BACKGROUND
porphyromonas gingivalis is an oral gram-negative, anaerobic, asaccharolytic and black-pigmented bacterium that is highly correlated with the development and progression of periodontal diseases and systemic comorbidities  <cit> .
the organism has been characterized as a ‘keystone’ pathogen whose interactions with other bacteria and the host are critical for the development of periodontitis  <cit> .
p. gingivalis utilizes multiple virulence factors, many of which have been identified and studied in vitro and in vivo such as proteinases , fimbriae, non-canonical lipopolysaccharide , capsular polysaccharide , and cytotoxic and hemolytic molecules  <cit> .
the identification of genes and proteins involved in pathogenesis has most commonly relied on analyzing genetic variations between strains or by directly isolating then genetically and biochemically characterizing specific mutants  <cit> .
in contrast, the identification of essential genes in this organism has lagged.
essential genes can be used as targets for antimicrobial drug development, and through bioinformatic and experimental study of essential gene, may reveal unique aspects of the physiology and metabolism of p. gingivalis.
high-throughput strategies to screen for genetic determinants of virulence and identify essential genes have been limited due to a paucity of tools for genetic manipulation  <cit> .
transposon mutagenesis has been used to identify genes involved in pathogenesis and other bacterial functions  <cit> .
the utility of transposon mutagenesis depends on the ability of the transposable element to insert randomly into different sites in the host genome in a one insertion per strain manner.
two previous transposon mutagenesis systems for p. gingivalis were based on tn <dig> and tn <dig> <cit> .
while the use of the mutant libraries generated with these systems led to important insights into p. gingivalis pathogenesis, both elements inserted preferentially into ‘hot-spots’ in the genome thus limiting the distribution of interrupted genes and were also limited to which strains could be mutagenized.
the lack of insertion saturation with these transposons into p. gingivalis genes resulted in libraries that were not suitable for the genome-wide identification of essential genes.
mariner-family transposons have been used to generate highly-saturated mutant libraries in numerous phylogenetically distinct bacterial species  <cit> .
mariner transposons preferentially insert into ‘ta’ nucleotide sequences, which are abundant throughout genomes; the p. gingivalis atcc  <dig> genome only has four ncbi annotated genes that lack a ta site, all of which are hypothetical proteins and are less than  <dig> amino acids in length  <cit> .
no other constraints or preferences for mariner transposon insertion have been identified.
recently, several investigators paired mutagenesis with mini-transposon derivatives of the himar  <dig> mariner transposon with massively-parallel sequencing technology in strategies variously named tn-seq, in-seq and hits  <cit> .
these strategies allow for quantitative assessment of individual mutants in a library by sequencing the transposon-genome junctions.
complex mariner transposon libraries, in some cases saturating, have been used to define essential genes of several bacterial species including bacteroides thetaiotaomicron, campylobacter jejuni, haemophilus influenzae, staphylococcus aureus and streptococcus pneumoniae <cit> .
data from these studies have been collated into a database of essential genes   <cit> .
comparison of essential genes between bacterial species included in the deg reveals that a large fraction of essential genes are species-specific.
p. gingivalis essential genes cannot simply be inferred from studies in other bacteria, and such studies in p. gingivalis have not been performed to date, although a ‘core’ genome has been described by comparing ten strains by dna/dna-hybridization using microarray technology  <cit> .
however, while there is likely to be overlap, a core genome does not equate with the set of genes essential for survival, and likely includes both essential and non-essential genes.
we have successfully adapted a mariner-based transposon mutagenesis system to create highly-saturated mutant libraries in p. gingivalis.
here we describe our construction and analysis of this mutant library to identify essential genes in p. gingivalis and compare these genes to those identified in other bacteria.
RESULTS
generation of the mutant library
we generated transposon insertion libraries in p. gingivalis using a himar  <dig> mariner mini-transposon system created for use in bacteroides thetaiotaomicron <cit> .
the b. thetaiotaomicron promoter of bt <dig> that drives expression of himar1c9a transposase is recognized by p. gingivalis, allowing us to use the b. thetaiotaomicron plasmid vector psam_bt with modifications in growth media and antibiotic selection conditions.
this mini-transposon is constructed with two translational terminators downstream of the gene for antibiotic selection, thus eliminating read-through downstream from the insertion.
we performed mutagenesis using psam_bt with p. gingivalis strain atcc  <dig>  the  <dig>  kb psam_bt vector containing the mariner mini-transposon cannot replicate in p. gingivalis and, in addition, the plasmid lacks sequence homology with the p. gingivalis genome.
therefore, after the plasmid enters p. gingivalis by transformation, transposition from the plasmid into the genome occurs without significant background insertion of the plasmid into the genome by illegitimate recombination.
this system allows for single, stable transposition events since transposase activity is lost along with the plasmid.
we collected  <dig>  transposon insertion strains  from six separate transformation experiments.
variable colony sizes were observed among the mutants harvested and pooled following  <dig> days of growth.
however, the majority of macroscopically visible colonies were similar in size to those of wild-type p. gingivalis strain atcc  <dig> after  <dig> days of growth.
to confirm that the strains contained transposon insertions and not cryptic or full plasmid integrations, we performed pcr specific for the transposon  as well as for two portions of the vector backbone  .
of  <dig> colonies that were screened, all showed positive pcr reactions for the transposon gene and negative reactions for the vector backbone, indicating ‘correct’ transposition.
‘incorrect’ transpositions can include portions of the vector backbone inserting with the transposon, the vector being stably maintained within the bacterium extra-chromosomally or multiple insertions within the same genome; such transposition events were not detected in the subset of mutants tested .
to determine whether the transposon inserted into different genes and not preferentially into genetic ‘hot-spots’, we performed nested semi-random pcr followed by sequencing which confirmed that insertions occurred in multiple locations across the genome   <cit> .
this traditional sequencing method is effective for targeted sequencing a subset of mutants from the mutant library if massively-parallel high-throughput are neither desired nor necessary.
validation of tn-seq of the p. gingivalis library
having confirmed via nested semi-random pcr and subsequent sequencing that the libraries contained different transposon insertions scattered throughout the genome, we identified the location of each insertion in the library by tn-seq analysis  <cit> .
this method couples transposon mutagenesis with massively-parallel, next-generation sequencing technology to identify the location of each insertion and quantitate the relative abundance of each insertion mutant in the library.
prior tn-seq experiments using mariner libraries have taken advantage of an engineered mmei restriction site that cuts 18– <dig> base pairs away from the recognition site into the genomic dna  <cit> .
this method has been successfully employed to evaluate library sequences in a variety of settings, however, it suffers from a number of disadvantages including: 1) yielding small sequencing reads limited to 16– <dig> nucleotides in length.
2) requiring the use of a mutant transposon and hence existing transposition vectors must be mutated.
