extfieldsofstudy: Medicine Biology

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Accumulation of L-type Bovine Prions in Peripheral Nerve Tissues

We recently reported the intraspecies transmission of L-type atypical bovine spongiform encephalopathy (BSE). To clarify the peripheral pathogenesis of L-type BSE, we studied prion distribution in nerve and lymphoid tissues obtained from experimentally challenged cattle. As with classical BSE prions, L-type BSE prions accumulated in central and peripheral nerve tissues.

B ovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of cattle characterized by accumulation of a protease-resistant form of a normal cellular prion protein (PrPres) in the central nervous system. The scientifi c literature in general has assumed that BSE in cattle is caused by a uniform strain (classical BSE). However, different neuropathologic and molecular phenotypes of BSE (atypical BSEs) have recently been reported from various countries (1). Recent data from Western blot analyses of fi eld cases of atypical BSEs are characterized by a higher (H-type BSE) or lower (L-type BSE) molecular mass of the unglycosylated form of PrPres than is classical BSE (2). The origins of atypical BSEs remain obscure; unlike classical BSE, atypical BSE has been detected mainly in aged cattle and suggested as a possible sporadic form of BSE (3).
Several lines of evidence demonstrate that classical BSE and a variant form of Creutzfeldt-Jacob disease are most likely caused by the same agent (4,5). Transmission of classical BSE to humans has been proposed to result from ingestion of contaminated food. Whether atypical BSEs are transmissible to humans remains uncertain; however, human susceptibility to L-type BSEs is suggested by recent experimental transmission in primates (6) and mice transgenic for human prion protein (PrP) (7) by using the most effective route of intracerebral inoculations of prions. The L-type BSE prion is much more virulent in primates and in humanized mice than is the classical BSE prion, which suggests the possibility of zoonotic risk associated with the L-type BSE prion. These fi ndings emphasize the critical importance of understanding tissue distribution of L-type BSE prions in cattle because, among the current administrative measures for BSE controls, the specifi ed risk materials removal policy plays a crucial role in consumer protection.
In Japan, atypical BSE was detected in an aged Japanese Black cow (BSE/JP24) (8). We recently reported the successful transmission of BSE/JP24 prions to cattle and showed that the characteristics of these prions closely resemble those of L-type BSE prions found in Italy (9). In this study, we report the peripheral distribution of L-type BSE prions in experimentally challenged cattle.

The Study
The Animal Ethics Committee and Animal Care and Use Committee of the National Institute of Animal Health approved the study. Five Holstein calves 2-3 months of age were intracerebrally injected with 1 mL of 10% (w/v) brain homogenates prepared from the medulla oblongata of BSE/ JP24. In our earlier report, experimentally challenged cattle appeared to display clinical signs indicative of BSE at 11 months postinoculation (mpi) (9). Animals were sequentially euthanized before and after the onset of clinical signs (cattle identifi cation codes 8515 and 496 at 10 and 12 mpi, respectively) and at the terminal stage of the disease (cattle identifi cation codes 528, 1061, and 5566 at 16 mpi). A wide range of tissues was sampled at subsequent necropsy. We provisionally categorized the adrenal gland as nerve tissue because of the presence of chromaffi n cells in the medulla of the gland.
Western blot analysis for PrPres was performed on obex tissue samples as described previously by using anti-PrP monoclonal antibody T2 (9). PrPres was detectable in all obex samples obtained 10, 12, and 16 mpi, suggesting that transmission of L-type BSE prions to these animals was successful. Dilution of the protease-treated brain sample and analysis of Western blot results showed that the detection threshold for PrPres was 1.25 μg of brain tissue equivalent (data not shown).
A variety of nerve and lymphoid tissue samples were investigated for accumulation of PrPres by Western blot analysis by using phosphotungstic acid precipitation, as described previously (10) Figure 1. In cattle at the preclinical stage, PrPres was detectable in all tested ganglia and barely detectable in the vagus nerve and vagosympathic trunk. In cattle at the terminal stage, PrPres was barely detectable in the forelimb nerves (suprascapular nerve, brachial nerve plexus, median nerve, and radial nerve), whereas substantial amounts of PrPres were present in other nerve tissues except for facial and hypoglossal nerves (Table). A broader nerve tissue distribution of PrPres was observed in cattle at 16 mpi than at 10 and 12 mpi. Contrary to what we found in nerve tissues, we detected no PrPres from tests performed on lymphoid tissues obtained from any of the 5 cattle studied. Infectivity of selected nerve tissues (including the obex, sciatic nerve, adrenal gland, brachial nerve plexus, and vagus nerve) obtained from cattle euthanized at 10, 12, and 16 mpi (codes 8515, 498, and 5566, respectively) was analyzed by intracerebral injection into mice transgenic for bovine prion protein, as described previously (11). As a negative control, mice were injected with cells from the brainstem of a normal cow. The presence of PrPres in the brains of all mice used in the experiment was determined by Western blot analysis. Infectivity was detected in all nerve tissues tested, regardless of the presence of detectable PrPres (Figure 2). Control mice showed no apparent abnormality >500 days postinoculation.

