BACKGROUND
variations in protein concentrations are not solely related to transcriptional activity.
this is also true in bacteria.
recent studies with escherichia coli and desulfovibrio vulgaris described only moderate correlations between mrna levels and protein concentrations with a pearson coefficient varying from  <dig>  to  <dig>  according to culture conditions
<cit> .
in lactococcus lactis, a gram positive lactic acid bacterium, the pearson coefficient was even lower ranging from  <dig>  to  <dig>
<cit> .
post-transcriptional events  are proposed to be the principle causes of these weak correlations
<cit> .
in l. lactis, we identified protein abundance determinants by statistical modeling of proteome data: gene sequence features such as the codon adaptation index , gene length and functional categories were demonstrated to strongly influence mrna translation levels
<cit> .
in addition, individual mrna translation efficiency was estimated by a mechanistic modeling approach, highlighting significant variability in translation efficiencies among the different genes of the genome
<cit> .
consequently in l. lactis, not all mrnas are believed to be translated with the same efficacy and important regulations are expected to occur at the translational level.
an experimental approach for studying genome scale translational regulation is translatome analysis which provides the detailed translational status of all mrnas.
translation consists of three steps: initiation, elongation and termination.
translation initiation occurs with a ribosome binding to the mrna template and begins at the start codon with incorporation of the first amino acid of the polypeptide.
the ribosome continues to translate the coding sequence catalyzing polypeptide chain elongation until it reaches the stop codon.
then, translation termination occurs and the complete polypeptide chain is released.
translation is carried out by more than one ribosome simultaneously forming a polysomal structure.
translatome experiments provide the ribosome number on each mrna molecule in the cell.
translatome determination combines size separation of ribosome-mrna complexes according to the number of ribosomes loaded  and measurement of mrna levels in fractions by microarray techniques
<cit> .
most translatomes reported so far aimed at identifying translationally regulated genes in response to stress
<cit>  or changing growth phase
<cit> .
however, these studies were based on low-resolution polysomal profile analysis in which messengers were classified into only two fractions: polysomal versus non-polysomal .
in these conditions, the cellular translational status of each mrna molecule was not fully described since the number of loaded ribosomes was not quantified.
however, this information is required to study the diversity of translational regulations between all mrna species present in a cell and then to understand translation efficiency of individual mrnas.
to date, only a few studies described high resolution translatome analysis in microorganisms with mrnas classified with respect to the precise number of loaded ribosomes
<cit> .
unfortunately all high resolution translatome studies were carried out in yeast, leaving the understanding of detailed translational regulation in bacteria incomplete.
therefore we present here the first high resolution translatome analysis in the bacterium l. lactis.
this model lactic acid bacterium is a gram-positive non spore forming bacterium with a low gc content .
l. lactis is therefore phylogenetically closer to bacillus subtilis than e. coli.
l. lactis was grown under maximum growth rate conditions .
the profile of mrna-ribosome association led to the definition of two translational variables for each mrna species: the fraction engaged in translation  and the ribosome density.
by comparing the translational states of all mrnas, the regulation of both ribosome occupancy and ribosome density levels were explored.
in addition, the influence of ribosome occupancy and ribosome density on the final protein expression level was quantified.
this demonstrated the key role of the mrna translational status in the complex processes linking gene expression to protein synthesis.
RESULTS
polysomal profile description
the translatome of l. lactis was studied by coupling polysome profile determination, transcriptomics and statistical analyses .
a typical polysome profile is shown in figure
<dig>  after peak assignment, pooling of the two first fractions corresponding to mrnas not engaged in translation and pooling of the last four fractions representing the most highly ribosome-loaded transcripts, the seven resulting fractions were hybridized to the microarrays b to h, respectively .
<dig> genes were selected according to the cutoff criterion .
figure
<dig> shows sample distributions of mrna proportion between fractions b to h for six selected genes.
when considering the entire gene set, 61% and 37% of genes exhibited the highest mrna abundance in fraction c  or in fraction h , respectively.
the number of ribosomes per transcript was one for fraction d, and in the range  <dig> - <dig>  ,  <dig> - <dig>  ,  <dig> - <dig>   and  <dig> - <dig>   for fractions e, f, g and h, respectively .
a maximum of  <dig> ribosomes per transcript was thus obtained in l. lactis cells grown in the exponential phase.
the percentage of ribosomes engaged in translation was estimated by area integration of the polysomal profile  and an average ratio of  <dig> ± 2% of total ribosomes engaged in translation was obtained.
since about one third of the total ribosomal content was not associated with mrna, ribosomal content would appear in excess for protein synthesis under the growth conditions used in this study.
translatome variable determination and physiological analysis
two translatome variables were determined, the ribosome occupancy corresponding to the fraction of mrna engaged in translation and the ribosome density, the number of loaded ribosomes per  <dig> nucleotides.
ribosome occupancy reflects the efficiency of translation initiation while the interpretation of ribosome density can vary according to the limiting step of translation.
for genes exhibiting initiation-limited translation, high density is correlated with high translation levels, but for genes with elongation limitation, high density can result from ribosome congestion leading to low translation efficiency.
ribosome occupancy values were obtained for  <dig> genes, and a gaussian distribution was observed with a median value of  <dig> ± 6% .
high variability in ribosome occupancy was observed among genes with values ranging from 41% to 84%.
therefore, for all genes at least part of the corresponding mrna molecules was involved in translation but never the entire mrna population.
this suggests that for individual genes, the mrna concentration in exponentially-grown cells of l. lactis was in excess.
thus under the conditions used, both ribosome availability  and mrna abundancies could conceivably support higher protein synthesis.
a peak fraction, corresponding to the most frequently found number of ribosomes bound on each mrna, was assigned to  <dig> genes with a 95% bootstrap confidence interval.
except one gene  with a peak fraction in fraction f , all the other genes grouped into two classes of the peak fraction.
the first class was composed of  <dig> genes translated with a low number of ribosomes with a peak fraction in the monosome fraction d. the second class included  <dig> more highly loaded genes with a peak fraction in fraction h, corresponding to  <dig> - <dig>  loaded ribosomes.
we verified that the bacterial specificity of polycistronic structures did not introduce any bias in ribosome number determination.
