should readily be transferable to other archaeal species.  plasmid library with h. volcanii inserts tagged with transposon dna initially, we wanted to generate a plasmid library, which would contain transposon-tagged h. volcanii genomic dna segments as inserts, altogether covering the entire genome as overlapping fragments. for the tagging we made a shuttle transposon, trpa-cat-mu, which includes two marker genes, cat and trpa, allowing selection in escherichia coli and h. volcanii, respectively. initially, h. volcanii genomic dna was partially digested, in separate reactions, with three restriction enzymes, each recognizing a 4-bp sequence and generating a protruding 5′-gc. trpa-cat-mu was then integrated into the generated fragments using an in vitro transposition reaction, and the fragments were pooled. following chromatographic size-selection, to 6 kb fragments were cloned into a unique clai site of psuperscript sk+, ultimately yielding a plasmid library with independently generated member clones. as h. volcanii harbors approximately genes, on the average, each transposon-tagged gene should be present roughly seven times in this plasmid library.figure transposon trpa-cat-mu and overview of the transposon mutagenesis strategy. the transposon was made from cat-mu by the addition of a gene cassette containing trpa and pfdx. orange rectangles indicate 50 bp of mu r-end dna. the trpa gene under the control of pfdx promoter was used for the selection in h. volcanii; the cat gene under the cat promoter was used for the selection in e. coli. the transposon was released from its carrier plasmid by bglii digestion. h. volcanii h dna was partially digested with hpaii, acii, and taqi and used as a target in an in vitro mu transposition reaction with trpa-cat-mu as a donor dna. from transposition products, to 6 kb fragments were isolated and cloned into the clai site of pbluesript sk+ to yield a plasmid library. inserts were released by digesting with xhoi + hindiii or kpni + ecorv, gel-purified, and used to transform h. volcanii h cells to generate a transposon insertion mutant library. to validate the plasmid library, we isolated plasmid dna from three randomly selected clones. the h. volcanii dna segment present in each of these plasmids was identified by sequencing. plasmid pskt harbored transposon-tagged dna from the minor chromosome phv whereas pskt and pskt contained tagged dna from the main chromosome. these data and the applied cloning strategy with strong biochemical and biological selections imply that each clone in the library includes transposon-tagged h. volcanii dna.table plasmids used in the work plasmid relevant feature reference/source e. coli cloning vector; ampr e. coli cloning vector; ampr e. coli – h. volcanii shuttle vector; ampr, trp+ e. coli – h. volcanii shuttle vector; ampr, trp+ anumbering according to h. volcanii d phv and genomic sequence, respectively. amp: ampicillin; cam: chloramphenicol. validation of gene targeting procedure our initial targeting attempts with circular plasmid library dna were problematic, yielding h. volcanii clones with no genomic transposon insertions but harboring replicative plasmids. therefore, we decided to use linear dna fragments for targeting en masse. prior to the actual experiments, we wanted to validate the targeting procedure using known individual fragments. accordingly, we isolated and gel-purified the inserts from the abovementioned plasmids, pskt pskt and pskt using xhoi + hindiii double digestion. these fragments, named iskt iskt and iskt were then used to transform h. volcanii h for gene targeting. this strain was chosen, as it portrays an elevated level of homologous recombination activity. as dam methylation potentially influenced the outcome, we used both methylated and unmethylated dna. the replicative plasmid pta served as a control for the transformation of circular dna. while dam-methylation reduced the transformation efficiency of circular dna by tenfold, the methylation status, surprisingly, did not influence the number of produced colonies in transformations with linear dna. the efficiency of transformation varied among fragments, being to colony-forming units /μg dna with iskt and iskt but only 102 cfu/μg dna with iskt these results likely directly reflect the differences in the length of the homology regions flanking the transposon dna in these fragments. the novel finding that methylation status had no effect on the transformation efficiency of linear fragments demonstrates that there is no need to passage the dna to be transformed via a dam- bacterial strain. accordingly, methylated dna isolated from e. coli dh5α was used for further experiments.table microbial strains used in the work strain genotype use reference/source enda reca gale galk nupg rpsl Δlacx 80laczΔm arad Δ mcra arad Δ gale galk Δlacx rpsl hsdr mcra mcrb1 enda supe thi- reca rela gyra deor nupg 80laczΔm Δu hsdr ara- leub thi- fhua lacy tsx- galk galt