BACKGROUND
the dimorphic fungal pathogen, histoplasma capsulatum, parasitizes phagocytic cells of the mammalian immune system and causes one of the most common respiratory fungal infections world wide  <cit> .
the mycelia-produced histoplasma conidia are acquired by inhalation into the respiratory tract where exposure to mammalian body temperatures triggers their differentiation into pathogenic yeast cells  <cit> .
histoplasma virulence requires this transition to the yeast phase and expression of the corresponding yeast-phase regulon  <cit> .
this transcriptional profile includes genes encoding specific factors that promote histoplasma virulence  <cit> .
while mammalian alveolar macrophages efficiently phagocytose histoplasma cells, they are unable to kill the yeast  <cit> .
within the macrophage, histoplasma modifies the intracellular compartment to promote its survival and replication.
the ability to subvert immune defenses and to survive within phagocytes enables histoplasma to cause disease in both immunocompromised and immunocompetent individuals.
this high potential for infection is reflected in the fact that histoplasmosis is one of the most common pulmonary fungal infections among healthy individuals  <cit> .
the mechanistic details that underlie histoplasma pathogenesis are still largely unknown owing to limited or inefficient genetic methodologies.
the genome sequences of three phylogenetically distinct strains of histoplasma have been completed   <cit>  which has accelerated the ability to identify, define, and analyze histoplasma genes.
however, demonstration that a gene product contributes to a particular facet of biology requires specific depletion of the candidate factor and comparison to a factor-replete strain in functional tests.
targeted deletion of candidate factors is most often accomplished through genetic means, employing homologous recombination to replace the wild-type gene with an engineered deletion or disruption allele.
in saccharomyces cerevisiae, homologous recombination is so efficient that gene deletion libraries have been compiled with mutants representing entire sets of genes or even the majority of the genes in the genome  <cit> .
in contrast, non-homologous or illegitimate recombination dominates in the dimorphic fungal pathogens  <cit> , frustrating gene deletion attempts and impeding advancement of our molecular understanding of these fungi.
furthermore, histoplasma can maintain introduced dna  as an extrachromosomal element which impedes efforts to incorporate alleles into the genome  <cit> .
despite these obstacles, genes have been deleted in histoplasma following development of a two-step procedure  <cit> .
realization of the rare homologous recombination event necessitates a very large population as the frequency of allelic replacement is on the order of  <dig> in  <dig> transformants  <cit> .
as typical transformation frequencies are insufficient, individual transformants harboring recombination substrates are instead cultured and repeatedly passaged to generate a large number of potential recombination events.
in the second step, a dual positive and negative selection scheme enriches the population for the desired recombinant.
in practice, only a portion of the isolated clones harbor the deletion requiring screening of many potential isolates.
in histoplasma, this process of reverse genetics  has been successfully accomplished for only six genes to date, the vast majority in the panama phylogenetic group   <cit> .
for reasons not well understood, this procedure has not been very successful in the histoplasma nam  <dig> lineage despite numerous attempts.
recently, a deletion of the gene encoding dppiva has been reported in the nam  <dig> lineage  <cit> .
the inefficient and laborious process of deleting genes in histoplasma prompted development of rna interference  as an alternative method to determine the role of gene products in histoplasma biology  <cit> .
to date, eight genes have been functionally defined by rnai   <cit> .
however, rnai can not generate a complete loss of function, and this potential for residual function imposes difficulties in interpreting negative results with rnai .
unlike chromosomal mutations which are more permanent, plasmid-based rnai effects must be constantly maintained with selection.
for studies of histoplasma virulence, this in vivo selection of plasmids requires use of the ura <dig> selection marker and ura <dig> auxotrophic histoplasma strains.
to overcome these obstacles and limitations in our current techniques for genetic analysis of histoplasma, we have developed a procedure for isolating chromosomally-located gene mutants without reliance on homologous recombination.
we employed random mutagenesis to create collections of mutants.
one approach to identify the desired gene disruption would be to characterize the mutation of each isolate in the collection of random mutants.
however, this requires many resources, substantial time and effort, and thus is not well suited for studies targeting a particular gene.
in forward genetics, random mutagenesis is successful because the desired mutant can be typically isolated or identified out of the much larger collection of mutants by growth phenotype or morphological changes.
in reverse genetics, the mutant phenotype is the very aspect under investigation and thus mutants can not be identified by predicted changes.
to enable reverse genetics following random mutagenesis in histoplasma, we adapted pcr-based procedures employed for large scale screening in arabidopsis and c. elegans  <cit> .
we optimized a mutant pooling strategy and utilized pcr to efficiently identify mutant pools which contain the strain with the disrupted gene.
to extract the strain with the targeted mutation, the pool is subsequently subdivided and individual clones addressed and screened by pcr.
we demonstrate the effectiveness of this method by employing it to isolate a cbp <dig> mutant in the nam  <dig> histoplasma strain background.
