BACKGROUND
in the face of the global emergence of multi-drug resistant bacterial pathogens, the search for new classes of antimicrobial agents is one of the most important challenges of modern medicine.
novel potential anti-bacterial drugs have mainly been discovered by conventional screening methods.
these methods involved the testing of natural products or synthetic chemicals for growth inhibition or killing of wild-type test organisms, with the specific mode of action being worked out later  <cit> .
however, recent advances in in silico genomic approaches have provided an opportunity to specifically highlight potential drug targets and have facilitated a paradigm shift from direct antimicrobial screening programs toward rational target-based strategies, where drug discovery starts at the level of the gene  <cit> .
fundamental improvements of genome-based technologies such as whole genome expression- and protein-profiling as well as whole genome sequencing has lead to further changes in the drug discovery process.
this is due to the fact that large amounts of relevant biological information have become available to address highly complex biological questions  <cit> .
as essential genes provide perfect potential drug targets, it has been claimed that an important task of rational target validation would be the identification of the essentiality of the genes within the genome of one organism  <cit> .
there are several techniques to identify essential genes.
first, experimental genetic inactivation of a potential target can be accomplished by gene disruption  <cit> , either in a case-to-case approach  <cit>  or in a high throughput mode  <cit>  in order to provide a genome-wide assessment of essential genes in an organism.
when interpreting genetic inactivation data it should, however, be recognized that the inability to isolate a viable stain under standard laboratory conditions is generally judged as evidence of essentiality, albeit these conditions might not reflect the growth conditions in, e.g., the host environment.
in addition to the experimental validation of gene essentiality, information can be received by applying comparative genomics which involves the comparison of multiple fully sequenced genomes in order to identify a minimal genome set necessary to support bacterial viability  <cit> .
this bioinformatic strategy assumes that bacteria accomplish essential functions through common mechanisms and that the genes encoding these functions would be highly conserved.
an alternative approach for the prediction of gene essentiality would be to draw information from the sequence of the gene itself.
the detailed bioinformatic analysis of the genomic makeup of one organism might give all the information necessary to classify a gene.
although it was suggested that there is no difference between the rates of evolution for essential and non-essential genes in eukaryotes, jordan et al.
<cit>  demonstrated that essential genes appeared to be more conserved than non-essential genes, based on the analysis of multiple complete genomic sequences and experimental knock out data of three bacterial species.
however, other studies have suggested an important direct influence of the expression levels on the rate of nonsynonymous substitutions in bacteria and demonstrated that when a control for this variable was included, essentiality played no significant role in the rate of protein evolution  <cit> .
in this study we aimed to address the main issue of whether essential genes are more evolutionarily conserved than nonessential ones in bacteria and analyzed the impact of the gene expression rate and subcellular localization of the encoded proteins as additional factors that are linked to protein evolution.
a remarkable collection of genomic data is already available for the opportunistic pathogen pseudomonas aeruginosa  <cit> .
sequence information of six strains has been published  <cit> , and there is a large collection of transcriptional profiles that have been recorded under various environmental conditions  <cit> .
furthermore, comprehensive experimental knock out libraries of two p. aeruginosa strains have been established  <cit> .
in this study we amended the existing collection of p. aeruginosa genomic data with whole-genome sequence data from  <dig> clinical p. aeruginosa strains in order to accurately and definitively revisit the interdependence of gene essentiality, rate of nonsynonymous substitutions, gene expression and subcellular localization.
RESULTS
pooled sequencing of  <dig> clinical p. aeruginosa isolates
in order to analyze the sequence variation of p. aeruginosa on a genome wide scale, a collection of  <dig> strains, merged to three 'sequence pools' , was sequenced on an illumina genome analyzer.
additionally, four single strains  were sequenced individually, so that the accuracy of results obtained from the pooled samples could be analyzed.
pooling dna from different strains - instead of sequencing all  <dig> genomes individually - was demonstrated to efficiently yield a large amount of data with an economically reasonable effort.
since we were mainly interested in the variation of the p. aeruginosa core genome  <cit> , the obtained sequence fragments  were mapped to the genome of the reference strain pao <dig>  <cit> .
genome coverage was sufficient for all sequencing runs with read depths  of  <dig>  to  <dig>  , with the exception of some regions showing significantly decreased read depths.
such sequencing 'holes' indicate regions that are absent from the respective strain or - in case of sequence pools - from a fraction of the pooled strains.