glnv hisg rpsl xyl- mtl- dam13::tn dcm- mcrb hsdr mcra recf143 a,b dna right flank left flank dam + dam - aresults from one representative experiment are shown; befficiency in cfu/μg dna; cn.a., not applicable. pcr was then used to verify proper targeting. initially, genomic dna was isolated from four clones obtained from each of the three transformations, and dna was amplified from the corresponding loci. properly targeted clones were expected to generate a pcr fragment with a  kb size increase when compared to the corresponding fragment from the parental strain h diagnosing successful targeting, the clones from transformations h295/iskt and h295/iskt produced prominent  kb and  kb fragments, respectively. however, most clones also portrayed faint parental-size fragments, likely indicating the presence of a small number of non-mutated chromosome copies in the studied clonal cell populations. the results with clones from h295/iskt transformation were less uniform, as a prominent diagnostic  kb fragment was produced by one of the clones. the other three clones portrayed a major parental fragment of  kb, although a faint diagnostic fragment was also visible with two of them. thus, it seemed that the distribution of mutated versus parental minichromosomes was not uniform among the clones studied. this finding was somewhat surprising, as all of these clones grew well without tryptophan, indicating the obligate presence of the trpa selection marker. we hypothesized that correctly targeted minichromosomes nevertheless were present in all of these clones studied, but with a very low copy number when compared with the copy number of the wild type phv to test this hypothesis, we devised a pcr strategy that amplified dna only from a properly targeted minichromosome. all of the four clones studied exhibited a diagnostic  kb fragment, indicating proper targeting. taken together, the data from these experiments showed that linear selection-marker-containing genomic fragments can be targeted in a single step into specific loci, not only in the main chromosome but also in the minichromosome. the results also showed that a mixture of mutated and non-mutated chromosomes was present in cells even after the selection regime applied, and this was particularly evident with the minichromosome phv we suspect these findings directly reflect the multicopy nature of h. volcanii chromosomes and processes involved in the segregation and homogenotization of its genome.figure pcr analysis of transposon insertions. linear fragments containing transposon trpa-cat-mu and flanking h. volcanii dna were released from plasmid vectors by restriction digestion and transformed into h cells. four clones from three transformations were isolated, their dna was extracted, and pcr was used to detect the presence of the inserted transposon. h295/iskt clones were analyzed with primers hsp and hsp h295/iskt clones were analyzed with primers hsp and hsp h295/iskt clones were analyzed with primers hsp and hsp as well as with primers hsp and hsp white rectangles indicate the  kb transposon. flanking dna from the integrated fragments is shown with purple. the primer recognition sites are shown with arrows. h indicates control pcr with dna from the parental strain. crtl indicates template-free control reactions. a 1 kb dna ladder was used as a size standard. h. volcanii mutant libraries with genomic insertions to generate h. volcanii insertion mutants, a 4-6 kb fragment mixture was isolated from the plasmid library, and the mixture was used to transform h cells for chromosomal targeting en masse, in which each fragment would target its counterpart genomic locus. in order to avoid a bias caused by restriction enzyme recognition site distribution, two enzyme pairs were used. fragments released with xhoi + hindiii and kpni + ecorv double digestions both produced transformants with the efficiency of approximately × 104 cfu/μg dna. for these two sets of fragments, we performed two transformations each, in total yielding approximately × and approximately × colonies. to detect the presence and copy number of the transposon dna, of these clones were then studied by southern hybridization using a transposon-specific probe. a single fragment was detected with all of the clones, indicating that at least the majority of the library clones harbored the transposon as a single copy. these data are fully consistent with proper targeting via homologous recombination.figure southern analysis of transposon insertion mutants. genomic dna was digested with ecori + kpni and probed with a transposon-specific probe. purified trpa-cat-mu transposon and parental h dna were used as positive and negative controls, respectively. lanes and represent pigmentation mutants, lanes and auxotrophic mutants, and lanes to randomly selected uncharacterized mutants. faint bands in lanes and are due to a smaller amount of dna in these two samples. pigmentation-defective mutants h. volcanii