RESULTS
insertion mutant screening
to generate insertion mutations in the histoplasma genome, we used agrobacterium tumefaciens-mediated transformation.
this mutagen was selected because agrobacterium-mediated transfer of t-dna is efficient in producing random insertional mutations in histoplasma yeast cells  <cit> .
the majority of t-dna insertions are single integration events  <cit>  and thus the chance of secondary background mutations is minimized.
other mutagens such as uv or chemical agents result in multiple changes to the genome, and while these background mutations can be removed by repeated backcrossing of mutants to wild type, no reliable techniques for crossing laboratory strains have been developed for histoplasma  <cit> .
additionally, insertional mutagens provide a molecular tag with known sequence  which we can exploit in pcr-based screening for mutations in particular chromosomal loci using a t-dna specific primer in conjunction with a primer specific for the targeted gene .
the molecular weight of the pcr amplicon provides an estimate of the distance from the gene specific primer to the t-dna insertion, and this distance can be used to determine whether the t-dna element disrupts the targeted gene.
optimization of pool size for reliable detection of targeted mutations
as the successful isolation of a mutant in a targeted gene depends critically on the ability to identify a positive individual among a much larger population, we determined the pcr detection limit for different pool sizes.
histoplasma strain osu <dig> harbors a t-dna insertion in the ags <dig> gene in which the t-dna right border is oriented towards the 3' end of the ags <dig> gene.
performance of pcr using a right border t-dna primer and an ags <dig> gene-specific primer produces a pcr amplicon of  <dig> bp.
to estimate the detection limit afforded by pcr in which a single strain could be found among a population of  <dig>   <dig>  or  <dig> mutants,  <dig> ng of nucleic acid purified from osu  <dig> were diluted 1: <dig>  1: <dig>  and 1: <dig> with te buffer and pcr performed on these templates with rb <dig> and ags1- <dig> primers.
with  <dig> cycles, pcr could consistently detect the osu <dig> template when diluted as much as 1: <dig> .
to better approximate the condition where the desired mutant would be present among a much larger population of other t-dna insertions, we mixed osu <dig> with a pool of random t-dna insertion mutants at a osu <dig> yeast-to-mutant pool ratio of 1: <dig>  1: <dig>  and 1: <dig>  nucleic acids were purified from each pool and pcr was performed as before with  <dig> ng of total nucleic acid as templates.
the positive  <dig> bp amplicon was detected when osu <dig> was present in as little as 1/800th of the total population of yeast .
a faint band representing the ags1::t-dna pcr product was observed when osu <dig> constituted 1/3200th of the template.
no ags1::t-dna pcr product was detected in templates prepared from the mutant pool alone or wild-type histoplasma.
a smaller pcr amplicon which is not specific to the ags1::t-dna template was detected in all reactions derived from the random insertion mutant pool.
nested pcr to reduce false-positives
to discriminate between true- and false-positive pcr products, we employed a secondary pcr reaction using a set of nested primers.
nested primers that do not overlap with the primary pcr primers were designed for both the t-dna anchor and the ags <dig> gene.
primary pcr reactions in which osu <dig> represented 1/200th or 1/800th of the population were used as templates after 1: <dig>  1: <dig> , and 1: <dig>  dilution in h2o.
as shown in figure 1c, this process eliminated the false-positive band observed in the primary pcr reactions.
the ags1::t-dna specific amplicon could be detected after either 1: <dig> or 1: <dig>  dilution of the primary pcr reaction.
no ags1::t-dna amplicon was produced when osu <dig> was absent in the primary reaction template dna.
these data demonstrate that pcr can be an efficient screening technique to probe mutant pools for a clone in which a t-dna element has inserted into a target gene.
we selected a target pool size of approximately  <dig> insertion mutants as a balance between increased throughput afforded by larger pools but easier subdivision of smaller pools into individual clones to recover the detected mutant strain .
establishment of a bank of insertion mutants
optimization of freezing conditions
as the generation of t-dna insertion mutants in histoplasma is not trivial, establishment of a frozen bank of insertion mutants would facilitate future screens without having to produce new mutant pools as additional target genes are identified.
maintaining the mutant representation in the pool after freezing necessitates efficient recovery of viable cells following thawing.
to maximize the recovery of cells after freezing we examined two parameters: the cryoprotectant used and the method of freezing.
glycerol- or dmso-containing solutions are used for freezing eukaryotic cells as these chemicals reduce membrane-damaging ice crystal formation.
we also tested whether slowing the freezing rate using an insulated container also improved recovery from frozen stocks.
histoplasma wu <dig> yeast cells were frozen and stored at -80°c for  <dig> days or  <dig> weeks to determine the short and long term storage recovery rates, respectively.
recovered cfu counts were compared to those before freezing.
with glycerol as the cryoprotectant, slowing the freezing rate dramatically improved recovery of viable yeast , probably resulting from the increased time to allow for penetration of glycerol into cells during cooling.
dmso was a superior cryoprotectant than glycerol for histoplasma yeast when present at concentrations from 4% to 10% .
freezing with dmso was less sensitive to the rate of freezing but as with glycerol, slower rates of cooling resulted in greater recovery of viable yeast cells .
nearly 40% of the starting suspension of yeast cells were recovered when cells were slowly frozen in an 8% dmso-containing solution and this procedure was selected for long term storage of mutant pools.