3) mmei generates  <dig> base pair 3’ overhangs at adjacent sequences to which adapters are ligated.
it is possible that the enzyme cleaves these adjacent sequences with varying efficiency.
furthermore, t <dig> dna ligase is likely to join adapters to these varying overhangs with differential efficiency .
such variations in efficiencies, if they exist, would lead to unequal representation of sequenced insertions.
an alternate method for sequencing from junctions in transposon libraries involves the ligation of adapters to sequences near transposon junctions  <cit> .
however, the method is labor intensive, requires gel purification of ligated products, and is prone to the unintended creation of inhibitory adapter dimers and other side products.
here we report a new method, without the abovementioned disadvantages, for the construction of high-throughput sequencing libraries from transposon, retrovirus or repetitive element insertions sites in any genome.
for details see the materials and methods section.
in brief, genomic dna containing the insertion element of interest is sheared, and then terminal deoxynucleotidyl transferase  is used to add an average of twenty deoxycytidine nucleotides to the 3’ ends of all molecules.
two rounds of pcr using a poly-c-specific and an insertion element-specific primer pair are then used to amplify one of the two insertion element-genomic dna junctions and append all user-defined sequences needed for high-throughput sequencing and indexing.
this new method does not require a ligation reaction, does not produce adapter dimers, does not require gel purification and is compatible with long sequencing reads the size of which is only limited by the length of library fragments and the sequencing technology.
here, in contrast to the 16– <dig> nucleotide reads obtained with the mmei method we used  <dig> nucleotide reads allowing for significantly more effective and precise mapping of sequences to regions with nucleotide repeats as well as genes that contain nucleotide homology .
this is particularly important given that the current illumina hiseq <dig> base-calling algorithm gives poor quality scores for the first few bases .
two replicate samples derived from the same master mutant library, but processed separately for sequencing, were compared.
sequencing revealed  <dig>  and  <dig>  distinct insertions  respectively .
of the total insertions,  <dig>  and  <dig>  in the respective replicate runs were in putative intergenic regions.
after quality filtering sequencing reads an average  <dig> , <dig> reads could be attributed to an average of  <dig>  insertions mapped to the genome.
of note, during multiplexed illumina sequencing runs between 10– <dig> percent of sequencing reads are ‘thrown out’ during quality control analyses.
this level of ‘discarded’ read data is seen by all groups performing permutations of tn-seq, rna-seq, chip-seq and other massively-parallel adapted methods.
sequencing data removed during our quality control analyses was within the 10– <dig> percent range previously noted.
the number of insertions per gene and the number of reads per gene when comparing the technical replicates gave r <dig> values of  <dig>  and  <dig>  respectively .
the similarity between the two technical replicates demonstrates that aliquot production from the master library, processing of the samples as well as sequencing and analyses are highly reproducible.
identifying putative essential genes of p. gingivalis by tn-seq
the genome of p. gingivalis strain atcc  <dig> comprises  <dig>  mb of chromosomal dna and no plasmids.
with a gc-content of  <dig> %, there are  <dig>  genes,  <dig>  protein-coding sequences,  <dig> trna, and  <dig> rrna   <cit> .
an important factor for mariner transposition is that the genome contains  <dig>  informative ‘ta’ sites, the only known specific motif ‘required’ for mariner transposition.
in previous studies and in agreement with our sequencing results, approximately 98% of mariner insertions occur at ta sites .
the presence in our library of a mutant bacterium, in which a gene or intergenic region has been interrupted by insertion of the mariner transposon, would indicate that it is unlikely that the gene or region is essential for growth in vitro on blood agar plates, provided that the insertion was likely to have inactivated the function.
insertions into the first or last five percent of a gene have a higher likelihood of generating a functional gene product relative to insertions in the middle portions of a gene, therefore these mutants were eliminated from consideration.
in addition, we required a minimum of three sequencing ‘reads’ of the mutant locus be present in both technical replicates to exclude nonexistent insertions introduced by mapping of incorrectly sequenced reads, and lower rates may be due to mis-assignment by the reference assembly software.
by these criteria, we determined that  <dig>  genes are non-essential for growth in vitro.
sixteen of these genes contained  <dig> or greater insertions, notably the proteinases/adhesins kgp , rgpa , rgpb  and haga  as well as the minor fibrilin mfa <dig> and four of the twelve 23s rrna genes.
eighty-eight genes contained  <dig> or more insertions and  <dig> contained  <dig> or more insertions, with a median number of  <dig> insertions per gene.
there is a direct, but not completely exclusive, correlation between number of insertions and sequencing reads as  <dig> of the top  <dig> highest reads from the library belong to genes with more than  <dig> insertions; thus kgp, rgpa, rgpb and mfa <dig> are in the top ten most-read genes.
nine hundred and twenty genes had transposon insertions in at least 25% of their reported ta sites, while a remaining  <dig> genes had insertions in at least 10% of their reported ta sites.
the average number of ta sites per gene, when including all  <dig> genes , is  <dig>  a total of  <dig> genes were fully saturated with at least one mutant insertion into every available ta site in the gene.
full saturation results in a ta insertion ratio  of  <dig>  a ta site to insertion ratio of greater than  <dig> demonstrates that at low frequency mariner will insert into non-ta sites, most likely due to medium composition such as salt concentration that alleviate transposon specificity, local dna structure, nucleotide composition and/or dna-binding proteins.
of the  <dig> fully saturated genes having on average have  <dig> ta sites,  <dig> are present in multiple copies throughout the genome  and include rrna genes, ispg <dig>  ispg <dig>  gingipains, and hypothetical proteins .
all of the rrna genes  are located in spatially separated clusters of three and are fully saturated.
efforts are currently underway to determine whether there is conservation among non-ta insertion sites.
for the remaining putative essential genes we applied the following rules to identify those most likely to be essential for in vitro survival.
the rules are similar to those used in previous essential gene analyses of other bacteria and contend that  <cit> : 1) a gene must contain at least  <dig> ta sites.
genes with less sites could be under-inserted due to random chance.
of the  <dig> genes in the p. gingivalis atcc  <dig> genome with less than  <dig> ta sites,  <dig>  are annotated as hypothetical.
2) genes found to have an actual to theoretical insertion ratio of 50-fold or greater under-insertion were considered putatively essential .
applying these rules, out of a total  <dig>  genes in the atcc  <dig> genome , we identified  <dig>  genes as putatively essential for in vitro survival   .
twenty-two percent of a  <dig>  mb genome containing  <dig>  genes is within the range of essential genes determined by transposon mutagenesis, single gene deletions and in silico analyses of other bacterial genomes, as described below  <cit> .
prior to applying any cutoffs described above we found that  <dig> putative essential genes contained zero insertions.
given that these genes were a minimum of  <dig> base pairs in length and contained at least  <dig> ta sites the confidence level for concluding these as essential is high.
of the remaining  <dig> putative essential genes,  <dig> were found to have a ratio of between  <dig> - <dig> , 100-fold or less under-inserted, and  <dig> had a ratio between  <dig> - <dig> , 50-fold or less under-inserted.
in most cases these genes had a single insertion over a gene length of  <dig> - <dig>  kb.
fifty genes had ratios between  <dig> - <dig> , however, these insertions were found to fall under the constraints outlined above and also met our qualifications for putative essentiality as well.
of note, of these  <dig> genes the majority  have homology to genes of other bacteria identified in previous essential gene studies  <cit> .
in addition to identifying the essential nature of a gene, more detailed analysis, specifically mapping domains of proteins and intergenic regions, can provide valuable information about protein functional domains, promoter regions, mis-annotations, operon structure and regulatory rnas .
simply mapping the insertions onto the genome to view saturation of specific genes provides a qualitative understanding of library complexity .
annotations of genomes identify gene/coding-sequence start and stop codons, spatial relationships to other genes, operon structure, number of possible amino acids and amino acid composition.
such bioinformatic analyses are not perfect because they are based on coding-sequences from model organisms, e.g.
escherichia coli, and not adapted to less well-known bacterial species.
detailed insertion mapping allows for the determination of essential genes on a visual scale .
in addition, transposon mutagenesis mapping may reveal previously mis-annotated start and stop sites for genes as well as putative internal start sites, providing information on potential operon structure.
furthermore, essentiality of function domains can be determined .
although intergenic regions are far less abundant in prokaryotic genomes, mapping of insertions, or a lack thereof, to a specific intergenic region within the genome can provide insights on regulatory features within non-coding dna sequences.