short read data from all sequencing runs have been deposited at the ncbi sequence read archive  with the accession number 'srp001802'.
substitution
a median value over all genes
b normalized read depth ≤  <dig>
c mean substitution rate per  <dig> sites
d data set including all  <dig> strains obtained by merging short reads of the three padj sets.
sequence variation in p. aeruginosa
the median sequence variation  was calculated to be ~ <dig> %, which is consistent with previous reports that found sequence variation in p. aeruginosa to be about one order of magnitude lower than in other γ-proteobacteria  <cit> .
nucleotide substitutions in genes coding for proteins can be either synonymous , or non-synonymous .
the median number of non-synonymous differences dn for all protein coding genes was  <dig>  × 10- <dig> with a ratio of nonsynonymous to synonymous  substitutions of dn:ds =  <dig> , indicating that variations of amino acid sequences are generally suppressed by selection.
it is well known that the p. aeruginosa core genome is highly conserved and there are only few exceptions of highly variable genes , while inter-strain variation is mostly restricted to the accessory genome including pathogenicity islands and prophages  <cit> .
consequently, sequence variation among the  <dig> clinical p. aeruginosa strains was markedly increased  for genes belonging to these regions of genomic plasticity .
essential genes in p. aeruginosa
according to the generally used definition, essential genes cannot be deleted from an organism without a lethal effect.
following this definition, the essential subset of genes within the p. aeruginosa genome has been determined  <cit>  from a comparison of the two comprehensive transposon mutant libraries that are currently available for the two reference strains pao <dig>  <cit>  and pa <dig>  <cit> .
for a total number of  <dig> genes, no transposon insertion was detected in either project  <cit> .
however, three of these  <dig> putative essential genes were absent in at least two of the four individually sequenced genomes : pa <dig>  which belongs to a cluster of genes  encoding hypothetical phage-related proteins; pa <dig> , encoding a transcriptional regulator involved in pyoverdin mediated iron-uptake; and pa <dig>  encoding an unclassified hypothetical protein.
furthermore, we found that the normalized read depth was significantly reduced for these three genes in at least one of the sequence pools indicating that these three genes are not ubiquitously present in all strains and thus are most likely not essential for p. aeruginosa.
a within the scope of the two comprehensive libraries  <cit> .
b genes were assumed to be absent if the normalized read depth was below  <dig> .
in the pooled sequence data sets, the read depth gives only a rough estimate for the fraction of strains that harbor the gene.
codon adaptation index in p. aeruginosa
in a previous attempt to link gene essentiality with the gene expression level, the codon adaptation index  was used as an approximation for gene expression  <cit>  which is based on the assumption that highly expressed genes are generally optimized for translation speed and accuracy  <cit> .
essential genes were found to have a generally increased cai as an indicator for their increased expression in e. coli and b. subtilis  <cit> .
here we used expression data of  <dig> microarrays from  <dig> independent experiments  website  <cit> ) to get an estimate of the average gene expression level of each p. aeruginosa gene and compared gene expression and cai for all genes of the p. aeruginosa genome.
the bias caused by the specific setup of individual transcriptome experiments was assumed to be minimized by the large number of independent data sets that where included in this calculation.
indeed, the cai is generally very high , which is not reflected in a global increase in gene expression .
additionally, correlation of cai and gene expression was found to be weak , supporting the previous findings of kiewitz and tümmler  <cit>  who showed that the codon usage is globally optimized in p. aeruginosa and not restricted to only highly expressed genes pointing towards a generally high level of adaptation throughout the genome that would ensure optimal gene expression under various environmental and metabolic conditions.
in consistence, we found the cai to be significantly reduced in genes of the accessory genome , which in many cases were acquired horizontally and also differ from the core genome in several other aspects .
gene essentiality and expression level are correlated with genetic variability
it has previously been shown that essential genes are more evolutionarily conserved than nonessential genes  <cit> , and indeed determination of non-synonymous and synonymous substitution rates dn and ds for the  <dig> clinical p. aeruginosa strains confirmed this expected correlation of sequence variation and essentiality .
if genes are grouped by their functional classification, the variation is lowest for genes involved in 'transcription, rna processing, degradation', 'translation, post-translational modification, degradation' and 'cell division', which are enriched for essential genes .
in addition to gene essentiality, a high gene expression rate has previously been shown to correlate with low sequence variation  <cit> , and it was proposed that the underlying driving force for the slower evolution of essential genes is that most of the highly expressed genes are also generally indispensable.