although specialized cooling apparatuses can be used to control the freezing rate, we found that simple placement of vials of cells within readily and cheaply obtained styrofoam containers  was sufficient.
generation of mutant pools
insertion mutants were generated in the nam  <dig> histoplasma strain wu <dig> by co-cultivation of agrobacterium tumefaciens and histoplasma yeast cells.
co-cultures were plated onto filters and histoplasma transformants selected by transferring filters to medium containing hygromycin to which resistance is provided by sequences within the t-dna element  <cit> .
transformant yeast cells were collected and suspensions from individual plates combined to create pools derived from  <dig> to  <dig> independent mutant colonies.
yeast cell suspensions were diluted into fresh medium and allowed to grow for 24- <dig> hours.
twenty-four pools were prepared representing roughly  <dig> insertion mutants.
a portion of each culture was reserved for nucleic acid isolation and the remainder frozen in aliquots and stored at -80°c.
nucleic acids were purified from each pool, diluted to  <dig> ng/ul, and stored at -20°c until analysis by pcr.
with an estimated 9000- <dig>  genes encoded by the histoplasma genome, this collection does not represent the number of insertion mutants required for saturation of the genome.
we used two probability functions to estimate the size of the library required for a 95% chance of isolating an insertion in a particular locus in the  <dig> megabase nam  <dig> genome.
both calculations assume no bias in insertion sites.
based on the number of predicted genes, the poisson approach estimates a library of approximately  <dig>  insertions would be required.
the single study in which multiple alleles of a single locus were isolated in histoplasma  supports the poisson calculation; five alleles would be the most probable number of alleles based on a  <dig> or  <dig>  target estimate.
however, the ags <dig> gene is an unusually large target and thus this number is probably an underestimate.
an alternative calculation based solely on average gene size is provided by: p = 1-n where p is the probability of the t-dna inserting in a given target of size x in a genome of size g with n the total number of t-dna insertion mutants  <cit> .
assuming an average gene size of  <dig> nucleotides, this calculation estimates a library of nearly  <dig>  mutants would be required for a 95% probability of obtaining at least one insertion mutant in any given gene.
such a mutant bank would require  <dig> pools with an average pool size of  <dig> and pcr screening could be easily performed using three 96-well plates.
although our current collection of  <dig> mutants is inadequate for complete genome coverage, it was sufficient to demonstrate proof-of-concept through identification and recovery of a mutant at the cbp <dig> locus.
isolation of a cbp <dig> insertion mutant
detection of a t-dna insertion in cbp1
as no cbp <dig> mutant exists in the nam  <dig> background despite numerous attempts with allelic replacement, we screened our nam  <dig> mutant bank for t-dna insertions that disrupt the cbp <dig> gene.
the cbp <dig> protein was the first virulence factor demonstrated for histoplasma through deletion of the encoding gene in a panama class strain of histoplasma  <cit> .
two cbp <dig> gene-specific primers were designed at the 3' end of the cbp <dig> coding region and were oriented towards the 5' end of the gene.
as the t-dna element could insert with either the t-dna left border or the right border oriented towards the 3' end of the cbp <dig> gene, we screened each mutant pool by pcr with rb <dig> or with lb <dig> primers in combination with the cbp1- <dig> gene-specific primer.
while pcr reactions with the lb <dig> + cbp1- <dig> primer set did not produce any positive pcr products with any of the templates , reactions with rb <dig> and cbp1- <dig> primers produced amplicons in two different pools .
low abundance bands less than  <dig> bp are likely primer dimers or residual rna from the template nucleic acids and were thus not considered.
a nested pcr reaction was performed on the rb3-set of reactions .
the pcr product from pool  <dig> did not re-amplify in the nested pcr reaction suggesting that this product was a non-specific amplicon.
alternatively, the pool may indeed harbor an insertion of t-dna sequence in the cbp <dig> locus but the t-dna element could be truncated and the nested rb primer-binding site lost resulting in failure to amplify in the nested pcr.
the nested pcr reaction from pool  <dig> produced a very prominent, approximately  <dig> bp amplicon consistent with an insertion in the dna upstream of the cbp <dig> coding region .
sequencing of this amplicon confirmed insertion of the t-dna in the cbp <dig> promoter and localized the insertion  <dig> base pairs upstream of the cbp <dig> start codon .
although a faint band was observed in the secondary pcr from pool # <dig>  no product was detected in the primary reaction and it's abundance was much lower than that of the validated amplicon from pool # <dig>  thus, it was discarded as a candidate.
recovery of the cbp <dig> insertion mutant
to isolate the strain containing the cbp1::t-dna mutation, we recovered yeast cells from pool # <dig> and segregated the pool into individual clones.
the insertion was tracked using pcr with the primers described earlier.
pool # <dig> was thawed and dilutions plated to recover individual cfu's.
as each pool represents 100- <dig> clones, we screened  <dig> clones to increase the likelihood of recovering at least one strain with the detected cbp <dig> insertion mutation.
to drastically reduce the number of nucleic acid preparations and pcr tests required to screen nearly  <dig> individuals, we employed an addressing scheme .