comparison of p. gingivalis essential genes to core genome and transcriptome
the core genome of p. gingivalis previously proposed by brunner et al.
was derived from hybridization analysis of  <dig> different strains to a dna microarray of annotated genes from strain w <dig>  <cit> .
of note, any gene ‘missing’ from strain w <dig>  even if present in all other strains of p. gingivalis, would be considered not part of the ‘core’.
since both strains w <dig> and atcc  <dig> are now fully sequenced, it is known that  <dig> genes in the p. gingivalis atcc  <dig> essential list are missing in w <dig>  five of these genes have been identified in a third sequenced and annotated strain, tdc <dig>  there was nearly complete overlap between putative essential genes determined by tn-seq with the p. gingivalis core genome,  <dig> of  <dig>  putatively essential genes overlapped   <cit> .
also, several gene probes were left out of the core genome analysis due to low hybridization signals or redundancy; two of these are identified as essential in our study.
nearly half  of the essential genes not found in the p. gingivalis core genome had blast matches in the database of essential genes   <cit> .
the remaining small difference may be explained by the hypothesis that certain essential genes are strain- and not species-specific, and thus may not be identified in a core genome analysis.
in the circular genome representation of the base genome p. gingivalis atcc  <dig>  essential genes are depicted in arrows denoting directionality  and homologous coding sequences are shown as tick marks in strains w <dig> , w <dig>  and tdc <dig> .
the map also shows that areas of genetic aberrance between p. gingivalis strains are areas devoid of essential genes .
this would be hypothesized as essential genes should be conserved throughout a species unless duplication or gain-of-function mutation occur that can rescue the essential role of a give gene.
as more p. gingivalis strain genomes are sequenced, bioinformatic analyses that provide mapped read-outs will delineate putative essential, core and accessory genetic regions, thus giving insight into strain-based differences within the species.
such differences may be useful to identify strain phylogeny and aid in clinical treatment regimens based on knowledge of genotype-to-phenotype virulence attributes .
chen et al.
performed rna-seq analysis of mrna expression by p. gingivalis strain w <dig> from which  <dig> of the possible  <dig> atcc  <dig> essential genes were assayed  <cit> .
this analysis demonstrated that  <dig> of  <dig> p. gingivalis atcc  <dig> essential genes were expressed during growth on blood agar medium .
the  <dig> genes not expressed on blood agar plates as determined by rna-seq are annotated as ‘hypothetical’ proteins.
transcriptome analyses were also performed on p. gingivalis grown on minimal , tryptic soy  and blood agar  media, however, no essential genes were expressed solely on ba and not tsb or min despite some differences in levels of expression between the three media.
comparison of p. gingivalis essential genes with other essential gene analyses
of the  <dig> putative essential genes in p. gingivalis,  <dig>  have known essential gene homologues determined by blastp interrogation of the deg , version  <dig> , updated on november  <dig>   <dig>   <cit> .
the deg curates a searchable list of “essential genes  are those indispensable for the survival of an organism, and therefore are considered a foundation of life”.
p. gingivalis essential genes were determined to have deg homologues based strictly on blastp similarity.
blastp similarities that resulted in e-values of 1x10- <dig> or less were considered matches.
homologies were found in at least one of the following species which had previously undergone essential gene studies: bacillus subtilis, b. thetaiotaomicron, e. coli, francisella novicida, haemophilus influenzae, helicobacter pylori, mycobacterium tuberculosis, mycoplasma genitalium, mycoplasma pulminous, saccharomyces cerevisiae, salmonella typhimurium, staphylococcus aureus, streptococcus pneumoniae and vibrio cholerae <cit> .
for more than half of the  <dig> blast-matching essential genes there was homology within two or more species.
in cases where only one other species contained a blastp match to a p. gingivalis essential gene it was most frequently to a gene in b. thetaiotaomicron, h. influenzae or h. pylori, which are the most closely related species to p. gingivalis both based on phylogeny and ecology.
the remaining  <dig> % of putative essential genes that have no known homologue in the deg may be essential in a species-specific or niche-specific manner.
these  <dig> genes, many of which are functionally classified as containing known pfam protein motifs, ‘conserved domains’ or ‘hypothetical’ proteins, may reveal important aspects related to metabolism and physiology of porphyromonas species and closely related organisms.
of the  <dig> annotated as hypothetical proteins,  <dig> are among the  <dig> p. gingivalis essential genes not previously known to be essential from other studies.
of the organisms for which an essential gene set has been identified, h. influenzae, f. tularensis, acinetobacter, m. tuberculosis, salmonella typhimurium, s. aureus and b. thetaiotaomicron are the most relevant based on genome size, ecological niche and genetic relatedness to p. gingivalis.
the determined essential genes of the above species were  <dig>  genes with  <dig> essential ;  <dig>  genes with  <dig> essential ;  <dig>  genes with  <dig> essential ;  <dig>  genes with  <dig> essential ;  <dig>  genes with  <dig> essential ;  <dig>  genes with  <dig> ; and  <dig>  genes with  <dig> , respectively  <cit> .
p. gingivalis is a member of the bacteroidetes, and before reclassification was known as b. gingivalis.
there are no bacteroidetes species or other anaerobes represented in the deg, however, a putative list of b. thetaiotaomicron strain vpi- <dig> essential genes is available from the supplemental material of goodman et al.
<dig>  <cit> .
b. thetaiotaomicron strain vpi- <dig> was originally isolated from human feces.
the strain contains a  <dig>  mb chromosome and  <dig>  mb plasmid  with  <dig>  genes  and  <dig> ,  <dig>  protein coding sequences  and  <dig> ,  <dig> trna and  <dig> rrna genes  <cit>  .
in comparison, p. gingivalis atcc  <dig> has 43%  of protein coding sequences in a genome 37% of the size of that of b. thetaiotaomicron vpi- <dig>  it was estimated that b. thetaiotaomicron vpi- <dig> contains  <dig> “candidate essential genes”  <cit> .
maintaining a larger genome and gene set provides more opportunities for functional redundancy and alternative pathways which can lead to a relatively smaller number of essential genes.
thus,  <dig> of  <dig>  of b. thetaiotaomicron ‘essentials’ have blast homologues in p. gingivalis strain atcc  <dig> and of these, 78%  are also essential in both organisms .
fifty-nine b. thetaiotaomicron blast matches are not essential in p. gingivalis and  <dig> have no blast match at all in the organism .
a significant number of the shared essential genes  are not characterized in the deg  and of these  <dig> bacteroidetes-specific essentials, three are annotated as permeases and two appear to be regulatory.
three essentials, pgn_ <dig>  pgn_ <dig> and pgn_ <dig>  are likely associated with capsular polysaccharide biosynthesis based on pgn_ <dig> and pgn_ <dig> being involved in the dolichol pathway and pgn_ <dig> functionally annotated as polysaccharide biosynthesis related.
parsing out essential genes of specific groups of species, in this case bacteroidetes and/or anaerobes, can allow for specific drug targeting or directed nutrient supplementation.
in agreement with multiple previous studies on essential genes of bacteria, in p. gingivalis a significantly greater number of essential genes  are found on the negative dna strand, and  <dig>  are found on the positive dna strand   <cit> .
similarly, there is a greater than expected proportion of enzymes, especially those within multiple functions or involved in multiple pathways, within the essential gene groups  <cit> .
using the cluster of orthologous groups  functional class designations , we identified significant enrichment of essential genes within groups ‘d’ , ‘i’  and ‘j’ ; and a lack of enrichment was seen in ‘s’ , ‘p’  and ‘n’    <cit> .