synthesizes a distinct set of carotenoids, resulting in pink colonies and providing a convenient phenotype to validate a mutant library for gene discovery. accordingly, we screened the above two libraries for mutants deficient in color formation. a total of × colonies were visually inspected, and whitish clones were discovered. to identify the genes affected, we determined by sequencing the transposon integration site in of these clones. in each of them, the transposon was located in the crtb gene. crtb encodes phytoene synthase, which catalyzes the first specific step in the carotenoid synthesis pathway, logically explaining the observed phenotype and verifying that the library represents a potent tool for gene identification. four different insertion sites in crtb were identified, the number falling within the expectancy range of seven hits per gene on the average. the recovery of several identical sites was not surprising, as replicated sibling plasmids were necessarily present in the plasmid library prepared originally from e. coli colonies.table pigmentation-deficient mutants mutant integration site; orientation a gene protein athe first change compared to h. volcanii chromosomal sequence is indicated as the transposon integration site. the orientation is denoted as trpa direction compared to h. volcanii chromosome: +, same direction; -, opposite direction. auxotrophic mutants we screened a total of mutants for auxotrophy by initially testing their ability to grow on hv-min minimal medium plates. sixteen mutants did not grow, and their growth capacity was further studied on hv-min plates each supplemented with a single amino acid. all biologically relevant amino acids were tested, and auxotrophic mutants were found for arginine, histidine, proline, tyrosine, isoleucine, and phenylalanine. four of the sixteen mutants did not grow with any of these single amino acid supplements.table amino acid auxotrophic mutants mutant auxotrophy integration site; orientation a gene protein athe first change compared to h. volcanii chromosomal sequence is indicated as the transposon integration site. the orientation is denoted as trpa direction compared to h. volcanii chromosome: +, same direction; -, opposite direction. the transposon insertion sites in the growth-defective mutants were then determined. all the five arginine auxotrophs had the transposon inserted in the genes known to be involved in arginine biosynthesis: argb, argc, argd, and cara, of which argc was targeted at two different sites. the two histidine auxotrophs were identical with the transposon inserted in the same position in hvo_. one proline auxotroph had the insertion in hvo_ and the other in hvo_. in the tyrosine auxotroph the transposon interrupted hvo_. the isoleucine auxotroph harbored the transposon in leua proposed to encode either 2-isopropylmalate synthase or highly similar -citramalate synthase. in the phenylalanine auxotroph the transposon was inserted in phea. in summary, the transposon insertion sites pinpointed both genes known to be involved in amino acid biosyntesis and genes with no prior annotation. of the four mutants not growing with single amino acid supplements, three harbored the transposon in a gene involved in amino acid biosynthesis. two of them, ilvc and ilvb, are needed for valine, leucine, and isoleucine biosynthesis. indeed, the addition of the mixture of these three supplements restored the growth of these mutants. the third identified gene, thrc is required in glycine, serine, and threonine metabolism, and the addition of all three of these amino acids restored the mutant growth. the sequence data from the fourth growth-deficient mutant suggested that a larger scale genome rearrangement might have taken place, possibly due to an initial insertion of two transposons. as the revelation of the exact alteration would have required substantial extra efforts in sequencing, this mutant was not studied further. discussion more than sequenced archaeal genomes are currently available, and the number is rapidly increasing. although the sequence data are critically important for gene discovery, such data cannot be used to predict archaeal gene or protein functions if homologous counterparts have not been characterized in other organisms. for example, only about 66% of the predicted genes in seven sequenced haloarchaeal genomes was assigned with a hypothetical protein function in a recent study, leaving a significant portion of the genomes non-annotated. as simple sequence comparisons cannot reveal genes behind unique archaeal features, experimental approaches are needed to advance our knowledge about the biology of these intriguing organisms. various types of mutant libraries constitute an important resource for genetics/genomics studies, and those containing randomly distributed genomic insertions have been particularly useful for the identification of gene functions. transposon-mediated mutagenesis provides a