to test this for p. aeruginosa, we compared the gene expression data of p. aeruginosa  with the rate of nonsynonymous substitutions, dn .
applying robust regression to predict dn from gene expression clearly demonstrated a negative correlation , which means that highly expressed genes are indeed more conserved in p. aeruginosa.
furthermore, essential genes showed a significantly increased expression level in p. aeruginosa.
if the gene expression data are grouped by essentiality it becomes clear, that essential genes show a significant increased proportion of highly expressed genes .
with the aim of testing whether the observed correlation of gene essentiality and dn is the result of this overrepresentation of high expression rates among the essential genes or whether there is also an independent effect, we performed a statistical test for conditional independence.
therefore, the dn-values of the non-essential genes were normalized in order to compensate for their lower expression rates.
the null hypothesis for the test for conditional independence was that the distributions of the dn-values are identical for essential and non-essential genes after this normalization has been performed.
the null hypothesis of conditional independence had to be rejected for the averaged data set of all  <dig> strains .
this means that, besides the expression level, essentiality also accounts for dn in p. aeruginosa, although the statistical significance is not very high.
apparently, the effect of gene expression on protein evolution rates is dominant in p. aeruginosa, but still an independent  effect of essentiality could be observed.
extracellular proteins evolve at a faster rate
other parameters that have been identified to correlate with slow protein evolution include protein localization   <cit> , evolutionary age  <cit>  and protein connectivity  <cit> .
in p. aeruginosa genes encoding extracellular and outer membrane proteins  showed a significant increase in dn as compared to cytoplasmic genes .
we also repeated robust regression for the dn and expression data for each class of subcellular localization independently.
the aforementioned negative correlation of dn and gene expression could be found for all localization classes with the exception of extracellular genes.
on the contrary, extracellular genes even had a slightly positive coefficient indicating that they are even more variable if they are highly expressed .
highly conserved non-essential genes
as described above and depicted in figure  <dig>  essential genes were shown to be generally more conserved than non-essential ones in our collection of p. aeruginosa strains.
vice versa, many highly conserved genes were essential according to the standard definition  because no transposon mutants are available in either of the two comprehensive libraries  <cit> .
however, we also found a large subset of genes that are experimentally dispensable but highly conserved.
genes that are dispensable, but conserved throughout distantly related bacteria, have been termed persistent non-essential genes and are proposed to be regarded as truly essential from an evolutionary point of view  <cit> .
these genes might be dispensable for short term survival and growth under laboratory conditions but are in fact essential for successful survival of the population under varying environmental conditions in its native habitat.
interestingly, overall as many as  <dig> protein encoding genes  did not exhibit any variations in the protein sequence  in the  <dig> clinical isolates and  <dig> were even identical at the nucleotide level .
while this is consistent with the generally high conservation of the p. aeruginosa core genome, these genes could furthermore be interpreted as being 'evolutionarily essential' for the species p. aeruginosa in a sense similar to the aforementioned persistent non-essential genes  <cit> .
among the  <dig> completely conserved genes  we found many encoding ribosomal proteins, genes important for energy production  or dna replication and repair, e.g., dnan  or the helicase ruva/ruvb.
many of them also belong to the class of persistent non-essential genes in γ-proteobacteria  <cit> .
remarkably, among the set of completely conserved genes we also found the two paralogous operons phza1-g <dig> and phza2-g <dig> that encode enzymes necessary for phenazine synthesis  <cit> .
both operons are almost identical, not only within one strain  but furthermore no sequence variation could be detected for the whole collection of  <dig> clinical strains  - in most cases not even at the nucleotide level .
phenazines including pyocyanin, which is responsible for the well known blue-green color of p. aeruginosa cultures, and which is a major virulence factor  <cit> , have been shown to mediate extracellular electron transfer under microaerophilic conditions  <cit> .
other genes involved in phenazine synthesis and regulation  and many regulatory genes - vfr, algu, rsma, gaca, phop, mvat, algr, and phob, all of which play an important role in the environmental versatility of p. aeruginosa including virulence  <cit>  - also showed no protein variations.
additionally, we identified the quorum sensing  genes lasi, rhlr and rsal to be fully conserved at the protein level.
it has been reported that qs is frequently impaired in clinical isolates, especially those isolated from long term chronically infected cystic fibrosis  patients  <cit> .