enrichment  of essential genes in these categories has been reported previously, however, essential gene enrichment in specific cog categories appears to be a species-specific characteristic.
based on operon prediction and known essentials contained in the deg it was determined that  <dig> of the  <dig> putative essential genes of p. gingivalis identified by tn-seq may be the result of polar effects of the transposon insertion on downstream essential genes .
specifically, these  <dig> genes were identified as being upstream and potentially in an operon with one or more known essential genes, and additionally do not have blast matches in the deg.
further study of each of these genes would be required to confirm their essentiality.
bringing the deg and p. gingivalis core genome together in relation to p. gingivalis gene essentiality, we have determined that  <dig> genes within the core genome, ones not identified as essential in our study, have blast matches to genes within the deg .
within our mutant libraries we were able to identify transposon insertions into these genes such that they do not qualify as essential in p. gingivalis.
reasons for these genes being identified as essential in other species could be due to multiple variables such as in vitro selection media, species-specific essentiality, transposon type, library complexity, sequencing method, and criteria for essentiality.
such information gives importance to the distinction between a core gene set and an essential gene set as well as possible limitations of essential gene analyses based solely on in silico methodology.
characterization of p. gingivalis essential genes
metabolic pathways that lack redundancy or have critical functions have been identified previously through essential gene studies.
in our analysis of p. gingivalis we noted the presence of entire pathways as well as specific parts of pathways that are essential to p. gingivalis and to all other bacterial species.
a subset of p. gingivalis-specific essential genes, possibly related to the ecological niche of the species, have also been identified .
of pathways involved in ribosome function we identified the rpsa, rpma, rplb and rimp systems, which encode for 30s, 50s and maturation of ribosomes, respectively.
the three major protein translation regulatory pathways of infb, tsf and prfa, as well as translational machinery pathway rpoa were found to be essential in our study.
dna replication, recombination and repair pathways of dnaa and ruva as well as cell division pathways mreb, para and ftsa were also found to be essential.
multiple pathways involved in lps, cps, fatty acid and murein biosynthesis, including lpxa, rmla, mana, fabd and mura were also judged to be essential, as well as genes involved in secretion and chaperone pathways such as secd, groes/el and sura.
pathways involving nrfa, etfa, sufb, nadd and ribe associated with oxidation-reduction reactions, were found to be essential in p. gingivalis, which is not surprising for an anaerobic bacterium.
major metabolic pathways pura, pyrb, coaa, accb, pdxa, ispa, thif, sera and dapa, which encode nucleotide, amino acid and co-factor building blocks, respectively, were determined essential under our in vitro conditions.
all of the aforementioned systems and pathways have previously been identified as essential for in vitro growth of other bacterial species, which is not unexpected given that replication, transcription, translation, cell division, membrane stability and central metabolism are key to survival  <cit> .
hypothetical genes are an often-overlooked group within any bacterial genome, including those of ‘model’ organisms.
in our study we determined that approximately one-tenth of the essential genome of p. gingivalis encoded ‘hypothetical’ proteins, a few of which were homologous to other hypotheticals contained within the deg.
the majority of essential hypothetical genes are large and not within operons, suggesting that they encode functional proteins and are not essential due to a polar effect on a downstream essential gene.
the finding that certain hypothetical proteins are essential will stimulate the search for protein motifs, structural bioinformatic and spatial organizational data and studies to define their function.
although the notion that an essential gene within a given strain is likely to be essential in the species as a whole, intraspecies differences are known and often result in different phenotypes.
for example, in strain atcc  <dig> we found no insertions into raga and thus this gene was considered essential.
previous investigators also had difficulty making directed knockouts of raga in strain w50; however, these investigators were successful in deleting raga from strain wph <dig>  it is possible that raga is only essential within specific strains and those strains in which it is non-essential compensate for loss of its function through the presence of other genes.
limitations of essential gene analysis
limitations to essential gene studies should be addressed regardless of systems and methods utilized for their identification.
first, several studies have relied exclusively on in silico bioinformatic analyses to determine essentiality.
these analyses were based on programs designed to combine information from previous in vitro and in vivo mutagenesis studies with genome annotation and composition scripts without having carried out actual mutagenesis studies.
thus, any limitations of these experimental studies will be carried over into the new analyses and magnified by any inaccuracies of the program design itself.
second, in insertional mutagenesis methods to determine gene essentiality, genes may be misidentified as essential due to transposon insertion ‘cold-spots’.
there is no ideal transposon identified as yet that completely lacks any nucleotide specificity and which can create completely random and saturating mutant libraries.
thus, no matter what type of transposon is used, tn <dig>  tn <dig>  tn <dig>  a cryptic construct or mariner, all studies will have regions of the genome where fewer insertions occur.
third, genes that are actually non-essential but when mutated cause severe growth defects may be scored as essential due to practical limits to the depth of sequencing of transposon insertion junctions.
these ‘sick’ mutants could potentially be represented at levels below 1000-fold a neutral mutant due to the number of replications it could go through prior to being pooled from mutagenesis plates into the library.
fourth, non-essential genes immediately upstream of and co-transcribed with essentials may be incorrectly scored as essential due to polarity of the transposon insertion.
last, practical limits to library complexity can result in some genes that fail to get disrupted by the transposon and so are misidentified as essential.
this is particularly a problem for small genes or genes that are within cold spots for the transposon.
several studies, based mostly on the genome size of the species under investigation and the type of transposon, have attained different levels of saturation prior to analyses for essential genes.
the possible limitation of our library when combining the type of transposon and library complexity relates to genes that contain less than  <dig> ta sites in their coding-sequences.
of the  <dig> genes with fewer than  <dig> ta sites,  <dig> could potentially be scored as essential based on having zero insertions, but do not qualify, given our stringent criteria .
adding confidence to the notion that many of these are non-essential is that  <dig> of the  <dig> genes encode proteins of less than  <dig> amino acids in length.
since these are all characterized as ‘hypothetical’ and are rather short to encode functional proteins, we believe that some of these may simply be artifacts of annotation programs and thus not true protein-coding genes.
even complete gene deletion, non-transposon based studies of essential genes have limitations.
the keio collection of single and double gene deletions in escherichia coli is considered the most comprehensive essential gene study to date  <cit> .
genes that could not be deleted were scored as essential, however, failure to delete a gene is not a guarantee of essentiality and there a are few genes identified as essential in the keio collection that were successfully deleted by other labs.
furthermore, a handful of genes labeled non-essential were actually essential.
the keio deletions of those genes have second site suppressor mutations that compensate for the loss of the essential gene.
the best understanding of essential genes is likely to come from combining different modalities to confirm their essential nature and comparison of these databases both within and between species.
CONCLUSIONS
we have described a method for performing tn-seq with p. gingivalis using a mariner mini-transposon and illumina platform next-generation sequencing.
we have also invented a new method for creating sequencing libraries from bacterial transposon libraries that has many advantages compared to previous methodologies.
using that method we identified specific mutants quantitatively in a highly reproducible manner using massively-parallel sequencing techniques.
we used a near saturating insertion library generated in p. gingivalis strain atcc  <dig> to define the set of genes essential for growth on blood agar.
both the availability of a p. gingivalis mutant library and the ability to screen quantitatively are a marked advance in genetic and molecular tools for future studies of p. gingivalis biology and pathogenesis.
by applying different selective pressures to the library, it is now possible to identify genes critical for survival and growth under different conditions.
due to the quantitative nature of results provided by tn-seq, both positive and negative gene effects, including partial phenotypes, can now be readily identified.