in l. lactis,  <dig> genes have been experimentally confirmed in the literature as being organized as polycistronic operons
<cit> .
they were specifically examined to validate their eventual enrichment in the two subgroups of genes with peak fractions in d and h, respectively.
no significant over-representation of these genes was observed.
based on the peak fraction value, the second translatome variable, ribosome density, was calculated.
for each of the  <dig> genes, ribosome density was assessed as the ratio of the ribosome number in its peak fraction to the coding sequence length.
if we consider that one bound bacterial ribosome protects around  <dig> nucleotides
<cit> , the theoretical maximal ribosome density is  <dig>  ribosomes / <dig> nucleotides.
however,  <dig> genes within the  <dig> gene set exhibited experimental ribosome densities higher than  <dig> .
this  <dig> gene subgroup was significantly enriched in short monocistronic genes .
the presence of rather long 5’utrs  could contribute to ribosome density over-estimation, even more pronounced in the case of short genes.
this set of  <dig> genes with aberrant ribosome densities was omitted in the subsequent analyses leading to a new set of  <dig> genes.
figure
<dig> shows the distribution of ribosome density ranging from  <dig>  to  <dig>  ribosomes per  <dig> nucleotides.
the median ribosome density was  <dig>  ribosomes per  <dig> nucleotides  representing 2/ <dig> of the maximal theoretical ribosome density.
in l. lactis, for most of the genes, ribosome density was thus far from the maximal density as also reported in yeast
<cit> .
therefore, as proposed in yeast
<cit> , translation limitation appears to occur during ribosome loading on mrna in the initiation step even though ribosomes and mrna molecules were found in excess in l. lactis cells.
with a strictly mathematical modeling approach of translation control such low ribosome densities were previously demonstrated in bacteria to be related to a limitation of translation at the initiation step
<cit> .
thus in this context of initiation-limited translation, high ribosome density is expected to provoke high translation levels for most of the l. lactis genes.
homolactic lactic acid bacteria such as l. lactis are fermentative bacteria producing high concentrations of lactate during glucose catabolism .
the lactate production ensures cofactor  regeneration which is essential for bacterial activity but also induces the acidification of the ambient environment and thereby inhibition of bacterial growth
<cit> .
genes involved in important metabolic functional categories exhibited similar regulation of ribosome density and ribosome occupancy .
in agreement with the high growth rate, l. lactis favoured the translation of specific functions required for optimal growth.
a high glycolytic rate was favoured by the efficient translation of the first step of the glycolysis, the glucose transport system, which is believed to control the glycolysis rate in the studied il <dig> l. lactis strain
<cit> .
high ribosome occupancy and ribosome density were indeed observed for the main glucose transport system, mannose pts  and glucokinase , which are required when glucose enters through sugar permease.
the sugar permease system is still uncharacterised in l. lactis, however our finding of a high level of translation  of the gene ypch coding for a sugar permease suggests the involvement of this permease in glucose transport.
similarly, the last step of glycolysis, corresponding to conversion of pyruvate into lactate by lactate dehydrogenase , also corresponded to a very efficiently translated gene .
in order to counteract growth inhibition by cytoplasmic acidification under conditions of extracellular auto-acidification, translation was also increased for almost all genes coding for the atpase system  that catalyses the excretion of protons while consuming atp.
this proton expulsion system was efficient enough to prevent a strong acid stress since secondary pathways of the acid stress response were not favoured at the translational level.
genes of the arginine deiminase and glutamate decarboxylation pathways  exhibited low values of ribosome occupancy and ribosome density in comparison to the average.
in addition, positive regulation of translation of enzymes related to phosphate limitation , and to zinc and manganese transports  were observed, suggesting additional cation and anion requirements to fight against lactic acid inhibition.
determinants of translatome variables
in order to identify how ribosome occupancy and ribosome density levels can be regulated, we searched for their major biological determinants.
first, sequence analysis was performed to identify dna patterns which could be specifically associated with genes with high or low levels of ribosome occupancy or ribosome density, respectively.
we selected short nucleotide sequences  in the vicinity of the ribosome binding site and larger sections  containing the entire 5’utr .
according to their ribosome occupancy level, genes were first grouped into two sets containing the  <dig> genes with the highest  and the lowest  values, respectively.
in the nucleotide region from − <dig> to + <dig> relative to the start codon, we did not detect any discriminating sequence motif.
with enlarged nucleotide sequences , we detected, for high ribosome occupancies exclusively , a conserved dna pattern of  <dig> nucleotides whose sequence was atgacag.
the e-value of this pattern calculated with meme software  was significantly lower than that of the sequence ggagg, also identified as a conserved sequence in this subset of genes .
in l. lactis, when considering all gene sequences  the ggagg motif showed a e-value of  <dig>  x 10- <dig>  confirming its role as a shine-dalgarno sequence by base-pairing with its complementary sequence in the 3’end of l. lactis 16s rrna molecules.
<dig> genes of the high ribosome occupancy set had the atgacag sequence generally located upstream of aug, and  <dig> presented repeated patterns, including some inverted repeats suggesting formation of hairpin structures.
concerning the ribosome density level, no conserved dna pattern was found in the enlarged nucleotide region with genes of the first or fourth quartiles.
in order to identify other key systemic factors of translation, we used a more global approach and we statistically quantified the influence of various parameters related to gene, mrna and protein features on the two translatome variables.
all genome-scale data ) were obtained for l. lactis cells grown in identical standardized conditions in bioreactors to avoid introducing experimental artifacts linked to minor changes in growth conditions.
these omic data are indeed very sensitive to bacterial adaptation to changing conditions.
we used the statistical method of parameter selection developed previously
<cit> .
with this approach, we identified and classified the most significant parameters which could explain the different levels of translatome variables observed between all mrnas without any a priori subjective selection.
parameters included in the models were features related to gene , to mrna  and to protein sequence .
for this statistical modeling approach, the gene data set was reduced from  <dig> to  <dig> due to undetermined parameter values, mainly in the mrna half-life data set.
models of ribosome occupancy and ribosome density were obtained with adjusted squared-r equal to  <dig>  and  <dig> , respectively .
significant parameters were selected for both variables, and results were confirmed by simple linear regression analysis .