Conclusions
We report accumulation of L-type atypical BSE prions in peripheral nerve tissues sampled from intracerebrally chal-lenged cattle. Our study demonstrated that almost all of the peripheral nerve tissues tested became PrPres positive in a time-dependent manner, whereas no PrPres was detectable in lymphoid tissues, even in cattle with fatal atypical BSE. Our results suggest the possibility that, like classical BSE prions, L-type BSE prions propagated in the central nervous system and were spread centrifugally by nerve pathways (11,12). In   Italy, L-type BSE prions have been characterized in detail by using cattle challenged intracerebrally. However, PrPres was not detected in their peripheral tissues, including the peripheral nerves (13). The reason for the discrepancy in PrPres detection is unclear. In view of the similarities between the Ltype and BSE/JP24 prion characteristics (9), this discrepancy may result from differences in the methods used for PrPres detection.
We detected infectivity in the nerve tissue samples (including samples from the obex, sciatic nerve, adrenal gland, brachial nerve plexus, and vagus nerve) obtained 10, 12, and 16 mpi. On the basis of the incubation time of 223 ± 25 (mean ± SD) days in mice injected with a 1,000fold dilution of the obex homogenate, infectious titers in peripheral nerve tissues appeared to be 1,000 × lower than those estimated in the obex during endpoint titration of infectivity.
Our results demonstrate that L-type atypical BSE prions can be distributed in the peripheral nerve tissues of intracerebrally challenged cattle. These fi ndings are useful for understanding L-type BSE pathogenesis and accurately assessing the risks associated with this disease. Investigations of prion distribution in cattle that have been orally challenged with L-type BSE prions are critical.


extfieldsofstudy: Biology Medicine

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Genetic Analysis of Central Carbon Metabolism Unveils an Amino Acid Substitution That Alters Maize NAD-Dependent Isocitrate Dehydrogenase Activity

Background Central carbon metabolism (CCM) is a fundamental component of life. The participating genes and enzymes are thought to be structurally and functionally conserved across and within species. Association mapping utilizes a rich history of mutation and recombination to achieve high resolution mapping. Therefore, applying association mapping in maize (Zea mays ssp. mays), the most diverse model crop species, to study the genetics of CCM is a particularly attractive system. Methodology/Principal Findings We used a maize diversity panel to test the CCM functional conservation. We found heritable variation in enzyme activity for every enzyme tested. One of these enzymes was the NAD-dependent isocitrate dehydrogenase (IDH, E.C. 1.1.1.41), in which we identified a novel amino-acid substitution in a phylogenetically conserved site. Using candidate gene association mapping, we identified that this non-synonymous polymorphism was associated with IDH activity variation. The proposed mechanism for the IDH activity variation includes additional components regulating protein level. With the comparison of sequences from maize and teosinte (Zea mays ssp. Parviglumis), the maize wild ancestor, we found that some CCM genes had also been targeted for selection during maize domestication. Conclusions/Significance Our results demonstrate the efficacy of association mapping for dissecting natural variation in primary metabolic pathways. The considerable genetic diversity observed in maize CCM genes underlies heritable phenotypic variation in enzyme activities and can be useful to identify putative functional sites.