 porphyromonas gingivalis is an oral gram-negative, anaerobic, asaccharolytic and black-pigmented bacterium that is highly correlated with the development and progression of periodontal diseases and systemic comorbidities. the organism has been characterized as a ‘keystone’ pathogen whose interactions with other bacteria and the host are critical for the development of periodontitis. p. gingivalis utilizes multiple virulence factors, many of which have been identified and studied in vitro and in vivo such as proteinases, fimbriae, non-canonical lipopolysaccharide, capsular polysaccharide, and cytotoxic and hemolytic molecules. the identification of genes and proteins involved in pathogenesis has most commonly relied on analyzing genetic variations between strains or by directly isolating then genetically and biochemically characterizing specific mutants. in contrast, the identification of essential genes in this organism has lagged. essential genes can be used as targets for antimicrobial drug development, and through bioinformatic and experimental study of essential gene, may reveal unique aspects of the physiology and metabolism of p. gingivalis. high-throughput strategies to screen for genetic determinants of virulence and identify essential genes have been limited due to a paucity of tools for genetic manipulation. transposon mutagenesis has been used to identify genes involved in pathogenesis and other bacterial functions. the utility of transposon mutagenesis depends on the ability of the transposable element to insert randomly into different sites in the host genome in a one insertion per strain manner. two previous transposon mutagenesis systems for p. gingivalis were based on tn and tn. while the use of the mutant libraries generated with these systems led to important insights into p. gingivalis pathogenesis, both elements inserted preferentially into ‘hot-spots’ in the genome thus limiting the distribution of interrupted genes and were also limited to which strains could be mutagenized. the lack of insertion saturation with these transposons into p. gingivalis genes resulted in libraries that were not suitable for the genome-wide identification of essential genes. mariner-family transposons have been used to generate highly-saturated mutant libraries in numerous phylogenetically distinct bacterial species. mariner transposons preferentially insert into ‘ta’ nucleotide sequences, which are abundant throughout genomes; the p. gingivalis atcc genome only has four ncbi annotated genes that lack a ta site, all of which are hypothetical proteins and are less than amino acids in length. no other constraints or preferences for mariner transposon insertion have been identified. recently, several investigators paired mutagenesis with mini-transposon derivatives of the himar mariner transposon with massively-parallel sequencing technology in strategies variously named tn-seq, in-seq and hits. these strategies allow for quantitative assessment of individual mutants in a library by sequencing the transposon-genome junctions. complex mariner transposon libraries, in some cases saturating, have been used to define essential genes of several bacterial species including bacteroides thetaiotaomicron, campylobacter jejuni, haemophilus influenzae, staphylococcus aureus and streptococcus pneumoniae. data from these studies have been collated into a database of essential genes. comparison of essential genes between bacterial species included in the deg reveals that a large fraction of essential genes are species-specific. p. gingivalis essential genes cannot simply be inferred from studies in other bacteria, and such studies in p. gingivalis have not been performed to date, although a ‘core’ genome has been described by comparing ten strains by dna/dna-hybridization using microarray technology. however, while there is likely to be overlap, a core genome does not equate with the set of genes essential for survival, and likely includes both essential and non-essential genes. we have successfully adapted a mariner-based transposon mutagenesis system to create highly-saturated mutant libraries in p. gingivalis. here we describe our construction and analysis of this mutant library to identify essential genes in p. gingivalis and compare these genes to those identified in other bacteria.  generation of the mutant library we generated transposon insertion libraries in p. gingivalis using a himar mariner mini-transposon system created for use in bacteroides thetaiotaomicron. the b. thetaiotaomicron promoter of bt that drives expression of himar1c9a transposase is recognized by p. gingivalis, allowing us to use the b. thetaiotaomicron plasmid vector psam_bt with modifications in growth media and antibiotic selection conditions. this mini-transposon is constructed with two translational terminators downstream of the gene for antibiotic selection, thus eliminating read-through downstream from the insertion. we performed mutagenesis using psam_bt with p. gingivalis strain atcc the kb psam_bt vector containing the mariner mini-transposon cannot replicate in p. gingivalis and, in addition, the plasmid lacks sequence homology with the p. gingivalis genome. therefore, after the plasmid enters p. gingivalis by transformation, transposition from the plasmid into the genome occurs without significant background insertion of the plasmid into the genome by illegitimate recombination. this system allows for single, stable transposition events since transposase activity is lost along with the plasmid. we collected transposon insertion strains from six separate transformation experiments. variable colony sizes were observed among the mutants harvested and pooled following days of growth. however, the majority of macroscopically visible colonies were similar in size to those of wild-type p. gingivalis strain atcc after days of growth. to confirm that the strains contained transposon insertions and not cryptic or full plasmid integrations, we performed pcr specific for the transposon as well as for two portions of the vector backbone. of colonies that were screened, all showed positive pcr reactions for the transposon gene and negative reactions for the vector backbone, indicating ‘correct’ transposition. ‘incorrect’ transpositions can include portions of the vector backbone inserting with the transposon, the vector being stably maintained within the bacterium extra-chromosomally or multiple insertions within the same genome; such transposition events were not detected in the subset of mutants tested. to determine whether the transposon inserted into different genes and not preferentially into genetic ‘hot-spots’, we performed nested semi-random pcr followed by sequencing which confirmed that insertions occurred in multiple locations across the genome. this traditional sequencing method is effective for targeted sequencing a subset of mutants from the mutant library if massively-parallel high-throughput are neither desired nor necessary. validation of tn-seq of the p. gingivalis library having confirmed via nested semi-random pcr and subsequent sequencing that the libraries contained different transposon insertions scattered throughout the genome, we identified the location of each insertion in the library by tn-seq analysis. this method couples transposon mutagenesis with massively-parallel, next-generation sequencing technology to identify the location of each insertion and quantitate the relative abundance of each insertion mutant in the library. prior tn-seq experiments using mariner libraries have taken advantage of an engineered mmei restriction site that cuts 18– base pairs away from the recognition site into the genomic dna. this method has been successfully employed to evaluate library sequences in a variety of settings, however, it suffers from a number of disadvantages including: 1) yielding small sequencing reads limited to 16– nucleotides in length. 2) requiring the use of a mutant transposon and hence existing transposition vectors must be mutated. 3) mmei generates base pair 3’ overhangs at adjacent sequences to which adapters are ligated. it is possible that the enzyme cleaves these adjacent sequences with varying efficiency. furthermore, t dna ligase is likely to join adapters to these varying overhangs with differential efficiency. such variations in efficiencies, if they exist, would lead to unequal representation of sequenced insertions. an alternate method for sequencing from junctions in transposon libraries involves the ligation of adapters to sequences near transposon junctions. however, the method is labor intensive, requires gel purification of ligated products, and is prone to the unintended creation of inhibitory adapter dimers and other side products. here we report a new method, without the abovementioned disadvantages, for the construction of high-throughput sequencing libraries from transposon, retrovirus or repetitive element insertions sites in any genome. for details see the materials and methods section. in brief, genomic dna containing the insertion element of interest is sheared, and then terminal deoxynucleotidyl transferase is used to add an average of twenty deoxycytidine nucleotides to the 3’ ends of all molecules. two rounds of pcr using a poly-c-specific and an insertion element-specific primer pair are then used to amplify one of the two insertion element-genomic dna junctions and append all user-defined sequences needed for high-throughput sequencing and indexing. this new method does not require a ligation reaction, does not produce adapter dimers, does not require gel purification and is compatible with long sequencing reads the size of which is only limited by the length of library fragments and the sequencing technology. here, in contrast to the 16– nucleotide reads obtained with the mmei method we used nucleotide reads allowing for significantly more