first, mrna half-lives had a moderate negative estimated coefficient with both translatome variables, with a p-value <  <dig>  x 10- <dig> .
the ribosome occupancy and ribosome density were also positively related to the folding energy in the vicinity of the start and stop codons of mrna  .
these results indicated that less stable mrnas and mrnas with less stable secondary structures in 5’ and 3’utr regions could be better translated with higher level of ribosome occupancy.
furthermore, for the majority of the l. lactis genes translationally limited at the initiation step, increased ribosome density could also reflect higher level of translation.
two interpretations can be proposed for the striking inverse relationship between mrna stability and both translatome variables.
since the most stable mrnas were shown in l. lactis to be those present at low concentrations
<cit> , we can hypothesize that the low ribosome occupancy of stable transcripts could result from a low meeting probability between low concentrated mrna molecules and ribosomes.
however, in our  <dig> gene set, the top 10% less concentrated mrna displayed, as expected, increased stability compared to the average , but their ribosome occupancy was only slightly decreased .
a more probable interpretation would be related to the presence of a specific pattern in the group of genes with high mrna stability and low translational variables.
we have previously reported in l. lactis that an over-representation of the purine-rich sequence aggag was present, as in bacillus, in the 5’utr of stable transcripts
<cit> .
a hypothesis could be that this sequence blocks translation by inducing ribosome stalling.
for  <dig> genes of our  <dig> gene set having more than one aggag sequence in their 5’utr , we observed, indeed, a stabilization of the transcript  compared to the average, associated with a significant reduction  of the ribosome density.
Δgdown and Δgup: free energy of the predicted minimum free energy structure for upstream mrna sequences  and downstream sequences , respectively.
ami: amino acid biosynthesis, cof: biosynthesis of cofactors, int: central intermediary metabolism, reg: regulatory functions, trs: transcription.
while the previous parameters exhibited additive influence on both translatome variables, some parameters lead to antagonistic effects.
the length of the coding sequence had a negative coefficient for ribosome density  and this conclusion was valid for the two main classes of mrnas  .
in contrast, cds length had a positive coefficient for ribosome occupancy.
since translation of most l. lactis genes is expected to be initiation-limited, higher ribosome density of small mrnas could counteract their lower proportion involved in translation.
similarly, mrna concentration and also cai showed a positive coefficient for ribosome occupancy  but were negatively correlated with ribosome density .
cai is assessing translation elongation efficiency as a function of genes’ codon usage.
this statistical result confirms what is generally assumed.
for genes with elongation-limited translation, high cai values correspond to fast translation and therefore low ribosome density.
protein amino acid composition also interfered with the translation process but only at the ribosome density level.
our modeling approach also allowed to analyze the relationship between gene function
<cit>  and translatome variables.
genes involved in regulatory functions possessed higher ribosome occupancy and ribosome density than the average .
these regulatory genes coded mainly for transcriptional regulators involved in specific metabolisms , pyrimidine , biotin  or sugar ) but also for general transcriptional regulators  and central regulatory protein .
on the other hand, genes related to synthesis of both amino acids and cofactors exhibited significantly lower ribosome density than the other functional categories.
translatome variables as determinants of protein concentration
next we aimed to determine the influence of translational regulations downstream in the gene expression process.
more particularly, we analyzed protein concentration variations in relation with their mrna ribosome occupancy and ribosome density values.
protein level is generally considered as the final result of translation.
the amount of protein present results from complex multilevel regulations, some being independent of translation .
thus, protein concentrations are difficult to predict
<cit> .
in l. lactis, some determinants of protein levels were identified by statistical modelling but without taking into account any translational parameters
<cit> .
here, we added the two translation variables to the previously analyzed parameters to quantify the involvement of translation in the control of protein levels.
we took again special care to use proteomic data
<cit>  acquired in l. lactis under our standardized conditions .
due to the low number of proteins identified in this growth condition, the data set was reduced to  <dig> genes.
our understanding of protein concentration determinants was improved upon inclusion of the translatome data .
the adjusted squared-r of the model slightly increased with the translatome data from  <dig>  to  <dig> .
however, we observed a key influence of the ribosome occupancy on protein levels, with a positive estimated coefficient of  <dig> .
like in the original model without translational parameters, cai  and mrna concentration  also made a significant contribution to the protein level .
model  is the original model from
<cit> .
model  contains the parameters as model  plus two new explanatory parameters, ribosome occupancy and ribosome density.
discussion
in this study we have provided, for the first time, a detailed and complete picture of translation in a bacterium.
translatome experiments allowed the determination of the translational status of each mrna including its ribosome occupancy and ribosome density.
in the bacterium under study, l. lactis, the fraction of mrnas engaged in translation was on average 67% and the mean ribosome density reached 2/ <dig> of the maximal theoretical density.
similar results have been obtained in yeast
<cit> , although notable differences in the translation process itself  and in its regulation  are present between yeast and bacteria.
our data and data obtained in yeast
<cit> , show that low ribosome density and incomplete mrnas engaged in translation are two general translational rules in both kingdoms.
the low ribosome density indicated for most l. lactis genes that translation is limited at the initiation step.
in this particular case, higher ribosome density would result in higher translation level.
we have demonstrated that the two translational variables, ribosome occupancy and ribosome density were not constant for all the genes examined here.
a high level of variability was indeed demonstrated within the mrna population, indicating that even if global translation seems to be conserved between microorganisms, this process is also highly transcript-specific.
we showed in addition that genes involved in key metabolic pathways exhibited coordinated regulation of ribosome density and ribosome occupancy levels.
this result suggests the possibility for l. lactis to use fine translation tuning of selected transcripts during its adaptation to the external environment .
we have also demonstrated that ribosome occupancy was a major determinan of protein level, revealing the high influence of translational regulations in the process coupling gene expression to protein synthesis.
by exploring the diversity of ribosome occupancy and ribosome density values within the mrna population, systemic key factors involved in translation level regulation were sought.
the systemic influence of some factors , previously identified as protein expression regulators of specific mrnas
<cit>  or predicted as determinants of translational efficiency by transcriptome-proteome correlation analyses
<cit>  was seen to act directly on translatome variability.
in addition, less expected general factors influencing translation, such as gene function, mrna half-life and gene length were identified and organized hierarchically.