thereby, loss of qs is mainly caused by disruption of lasr that also showed considerable variation in this study, including the identification of premature stop-codons in a fraction of strains .
while the high conservation of the above described genes of the las and rhl system underlines the general importance of qs for p. aeruginosa, loss of qs - preferably by disruption of lasr - might be an important adaptive strategy to more specific habitats such as the chronically infected cf lung.
highly conserved genes linked to antibiotic resistance
assuming that high conservation of genes is positively correlated with their evolutionary importance, the  <dig> highly conserved genes described above might be potential novel drug targets.
to further examine this potential, we compared the list of genes with the results of three recently published screens for resistance determinants in p. aeruginosa  <cit> .
in total, of the  <dig> genes  <dig> were identified in at least one of the three screens as being positively linked to antibiotic resistance .
since non-specific drugs can cause more side-effects, potential new drug targets should be specific for the bacteria targeted.
thus, in order to determine the specificity of the highly conserved genes with respect to their phylogenetic distribution, we performed a blastp search of all  <dig> protein sequences against the database of non-redundant protein sequences  obtained from the ncbi blast website http://blast.ncbi.nlm.nih.gov/.
<dig> genes were found to be specific to pseudomonas, this included  <dig> species-specific genes for which no orthologs outside p. aeruginosa were identified .
for the majority of genes a large number of orthologs could be identified that belonged to a variety of species outside the pseudomonas genus indicating a broad phylogenetic distribution among different bacterial genera.
at least one human ortholog was identified for  <dig> of the  <dig> genes , which might be adversely affected by a drug.
a no orthologous genes were found outside the genus pseudomonas.
b no orthologous genes were found outside the species p. aeruginosa
CONCLUSIONS
thanks to recent advances in sequencing technologies it has been possible to analyze many genomes with little expense within a short space of time.
because whole-genome sequence data and bioinformatics provide potential for large scale comparative genomics and evolutionary inference, they will be an important sector in the future global approaches for deciphering the genomic make-up of an organism and for rational drug discovery programs.
in the presented work, we have demonstrated the combined use of short read sequencing and bioinformatic methods to analyze a whole collection of clinical p. aeruginosa strains.
the genome sequence data of  <dig> pooled bacterial strains combined with the availability of more than  <dig> gene expression profiles and the experimental genome-wide assessment of essential genes, gave the unique opportunity to revisit the central question of the interdependency of the effects of various factors on sequence variation in an accurate and definite manner.
here, we have confirmed that the rate of expression is a major determinant of how rapidly a protein evolves.
we were furthermore able to demonstrate an independent correlation of gene essentiality with protein evolution rates in p. aeruginosa, although essential genes on average also showed an increased expression.
additionally, we could demonstrate increased substitution rates for genes encoding for extracellular proteins.
furthermore, our comparison of whole genome sequence data of  <dig> p. aeruginosa strains revealed a subset of  <dig> proteins that were fully conserved, and did not show any variation in the amino acid sequence.
the full conservation of these protein sequences may indicate their evolutionary importance for p. aeruginosa as a species, comparable to the persistent nonessential genes on the inter-species level.
among the completely conserved proteins, we found many that are involved in the central cellular mechanisms like energy production, replication and protein synthesis, many of which were also identified by fang et al.
as persistent nonessential genes, or that are coding for regulatory and virulence factors .
in accordance with the lifestyle of p. aeruginosa as a versatile environmental organism and opportunistic pathogen, this demonstrates a) the high level of optimization of the genome  and b) the evolutionary importance of these genes for its survival in various habitats.
the genomic analysis of multiple strains will greatly enhance our knowledge on the distribution and variation of genes and their relation to the lifestyle of the particular organism.
a future expansion of this study, which is biased towards clinical isolates, would be to include soil and water isolated p. aeruginosa strains and to test whether they share the identified traits.
furthermore, the comparison of strain collections with specific virulence or resistance phenotypes will simplify the detection of genetic determinants that are responsible for the development of severe infections and/or multi-resistance and will uncover putative targets for novel anti-bacterial strategies.