each clone was picked into individual wells of three 96-well plates containing liquid medium.
for each 96-well plate, wells from each row and from each column were pooled to produce  <dig> yeast suspensions.
an aliquot of these row and column sub-pools from each plate were combined to create a yeast suspension representing the clones from the entire 96-well plate.
nucleic acids were isolated from the three 96-well plate suspensions and subjected to pcr.
the cbp1::t-dna insertion amplicon was detected in two of the three collections of  <dig> .
for one of these pools of  <dig> individual clones, nucleic acids were isolated from the corresponding row and column sub-pools and pcr was used to screen for the t-dna insertion.
positive amplicons were detected in sub-pools representing the clone located at b <dig> .
the suspension of yeast was recovered from well b <dig> and plated on solid medium to recover individual colonies.
pcr performed on nucleic acids prepared from one of these colonies confirmed the identity and recovery of the desired insertion at the cbp <dig> locus.
the strain with this insertion was designated osu <dig>
to further characterize the t-dna insertion in osu <dig>  we amplified and sequenced the dna flanking the t-dna element.
pcr amplicons were produced for both the left and right border flanking regions using t-dna specific primers and cbp <dig> specific primers .
alignment of the flanking regions with the histoplasma g217b genome and t-dna sequences showed truncation of the t-dna imperfect direct repeats by  <dig> bp from the left border and  <dig> bp from the right border.
additionally, the t-dna insertion event deleted  <dig> base pairs of the cbp <dig> promoter surrounding the site of insertion .
due to t-dna-induced genetic rearrangements that can occur, pcr-product sizes should be used only as an initial estimate of the location of t-dna integration and the precise location of the insertion confirmed by sequencing the dna flanking the t-dna element.
as our pcr screening method would not detect multiple t-dna integrations, we performed a southern blot using a t-dna-specific probe to determine how many t-dna elements were present in the osu <dig> mutagenized genome.
as shown in figure 3d, only one band is detected indicating the osu <dig> strain harbors a single t-dna insertion.
this  <dig>  kb t-dna probe-hybridizing fragment is the size predicted for the described insertion in the cbp <dig> promoter.
no t-dna sequences were detected in the parental wu <dig> strain.
validation of the cbp <dig> mutant
since the t-dna insertion in osu <dig> did not lie within the cbp <dig> gene but was instead located in the sequence upstream of the cbp <dig> coding sequence, we tested whether the recovered mutant had lost the ability to produce the cbp <dig> protein.
the t-dna insertion site lies within the region defined as the cbp <dig> promoter  <cit> ; transcriptional fusion constructs with less than  <dig> base pairs of sequence upstream of cbp <dig> do not support transcription.
thus, insertion of  <dig> kb of foreign sequence  into this region should disrupt promoter activity.
osu <dig> and the parent wu <dig> strain were grown to early stationary phase and cell-free supernatants were prepared.
to determine whether cbp <dig> production was impaired in osu <dig>  we separated supernatant proteins by poly-acrylamide gel electrophoresis and visualized the proteins by silver staining.
supernatants from the cbp <dig> wu <dig> strain had a prominent 9- <dig> kd protein which was not detected in supernatants harvested from the osu <dig> culture  indicating the cbp1::t-dna insertion disrupts production of cbp <dig> protein.
the identity of this protein was confirmed as cbp <dig> since supernatant from a strain in which cbp <dig> was independently depleted by rnai also specifically lacked this protein band.
thus, while the t-dna insertion does not interrupt the coding region, insertion into the cbp <dig> promoter effectively prevents production of cbp <dig> in osu <dig>
CONCLUSIONS
we have developed a reverse genetics procedure employing random mutagenesis and pcr-based screening techniques to identify insertion mutants in a targeted gene in histoplasma capsulatum without regard to a mutant phenotype.
since the mutagen creates a large insertion, the majority of mutations should reflect the knock-out mutant phenotype.
however, insertions within the promoter of a gene may allow some residual transcription resulting in hypomorphic but not null phenotypes.
in such cases, as demonstrated by our cbp1:t-dna mutant, delineation of the minimal promoter of a targeted gene could resolve what type of phenotype the insertion mutation would likely produce.
thus, the regions most likely to produce mutant phenotypes are the proximal promoter and the coding region of the targeted gene.
consequently, we routinely design our pcr screening primers at the 3' end of the gene to amplify these regions in particular and maximize the targeted site for insertions.
this process, as with any mutational approach, will not successfully isolate insertion mutations in genes essential for viability, and if the intended target is suspect to be such, rnai may prove more beneficial.
while integration of t-dna into the histoplasma genome appears relatively random, large scale studies in magnaporthe, leptosphaeria, and arabidopsis indicate there is a bias for insertion of the t-dna element into non-coding regions  <cit> .
in addition, occurrence of large-scale deletions or rearrangement mutations will be missed by this approach.
thus, more insertion mutants may be required for saturation mutagenesis of the histoplasma genome than calculated above.
the reverse genetics process detailed here increases the repertoire of methods available to disrupt gene functions in histoplasma capsulatum.
since agrobacterium-mediated transformation has been developed as an efficient mutagen for a variety of fungal species  <cit> , this procedure should be readily applicable to those microorganisms as well.
for intractable fungal systems where homologous recombination is very limited or allelic replacement unfeasible, this process provides the ability to disrupt gene functions necessary for functional genetic tests.
the only requirement is an efficient insertional mutagen.
the increased capability to disrupt gene functions in histoplasma and in other fungi will greatly improve our mechanistic understanding of fungal biology.