straightforward means to produce insertion libraries and has been widely applied in bacteria and eukaryotes. however, the use of insertion mutagenesis has been very limited with archaea. this is presumably due to the difficulties in methods development caused by the challenging biology of most archaeal species. furthermore, inadequately characterized native transposon systems and potential incompatibility problems with heterologous systems may have hindered progress towards the efficient utilization of transposition-based techniques in archaea. the aim of this study was to develop a robust insertion mutagenesis methodology for h. volcanii. we chose to use minimal-component mu in vitro transposition for the distribution of insertions in dna and homologous recombination machinery of h. volcanii for genomic integration. mu in vitro reaction yields transposition products highly efficiently and with a relatively low target site selectivity. in practice, the distribution of integrations is essentially random along any longer segment of dna. these characteristics have made the mu reaction ideal for a variety of molecular biology, protein engineering, and genomics applications. also in this study, the reaction provided a straightforward means to deliver selection cassettes into random positions along the h. volcanii chromosomal dna. in order to maximize the frequency of gene targeting via homologous recombination into the h. volcanii genome, we chose to use the strain h which is devoid of mre11and rad proteins. in the wild type strains these proteins form a complex restraining homologous recombination, and in the h mutant strain this activity is reported to be increased roughly 100-fold. as the targeting frequency with h in our experiments was approximately × 104 cfu/μg dna, it is likely that the generation of sizeable mutant libraries can also be accomplished with strains exhibiting the wild type level of homologous recombination activity. in order to produce a high quality insertion mutant library for h. volcanii, we paid careful attention to the details of the strategy design and optimized several key steps in the techniques used. three different restriction endonucleases and partial digestions were used to fragment h. volcanii genomic dna, effectively reducing the bias caused by restriction site distribution and guaranteeing an extensive coverage of the genome with overlapping fragments. the size-selection following the in vitro transposition reaction prior to ligation ensured that 4-6 kb genomic inserts were enriched in the plasmid library. accordingly, the effective elimination of short fragments guaranteed that practically all of the cloned inserts were of suitable length for gene targeting. our preliminary en masse targeting trials with circular plasmids resulted in a large fraction of h. volcanii clones that had not experienced genomic insertions but contained replicative plasmids harboring the selection cassette used and an h. volcanii origin of replication. these plasmids evidently represented molecules, in which the transposon was originally integrated in the vicinity of an h. volcanii origin of replication, and ultimately resulted in shuttle plasmids capable of replication in both e. coli and h. volcanii. this prompted us to use linear dna fragments for targeting and to eliminate even trace amounts of dna circles from the targeting preparation. as alkaline plasmid preparation methods produce a fraction of collapsed supercoiled plasmid forms, which enter the cells effectively and cannot be digested with restriction endonucleases, we removed these plasmid forms by purifying the plasmid library using cesium chloride gradient centrifugation. in addition, the dna to be transformed was size-selected prior to transformation using a preparative agarose gel, a technique which also selects against circular plasmid forms. we believe that the circle-elimination procedures were particularly pivotal for the successful generation of a high-quality insertion mutant library. the plasmid library was made from e. coli colonies. theoretically, assuming random transposon insertion into h. volcanii dna, the probability at which every gene is targeted at least once can be calculated using the formula p =  - n, where x is the average gene size, g is the genome size, and n is the number of mutants. the size of the h. volcanii genome is  mb, and the number of genes is. thus, the average gene size is 971 bp. with these numbers, the probability of every gene being tagged in the plasmid library was, indicating that all of the h. volcanii genes were represented in this library with a very high likelihood. note that these calculations do not take into account the known coding density of 86%. it should be emphasized that the plasmid library generated contains a full repertoire of both essential and non-essential h. volcanii genes. however, the original diversity of the plasmid library could not be maintained in the final