Introduction
Glycolysis and the tricarboxylic acid (TCA) cycle, known as CCM, are responsible for the production of accessible energy and the creation of primary building blocks of other metabolisms. Therefore, CCM is critical to plant growth and development. IDH is a TCA cycle enzyme that produces 2-oxoglutarate required by the glutamine synthetase and glutamate synthase cycle for nitrogen assimilation. To study the genetics of CCM, the main focus to date has been on scoring gene expression [1,2] or metabolite levels [3,4,5]. For technical reasons, relatively little attention has been paid to adding protein levels or enzyme activities. However, there is evidence that protein levels and enzyme activities are more heritable because they integrate over time, in a manner analogous to whole plant phenotypes [6]. In this study we adopted a robot-based platform to simultaneously measure multiple enzyme activities [6] in order to study the genetics of CCM enzyme activities in maize.
Linkage analysis and association mapping are two of the most commonly used tools for dissecting complex quantitative traits, known as quantitative trait loci (QTL) mapping. In plants, metabolic QTL has been reported in the studies in tomato [4], Arabidopsis [7,8] and maize [9,10,11,12,13,14]. These studies use biparental mapping populations (F 2 , RILs, NILs etc.) that obviously capture only a limited fraction of the variation and do not have sufficient resolution to identify candidate polymorphisms within structural genes. The other drawback lies in the fact that dissecting a quantitative trait down to a gene level using traditional linkage populations could be a long, challenging, and tedious process [15].
Previously, association mapping was used mainly in human genetics, but it is now becoming a common complement to biparental crosses in the genetic mapping of quantitative traits in plants [16,17,18]. Among the advantages of this approach are the ability to analyze diverse germplasm that represents multiple alleles for each locus, as well as the utilization of a rich history of mutation and recombination to achieve high resolution mapping.
Maize, a major crop worldwide, is a particularly attractive system for association mapping. Studies have shown that in maize, the advantages of association mapping can be efficiently exploited for starch metabolism in kernels [19,20,21], maysin [22], and carotenoids [23]. The enzymes from CCM are conserved at the molecular and functional levels. The availability of extensive genetic diversity is crucial when studying conserved or selected pathways [19], and maize meets this availability requirement because it is the most diverse model crop species. In addition, the unique pattern of linkage disequilibrium (LD) in maize, where LD decays within only a few kb in a population of diverse inbreds, makes it suitable for LDbased, high-resolution association mapping [24].
Searching for the signatures of domestication-related selection among CCM gene sequences will provide useful information to reveal fundamental metabolic properties. In the fish Fundulus heteroclitus, clinal variation in lactate dehydrogenase (Ldh, EC1.1.1.27) allozyme allele frequency along the environmental gradient has been found, with the LdhB b allele type having greater catalytic efficiency at low temperatures and the LdhB a allele type having greater catalytic efficiency at high temperatures [25,26]. Similar studies were also conducted in alcohol dehydrogenase (Adh, EC 1.1.1.1) for Drosophila [27,28]. Clinal variation in allozyme allele frequency along an environmental gradient is often taken as strong, indirect evidence for natural selection. Wright et al. [29] analyzed single-nucleotide polymorphisms (SNPs) in 774 genes in maize and indicated that 2 to 4% of these genes experienced artificial selection. In central starch production in the kernel of maize, four of six genes were selection targets [19]. The proportion of CCM genes under selection still remains to be revealed.
In this project, we used a maize diversity panel and accessions of teosinte to conduct the genetic analysis of CCM. We showed here the presence of substantial genetic variation among various CCM genes. Using association mapping, we identified that this variation in one case is related to heritable enzyme activity variation. We also evaluated selection at CCM loci by examining nucleotide diversity in maize and in its wild ancestor, teosinte. We demonstrated that association mapping combined with other approaches can be an efficient way to dissect natural variation in conserved metabolic pathways.