effective and precise mapping of sequences to regions with nucleotide repeats as well as genes that contain nucleotide homology. this is particularly important given that the current illumina hiseq base-calling algorithm gives poor quality scores for the first few bases. two replicate samples derived from the same master mutant library, but processed separately for sequencing, were compared. sequencing revealed and distinct insertions respectively. of the total insertions, and in the respective replicate runs were in putative intergenic regions. after quality filtering sequencing reads an average, reads could be attributed to an average of insertions mapped to the genome. of note, during multiplexed illumina sequencing runs between 10– percent of sequencing reads are ‘thrown out’ during quality control analyses. this level of ‘discarded’ read data is seen by all groups performing permutations of tn-seq, rna-seq, chip-seq and other massively-parallel adapted methods. sequencing data removed during our quality control analyses was within the 10– percent range previously noted. the number of insertions per gene and the number of reads per gene when comparing the technical replicates gave r values of and respectively. the similarity between the two technical replicates demonstrates that aliquot production from the master library, processing of the samples as well as sequencing and analyses are highly reproducible. identifying putative essential genes of p. gingivalis by tn-seq the genome of p. gingivalis strain atcc comprises mb of chromosomal dna and no plasmids. with a gc-content of %, there are genes, protein-coding sequences, trna, and rrna. an important factor for mariner transposition is that the genome contains informative ‘ta’ sites, the only known specific motif ‘required’ for mariner transposition. in previous studies and in agreement with our sequencing results, approximately 98% of mariner insertions occur at ta sites. the presence in our library of a mutant bacterium, in which a gene or intergenic region has been interrupted by insertion of the mariner transposon, would indicate that it is unlikely that the gene or region is essential for growth in vitro on blood agar plates, provided that the insertion was likely to have inactivated the function. insertions into the first or last five percent of a gene have a higher likelihood of generating a functional gene product relative to insertions in the middle portions of a gene, therefore these mutants were eliminated from consideration. in addition, we required a minimum of three sequencing ‘reads’ of the mutant locus be present in both technical replicates to exclude nonexistent insertions introduced by mapping of incorrectly sequenced reads, and lower rates may be due to mis-assignment by the reference assembly software. by these criteria, we determined that genes are non-essential for growth in vitro. sixteen of these genes contained or greater insertions, notably the proteinases/adhesins kgp, rgpa, rgpb and haga as well as the minor fibrilin mfa and four of the twelve 23s rrna genes. eighty-eight genes contained or more insertions and contained or more insertions, with a median number of insertions per gene. there is a direct, but not completely exclusive, correlation between number of insertions and sequencing reads as of the top highest reads from the library belong to genes with more than insertions; thus kgp, rgpa, rgpb and mfa are in the top ten most-read genes. nine hundred and twenty genes had transposon insertions in at least 25% of their reported ta sites, while a remaining genes had insertions in at least 10% of their reported ta sites. the average number of ta sites per gene, when including all genes, is a total of genes were fully saturated with at least one mutant insertion into every available ta site in the gene. full saturation results in a ta insertion ratio of a ta site to insertion ratio of greater than demonstrates that at low frequency mariner will insert into non-ta sites, most likely due to medium composition such as salt concentration that alleviate transposon specificity, local dna structure, nucleotide composition and/or dna-binding proteins. of the fully saturated genes having on average have ta sites, are present in multiple copies throughout the genome and include rrna genes, ispg ispg gingipains, and hypothetical proteins. all of the rrna genes are located in spatially separated clusters of three and are fully saturated. efforts are currently underway to determine whether there is conservation among non-ta insertion sites. for the remaining putative essential genes we applied the following rules to identify those most likely to be essential for in vitro survival. the rules are similar to those used in previous essential gene analyses of other bacteria and contend that: 1) a gene must contain at least ta sites. genes with less sites could be under-inserted due to random chance. of the genes in the p. gingivalis atcc genome with less than ta sites, are annotated as hypothetical. 2) genes found to have an actual to theoretical insertion ratio of 50-fold or greater under-insertion were considered putatively essential. applying these rules, out of a total genes in the atcc genome, we identified genes as putatively essential for in vitro survival. twenty-two percent of a mb genome containing genes is within the range of essential genes determined by transposon mutagenesis, single gene deletions and in silico analyses of other bacterial genomes, as described below. prior to applying any cutoffs described above we found that putative essential genes contained zero insertions. given that these genes were a minimum of base pairs in length and contained at least ta sites the confidence level for concluding these as essential is high. of the remaining putative essential genes, were found to have a ratio of between -, 100-fold or less under-inserted, and had a ratio between -, 50-fold or less under-inserted. in most cases these genes had a single insertion over a gene length of - kb. fifty genes had ratios between -, however, these insertions were found to fall under the constraints outlined above and also met our qualifications for putative essentiality as well. of note, of these genes the majority have homology to genes of other bacteria identified in previous essential gene studies. in addition to identifying the essential nature of a gene, more detailed analysis, specifically mapping domains of proteins and intergenic regions, can provide valuable information about protein functional domains, promoter regions, mis-annotations, operon structure and regulatory rnas. simply mapping the insertions onto the genome to view saturation of specific genes provides a qualitative understanding of library complexity. annotations of genomes identify gene/coding-sequence start and stop codons, spatial relationships to other genes, operon structure, number of possible amino acids and amino acid composition. such bioinformatic analyses are not perfect because they are based on coding-sequences from model organisms, e.g. escherichia coli, and not adapted to less well-known bacterial species. detailed insertion mapping allows for the determination of essential genes on a visual scale. in addition, transposon mutagenesis mapping may reveal previously mis-annotated start and stop sites for genes as well as putative internal start sites, providing information on potential operon structure. furthermore, essentiality of function domains can be determined. although intergenic regions are far less abundant in prokaryotic genomes, mapping of insertions, or a lack thereof, to a specific intergenic region within the genome can provide insights on regulatory features within non-coding dna sequences. comparison of p. gingivalis essential genes to core genome and transcriptome the core genome of p. gingivalis previously proposed by brunner et al. was derived from hybridization analysis of different strains to a dna microarray of annotated genes from strain w. of note, any gene ‘missing’ from strain w even if present in all other strains of p. gingivalis, would be considered not part of the ‘core’. since both strains w and atcc are now fully sequenced, it is known that genes in the p. gingivalis atcc essential list are missing in w five of these genes have been identified in a third sequenced and annotated strain, tdc there was nearly complete overlap between putative essential genes determined by tn-seq with the p. gingivalis core genome, of putatively essential genes overlapped. also, several gene probes were left out of the core genome analysis due to low hybridization signals or redundancy; two of these are identified as essential in our study. nearly half of the essential genes not found in the p. gingivalis core genome had blast matches in the database of essential genes. the remaining small difference may be explained by the hypothesis that certain essential genes are strain- and not species-specific, and thus may not be identified in a core genome analysis. in the circular genome representation of the base genome p. gingivalis atcc essential genes are depicted in arrows denoting directionality and homologous coding sequences are shown as tick marks in strains w, w and tdc. the map also shows that areas of genetic aberrance between p. gingivalis strains are areas devoid of essential genes. this would be hypothesized as essential genes should be conserved throughout a species unless duplication or gain-of-function mutation occur that can rescue the essential role of a give gene. as more p. gingivalis strain genomes are sequenced, bioinformatic analyses that provide mapped read-outs will delineate putative essential, core and accessory genetic regions, thus giving insight into strain-based differences within the species. such differences may be useful to identify strain phylogeny and aid in clinical treatment