 the dimorphic fungal pathogen, histoplasma capsulatum, parasitizes phagocytic cells of the mammalian immune system and causes one of the most common respiratory fungal infections world wide. the mycelia-produced histoplasma conidia are acquired by inhalation into the respiratory tract where exposure to mammalian body temperatures triggers their differentiation into pathogenic yeast cells. histoplasma virulence requires this transition to the yeast phase and expression of the corresponding yeast-phase regulon. this transcriptional profile includes genes encoding specific factors that promote histoplasma virulence. while mammalian alveolar macrophages efficiently phagocytose histoplasma cells, they are unable to kill the yeast. within the macrophage, histoplasma modifies the intracellular compartment to promote its survival and replication. the ability to subvert immune defenses and to survive within phagocytes enables histoplasma to cause disease in both immunocompromised and immunocompetent individuals. this high potential for infection is reflected in the fact that histoplasmosis is one of the most common pulmonary fungal infections among healthy individuals. the mechanistic details that underlie histoplasma pathogenesis are still largely unknown owing to limited or inefficient genetic methodologies. the genome sequences of three phylogenetically distinct strains of histoplasma have been completed which has accelerated the ability to identify, define, and analyze histoplasma genes. however, demonstration that a gene product contributes to a particular facet of biology requires specific depletion of the candidate factor and comparison to a factor-replete strain in functional tests. targeted deletion of candidate factors is most often accomplished through genetic means, employing homologous recombination to replace the wild-type gene with an engineered deletion or disruption allele. in saccharomyces cerevisiae, homologous recombination is so efficient that gene deletion libraries have been compiled with mutants representing entire sets of genes or even the majority of the genes in the genome. in contrast, non-homologous or illegitimate recombination dominates in the dimorphic fungal pathogens, frustrating gene deletion attempts and impeding advancement of our molecular understanding of these fungi. furthermore, histoplasma can maintain introduced dna as an extrachromosomal element which impedes efforts to incorporate alleles into the genome. despite these obstacles, genes have been deleted in histoplasma following development of a two-step procedure. realization of the rare homologous recombination event necessitates a very large population as the frequency of allelic replacement is on the order of in transformants. as typical transformation frequencies are insufficient, individual transformants harboring recombination substrates are instead cultured and repeatedly passaged to generate a large number of potential recombination events. in the second step, a dual positive and negative selection scheme enriches the population for the desired recombinant. in practice, only a portion of the isolated clones harbor the deletion requiring screening of many potential isolates. in histoplasma, this process of reverse genetics has been successfully accomplished for only six genes to date, the vast majority in the panama phylogenetic group. for reasons not well understood, this procedure has not been very successful in the histoplasma nam lineage despite numerous attempts. recently, a deletion of the gene encoding dppiva has been reported in the nam lineage. the inefficient and laborious process of deleting genes in histoplasma prompted development of rna interference as an alternative method to determine the role of gene products in histoplasma biology. to date, eight genes have been functionally defined by rnai. however, rnai can not generate a complete loss of function, and this potential for residual function imposes difficulties in interpreting negative results with rnai. unlike chromosomal mutations which are more permanent, plasmid-based rnai effects must be constantly maintained with selection. for studies of histoplasma virulence, this in vivo selection of plasmids requires use of the ura selection marker and ura auxotrophic histoplasma strains. to overcome these obstacles and limitations in our current techniques for genetic analysis of histoplasma, we have developed a procedure for isolating chromosomally-located gene mutants without reliance on homologous recombination. we employed random mutagenesis to create collections of mutants. one approach to identify the desired gene disruption would be to characterize the mutation of each isolate in the collection of random mutants. however, this requires many resources, substantial time and effort, and thus is not well suited for studies targeting a particular gene. in forward genetics, random mutagenesis is successful because the desired mutant can be typically isolated or identified out of the much larger collection of mutants by growth phenotype or morphological changes. in reverse genetics, the mutant phenotype is the very aspect under investigation and thus mutants can not be identified by predicted changes. to enable reverse genetics following random mutagenesis in histoplasma, we adapted pcr-based procedures employed for large scale screening in arabidopsis and c. elegans. we optimized a mutant pooling strategy and utilized pcr to efficiently identify mutant pools which contain the strain with the disrupted gene. to extract the strain with the targeted mutation, the pool is subsequently subdivided and individual clones addressed and screened by pcr. we demonstrate the effectiveness of this method by employing it to isolate a cbp mutant in the nam histoplasma strain background.  