h. volcanii genomic insertion mutant library. this is because an insertion into any essential gene would result in cell death upon mutant genome homogenotization, leading to a situation where the cell would not be able to survive following the loss of wild type chromosome copies. likewise, it should be remembered that not all of the insertions generate a loss-of-function mutation. as dam-methylation had earlier been shown to reduce transformation efficiency in h. volcanii, we used both dam+ and dam- dna in our experiments. the transformation efficiency with plasmid dna was shown to be dependent on its methylation status, being ten times higher with unmethylated than methylated dna. this difference is smaller than the 100- or -fold difference described earlier for h. volcanii. the reason for this apparent discrepancy is not known but may plausibly relate to the use of different h. volcanii strains. intriguingly, the methylation status had no effect on the transformation efficiency with linear fragments. this is a novel finding, as comparative transformation experiments with linear dna fragments have not previously been conducted with h. volcanii. these data might indicate that targeting is a relatively rapid process and/or that the homologous recombination machinery of h. volcanii protects the incoming dna from degradation. this conclusion is in line with earlier studies with h. volcanii, showing that integration into the chromosome can rescue methylated foreign dna from being destroyed. an important practical consequence of our findings is that the linear dna used for transformation can be purified from a variety of sources, and it does not need to be passaged through a dam- microbial strain. we performed important quality measures to control the library generation process. initially, we studied the accuracy of the targeting procedure by introducing three separate linear transposon-tagged h. volcanii dna fragments into cells. with each fragment, the transposon was found in the expected locus in the genome, confirming that targeting proceeded accurately via homologous recombination. thus, linear tagged dna fragments can be used effectively in a fast, one-step procedure to generate site-specific insertions into the h. volcanii genome. however, in theory there exists a possibility that the integration of incoming dna could also follow a non-homologous recombination pathway. on the basis of our data, we believe that such pathways, if operational, do not nevertheless prevail in h. volcanii. this is demonstrated by our results from the gene discovery experiments: sixteen out of eighteen phenotypically screened insertion sites identified a known gene on a logical biochemical pathway, a result incompatible with a dominating integration via non-homologous recombination machinery. as the second quality measure, we determined the number of transposon copies present in the genome of twelve insertion mutant library clones. in each case southern analysis revealed only one resident copy. as the stoichiometry in the transformation of linear fragments should favor single-copy genomic integrations, the result was not surprising. thus, each mutant clone should harbor only one tagged gene, establishing the genomic insertion library as a powerful tool for genetic screens. to validate the insertion mutant library for gene identification, we initially searched for mutants defective in pigment formation. the crtb gene, identified in the search, encodes phytoene synthase. this enzyme, which has been characterized in several bacterial, archaeal, and plant species, catalyzes the first specific step in the carotenoid biosynthesis pathway, that is, the formation of phytoene from two molecules of geranylgeranyl pyrophosphate. the loss of pigmentation was thus an expected outcome upon the interruption of crtb. we were not able to identify insertion mutations in the genes required for the synthesis of ggpp. this molecule is required not only for the carotenoid biosynthesis, but it is also a precursor of isoprenoid lipids, essential components of the archaeal cell membrane. thus, it is likely that insertions into any of the genes needed for ggpp biosynthesis would result in cell death, and our data are consistent with this supposition. in archaea, the colorless phytoene is converted into the red-colored lycopene by phytoene dehydrogenase. in h. volcanii, the crti gene has been annotated to encode this enzyme. if this gene is solely responsible for the phytoene hydrogenase activity in h. volcanii, we should have pinpointed it with our screen. a closer look at the h. volcanii genome annotation revealed hvo_ annotated to encode a flavin-containing amine-oxidoreductase. this enzyme family contains various amine oxidases, also including phytoene dehydrogenases and related enzymes. homology searches with other halophilic archaea indicated that the deduced protein sequence of hvo_ shares a high degree of similarity with haloferax