 in the face of the global emergence of multi-drug resistant bacterial pathogens, the search for new classes of antimicrobial agents is one of the most important challenges of modern medicine. novel potential anti-bacterial drugs have mainly been discovered by conventional screening methods. these methods involved the testing of natural products or synthetic chemicals for growth inhibition or killing of wild-type test organisms, with the specific mode of action being worked out later. however, recent advances in in silico genomic approaches have provided an opportunity to specifically highlight potential drug targets and have facilitated a paradigm shift from direct antimicrobial screening programs toward rational target-based strategies, where drug discovery starts at the level of the gene. fundamental improvements of genome-based technologies such as whole genome expression- and protein-profiling as well as whole genome sequencing has lead to further changes in the drug discovery process. this is due to the fact that large amounts of relevant biological information have become available to address highly complex biological questions. as essential genes provide perfect potential drug targets, it has been claimed that an important task of rational target validation would be the identification of the essentiality of the genes within the genome of one organism. there are several techniques to identify essential genes. first, experimental genetic inactivation of a potential target can be accomplished by gene disruption, either in a case-to-case approach or in a high throughput mode in order to provide a genome-wide assessment of essential genes in an organism. when interpreting genetic inactivation data it should, however, be recognized that the inability to isolate a viable stain under standard laboratory conditions is generally judged as evidence of essentiality, albeit these conditions might not reflect the growth conditions in, e.g., the host environment. in addition to the experimental validation of gene essentiality, information can be received by applying comparative genomics which involves the comparison of multiple fully sequenced genomes in order to identify a minimal genome set necessary to support bacterial viability. this bioinformatic strategy assumes that bacteria accomplish essential functions through common mechanisms and that the genes encoding these functions would be highly conserved. an alternative approach for the prediction of gene essentiality would be to draw information from the sequence of the gene itself. the detailed bioinformatic analysis of the genomic makeup of one organism might give all the information necessary to classify a gene. although it was suggested that there is no difference between the rates of evolution for essential and non-essential genes in eukaryotes, jordan et al. demonstrated that essential genes appeared to be more conserved than non-essential genes, based on the analysis of multiple complete genomic sequences and experimental knock out data of three bacterial species. however, other studies have suggested an important direct influence of the expression levels on the rate of nonsynonymous substitutions in bacteria and demonstrated that when a control for this variable was included, essentiality played no significant role in the rate of protein evolution. in this study we aimed to address the main issue of whether essential genes are more evolutionarily conserved than nonessential ones in bacteria and analyzed the impact of the gene expression rate and subcellular localization of the encoded proteins as additional factors that are linked to protein evolution. a remarkable collection of genomic data is already available for the opportunistic pathogen pseudomonas aeruginosa. sequence information of six strains has been published, and there is a large collection of transcriptional profiles that have been recorded under various environmental conditions. furthermore, comprehensive experimental knock out libraries of two p. aeruginosa strains have been established. in this study we amended the existing collection of p. aeruginosa genomic data with whole-genome sequence data from clinical p. aeruginosa strains in order to accurately and definitively revisit the interdependence of gene essentiality, rate of nonsynonymous substitutions, gene expression and subcellular localization.  pooled sequencing of clinical p. aeruginosa isolates in order to analyze the sequence variation of p. aeruginosa on a genome wide scale, a collection of strains, merged to three'sequence pools', was sequenced on an illumina genome analyzer. additionally, four single strains were sequenced individually, so that the accuracy of results obtained from the pooled samples could be analyzed. pooling dna from different strains - instead of sequencing all genomes individually - was demonstrated to efficiently yield a large amount of data with an economically reasonable effort. since we were mainly interested in the variation of the p. aeruginosa core genome, the obtained sequence fragments were mapped to the genome of the reference strain pao. genome coverage was sufficient for all sequencing runs with read depths of to, with the exception of some regions showing significantly decreased read depths. such sequencing'holes' indicate regions that are absent from the respective strain or - in case of sequence pools - from a fraction of the pooled strains. short read data from all sequencing runs have been deposited at the ncbi sequence read archive with the accession number'srp001802'. substitution a median value over all genes b normalized read depth ≤ c mean substitution rate per sites d data set including all strains obtained by merging short reads of the three padj sets. sequence variation in p. aeruginosa the median sequence variation was calculated to be ~ %, which is consistent with previous reports that found sequence variation in p. aeruginosa to be about one order of magnitude lower than in other γ-proteobacteria. nucleotide substitutions in genes coding for proteins can be either