contribution of these parameters to differentiated translational regulation in natural and stress conditions was also reported in yeast
<cit> .
linear covariance models established in l. lactis a link between translational efficiency and gene function.
first, genes involved in biosynthesis of amino acids and cofactors exhibited lower ribosome density than others.
l. lactis thus adapted its metabolism by attenuating translation of genes involved in these two anabolic pathways.
the chemically defined medium used in this study for growing the bacterium contains all the amino acids and vitaminic precursors of the cofactors.
in these growth conditions, l. lactis thus adapted its metabolism to ensure cofactor and amino acid supply via their import from external medium rather than via de novo synthesis.
this translational attenuation constitutes an adaptive response appropriate to limit energy wastage in the bacterium.
we have also observed high ribosome occupancy and high ribosome density for genes of the functional category “regulatory functions”.
important transcriptional regulators were concerned suggesting their efficient translation in vivo.
this finding illustrates the capacity of l. lactis cells to coordinate the control of the entire gene expression network through positive translational regulations of key regulators.
mechanisms of transcriptional control by translation have already been reported in e. coli.
for mrnas of some amino acid metabolic enzymes, ribosome deceleration leads to attenuation or antitermination of transcription
<cit> .
in a more general way, ribosome acceleration and deceleration were shown to directly result in corresponding changes in speed of the rna polymerase
<cit> .
in addition, an inverse relationship between mrna stability and both translatome variables was found.
as a consequence, less stable mrnas tended to exhibit rather efficient translation initiation.
for initiation-limited unstable genes, higher ribosome density would lead to higher level of translation.
mrna decay and translation would thus act in an antagonistic manner on gene expression regulation in l. lactis.
these results are in contrast with what was observed in yeast
<cit> .
a positive relationship between ribosome density and mrna half-life was expected in l. lactis, assuming the protective effect of ribosomes against rnases.
the absence of such a positive correlation indicates that protection of mrna against degradation should be more related to ribosome position than to ribosome density.
in b. subtilis, mrna protection by ribosomes located far upstream of cleavage sites  was previously demonstrated
<cit> .
translation in l. lactis was strongly dependent on coding sequence length, ribosome density being negatively correlated with gene length.
such an inverse correlation between ribosome density and gene length was also reported in yeast
<cit> .
it would be interesting to verify if an excess of ribosomes at the beginning of the coding sequence as reported in yeast
<cit>  is also present in l. lactis to explain why short mrnas tended to present higher ribosome density.
however, contrary to yeast
<cit> , a positive correlation was obtained between ribosome occupancy and gene length.
this correlation is probably necessary in bacteria to counteract the negative ribosome density effect in order to not penalize translation of long important mrnas.
in l. lactis the longest genes represent major functions involved for example in replication, energy metabolism or transport .
but more generally, in bacteria, rather long mrnas are expected in comparison to yeast as a consequence of numerous polycistronic structures.
using our translatome data, the genome-scale regulation of the initiation and elongation steps was analyzed.
mrna folding was identified as a key determinant of ribosome occupancy.
site-directed experiments at the initiation region have previously demonstrated in e. coli and in l. lactis the association between mrna folding and translation initiation efficiency
<cit> .
mrna folding is expected to interact with ribosome binding site accessibility and modify individual gene expression as demonstrated in e. coli <cit> .
in addition, for genes with high ribosome occupancy, we have identified a conserved nucleotide pattern  upstream of the start codon.
it is thus tempting to speculate that these sequence features could modulate ribosome binding.
specific translation-enhancing sequences upstream of the start codon have been reported in e. coli and proposed to act as additional interaction sites with either 16s rrna or s <dig> ribosomal protein
<cit> .
however, l. lactis 16s rrna does not contain a complementary sequence to our motif.
in addition, as in other gram-positive bacteria
<cit> , no homologous protein of the ribosomal s <dig> protein was found in l. lactis.
thus, mechanisms of enhancement of translation initiation based on improved mrna-rrna base pairing or on increased ribosomal protein-mrna interaction seem unlikely in l. lactis.
alternatively, the conserved nucleotide pattern found in l. lactis could be involved in secondary structure stability playing a role in translation initiation efficiency
<cit> .
in yeast, specific base usages around the start codon for genes with high ribosome occupancy were also reported
<cit> .
here two determinants of the initiation step were identified; they could be thus involved in the initiation limitation of translation of most of l. lactis genes.
nevertheless, two factors of the elongation step, codon usage and amino acid composition, were shown to influence translation in l. lactis .
cai is widely accepted as the key factor in the determination of the elongation rate
<cit> .
therefore, our results indicate that for some genes the elongation step could also contribute to translation control corroborating the current point of view of mixed control of translation by initiation and elongation
<cit> .
to further explore translation regulation, local mrna ribosome density could be explored via ribosome density mapping or ribosome footprint experiments
<cit> .
comparison of ribosome density in the 5’utr of mrnas exhibiting high or low ribosome occupancies could help to confirm the link between the sequence signatures identified in this work and efficient ribosome binding.
the determination of local ribosome densities within a coding sequence could contribute to a better understanding of the relationship between ribosome density and translation efficiency.
CONCLUSIONS
this study described a global analysis of translation-level regulation in a model bacterium .
studies describing the detailed translational status of an entire mrna population  are rare and were until this work only available in the yeast model.
for each mrna of l. lactis in the exponential growth phase, we have determined the number of ribosomes loaded as well as the proportion of mrna involved in translation.
a high variability in the translational status was demonstrated within the mrna population reflecting transcript-specific translational level regulation.
integration of translatome data with other relevant omic data  using statistical modeling let to the identification of key regulatory parameters required for efficient translation.
this approach significantly increased the knowledge of translational control in microorganisms and concurred in the understanding of how post-transcriptional regulation leads to weak correlations between transcriptomic and proteomic data.
the main conclusion of our work was that translational regulation plays, as transcriptional regulation, an important role in the control of protein expression levels.