mediterranei and halobacterium salinarum phytoene dehydrogenases: % and % identical amino acids with the products of crti and crti, respectively. thus, h. volcanii hvo_ most likely encodes a phytoene dehydrogenase. considering that the theoretical likelihood for an average gene not being tagged in our study was as low as, the fact that we were unable to pinpoint either hvo_ or hvo_ strongly suggests that these two genes encode overlapping functions and thus complement each other. as similar redundant phytoene dehydrogenase genes are present also in h. mediterranei and h. salinarum, such a gene arrangement may be a common feature among haloarchaea.figure carotenoid biosyntehesis pathway in haloarchaea. the pathway from geranylgeranyl pyrophosphate to lycopene is shown. the insert portrays the comparison of the deduced phytoene dehydrogenase sequences of haloferax volcanii to those of haloferax mediterranei and halobacterium salinarum. the numbers indicate the percentage of identical amino acids between the compared proteins. pairwise analyses were made by using emboss water. the screen for amino acid auxotrophic mutants mostly identified genes known to be involved in amino acid biosynthesis: argb, argc, argd, and cara for arginine, hvo_ for tyrosine, ilvc and ilvb for valine, leucine and isoleucine, and thr for glycine, serine and threonine. interestingly, the transposon insertion in the isoleucine auxotroph was in the leua gene, which has been proposed to encode either a 2-isopropylmalate synthase required for leucine biosynthesis or an –citramalate synthase required for isoleucine biosynthesis. our results thus support the latter hypothesis. the proline auxotroph had the transposon inserted in hvo_ which encodes glutamate synthase large chain. glutamate synthase is a key enzyme in ammonia assimilation, and the interruption of hvo_ is expected to result in unbalanced glutamate and nitrogen metabolism. more work would be needed to find out why the growth defect of this mutant is restored by the addition of proline, but not by any other amino acid. in addition to these known amino acid biosynthesis genes, we obtained two auxotrophic mutants having insertions in putative or unidentified genes: hvo_ for histidine and hvo_ for proline. our study thus proposes the involvement of these genes in amino acid biosynthesis pathways, illustrating the huge potential of the library for the discovery of novel genes. the copy number of the h. volcanii major chromosome is dependent on growth phase and conditions, being as high as while the minichromosome copy numbers are somewhat lower. a study with h. mediterranei minichromosome phm suggests that the replication of the major and minor chromosomes is controlled independently, and we suspect that this may be the case also in h. volcanii. in transposition mutagenesis, the transposon is integrated into the dna of one chromosomal copy, while others remain wild type. therefore, during the first few cell generations following mutagenesis, the transposon-containing genome copies are necessarily in a minority. as the mixture of mutated and non-mutated chromosomes may persist in a cell for some time, the cells thus being heterozygous, it may be intuitively somewhat surprising that loss-of function mutants could be isolated in our study relatively easily. our results suggest that chromosome homogenotization takes place under the selection pressure applied, and the pcr data on the targeting into the major chromosome supports this conclusion. the findings are consistent with the results of an earlier h. volcanii study, showing that genome copies will be equalized under selection pressure relatively rapidly. in the paper the authors suggested efficient gene conversion machinery as a primary driving force behind the phenomenon, although mutually non-exclusive uneven segregation could not entirely be ruled out. our phv targeting data showed variation in chromosome homogenotization, possibly indicating that this process may be slow or inefficient with minichromosomes. there are a few important issues that need to be considered when generating and using mutant libraries for microbes as described in this study: two copies of the transposon may be integrated in vitro into the same target dna fragment, although rarely, as the reaction stoichiometry favors single insertions. if such a fragment passes the size selection applied, it may experience recombination between the transposon copies. this can occur either in e. coli or in the target organism, and it could ultimately lead to a deletion or inversion, both relatively uninformative with regard to gene discovery. the mutagenesis strategy produces siblings in the plasmid library. thus, following transformation into the target organism, the recovery of identical genomic insertion clones is expected, which was particularly well illustrated with our crtb mutants. therefore, a large number of mutants with a desired phenotype should be