synonymous, or non-synonymous. the median number of non-synonymous differences dn for all protein coding genes was × 10- with a ratio of nonsynonymous to synonymous substitutions of dn:ds =, indicating that variations of amino acid sequences are generally suppressed by selection. it is well known that the p. aeruginosa core genome is highly conserved and there are only few exceptions of highly variable genes, while inter-strain variation is mostly restricted to the accessory genome including pathogenicity islands and prophages. consequently, sequence variation among the clinical p. aeruginosa strains was markedly increased for genes belonging to these regions of genomic plasticity. essential genes in p. aeruginosa according to the generally used definition, essential genes cannot be deleted from an organism without a lethal effect. following this definition, the essential subset of genes within the p. aeruginosa genome has been determined from a comparison of the two comprehensive transposon mutant libraries that are currently available for the two reference strains pao and pa. for a total number of genes, no transposon insertion was detected in either project. however, three of these putative essential genes were absent in at least two of the four individually sequenced genomes: pa which belongs to a cluster of genes encoding hypothetical phage-related proteins; pa, encoding a transcriptional regulator involved in pyoverdin mediated iron-uptake; and pa encoding an unclassified hypothetical protein. furthermore, we found that the normalized read depth was significantly reduced for these three genes in at least one of the sequence pools indicating that these three genes are not ubiquitously present in all strains and thus are most likely not essential for p. aeruginosa. a within the scope of the two comprehensive libraries. b genes were assumed to be absent if the normalized read depth was below. in the pooled sequence data sets, the read depth gives only a rough estimate for the fraction of strains that harbor the gene. codon adaptation index in p. aeruginosa in a previous attempt to link gene essentiality with the gene expression level, the codon adaptation index was used as an approximation for gene expression which is based on the assumption that highly expressed genes are generally optimized for translation speed and accuracy. essential genes were found to have a generally increased cai as an indicator for their increased expression in e. coli and b. subtilis. here we used expression data of microarrays from independent experiments website ) to get an estimate of the average gene expression level of each p. aeruginosa gene and compared gene expression and cai for all genes of the p. aeruginosa genome. the bias caused by the specific setup of individual transcriptome experiments was assumed to be minimized by the large number of independent data sets that where included in this calculation. indeed, the cai is generally very high, which is not reflected in a global increase in gene expression. additionally, correlation of cai and gene expression was found to be weak, supporting the previous findings of kiewitz and tümmler who showed that the codon usage is globally optimized in p. aeruginosa and not restricted to only highly expressed genes pointing towards a generally high level of adaptation throughout the genome that would ensure optimal gene expression under various environmental and metabolic conditions. in consistence, we found the cai to be significantly reduced in genes of the accessory genome, which in many cases were acquired horizontally and also differ from the core genome in several other aspects. gene essentiality and expression level are correlated with genetic variability it has previously been shown that essential genes are more evolutionarily conserved than nonessential genes, and indeed determination of non-synonymous and synonymous substitution rates dn and ds for the clinical p. aeruginosa strains confirmed this expected correlation of sequence variation and essentiality. if genes are grouped by their functional classification, the variation is lowest for genes involved in'transcription, rna processing, degradation','translation, post-translational modification, degradation' and'cell division', which are enriched for essential genes. in addition to gene essentiality, a high gene expression rate has previously been shown to correlate with low sequence variation, and it was proposed that the underlying driving force for the slower evolution of essential genes is that most of the highly expressed genes are also generally indispensable. to test this for p. aeruginosa, we compared the gene expression data of p. aeruginosa with the rate of nonsynonymous substitutions, dn. applying robust regression to predict dn from gene expression clearly demonstrated a negative correlation, which means that highly expressed genes are indeed more conserved in p. aeruginosa. furthermore, essential genes showed a significantly increased expression level in p. aeruginosa. if the gene expression data are grouped by essentiality it becomes clear, that essential genes show a significant increased proportion of highly expressed genes. with the aim of testing whether the observed correlation of gene essentiality and dn is the result of this overrepresentation of high expression rates among the essential genes or whether there is also an independent effect, we performed a statistical test for conditional independence. therefore, the dn-values of the non-essential genes were normalized in order to compensate for their lower expression rates. the null hypothesis for the test for conditional independence was that the distributions of the dn-values are identical for essential and non-essential genes after this normalization has been performed. the null hypothesis of conditional independence had to be rejected for the averaged data set of all strains. this means that, besides the expression level, essentiality also accounts for dn in p. aeruginosa, although the statistical significance is not very