 variations in protein concentrations are not solely related to transcriptional activity. this is also true in bacteria. recent studies with escherichia coli and desulfovibrio vulgaris described only moderate correlations between mrna levels and protein concentrations with a pearson coefficient varying from to according to culture conditions. in lactococcus lactis, a gram positive lactic acid bacterium, the pearson coefficient was even lower ranging from to. post-transcriptional events are proposed to be the principle causes of these weak correlations. in l. lactis, we identified protein abundance determinants by statistical modeling of proteome data: gene sequence features such as the codon adaptation index, gene length and functional categories were demonstrated to strongly influence mrna translation levels. in addition, individual mrna translation efficiency was estimated by a mechanistic modeling approach, highlighting significant variability in translation efficiencies among the different genes of the genome. consequently in l. lactis, not all mrnas are believed to be translated with the same efficacy and important regulations are expected to occur at the translational level. an experimental approach for studying genome scale translational regulation is translatome analysis which provides the detailed translational status of all mrnas. translation consists of three steps: initiation, elongation and termination. translation initiation occurs with a ribosome binding to the mrna template and begins at the start codon with incorporation of the first amino acid of the polypeptide. the ribosome continues to translate the coding sequence catalyzing polypeptide chain elongation until it reaches the stop codon. then, translation termination occurs and the complete polypeptide chain is released. translation is carried out by more than one ribosome simultaneously forming a polysomal structure. translatome experiments provide the ribosome number on each mrna molecule in the cell. translatome determination combines size separation of ribosome-mrna complexes according to the number of ribosomes loaded and measurement of mrna levels in fractions by microarray techniques. most translatomes reported so far aimed at identifying translationally regulated genes in response to stress or changing growth phase. however, these studies were based on low-resolution polysomal profile analysis in which messengers were classified into only two fractions: polysomal versus non-polysomal. in these conditions, the cellular translational status of each mrna molecule was not fully described since the number of loaded ribosomes was not quantified. however, this information is required to study the diversity of translational regulations between all mrna species present in a cell and then to understand translation efficiency of individual mrnas. to date, only a few studies described high resolution translatome analysis in microorganisms with mrnas classified with respect to the precise number of loaded ribosomes. unfortunately all high resolution translatome studies were carried out in yeast, leaving the understanding of detailed translational regulation in bacteria incomplete. therefore we present here the first high resolution translatome analysis in the bacterium l. lactis. this model lactic acid bacterium is a gram-positive non spore forming bacterium with a low gc content. l. lactis is therefore phylogenetically closer to bacillus subtilis than e. coli. l. lactis was grown under maximum growth rate conditions. the profile of mrna-ribosome association led to the definition of two translational variables for each mrna species: the fraction engaged in translation and the ribosome density. by comparing the translational states of all mrnas, the regulation of both ribosome occupancy and ribosome density levels were explored. in addition, the influence of ribosome occupancy and ribosome density on the final protein expression level was quantified. this demonstrated the key role of the mrna translational status in the complex processes linking gene expression to protein synthesis.  polysomal profile description the translatome of l. lactis was studied by coupling polysome profile determination, transcriptomics and statistical analyses. a typical polysome profile is shown in figure after peak assignment, pooling of the two first fractions corresponding to mrnas not engaged in translation and pooling of the last four fractions representing the most highly ribosome-loaded transcripts, the seven resulting fractions were hybridized to the microarrays b to h, respectively. genes were selected according to the cutoff criterion. figure shows sample distributions of mrna proportion between fractions b to h for six selected genes. when considering the entire gene set, 61% and 37% of genes exhibited the highest mrna abundance in fraction c or in fraction h, respectively. the number of ribosomes per transcript was one for fraction d, and in the range -, -, - and - for fractions e, f, g and h, respectively. a maximum of ribosomes per transcript was thus obtained in l. lactis cells grown in the exponential phase. the percentage of ribosomes engaged in translation was estimated by area integration of the polysomal profile and an average ratio of ± 2% of total ribosomes engaged in translation was obtained. since about one third of the total ribosomal content was not associated with mrna, ribosomal content would appear in excess for protein synthesis under the growth conditions used in this study. translatome variable determination and physiological analysis two translatome variables were determined, the ribosome occupancy corresponding to the fraction of mrna engaged in translation and the ribosome density, the number of loaded ribosomes per nucleotides. ribosome occupancy reflects the efficiency of translation initiation while the interpretation of ribosome density can vary according to the limiting step of translation. for genes exhibiting initiation-limited translation, high density is correlated with high translation levels, but for genes with elongation limitation, high density can result from ribosome congestion leading to low translation efficiency. ribosome occupancy values were obtained for genes, and a gaussian distribution was observed with a median value of ± 6%. high variability in ribosome occupancy was observed among genes with values ranging from 41% to 84%. therefore, for all genes at least part of the corresponding mrna molecules was involved in translation but never the entire mrna population. this suggests that for individual genes, the mrna concentration in exponentially-grown cells of l. lactis was in excess. thus under the conditions used, both ribosome availability and mrna abundancies could conceivably support higher protein synthesis. a peak fraction, corresponding to the most frequently found number of ribosomes bound on each mrna, was assigned to genes with a 95% bootstrap confidence interval. except one gene with a peak fraction in fraction f, all the other genes grouped into two classes of the peak fraction. the first class was composed of genes translated with a low number of ribosomes with a peak fraction in the monosome fraction d. the second class included more highly loaded genes with a peak fraction in fraction h, corresponding to - loaded ribosomes. we verified that the bacterial specificity of polycistronic structures did not introduce any bias in ribosome number determination. in l. lactis, genes have been experimentally confirmed in the literature as being organized as polycistronic operons. they were specifically examined to validate their eventual enrichment in the two subgroups of genes with peak fractions in d and h, respectively. no significant over-representation of these genes was observed. based on the peak fraction value, the second translatome variable, ribosome density, was calculated. for each of the genes, ribosome density was assessed as the ratio of the ribosome number in its peak fraction to the coding sequence length. if we consider that one bound bacterial ribosome protects around nucleotides, the theoretical maximal ribosome density is ribosomes / nucleotides. however, genes within the gene set exhibited experimental ribosome densities higher than. this gene subgroup was significantly enriched in short monocistronic genes. the presence of rather long 5’utrs could contribute to ribosome density over-estimation, even more pronounced in the case of short genes. this set of genes with aberrant