regimens based on knowledge of genotype-to-phenotype virulence attributes. chen et al. performed rna-seq analysis of mrna expression by p. gingivalis strain w from which of the possible atcc essential genes were assayed. this analysis demonstrated that of p. gingivalis atcc essential genes were expressed during growth on blood agar medium. the genes not expressed on blood agar plates as determined by rna-seq are annotated as ‘hypothetical’ proteins. transcriptome analyses were also performed on p. gingivalis grown on minimal, tryptic soy and blood agar media, however, no essential genes were expressed solely on ba and not tsb or min despite some differences in levels of expression between the three media. comparison of p. gingivalis essential genes with other essential gene analyses of the putative essential genes in p. gingivalis, have known essential gene homologues determined by blastp interrogation of the deg, version, updated on november. the deg curates a searchable list of “essential genes are those indispensable for the survival of an organism, and therefore are considered a foundation of life”. p. gingivalis essential genes were determined to have deg homologues based strictly on blastp similarity. blastp similarities that resulted in e-values of 1x10- or less were considered matches. homologies were found in at least one of the following species which had previously undergone essential gene studies: bacillus subtilis, b. thetaiotaomicron, e. coli, francisella novicida, haemophilus influenzae, helicobacter pylori, mycobacterium tuberculosis, mycoplasma genitalium, mycoplasma pulminous, saccharomyces cerevisiae, salmonella typhimurium, staphylococcus aureus, streptococcus pneumoniae and vibrio cholerae. for more than half of the blast-matching essential genes there was homology within two or more species. in cases where only one other species contained a blastp match to a p. gingivalis essential gene it was most frequently to a gene in b. thetaiotaomicron, h. influenzae or h. pylori, which are the most closely related species to p. gingivalis both based on phylogeny and ecology. the remaining % of putative essential genes that have no known homologue in the deg may be essential in a species-specific or niche-specific manner. these genes, many of which are functionally classified as containing known pfam protein motifs, ‘conserved domains’ or ‘hypothetical’ proteins, may reveal important aspects related to metabolism and physiology of porphyromonas species and closely related organisms. of the annotated as hypothetical proteins, are among the p. gingivalis essential genes not previously known to be essential from other studies. of the organisms for which an essential gene set has been identified, h. influenzae, f. tularensis, acinetobacter, m. tuberculosis, salmonella typhimurium, s. aureus and b. thetaiotaomicron are the most relevant based on genome size, ecological niche and genetic relatedness to p. gingivalis. the determined essential genes of the above species were genes with essential; genes with essential; genes with essential; genes with essential; genes with essential; genes with; and genes with, respectively. p. gingivalis is a member of the bacteroidetes, and before reclassification was known as b. gingivalis. there are no bacteroidetes species or other anaerobes represented in the deg, however, a putative list of b. thetaiotaomicron strain vpi- essential genes is available from the supplemental material of goodman et al.. b. thetaiotaomicron strain vpi- was originally isolated from human feces. the strain contains a mb chromosome and mb plasmid with genes and, protein coding sequences and, trna and rrna genes. in comparison, p. gingivalis atcc has 43% of protein coding sequences in a genome 37% of the size of that of b. thetaiotaomicron vpi- it was estimated that b. thetaiotaomicron vpi- contains “candidate essential genes”. maintaining a larger genome and gene set provides more opportunities for functional redundancy and alternative pathways which can lead to a relatively smaller number of essential genes. thus, of of b. thetaiotaomicron ‘essentials’ have blast homologues in p. gingivalis strain atcc and of these, 78% are also essential in both organisms. fifty-nine b. thetaiotaomicron blast matches are not essential in p. gingivalis and have no blast match at all in the organism. a significant number of the shared essential genes are not characterized in the deg and of these bacteroidetes-specific essentials, three are annotated as permeases and two appear to be regulatory. three essentials, pgn_ pgn_ and pgn_ are likely associated with capsular polysaccharide biosynthesis based on pgn_ and pgn_ being involved in the dolichol pathway and pgn_ functionally annotated as polysaccharide biosynthesis related. parsing out essential genes of specific groups of species, in this case bacteroidetes and/or anaerobes, can allow for specific drug targeting or directed nutrient supplementation. in agreement with multiple previous studies on essential genes of bacteria, in p. gingivalis a significantly greater number of essential genes are found on the negative dna strand, and are found on the positive dna strand. similarly, there is a greater than expected proportion of enzymes, especially those within multiple functions or involved in multiple pathways, within the essential gene groups. using the cluster of orthologous groups functional class designations, we identified significant enrichment of essential genes within groups ‘d’, ‘i’ and ‘j’; and a lack of enrichment was seen in ‘s’, ‘p’ and ‘n’. enrichment of essential genes in these categories has been reported previously, however, essential gene enrichment in specific cog categories appears to be a species-specific characteristic. based on operon prediction and known essentials contained in the deg it was determined that of the putative essential genes of p. gingivalis identified by tn-seq may be the result of polar effects of the transposon insertion on downstream essential genes. specifically, these genes were identified as being upstream and potentially in an operon with one or more known essential genes, and additionally do not have blast matches in the deg. further study of each of these genes would be required to confirm their essentiality. bringing the deg and p. gingivalis core genome together in relation to p. gingivalis gene essentiality, we have determined that genes within the core genome, ones not identified as essential in our study, have blast matches to genes within the deg. within our mutant libraries we were able to identify transposon insertions into these genes such that they do not qualify as essential in p. gingivalis. reasons for these genes being identified as essential in other species could be due to multiple variables such as in vitro selection media, species-specific essentiality, transposon type, library complexity, sequencing method, and criteria for essentiality. such information gives importance to the distinction between a core gene set and an essential gene set as well as possible limitations of essential gene analyses based solely on in silico methodology. characterization of p. gingivalis essential genes metabolic pathways that lack redundancy or have critical functions have been identified previously through essential gene studies. in our analysis of p. gingivalis we noted the presence of entire pathways as well as specific parts of pathways that are essential to p. gingivalis and to all other bacterial species. a subset of p. gingivalis-specific essential genes, possibly related to the ecological niche of the species, have also been identified. of pathways involved in ribosome function we identified the rpsa, rpma, rplb and rimp systems, which encode for 30s, 50s and maturation of ribosomes, respectively. the three major protein translation regulatory pathways of infb, tsf and prfa, as well as translational machinery pathway rpoa were found to be essential in our study. dna replication, recombination and repair pathways of dnaa and ruva as well as cell division pathways mreb, para and ftsa were also found to be essential. multiple pathways involved in lps, cps, fatty acid and murein biosynthesis, including lpxa, rmla, mana, fabd and mura were also judged to be essential, as well as genes involved in secretion and chaperone pathways such as secd, groes/el and sura. pathways involving nrfa, etfa, sufb, nadd and ribe associated with oxidation-reduction reactions, were found to be essential in p. gingivalis, which is not surprising for an anaerobic bacterium. major metabolic pathways pura, pyrb, coaa, accb, pdxa, ispa, thif, sera and dapa, which encode nucleotide, amino acid and co-factor building blocks, respectively, were determined essential under our in vitro conditions. all of the aforementioned systems and pathways have previously been identified as essential for in vitro growth of other bacterial species, which is not unexpected given that replication, transcription, translation, cell division, membrane stability and central metabolism are key to survival. hypothetical genes are an often-overlooked group within any bacterial genome, including those of ‘model’ organisms. in our study we determined that approximately one-tenth of the essential genome of p. gingivalis encoded ‘hypothetical’ proteins, a few of which were homologous to other hypotheticals contained within the deg. the majority of essential hypothetical genes are large and not within operons, suggesting that they encode functional proteins and are not essential due to a polar effect on a downstream essential gene. the finding that certain hypothetical proteins are essential will stimulate the search for protein motifs, structural bioinformatic and spatial organizational data and studies to define their function. although the notion that an essential gene within a given strain is likely to be essential