high. apparently, the effect of gene expression on protein evolution rates is dominant in p. aeruginosa, but still an independent effect of essentiality could be observed. extracellular proteins evolve at a faster rate other parameters that have been identified to correlate with slow protein evolution include protein localization, evolutionary age and protein connectivity. in p. aeruginosa genes encoding extracellular and outer membrane proteins showed a significant increase in dn as compared to cytoplasmic genes. we also repeated robust regression for the dn and expression data for each class of subcellular localization independently. the aforementioned negative correlation of dn and gene expression could be found for all localization classes with the exception of extracellular genes. on the contrary, extracellular genes even had a slightly positive coefficient indicating that they are even more variable if they are highly expressed. highly conserved non-essential genes as described above and depicted in figure essential genes were shown to be generally more conserved than non-essential ones in our collection of p. aeruginosa strains. vice versa, many highly conserved genes were essential according to the standard definition because no transposon mutants are available in either of the two comprehensive libraries. however, we also found a large subset of genes that are experimentally dispensable but highly conserved. genes that are dispensable, but conserved throughout distantly related bacteria, have been termed persistent non-essential genes and are proposed to be regarded as truly essential from an evolutionary point of view. these genes might be dispensable for short term survival and growth under laboratory conditions but are in fact essential for successful survival of the population under varying environmental conditions in its native habitat. interestingly, overall as many as protein encoding genes did not exhibit any variations in the protein sequence in the clinical isolates and were even identical at the nucleotide level. while this is consistent with the generally high conservation of the p. aeruginosa core genome, these genes could furthermore be interpreted as being'evolutionarily essential' for the species p. aeruginosa in a sense similar to the aforementioned persistent non-essential genes. among the completely conserved genes we found many encoding ribosomal proteins, genes important for energy production or dna replication and repair, e.g., dnan or the helicase ruva/ruvb. many of them also belong to the class of persistent non-essential genes in γ-proteobacteria. remarkably, among the set of completely conserved genes we also found the two paralogous operons phza1-g and phza2-g that encode enzymes necessary for phenazine synthesis. both operons are almost identical, not only within one strain but furthermore no sequence variation could be detected for the whole collection of clinical strains - in most cases not even at the nucleotide level. phenazines including pyocyanin, which is responsible for the well known blue-green color of p. aeruginosa cultures, and which is a major virulence factor, have been shown to mediate extracellular electron transfer under microaerophilic conditions. other genes involved in phenazine synthesis and regulation and many regulatory genes - vfr, algu, rsma, gaca, phop, mvat, algr, and phob, all of which play an important role in the environmental versatility of p. aeruginosa including virulence - also showed no protein variations. additionally, we identified the quorum sensing genes lasi, rhlr and rsal to be fully conserved at the protein level. it has been reported that qs is frequently impaired in clinical isolates, especially those isolated from long term chronically infected cystic fibrosis patients. thereby, loss of qs is mainly caused by disruption of lasr that also showed considerable variation in this study, including the identification of premature stop-codons in a fraction of strains. while the high conservation of the above described genes of the las and rhl system underlines the general importance of qs for p. aeruginosa, loss of qs - preferably by disruption of lasr - might be an important adaptive strategy to more specific habitats such as the chronically infected cf lung. highly conserved genes linked to antibiotic resistance assuming that high conservation of genes is positively correlated with their evolutionary importance, the highly conserved genes described above might be potential novel drug targets. to further examine this potential, we compared the list of genes with the results of three recently published screens for resistance determinants in p. aeruginosa. in total, of the genes were identified in at least one of the three screens as being positively linked to antibiotic resistance. since non-specific drugs can cause more side-effects, potential new drug targets should be specific for the bacteria targeted. thus, in order to determine the specificity of the highly conserved genes with respect to their phylogenetic distribution, we performed a blastp search of all protein sequences against the database of non-redundant protein sequences obtained from the ncbi blast website http://blast.ncbi.nlm.nih.gov/. genes were found to be specific to pseudomonas, this included species-specific genes for which no orthologs outside p. aeruginosa were identified. for the majority of genes a large number of orthologs could be identified that belonged to a variety of species outside the pseudomonas genus indicating a broad phylogenetic distribution among different bacterial genera. at least one human ortholog was identified for of the genes, which might be adversely affected by a drug. a no orthologous genes were found outside the genus pseudomonas. b no orthologous genes were found outside the species p. aeruginosa  thanks to recent advances in sequencing technologies it has been possible to analyze many genomes with little expense within a short space of time. because whole-genome sequence data and bioinformatics provide potential for large scale