ribosome densities was omitted in the subsequent analyses leading to a new set of genes. figure shows the distribution of ribosome density ranging from to ribosomes per nucleotides. the median ribosome density was ribosomes per nucleotides representing 2/ of the maximal theoretical ribosome density. in l. lactis, for most of the genes, ribosome density was thus far from the maximal density as also reported in yeast. therefore, as proposed in yeast, translation limitation appears to occur during ribosome loading on mrna in the initiation step even though ribosomes and mrna molecules were found in excess in l. lactis cells. with a strictly mathematical modeling approach of translation control such low ribosome densities were previously demonstrated in bacteria to be related to a limitation of translation at the initiation step. thus in this context of initiation-limited translation, high ribosome density is expected to provoke high translation levels for most of the l. lactis genes. homolactic lactic acid bacteria such as l. lactis are fermentative bacteria producing high concentrations of lactate during glucose catabolism. the lactate production ensures cofactor regeneration which is essential for bacterial activity but also induces the acidification of the ambient environment and thereby inhibition of bacterial growth. genes involved in important metabolic functional categories exhibited similar regulation of ribosome density and ribosome occupancy. in agreement with the high growth rate, l. lactis favoured the translation of specific functions required for optimal growth. a high glycolytic rate was favoured by the efficient translation of the first step of the glycolysis, the glucose transport system, which is believed to control the glycolysis rate in the studied il l. lactis strain. high ribosome occupancy and ribosome density were indeed observed for the main glucose transport system, mannose pts and glucokinase, which are required when glucose enters through sugar permease. the sugar permease system is still uncharacterised in l. lactis, however our finding of a high level of translation of the gene ypch coding for a sugar permease suggests the involvement of this permease in glucose transport. similarly, the last step of glycolysis, corresponding to conversion of pyruvate into lactate by lactate dehydrogenase, also corresponded to a very efficiently translated gene. in order to counteract growth inhibition by cytoplasmic acidification under conditions of extracellular auto-acidification, translation was also increased for almost all genes coding for the atpase system that catalyses the excretion of protons while consuming atp. this proton expulsion system was efficient enough to prevent a strong acid stress since secondary pathways of the acid stress response were not favoured at the translational level. genes of the arginine deiminase and glutamate decarboxylation pathways exhibited low values of ribosome occupancy and ribosome density in comparison to the average. in addition, positive regulation of translation of enzymes related to phosphate limitation, and to zinc and manganese transports were observed, suggesting additional cation and anion requirements to fight against lactic acid inhibition. determinants of translatome variables in order to identify how ribosome occupancy and ribosome density levels can be regulated, we searched for their major biological determinants. first, sequence analysis was performed to identify dna patterns which could be specifically associated with genes with high or low levels of ribosome occupancy or ribosome density, respectively. we selected short nucleotide sequences in the vicinity of the ribosome binding site and larger sections containing the entire 5’utr. according to their ribosome occupancy level, genes were first grouped into two sets containing the genes with the highest and the lowest values, respectively. in the nucleotide region from − to + relative to the start codon, we did not detect any discriminating sequence motif. with enlarged nucleotide sequences, we detected, for high ribosome occupancies exclusively, a conserved dna pattern of nucleotides whose sequence was atgacag. the e-value of this pattern calculated with meme software was significantly lower than that of the sequence ggagg, also identified as a conserved sequence in this subset of genes. in l. lactis, when considering all gene sequences the ggagg motif showed a e-value of x 10- confirming its role as a shine-dalgarno sequence by base-pairing with its complementary sequence in the 3’end of l. lactis 16s rrna molecules. genes of the high ribosome occupancy set had the atgacag sequence generally located upstream of aug, and presented repeated patterns, including some inverted repeats suggesting formation of hairpin structures. concerning the ribosome density level, no conserved dna pattern was found in the enlarged nucleotide region with genes of the first or fourth quartiles. in order to identify other key systemic factors of translation, we used a more global approach and we statistically quantified the influence of various parameters related to gene, mrna and protein features on the two translatome variables. all genome-scale data ) were obtained for l. lactis cells grown in identical standardized conditions in bioreactors to avoid introducing experimental artifacts linked to minor changes in growth conditions. these omic data are indeed very sensitive to bacterial adaptation to changing conditions. we used the statistical method of parameter selection developed previously. with this approach, we identified and classified the most significant parameters which could explain the different levels of translatome variables observed between all mrnas without any a priori subjective selection. parameters included in the models were features related to gene, to mrna and to protein sequence. for this statistical modeling approach, the gene data set was reduced from to due to undetermined parameter values, mainly in the mrna half-life data set. models of ribosome occupancy and ribosome density were obtained with adjusted squared-r equal to and, respectively. significant parameters were selected for both variables, and results were confirmed by simple linear regression analysis. first, mrna half-lives had a moderate negative estimated coefficient with both translatome variables, with a p-value < x 10-. the ribosome occupancy and ribosome density were also positively related to the folding energy in the vicinity of the start and stop codons of mrna. these results indicated that less stable mrnas and mrnas with less stable secondary structures in 5’ and 3’utr regions could be better translated with higher level of ribosome occupancy. furthermore, for the majority of the l. lactis genes translationally limited at the initiation step, increased ribosome density could also reflect higher level of translation. two interpretations can be proposed for the striking inverse relationship between mrna stability and both translatome variables. since the most stable mrnas were shown in l. lactis to be those present at low concentrations, we can hypothesize that the low ribosome occupancy of stable transcripts could result from a low meeting probability between low concentrated mrna molecules and ribosomes. however, in our gene set, the top 10% less concentrated mrna displayed, as expected, increased stability compared to the average, but their ribosome occupancy was only slightly decreased. a more probable interpretation would be related to the presence of a specific pattern in the group of genes with high mrna stability and low translational variables. we have previously reported in l. lactis that an over-representation of the purine-rich sequence aggag was present, as in bacillus, in the 5’utr of stable transcripts. a hypothesis could be that this sequence blocks translation by inducing ribosome stalling. for genes of our gene set having more than one aggag sequence in their 5’utr, we observed, indeed, a stabilization of the transcript compared to the average, associated with a significant reduction of the ribosome density. Δgdown and Δgup: free energy of the predicted minimum free energy structure for upstream mrna sequences and downstream sequences, respectively. ami: amino acid biosynthesis, cof: biosynthesis of cofactors, int: central intermediary metabolism, reg: regulatory functions, trs: transcription. while the previous parameters exhibited additive influence on both translatome variables, some parameters