Activities of CCM Enzymes Are Heritable and Co-regulated
We tested if the polymorphisms within CCM genes were associated with variation in the corresponding enzyme activities. A preliminary panel of eight diverse maize inbred lines, grown in five replications, was used to assess the activity variation across nine CCM enzymes. We listed the mean and standard error of activities of the nine CCM enzymes in Table S1. The results showed that all nine enzymes tested from the glycolytic and TCA pathways had substantial differences in activity and significant genetic effects in leaf samples ( Table 1). The extent of such heritable activity variation is surprising for core metabolic enzymes, and might indicate the occurrence of selection during adaptation. Positive correlations were found between most enzyme activities ( Table 2), suggesting some form of co-regulation acting on these enzymes. Therefore, we also analyzed the residual variation not controlled by co-regulation (i.e. normalized). The normalized heritabilities were still high (Table 1). Based on these analyses and follow-up studies, we focused on IDH, which had the most significant heritable variation.

Phe/Tyr 209 Polymorphism Is Associated with the IDH Activity
Since previous studies showed that activities of Ldh and Adh are affected by both genetic and environmental differences and their Broad-sense heritability, the proportion of the genetic variation from the total phenotypic variation (s 2 G /(s 2 G +s 2 E )). d Normalized heritability was calculated in the same way as broad-sense heritability but using normalized enzyme activity values that are the difference between each individual standardized activity value of a sample and the mean standardized activity of each sample across the 10 enzymes. doi:10.1371/journal.pone.0009991.t001 G X E interaction in fish Fundulus heteroclitus [25,26] and Drosophila [27,28], respectively, we wanted to see if the same pattern applies to IDH. Therefore, we conducted IDH activity assay at 25uC and at 35uC in 100 diverse inbred lines. However, while the temperature effect on IDH activity was highly significant, there was no genotypic interaction with temperature ( Figure S1). Initial association analysis on the two maize IDH paralogs indicated that variation in only one of the paralogs associated with enzyme activity while the second paralog did not (data not shown). Four overlapping regions, covering approximately 3.5 kb of genomic sequence from the IDH gene (the significant paralog), were sequenced across the 288 inbred line set. An additional 0.75 kb region was sequenced across the 100 line subset. This extensive sequence analysis detected 68 SNPs and 17 INDELs with minor allele frequencies (MAF) .0.1 ( Figure 1A). The pattern of LD within the idh gene decayed relatively quickly (average r 2 drops below 0.1 within ,2 kb), but there were some haplotypes with a longer range LD of 4 kb ( Figure 1B). In general, this panel provided gene to subgene resolution for this region of the genome. Mixed-model association tests that controlled for population structure were performed for all 68 SNPs and 17 INDELs with MAF .0.1 against IDH activity. One of the major concerns in association mapping experiments is the possible detection of nonfunctional, spurious associations resulting from population stratification [30]. Although there are several methods to control for population structure in association mapping that were also applied here [31], it is still useful to estimate the extent of false positives using a null distribution of association tests P-values from a random set of markers. Comparison of the P-values obtained from SNPs in a candidate gene to the distribution of P-values from random markers can put the candidate SNPs' P-values in a more realistic perspective, irrespective to the method that was used to control for population structure. Figure 2A shows the distribution of P-values for 553 random SNPs tested against IDH activity together with the P-value obtained for IDH SNP3593 under the same rigorous statistic test. The set of 553 random markers that are used here are not expected to capture true associations due to the fast-decaying LD structure in our population. Indeed, the distribution of P-values for the 553 random SNP was not different from the expected null distribution.
The association analyses showed that thirteen SNPs and five INDELs from idh were significant at P,10 -4 ( Figure 1A), which greatly exceeded the significance levels of 553 SNPs in a random panel ( Figure 2A). Only one of the significant SNPs (idh SNP3593) encoded a non-synonymous polymorphism, resulting in a tyrosine (TAC) (polar amino acid) to phenylalanine (TTC) (non-polar amino acid) substitution (Phe/Tyr 209 ). The effect of this SNP on IDH activity was 32% at 25uC and 25% at 35uC (R 2 SNP = 0.11, P = 10 25 , Figure 2B).
Since all 13 SNPs and 5 INDELs in idh showed a similar magnitude of effect, we inspected their LD in an attempt to determine whether they were likely to represent one or more functional sites. The r 2 between almost every pair of significant SNPs was higher than 0.4 ( Figure 1C). Only a limited number of recombinations were found, creating three major haplotypes and several rare ones. While this LD and haplotype structure restricted the mapping resolution, it was consistent with the SNPs being in LD with one causative polymorphism. Therefore, we hypothesized that Phe/Tyr 209 is most likely the functional SNP. Further statistical support was provided by the finding that none of the other significant SNPs improved the R 2 value when tested in a stepwise model alongside Phe/Tyr 209 .
To further investigate this QTL we tested the effect of the Phe/ Tyr 209 SNP on IDH activity at 25uC in a separate experiment across 26 diverse inbreds and their F 1 hybrids, with 3 different testers from different germplasm groups. Combined two-way ANOVA revealed significant Phe/Tyr 209 and genetic background effects (p = 0.0017 and p,0.0001, respectively), but a non-significant Phe/Tyr 209 by genetic background interaction ( Figure 3B). A significant Phe/Tyr 209 effect was found across the inbreds (p = 0.005) and across hybrids with the Mo17 tester (p = 0.03, Figure 3A). A similar trend (p = 0.1) was found across the CML323 hybrids. However, the effect in B73 hybrids was not significant, suggesting a weak interaction. These results confirm the basic results and suggest that while background has an effect on this gene, there is no statistical interaction with that background.
Normalization of the IDH activity data by genetic background allowed us to test the mode of action of Phe/Tyr 209 across this diverse sample. The mode of action was not significantly different from additive; the heterozygote value fell between both homozygous classes, and the dominance effect was not significant (Table 3 and 4).