insertion mutant screening to generate insertion mutations in the histoplasma genome, we used agrobacterium tumefaciens-mediated transformation. this mutagen was selected because agrobacterium-mediated transfer of t-dna is efficient in producing random insertional mutations in histoplasma yeast cells. the majority of t-dna insertions are single integration events and thus the chance of secondary background mutations is minimized. other mutagens such as uv or chemical agents result in multiple changes to the genome, and while these background mutations can be removed by repeated backcrossing of mutants to wild type, no reliable techniques for crossing laboratory strains have been developed for histoplasma. additionally, insertional mutagens provide a molecular tag with known sequence which we can exploit in pcr-based screening for mutations in particular chromosomal loci using a t-dna specific primer in conjunction with a primer specific for the targeted gene. the molecular weight of the pcr amplicon provides an estimate of the distance from the gene specific primer to the t-dna insertion, and this distance can be used to determine whether the t-dna element disrupts the targeted gene. optimization of pool size for reliable detection of targeted mutations as the successful isolation of a mutant in a targeted gene depends critically on the ability to identify a positive individual among a much larger population, we determined the pcr detection limit for different pool sizes. histoplasma strain osu harbors a t-dna insertion in the ags gene in which the t-dna right border is oriented towards the 3' end of the ags gene. performance of pcr using a right border t-dna primer and an ags gene-specific primer produces a pcr amplicon of bp. to estimate the detection limit afforded by pcr in which a single strain could be found among a population of or mutants, ng of nucleic acid purified from osu were diluted 1: 1: and 1: with te buffer and pcr performed on these templates with rb and ags1- primers. with cycles, pcr could consistently detect the osu template when diluted as much as 1:. to better approximate the condition where the desired mutant would be present among a much larger population of other t-dna insertions, we mixed osu with a pool of random t-dna insertion mutants at a osu yeast-to-mutant pool ratio of 1: 1: and 1: nucleic acids were purified from each pool and pcr was performed as before with ng of total nucleic acid as templates. the positive bp amplicon was detected when osu was present in as little as 1/800th of the total population of yeast. a faint band representing the ags1::t-dna pcr product was observed when osu constituted 1/3200th of the template. no ags1::t-dna pcr product was detected in templates prepared from the mutant pool alone or wild-type histoplasma. a smaller pcr amplicon which is not specific to the ags1::t-dna template was detected in all reactions derived from the random insertion mutant pool. nested pcr to reduce false-positives to discriminate between true- and false-positive pcr products, we employed a secondary pcr reaction using a set of nested primers. nested primers that do not overlap with the primary pcr primers were designed for both the t-dna anchor and the ags gene. primary pcr reactions in which osu represented 1/200th or 1/800th of the population were used as templates after 1: 1:, and 1: dilution in h2o. as shown in figure 1c, this process eliminated the false-positive band observed in the primary pcr reactions. the ags1::t-dna specific amplicon could be detected after either 1: or 1: dilution of the primary pcr reaction. no ags1::t-dna amplicon was produced when osu was absent in the primary reaction template dna. these data demonstrate that pcr can be an efficient screening technique to probe mutant pools for a clone in which a t-dna element has inserted into a target gene. we selected a target pool size of approximately insertion mutants as a balance between increased throughput afforded by larger pools but easier subdivision of smaller pools into individual clones to recover the detected mutant strain. establishment of a bank of insertion mutants optimization of freezing conditions as the generation of t-dna insertion mutants in histoplasma is not trivial, establishment of a frozen bank of insertion mutants would facilitate future screens without having to produce new mutant pools as additional target genes are identified. maintaining the mutant representation in the pool after freezing necessitates efficient recovery of viable cells following thawing. to maximize the recovery of cells after freezing we examined two parameters: the cryoprotectant used and the method of freezing. glycerol- or dmso-containing solutions are used for freezing eukaryotic cells as these chemicals reduce membrane-damaging ice crystal formation. we also tested whether slowing the freezing rate using an insulated container also improved recovery from frozen stocks. histoplasma wu yeast cells were frozen and stored at -80°c for days or weeks to determine the short and long term storage recovery rates, respectively. recovered cfu counts were compared to those before freezing. with glycerol as the cryoprotectant, slowing the freezing rate dramatically improved recovery of viable yeast, probably resulting from the increased time to allow for penetration of glycerol into cells during cooling. dmso was a superior cryoprotectant than glycerol for histoplasma yeast when present at concentrations from 4% to 10%. freezing with dmso was less sensitive to the rate of freezing but as with glycerol, slower rates of cooling resulted in greater recovery of viable yeast cells. nearly 40% of the starting suspension of yeast cells were recovered when cells were slowly frozen in an 8% dmso-containing solution and this procedure was selected for long term storage of mutant pools. although specialized cooling apparatuses can be used to control the freezing rate, we found that simple placement of vials of cells within readily and cheaply obtained styrofoam containers was sufficient. generation of mutant pools insertion mutants were generated in the nam histoplasma strain wu by co-cultivation of agrobacterium tumefaciens and histoplasma yeast cells. co-cultures were plated onto filters and histoplasma transformants selected by transferring filters to medium containing hygromycin to which resistance is provided by sequences within the t-dna element. transformant yeast cells were collected and suspensions from individual plates combined to create pools derived from to independent mutant colonies. yeast cell suspensions were diluted into fresh medium and allowed to grow for 24- hours. twenty-four pools were prepared representing roughly insertion mutants. a portion of each culture was reserved for nucleic acid isolation and the remainder frozen in aliquots and stored at -80°c. nucleic acids were purified from each pool, diluted to ng/ul, and stored at -20°c until analysis by pcr. with an estimated 9000- genes encoded by the histoplasma genome, this collection does not represent the number of insertion mutants required for saturation of the genome. we used two probability functions to estimate the size of the library required for a 95% chance of isolating an insertion in a particular locus in the megabase nam genome. both calculations assume no bias in insertion sites. based on the number of predicted genes, the poisson approach estimates a library of approximately insertions would be required. the single study in which multiple alleles of a single locus were isolated in histoplasma supports the poisson calculation; five alleles would be the most probable number of alleles based on a or target estimate. however, the ags gene is an unusually large target and thus this number is probably an underestimate. an alternative calculation based solely on average gene size is provided by: p = 1-n where p is the probability of the t-dna inserting in a given target of size x in a genome of size g with n the total number of t-dna insertion mutants. assuming an average gene size of nucleotides, this calculation estimates a library of nearly mutants would be required for a 95% probability of obtaining at least one insertion mutant in any given gene. such a mutant bank would require pools with an average pool size of and pcr screening could be easily performed using three 96-well plates. although our current collection of mutants is inadequate for complete genome coverage, it was sufficient to demonstrate proof-of-concept through identification and recovery of a mutant at the cbp locus. isolation of a cbp insertion mutant detection of a t-dna insertion in cbp1 as no cbp mutant exists in the nam background despite numerous attempts with allelic replacement, we screened our nam mutant bank for t-dna insertions that disrupt the cbp gene. the cbp protein was the first virulence factor demonstrated for histoplasma through deletion of the encoding gene in a panama class strain of histoplasma. two cbp gene-specific primers were designed at the 3' end of the cbp coding region and were oriented towards the 5' end of the gene. as the t-dna element could insert with either the t-dna left border or the right border oriented towards the 3' end of the cbp gene, we screened each mutant pool by pcr with rb or with lb primers in combination with the cbp1- gene-specific primer. while pcr reactions with the lb + cbp1- primer set did not produce any positive pcr products with any of the templates, reactions with rb and cbp1- primers produced amplicons in two different pools. low abundance bands less than bp are likely primer dimers or residual rna from the template nucleic acids and were thus not considered. a nested pcr reaction was performed on the rb3-set of reactions. the pcr product from pool did not re-amplify in the nested pcr reaction suggesting that this product was a non-specific amplicon. alternatively, the pool may indeed harbor an insertion of t-dna sequence in the cbp locus but the t-dna element could be truncated and the nested rb primer-binding site lost resulting in failure to amplify in the nested pcr. the nested pcr reaction from pool produced a very prominent, approximately bp amplicon consistent with an insertion in the dna upstream of the cbp coding region. sequencing of this amplicon confirmed insertion of the t-dna in the cbp promoter and localized the insertion base pairs upstream of the cbp start codon. although a faint band was observed in the secondary pcr from pool # no product was detected in the primary reaction and it's abundance was much lower than that of the validated amplicon from pool # thus, it was discarded as a candidate. recovery of the cbp insertion mutant to isolate the strain containing the cbp1::t-dna mutation, we recovered yeast cells from pool # and segregated the pool into individual clones. the insertion was tracked using pcr with the primers described earlier. pool # was thawed and dilutions plated to recover individual cfu's. as each pool represents 100- clones, we screened clones to increase the likelihood of recovering at least one strain with the detected cbp insertion mutation. to drastically reduce the number of nucleic acid preparations and pcr tests required to screen nearly individuals, we employed an addressing scheme. each clone was picked into individual wells of three 96-well plates containing liquid medium. for each 96-well plate, wells from each row and from each column were pooled to produce yeast suspensions. an aliquot of these row and column sub-pools from each plate were combined to create a yeast suspension representing the clones from the entire 96-well plate. nucleic acids were isolated from the three 96-well plate suspensions and subjected to pcr. the cbp1::t-dna insertion amplicon was detected in two of the three collections of. for one of these pools of individual clones, nucleic acids were isolated from the corresponding row and column sub-pools and pcr was used to screen for the t-dna insertion. positive amplicons were detected in sub-pools representing the clone located at b. the suspension of yeast was recovered from well b and plated on solid medium to recover individual colonies. pcr performed on nucleic acids prepared from one of these colonies confirmed the identity and recovery of the desired insertion at the cbp locus. the strain with this insertion was designated osu to further characterize the t-dna insertion in osu we amplified and sequenced the dna flanking the t-dna element. pcr amplicons were produced for both the left and right border flanking regions using t-dna specific primers and cbp specific primers. alignment of the flanking regions with the histoplasma g217b genome and t-dna sequences showed truncation of the t-dna imperfect direct repeats by bp from the left border and bp from the right border. additionally, the t-dna insertion event deleted base pairs of the cbp promoter surrounding the site of insertion. due to t-dna-induced genetic rearrangements that can occur, pcr-product sizes should be used only as an initial estimate of the location of t-dna integration and the precise location of the insertion confirmed by sequencing the dna flanking the t-dna