comparative genomics and evolutionary inference, they will be an important sector in the future global approaches for deciphering the genomic make-up of an organism and for rational drug discovery programs. in the presented work, we have demonstrated the combined use of short read sequencing and bioinformatic methods to analyze a whole collection of clinical p. aeruginosa strains. the genome sequence data of pooled bacterial strains combined with the availability of more than gene expression profiles and the experimental genome-wide assessment of essential genes, gave the unique opportunity to revisit the central question of the interdependency of the effects of various factors on sequence variation in an accurate and definite manner. here, we have confirmed that the rate of expression is a major determinant of how rapidly a protein evolves. we were furthermore able to demonstrate an independent correlation of gene essentiality with protein evolution rates in p. aeruginosa, although essential genes on average also showed an increased expression. additionally, we could demonstrate increased substitution rates for genes encoding for extracellular proteins. furthermore, our comparison of whole genome sequence data of p. aeruginosa strains revealed a subset of proteins that were fully conserved, and did not show any variation in the amino acid sequence. the full conservation of these protein sequences may indicate their evolutionary importance for p. aeruginosa as a species, comparable to the persistent nonessential genes on the inter-species level. among the completely conserved proteins, we found many that are involved in the central cellular mechanisms like energy production, replication and protein synthesis, many of which were also identified by fang et al. as persistent nonessential genes, or that are coding for regulatory and virulence factors. in accordance with the lifestyle of p. aeruginosa as a versatile environmental organism and opportunistic pathogen, this demonstrates a) the high level of optimization of the genome and b) the evolutionary importance of these genes for its survival in various habitats. the genomic analysis of multiple strains will greatly enhance our knowledge on the distribution and variation of genes and their relation to the lifestyle of the particular organism. a future expansion of this study, which is biased towards clinical isolates, would be to include soil and water isolated p. aeruginosa strains and to test whether they share the identified traits. furthermore, the comparison of strain collections with specific virulence or resistance phenotypes will simplify the detection of genetic determinants that are responsible for the development of severe infections and/or multi-resistance and will uncover putative targets for novel anti-bacterial strategies. 
the detailed analysis of a comprehensive set of p. aeruginosa genomes in this study clearly disclosed detailed information of the genomic makeup and revealed a large set of highly conserved genes that play an important role for the lifestyle of this microorganism. sequencing strain collections enables for a detailed and extensive identification of sequence variations as potential bacterial adaptation processes, e.g., during the development of antibiotic resistance in the clinical setting and thus may be the basis to uncover putative targets for novel treatment strategies. among the conserved nonessential genes we found several that are involved in regulation, motility and virulence, indicating that they represent factors of evolutionary importance for the lifestyle of a successful environmental bacterium and opportunistic pathogen. new sequencing technologies are now on the horizon that will yield massive increases in our capacity for dna sequencing and will revolutionize the drug discovery process. since essential genes are promising novel antibiotic targets, the prediction of gene essentiality based on genomic information has become a major focus. in this study we demonstrate that pooled sequencing is applicable for the analysis of sequence variations of strain collections with more than individual isolates. we furthermore refined the list of experimentally essential p. aeruginosa genes, and identified genes that show no sequence variation at all.
 the constant increase in development and spread of bacterial resistance to antibiotics poses a serious threat to human health. new sequencing technologies are now on the horizon that will yield massive increases in our capacity for dna sequencing and will revolutionize the drug discovery process. since essential genes are promising novel antibiotic targets, the prediction of gene essentiality based on genomic information has become a major focus.  in this study we demonstrate that pooled sequencing is applicable for the analysis of sequence variations of strain collections with more than individual isolates. pooled sequencing of clinical pseudomonas aeruginosa isolates revealed that essential and highly expressed proteins evolve at lower rates, whereas extracellular proteins evolve at higher rates. we furthermore refined the list of experimentally essential p. aeruginosa genes, and identified genes that show no sequence variation at all. among the conserved nonessential genes we found several that are involved in regulation, motility and virulence, indicating that they represent factors of evolutionary importance for the lifestyle of a successful environmental bacterium and opportunistic pathogen.  the detailed analysis of a comprehensive set of p. aeruginosa genomes in this study clearly disclosed detailed information of the genomic makeup and revealed a large set of highly conserved genes that play an important role for the lifestyle of this microorganism. sequencing strain collections enables for a detailed and extensive identification of sequence variations as potential bacterial adaptation processes, e.g., during the development of antibiotic resistance in the clinical setting and thus may be the basis to uncover putative targets for novel treatment strategies. 