IDH Protein Quantity Also Contributes to the IDH Activity Variation
The Tyr 209 residue was completely conserved in idh orthologs in other sequenced plant species ( Figure 4A), as were most of the surrounding residues in this region. Alignment of the maize IDH protein with those of other organisms for which the IDH crystal structures have been defined allowed us to predict that Phe/Tyr 209 is located on the protein surface, but not in the active site [32].
To address the biochemical mode of action of this quantitative trait nucleotide (QTN), we quantified IDH protein content for a subset of 35 selected lines carrying different alleles at Phe/Tyr 209 , using quantitative scanning of immunoblotted gels. IDH protein quantity was significantly different between the two Phe/Tyr 209 alleles (P = 8.3610 27 ). There was a positive correlation between protein quantity and enzyme activity (R = 0.72; Figure 2C). This result suggests that at least some of the variation in total IDH activity is explained by differences in IDH protein quantity.
We re-tested the Phe/Tyr 209 effect on IDH activity after normalizing the activity for IDH protein quantity. The SNP effect on the normalized specific activity was still significant (P = 3.6610 27 ), suggesting that at least two independent factors contribute to the variation in total activity. A stepwise regression where both protein content and Phe/Tyr 209 were used as factors in the model against IDH total activity confirmed that both independently contribute to the activity variation, with the Phe/ Tyr 209 effect being stronger (Table 5).
Based on these results we suggest a two-factor model to explain the variation in IDH total activity; the first component is most likely an element regulating IDH protein level and the second component is Phe/Tyr 209 , which most likely affects enzymatic properties including the specific activity.

Low-activity Allele (Phe 209 ) of IDH Is More Frequent in Domesticated Maize
The Phe/Tyr 209 residue segregates in maize and also in its wild ancestor, teosinte ( Figure 4B). The frequency of the tyrosine allele is still high among teosinte accessions (Freq of Tyr = 80%, N = 16) but is substantially reduced among maize inbreds (Freq of Tyr = 30%, N = 267; P(x 2 ) = 0.004) ( Figure 4B). No significant difference in allele frequency was found between tropical and nontropical maize germplasm (data not shown). This significant shift in allele frequency between teosinte and maize could be a result of selection. Our data therefore indicate that the low-activity allele (Phe) was favored among domesticated maize. A negative correlation between IDH activity and plant biomass was also identified in a separate experiment across 60 diverse maize hybrids in five replications (R = 20.46, P = 0.0002) suggesting that reduced IDH activity in leaves can have a positive effect on growth rate and, thus, can be a target for indirect selection.