collected in order to guarantee the finding of all possible insertion sites. the target genome may contain regions that cannot be cloned into a plasmid vector, and such regions will not be recovered as transposon-tagged clones in the final insertion library. the possibility of polar effects should be remembered when working with insertion mutant libraries of microbes. although rarely, spontaneous mutations may generate false positive results, in which case the transposon tag will identify a gene not involved in the observed phenotype. the genetic screens should always be regarded as a first step towards gene discovery. in order to verify the genotype-phenotype connection, complementation or site-directed mutagenesis studies should be executed with each potential candidate gene.  to conclude, we present here the generation of the first genome-wide insertion mutant library for haloarchaea using the model archaeon h. volcanii. the library includes clones that harbor a single transposon insertion in their genome, and it can be used for gene identification in h. volcanii. the strategy used provides a straightforward general means to generate comprehensive mutant libraries, is characteristically species non-specific and should readily be transferable to other archaeal species. thus, the library represents a significant step towards advanced genetics studies in archaea. 
archaea share fundamental properties with bacteria and eukaryotes. it serves as an excellent archaeal model organism to study the molecular mechanisms of biological processes and cellular responses to changes in the environment. from the library, we isolated pigmentation-defective and auxotrophic mutants, and the respective insertions pinpointed a number of genes previously known to be involved in carotenoid and amino acid biosynthesis pathways, thus validating the performance of the methodologies used. we also identified mutants that had a transposon insertion in a gene encoding a protein of unknown or putative function, demonstrating that novel roles for non-annotated genes could be assigned. it represents a significant step towards achieving a more complete understanding of the unique characteristics of halophilic archaea. haloferax volcaniihalophilic archaeainsertion mutant librarymu transpositionmua proteingene discoveryissue-copyright-statement© the author 2014 haloferax volcanii is an aerobic, moderately halophilic archaeon that can be grown in defined media. we generated an insertion mutant library, in which the clones contained a single genomic insertion. we have generated, for the first time, a random genomic insertion mutant library for a halophilic archaeon and used it for efficient gene discovery. here, we devised an insertion mutagenesis strategy that combined mu in vitro dna transposition and homologous-recombination-based gene targeting in h. volcanii. studies on haloarchaea have been impeded by the lack of efficient genetic screens that would facilitate the identification of protein functions and respective metabolic pathways.
 archaea share fundamental properties with bacteria and eukaryotes. yet, they also possess unique attributes, which largely remain poorly characterized. haloferax volcanii is an aerobic, moderately halophilic archaeon that can be grown in defined media. it serves as an excellent archaeal model organism to study the molecular mechanisms of biological processes and cellular responses to changes in the environment. studies on haloarchaea have been impeded by the lack of efficient genetic screens that would facilitate the identification of protein functions and respective metabolic pathways.  here, we devised an insertion mutagenesis strategy that combined mu in vitro dna transposition and homologous-recombination-based gene targeting in h. volcanii. we generated an insertion mutant library, in which the clones contained a single genomic insertion. from the library, we isolated pigmentation-defective and auxotrophic mutants, and the respective insertions pinpointed a number of genes previously known to be involved in carotenoid and amino acid biosynthesis pathways, thus validating the performance of the methodologies used. we also identified mutants that had a transposon insertion in a gene encoding a protein of unknown or putative function, demonstrating that novel roles for non-annotated genes could be assigned.  we have generated, for the first time, a random genomic insertion mutant library for a halophilic archaeon and used it for efficient gene discovery. the library will facilitate the identification of non-essential genes behind any specific biochemical pathway. it represents a significant step towards achieving a more complete understanding of the unique characteristics of halophilic archaea. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords haloferax volcaniihalophilic archaeainsertion mutant librarymu transpositionmua proteingene discoveryissue-copyright-statement© the author 2014 