lead to antagonistic effects. the length of the coding sequence had a negative coefficient for ribosome density and this conclusion was valid for the two main classes of mrnas. in contrast, cds length had a positive coefficient for ribosome occupancy. since translation of most l. lactis genes is expected to be initiation-limited, higher ribosome density of small mrnas could counteract their lower proportion involved in translation. similarly, mrna concentration and also cai showed a positive coefficient for ribosome occupancy but were negatively correlated with ribosome density. cai is assessing translation elongation efficiency as a function of genes’ codon usage. this statistical result confirms what is generally assumed. for genes with elongation-limited translation, high cai values correspond to fast translation and therefore low ribosome density. protein amino acid composition also interfered with the translation process but only at the ribosome density level. our modeling approach also allowed to analyze the relationship between gene function and translatome variables. genes involved in regulatory functions possessed higher ribosome occupancy and ribosome density than the average. these regulatory genes coded mainly for transcriptional regulators involved in specific metabolisms, pyrimidine, biotin or sugar ) but also for general transcriptional regulators and central regulatory protein. on the other hand, genes related to synthesis of both amino acids and cofactors exhibited significantly lower ribosome density than the other functional categories. translatome variables as determinants of protein concentration next we aimed to determine the influence of translational regulations downstream in the gene expression process. more particularly, we analyzed protein concentration variations in relation with their mrna ribosome occupancy and ribosome density values. protein level is generally considered as the final result of translation. the amount of protein present results from complex multilevel regulations, some being independent of translation. thus, protein concentrations are difficult to predict. in l. lactis, some determinants of protein levels were identified by statistical modelling but without taking into account any translational parameters. here, we added the two translation variables to the previously analyzed parameters to quantify the involvement of translation in the control of protein levels. we took again special care to use proteomic data acquired in l. lactis under our standardized conditions. due to the low number of proteins identified in this growth condition, the data set was reduced to genes. our understanding of protein concentration determinants was improved upon inclusion of the translatome data. the adjusted squared-r of the model slightly increased with the translatome data from to. however, we observed a key influence of the ribosome occupancy on protein levels, with a positive estimated coefficient of. like in the original model without translational parameters, cai and mrna concentration also made a significant contribution to the protein level. model is the original model from. model contains the parameters as model plus two new explanatory parameters, ribosome occupancy and ribosome density. discussion in this study we have provided, for the first time, a detailed and complete picture of translation in a bacterium. translatome experiments allowed the determination of the translational status of each mrna including its ribosome occupancy and ribosome density. in the bacterium under study, l. lactis, the fraction of mrnas engaged in translation was on average 67% and the mean ribosome density reached 2/ of the maximal theoretical density. similar results have been obtained in yeast, although notable differences in the translation process itself and in its regulation are present between yeast and bacteria. our data and data obtained in yeast, show that low ribosome density and incomplete mrnas engaged in translation are two general translational rules in both kingdoms. the low ribosome density indicated for most l. lactis genes that translation is limited at the initiation step. in this particular case, higher ribosome density would result in higher translation level. we have demonstrated that the two translational variables, ribosome occupancy and ribosome density were not constant for all the genes examined here. a high level of variability was indeed demonstrated within the mrna population, indicating that even if global translation seems to be conserved between microorganisms, this process is also highly transcript-specific. we showed in addition that genes involved in key metabolic pathways exhibited coordinated regulation of ribosome density and ribosome occupancy levels. this result suggests the possibility for l. lactis to use fine translation tuning of selected transcripts during its adaptation to the external environment. we have also demonstrated that ribosome occupancy was a major determinan of protein level, revealing the high influence of translational regulations in the process coupling gene expression to protein synthesis. by exploring the diversity of ribosome occupancy and ribosome density values within the mrna population, systemic key factors involved in translation level regulation were sought. the systemic influence of some factors, previously identified as protein expression regulators of specific mrnas or predicted as determinants of translational efficiency by transcriptome-proteome correlation analyses was seen to act directly on translatome variability. in addition, less expected general factors influencing translation, such as gene function, mrna half-life and gene length were identified and organized hierarchically. contribution of these parameters to differentiated translational regulation in natural and stress conditions was also reported in yeast. linear covariance models established in l. lactis a link between translational efficiency and gene function. first, genes involved in biosynthesis of amino acids and cofactors exhibited lower ribosome density than others. l. lactis thus adapted its metabolism by attenuating translation of genes involved in these two anabolic pathways. the chemically defined medium used in this study for growing the bacterium contains all the amino acids and vitaminic precursors of the cofactors. in these growth conditions, l. lactis thus adapted its metabolism to ensure cofactor and amino acid supply via their import from external medium rather than via de novo synthesis. this translational attenuation constitutes an adaptive response appropriate to limit energy wastage in the bacterium. we have also observed high ribosome occupancy and high ribosome density for genes of the functional category “regulatory functions”. important transcriptional regulators were concerned suggesting their efficient translation in vivo. this finding illustrates the capacity of l. lactis cells to coordinate the control of the entire gene expression network through positive translational regulations of key regulators. mechanisms of transcriptional control by translation have already been reported in e. coli. for mrnas of some amino acid metabolic enzymes, ribosome deceleration leads to attenuation or antitermination of transcription. in a more general way, ribosome acceleration and deceleration were shown to directly result in corresponding changes in speed of the rna polymerase. in addition, an inverse relationship between mrna stability and both translatome variables was found. as a consequence, less stable mrnas tended to exhibit rather efficient translation initiation. for initiation-limited unstable genes, higher ribosome density would lead to higher level of translation. mrna decay and translation would thus act in an antagonistic manner on gene expression regulation in l. lactis. these results are in contrast with what was observed in yeast. a positive relationship between ribosome density and mrna half-life was expected in l. lactis, assuming the protective effect of ribosomes against rnases. the absence of such a positive correlation indicates that protection of mrna against degradation should be more related to ribosome position than to ribosome density. in b. subtilis, mrna protection by ribosomes located far upstream of cleavage sites was previously demonstrated. translation in l. lactis was strongly dependent on coding sequence length, ribosome density being negatively correlated with gene length. such an inverse