Some CCM Genes Had Been Targeted for Selection during Maize Domestication
In addition to directly testing for associations between polymorphisms within CCM genes with enzyme activities, another way to reveal fundamental metabolic properties is to search for the signatures of domestication-related selection among CCM gene sequences, as previously shown across a large set of random genes in maize [29]. Here, we sequenced 17 CCM loci across 28 diverse maize inbred lines and 16 teosinte accessions and examined nucleotide diversity in maize relative to that in teosinte ( Table 6). The pi-maize vs. pi-teosinte ratio for CCM genes was compared with that of random genes [29] (Table 6).
The results showed that CCM genes were within normal range of bottleneck associated domestication. One of our 17 CCM genes, hexokinase (hex), was also included in the 774 random genes and is one of the 30 selected genes [29]. However, we did not find selection signature on it. The discrepancy could be explained by the different regions sequenced and different maize lines and teosinte accessions used. Another CCM gene, aconitate hydratase (aconitase, aco), which is a different paralog from the one we studied in the 17 CCM genes, is also one of the 30 selected genes [29] though we did not find artificial selection signature on the paralog we studied. Therefore, like other genes being selected, some CCM genes had also been targeted for selection during maize domestication.
In addition, we found the presence of teosinte-specific nonsynonymous (results not shown) SNPs that can be tested for their association with enzyme activity. Such an approach can be a direct way to identify domestication-related metabolic QTNs.

Discussion
In this study, we used an association-mapping strategy that utilizes diverse, multi-allelic germplasm in a single mapping experiment. For conserved metabolic pathways such as glycolysis and the TCA cycle, such an approach is necessary in order to capture enough functional diversity within candidate genes. We were able to zoom-in to the gene level and identify a putative functional SNP that is associated with variation in a metabolic quantitative trait.
We found that CCM enzyme activities were positively correlated, therefore suggesting some form of co-regulation acting on these enzymes. Moreover, CCM enzyme activities showed great genetic effects and relatively high heritabilites. Similar results were also shown in other studies [7,8,33]. In addition, enzyme activities may have simpler mode of genetic control than other complex quantitative traits [7]. Therefore, enzyme activities were mappable traits, ideal for studying the mechanism of underlying QTL [7].
In this study, we identified a novel amino-acid substitution in a phylogenetically conserved site (Phe/Tyr 209 ) which was strongly associated with changes in IDH activity. Given its conservation across plants, this amino acid is extremely likely to play an important role in the enzyme. Similar large amino acid changes are responsible for phenotypic variation in sweet corn [19,21]. Moreover, we found that IDH protein quantity also contributed to the IDH activity. Since SNP3593 resulting in Phe/Tyr 209 is in LD with the potential transcription related site SNP10 ( Figure 1C), it is possible that the protein level difference is due to polymorphisms in the promoter that are in LD with SNP3593. To test this hypothesis, we need to look at the transcription levels and sequence cis-regulatory regions (such as the promoter region) to see if the transcript abundance is correlated with protein levels and if the functional sequence variations in cis-regulatory regions are in LD with SNP3593. However, predicting protein quantity from transcriptional levels is not that straightforward sometimes because of post-transcriptional, translational or post-translational modification. For example, Gibon et al. [6] found that there was no relation between the amplitudes of the diurnal changes of transcript and enzyme activity for many Arabidopsis enzymes. Nevertheless, neither IDH protein quantity nor Phe/Tyr 209 alone could account for all the IDH activity. Therefore, we proposed a two-factor model to explain the variation in IDH total activity: an element regulating IDH protein level and a second element, Phe/ Tyr 209 , affecting enzymatic properties.
The SNPs significantly associated with IDH activity are in LD and span across the whole gene. Similar allele series are present in the maize Lycopene Epsilon Cyclase (lcyE) for carotenoid metabolism [23]. Fine mapping within the 4.8 kb of idh is still very challenging, but teosinte introgression (Flint-Garcia in prep), teosinte association [34,35], or landraces will provide more recombination to evaluate these biochemical hypotheses. Recently, the Maize Diversity Project (www.panzea.org) developed the maize nested association mapping (NAM) panel for dissecting complex quantitative traits with high resolution and statistical power [36]. The NAM design integrates linkage and association approaches, by using a population that is comprised of 5000 recombinant inbred lines (RIL) derived from crosses between B73 and 25 highly diverse maize lines (200 RILs for each cross) [36]. In the NAM panel, there are nine of 25 populations segregating for the idh SNP3593 and its associated haplotype (data not shown). Therefore, it's possible to use the NAM panel to dissect the IDH activity.
While Wright et al. (2005) [29] found that 2 to 4% of 774 maize genes experienced artificial selection and Whitt et al. (2002) [19]    showed that four of six genes in the central starch production pathway in maize kernels were targets of selection, our study showed that some CCM genes had also been targeted for selection during maize domestication process. We detected significant Phe/ Tyr 209 allele frequency shift in idh between teosinte and maize. The low-activity idh allele (Phe) was favored among domesticated maize and a negative correlation between IDH activity and plant biomass was also identified. Previous studies in tomato showed that increased plant growth as a result of reduced activities of two other TCA enzymes: mitochondrial malate-dehydrogenase [37] and aconitase [38]. The negative correlation between IDH activity and plant growth may be due to increase in photosynthesis, a compensation for the reduction in energy production by respiration [37] or ''over'' compensation by other isoforms of IDH. Though we found no significant G x E effects for IDH activity in the present study ( Figure S1), the reason that CCM genes were targets of selection might still be because of changing temperatures and/or a centralization of the source-sink relationships during domestication. Temperatures in Mexico differ greatly from environments throughout North America and South America. Unlike teosinte, which has long lateral branches terminated by male tassels, maize concentrates all its resources in one inflorescence, and maize has little competition. This suggests that if all CCM enzyme activities parallel IDH, maize CCM activity may be reduced relative to teosinte. The maize panel used in this study represents a considerable portion of the genetic diversity found in the domesticated maize breeding germplasm [24]. Our results have demonstrated that association mapping is an efficient way to study the genetics of CCM and suggested that there is still enough genetic diversity and phenotypic variation among those enzyme activities and gene sequences in maize. With emerging and anticipated improvements in proteomics and enzymatic measurements for throughput and accuracy, along with robust genotyping platforms, scientists will soon be able to use natural variation to better understand the genetics of metabolism and the importance of metabolism to plant growth and development.