in the species as a whole, intraspecies differences are known and often result in different phenotypes. for example, in strain atcc we found no insertions into raga and thus this gene was considered essential. previous investigators also had difficulty making directed knockouts of raga in strain w50; however, these investigators were successful in deleting raga from strain wph it is possible that raga is only essential within specific strains and those strains in which it is non-essential compensate for loss of its function through the presence of other genes. limitations of essential gene analysis limitations to essential gene studies should be addressed regardless of systems and methods utilized for their identification. first, several studies have relied exclusively on in silico bioinformatic analyses to determine essentiality. these analyses were based on programs designed to combine information from previous in vitro and in vivo mutagenesis studies with genome annotation and composition scripts without having carried out actual mutagenesis studies. thus, any limitations of these experimental studies will be carried over into the new analyses and magnified by any inaccuracies of the program design itself. second, in insertional mutagenesis methods to determine gene essentiality, genes may be misidentified as essential due to transposon insertion ‘cold-spots’. there is no ideal transposon identified as yet that completely lacks any nucleotide specificity and which can create completely random and saturating mutant libraries. thus, no matter what type of transposon is used, tn tn tn a cryptic construct or mariner, all studies will have regions of the genome where fewer insertions occur. third, genes that are actually non-essential but when mutated cause severe growth defects may be scored as essential due to practical limits to the depth of sequencing of transposon insertion junctions. these ‘sick’ mutants could potentially be represented at levels below 1000-fold a neutral mutant due to the number of replications it could go through prior to being pooled from mutagenesis plates into the library. fourth, non-essential genes immediately upstream of and co-transcribed with essentials may be incorrectly scored as essential due to polarity of the transposon insertion. last, practical limits to library complexity can result in some genes that fail to get disrupted by the transposon and so are misidentified as essential. this is particularly a problem for small genes or genes that are within cold spots for the transposon. several studies, based mostly on the genome size of the species under investigation and the type of transposon, have attained different levels of saturation prior to analyses for essential genes. the possible limitation of our library when combining the type of transposon and library complexity relates to genes that contain less than ta sites in their coding-sequences. of the genes with fewer than ta sites, could potentially be scored as essential based on having zero insertions, but do not qualify, given our stringent criteria. adding confidence to the notion that many of these are non-essential is that of the genes encode proteins of less than amino acids in length. since these are all characterized as ‘hypothetical’ and are rather short to encode functional proteins, we believe that some of these may simply be artifacts of annotation programs and thus not true protein-coding genes. even complete gene deletion, non-transposon based studies of essential genes have limitations. the keio collection of single and double gene deletions in escherichia coli is considered the most comprehensive essential gene study to date. genes that could not be deleted were scored as essential, however, failure to delete a gene is not a guarantee of essentiality and there a are few genes identified as essential in the keio collection that were successfully deleted by other labs. furthermore, a handful of genes labeled non-essential were actually essential. the keio deletions of those genes have second site suppressor mutations that compensate for the loss of the essential gene. the best understanding of essential genes is likely to come from combining different modalities to confirm their essential nature and comparison of these databases both within and between species.  we have described a method for performing tn-seq with p. gingivalis using a mariner mini-transposon and illumina platform next-generation sequencing. we have also invented a new method for creating sequencing libraries from bacterial transposon libraries that has many advantages compared to previous methodologies. using that method we identified specific mutants quantitatively in a highly reproducible manner using massively-parallel sequencing techniques. we used a near saturating insertion library generated in p. gingivalis strain atcc to define the set of genes essential for growth on blood agar. both the availability of a p. gingivalis mutant library and the ability to screen quantitatively are a marked advance in genetic and molecular tools for future studies of p. gingivalis biology and pathogenesis. by applying different selective pressures to the library, it is now possible to identify genes critical for survival and growth under different conditions. due to the quantitative nature of results provided by tn-seq, both positive and negative gene effects, including partial phenotypes, can now be readily identified. 
previously, the p. gingivalis core genome was shown to encode proteins out of a possible; of essential genes are contained within the core genome. genetic tools for the manipulation of bacterial genomes allow for in-depth mechanistic studies of metabolism, physiology, interspecies and host-pathogen interactions. porphyromonas gingivalis is a gram-negative anaerobic bacterium associated with periodontal disease onset and progression. analysis of the essential genes, protein-coding sequences necessary for survival of p. gingivalis by transposon mutagenesis has not previously been attempted due to the limitations of available transposon systems for the organism. a mariner transposon system can be adapted to create mutant libraries in p. gingivalis amenable to analysis by next-generation sequencing technologies. twenty-five genes are known to be essential in p. gingivalis and b. thetaiotaomicron only. in silico analysis of genes essential for in vitro growth demonstrates that although the majority are homologous across bacterial species as a whole, species and strain-specific subsets are apparent. comparing the p. gingivalis essential genes with previous essential gene studies, of the are homologues to essential genes in other species; are shared with more than one other species. significant enrichment of essential genes within cluster of orthologous groups ‘d’, ‘i’ and ‘j’ were identified. understanding the putative essential genes of p. gingivalis will provide insights into metabolic pathways and niche adaptations as well as clinical therapeutic strategies. by analyzing the location of insertions using massively-parallel sequencing technology we used this mutant library to define genes essential for p. gingivalis survival under in vitro conditions. we adapted a mariner transposon system for mutagenesis of p. gingivalis and created an insertion mutant library. in mutagenesis experiments we identified genes in p. gingivalis strain atcc that are putatively essential for viability in vitro. thus, for the species p. gingivalis twenty-two, seventy-seven and twenty-three percent of the genome respectively are devoted to essential, core and accessory functions.
 porphyromonas gingivalis is a gram-negative anaerobic bacterium associated with periodontal disease onset and progression. genetic tools for the manipulation of bacterial genomes allow for in-depth mechanistic studies of metabolism, physiology, interspecies and host-pathogen interactions. analysis of the essential genes, protein-coding sequences necessary for survival of p. gingivalis by transposon mutagenesis has not previously been attempted due to the limitations of available transposon systems for the organism. we adapted a mariner transposon system for mutagenesis of p. gingivalis and created an insertion mutant library. by analyzing the location of insertions using massively-parallel sequencing technology we used this mutant library to define genes essential for p. gingivalis survival under in vitro conditions.  in mutagenesis experiments we identified genes in p. gingivalis strain atcc that are putatively essential for viability in vitro. comparing the p. gingivalis essential genes with previous essential gene studies, of the are homologues to essential genes in other species; are shared with more than one other species. twenty-five genes are known to be essential in p. gingivalis and b. thetaiotaomicron only. significant enrichment of essential genes within cluster of orthologous groups ‘d’, ‘i’ and ‘j’ were identified. previously, the p. gingivalis core genome was shown to encode proteins out of a possible; of essential genes are contained within the core genome. thus, for the species p. gingivalis twenty-two, seventy-seven and twenty-three percent of the genome respectively are devoted to essential, core and accessory functions.  a mariner transposon system can be adapted to create mutant libraries in p. gingivalis amenable to analysis by next-generation sequencing technologies. in silico analysis of genes essential for in vitro growth demonstrates that although the majority are homologous across bacterial species as a whole, species and strain-specific subsets are apparent. understanding the putative essential genes of p. gingivalis will provide insights into metabolic pathways and niche adaptations as well as clinical therapeutic strategies. porphyromonas gingivalistransposon mutagenesisessential genestn-seqperiodontal disease 