correlation between ribosome density and gene length was also reported in yeast. it would be interesting to verify if an excess of ribosomes at the beginning of the coding sequence as reported in yeast is also present in l. lactis to explain why short mrnas tended to present higher ribosome density. however, contrary to yeast, a positive correlation was obtained between ribosome occupancy and gene length. this correlation is probably necessary in bacteria to counteract the negative ribosome density effect in order to not penalize translation of long important mrnas. in l. lactis the longest genes represent major functions involved for example in replication, energy metabolism or transport. but more generally, in bacteria, rather long mrnas are expected in comparison to yeast as a consequence of numerous polycistronic structures. using our translatome data, the genome-scale regulation of the initiation and elongation steps was analyzed. mrna folding was identified as a key determinant of ribosome occupancy. site-directed experiments at the initiation region have previously demonstrated in e. coli and in l. lactis the association between mrna folding and translation initiation efficiency. mrna folding is expected to interact with ribosome binding site accessibility and modify individual gene expression as demonstrated in e. coli. in addition, for genes with high ribosome occupancy, we have identified a conserved nucleotide pattern upstream of the start codon. it is thus tempting to speculate that these sequence features could modulate ribosome binding. specific translation-enhancing sequences upstream of the start codon have been reported in e. coli and proposed to act as additional interaction sites with either 16s rrna or s ribosomal protein. however, l. lactis 16s rrna does not contain a complementary sequence to our motif. in addition, as in other gram-positive bacteria, no homologous protein of the ribosomal s protein was found in l. lactis. thus, mechanisms of enhancement of translation initiation based on improved mrna-rrna base pairing or on increased ribosomal protein-mrna interaction seem unlikely in l. lactis. alternatively, the conserved nucleotide pattern found in l. lactis could be involved in secondary structure stability playing a role in translation initiation efficiency. in yeast, specific base usages around the start codon for genes with high ribosome occupancy were also reported. here two determinants of the initiation step were identified; they could be thus involved in the initiation limitation of translation of most of l. lactis genes. nevertheless, two factors of the elongation step, codon usage and amino acid composition, were shown to influence translation in l. lactis. cai is widely accepted as the key factor in the determination of the elongation rate. therefore, our results indicate that for some genes the elongation step could also contribute to translation control corroborating the current point of view of mixed control of translation by initiation and elongation. to further explore translation regulation, local mrna ribosome density could be explored via ribosome density mapping or ribosome footprint experiments. comparison of ribosome density in the 5’utr of mrnas exhibiting high or low ribosome occupancies could help to confirm the link between the sequence signatures identified in this work and efficient ribosome binding. the determination of local ribosome densities within a coding sequence could contribute to a better understanding of the relationship between ribosome density and translation efficiency.  this study described a global analysis of translation-level regulation in a model bacterium. studies describing the detailed translational status of an entire mrna population are rare and were until this work only available in the yeast model. for each mrna of l. lactis in the exponential growth phase, we have determined the number of ribosomes loaded as well as the proportion of mrna involved in translation. a high variability in the translational status was demonstrated within the mrna population reflecting transcript-specific translational level regulation. integration of translatome data with other relevant omic data using statistical modeling let to the identification of key regulatory parameters required for efficient translation. this approach significantly increased the knowledge of translational control in microorganisms and concurred in the understanding of how post-transcriptional regulation leads to weak correlations between transcriptomic and proteomic data. the main conclusion of our work was that translational regulation plays, as transcriptional regulation, an important role in the control of protein expression levels. 
gene function was a major translational regulatory determinant. the fraction of mrna engaged in translation and ribosome density were not constant for all genes. for most of the genes, the ribosome density was lower than the maximum value revealing major control of translation by initiation. both ribosome occupancy and ribosome density were particularly high for transcriptional regulators, demonstrating the positive role of translational regulation in the coordination of transcriptional networks. mrna stability was a negative regulatory factor of ribosome occupancy and ribosome density, suggesting antagonistic regulation of translation and mrna stability. we have demonstrated experimentally the high diversity of translational states allowing individual gene differentiation and the importance of translation-level regulation in the complex process linking gene expression to protein synthesis. results demonstrated that only part of the entire population of each mrna species was engaged in translation. furthermore, ribosome occupancy was identified as a key component of intracellular protein levels underlining the importance of translational regulation. in bacteria, the weak correlations at the genome scale between mrna and protein levels suggest that not all mrnas are translated with the same efficiency. this high degree of variability was analyzed by bioinformatics and statistical modeling in order to identify general rules of translational regulation. to experimentally explore mrna translational level regulation at the systemic level, the detailed translational status of all mrnas was measured in the model bacterium lactococcus lactis in exponential phase growth. we have determined, for the first time in a bacterium, the detailed translational status for all mrnas present in the cell.
 in bacteria, the weak correlations at the genome scale between mrna and protein levels suggest that not all mrnas are translated with the same efficiency. to experimentally explore mrna translational level regulation at the systemic level, the detailed translational status of all mrnas was measured in the model bacterium lactococcus lactis in exponential phase growth.  results demonstrated that only part of the entire population of each mrna species was engaged in translation. for transcripts involved in translation, the polysome size reached a maximum of ribosomes. the fraction of mrna engaged in translation and ribosome density were not constant for all genes. this high degree of variability was analyzed by bioinformatics and statistical modeling in order to identify general rules of translational regulation. for most of the genes, the ribosome density was lower than the maximum value revealing major control of translation by initiation. gene function was a major translational regulatory determinant. both ribosome occupancy and ribosome density were particularly high for transcriptional regulators, demonstrating the positive role of translational regulation in the coordination of transcriptional networks. mrna stability was a negative regulatory factor of ribosome occupancy and ribosome density, suggesting antagonistic regulation of translation and mrna stability. furthermore, ribosome occupancy was identified as a key component of intracellular protein levels underlining the importance of translational regulation.  we have determined, for the first time in a bacterium, the detailed translational status for all mrnas present in the cell. we have demonstrated experimentally the high diversity of translational states allowing individual gene differentiation and the importance of translation-level regulation in the complex process linking gene expression to protein synthesis. translational regulationmrna ribosometranslatomestatistical modelinglactococcus lactis 