element. as our pcr screening method would not detect multiple t-dna integrations, we performed a southern blot using a t-dna-specific probe to determine how many t-dna elements were present in the osu mutagenized genome. as shown in figure 3d, only one band is detected indicating the osu strain harbors a single t-dna insertion. this kb t-dna probe-hybridizing fragment is the size predicted for the described insertion in the cbp promoter. no t-dna sequences were detected in the parental wu strain. validation of the cbp mutant since the t-dna insertion in osu did not lie within the cbp gene but was instead located in the sequence upstream of the cbp coding sequence, we tested whether the recovered mutant had lost the ability to produce the cbp protein. the t-dna insertion site lies within the region defined as the cbp promoter; transcriptional fusion constructs with less than base pairs of sequence upstream of cbp do not support transcription. thus, insertion of kb of foreign sequence into this region should disrupt promoter activity. osu and the parent wu strain were grown to early stationary phase and cell-free supernatants were prepared. to determine whether cbp production was impaired in osu we separated supernatant proteins by poly-acrylamide gel electrophoresis and visualized the proteins by silver staining. supernatants from the cbp wu strain had a prominent 9- kd protein which was not detected in supernatants harvested from the osu culture indicating the cbp1::t-dna insertion disrupts production of cbp protein. the identity of this protein was confirmed as cbp since supernatant from a strain in which cbp was independently depleted by rnai also specifically lacked this protein band. thus, while the t-dna insertion does not interrupt the coding region, insertion into the cbp promoter effectively prevents production of cbp in osu  we have developed a reverse genetics procedure employing random mutagenesis and pcr-based screening techniques to identify insertion mutants in a targeted gene in histoplasma capsulatum without regard to a mutant phenotype. since the mutagen creates a large insertion, the majority of mutations should reflect the knock-out mutant phenotype. however, insertions within the promoter of a gene may allow some residual transcription resulting in hypomorphic but not null phenotypes. in such cases, as demonstrated by our cbp1:t-dna mutant, delineation of the minimal promoter of a targeted gene could resolve what type of phenotype the insertion mutation would likely produce. thus, the regions most likely to produce mutant phenotypes are the proximal promoter and the coding region of the targeted gene. consequently, we routinely design our pcr screening primers at the 3' end of the gene to amplify these regions in particular and maximize the targeted site for insertions. this process, as with any mutational approach, will not successfully isolate insertion mutations in genes essential for viability, and if the intended target is suspect to be such, rnai may prove more beneficial. while integration of t-dna into the histoplasma genome appears relatively random, large scale studies in magnaporthe, leptosphaeria, and arabidopsis indicate there is a bias for insertion of the t-dna element into non-coding regions. in addition, occurrence of large-scale deletions or rearrangement mutations will be missed by this approach. thus, more insertion mutants may be required for saturation mutagenesis of the histoplasma genome than calculated above. the reverse genetics process detailed here increases the repertoire of methods available to disrupt gene functions in histoplasma capsulatum. since agrobacterium-mediated transformation has been developed as an efficient mutagen for a variety of fungal species, this procedure should be readily applicable to those microorganisms as well. for intractable fungal systems where homologous recombination is very limited or allelic replacement unfeasible, this process provides the ability to disrupt gene functions necessary for functional genetic tests. the only requirement is an efficient insertional mutagen. the increased capability to disrupt gene functions in histoplasma and in other fungi will greatly improve our mechanistic understanding of fungal biology. 
we describe a reverse genetics process that combines the random mutagenesis of agrobacterium-mediated transformation with screening techniques to identify targeted gene disruptions in a collection of insertion mutants. isolation of the desired mutant is accomplished by arraying individual clones from a pool and employing a pcr-addressing method. application of this procedure facilitated the isolation of a cbp mutant in a north american type strain, a histoplasma strain recalcitrant to gene knock-outs through homologous recombination. the procedures described are widely applicable to many fungal systems and will be of particular interest to those for which homologous recombination techniques are inefficient or do not currently exist.
 the dimorphic fungal pathogen histoplasma capsulatum causes respiratory and systemic disease in humans and other mammals. progress in understanding the mechanisms underlying the biology and the pathogenesis of histoplasma has been hindered by a shortage of methodologies for mutating a gene of interest.  we describe a reverse genetics process that combines the random mutagenesis of agrobacterium-mediated transformation with screening techniques to identify targeted gene disruptions in a collection of insertion mutants. isolation of the desired mutant is accomplished by arraying individual clones from a pool and employing a pcr-addressing method. application of this procedure facilitated the isolation of a cbp mutant in a north american type strain, a histoplasma strain recalcitrant to gene knock-outs through homologous recombination. optimization of cryopreservation conditions allows pools of mutants to be banked for later analysis and recovery of targeted mutants.  this methodology improves our ability to isolate mutants in targeted genes, thereby facilitating the molecular genetic analysis of histoplasma biology. the procedures described are widely applicable to many fungal systems and will be of particular interest to those for which homologous recombination techniques are inefficient or do not currently exist. 