Plant Materials and Green-house Experiment
For the enzymatic measurements, a preliminary panel of eight diverse maize inbred lines, grown in five replications, was used to assess the phenotypic variation across nine CCM enzymes   (Table 1). A subset of 100 inbred lines was selected from the core 300-line association-panel [24] for association mapping. Line selection was based on genotypic data from 553 random SNPs in order to capture maximal genetic diversity and was conducted using the PowerMarker software [39]. The hybrid experiment included 26 diverse inbreds that were selected from the core set to capture maximal genetic diversity. Each of these inbreds was crossed with 3 different testers (B73, Mo17 and CML323) to create F1 hybrids. For enzymatic measurements, plants were grown in five replications in cell-packs in the green-house in a completely randomized design. Three seeds from each line were sown in each cell and thinned five days after germination to one plant per cell to ensure uniform germination across the experiment. Tissue was collected from the youngest expanded leaf 35 days after germination and immediately frozen in liquid nitrogen. Tissue was stored at -80uC until analysis.

Enzymatic Assays
The activities of the nine CCM enzymes in Table 1 (their pathway locations demonstrated in Figure S2) [42]. Extracts, as well as NADH standards ranging from 0 to 1 nmol, freshly prepared in an extraction buffer [6] containing 20% (v/v) glycerol, 1% (v/v) Triton-X100 and 0.5 mM dithiothreitol were incubated in a medium containing: 50 mM MOPS/KOH pH 7.5; 5 mM MgSO 4; and 2 mM NAD + . The reaction was started by the addition of isocitrate to a final concentration of 0 (blank) or 4 mM ('maximal' activity). The reaction was stopped with 20 ml of 0.5 M NaOH. NADH was then determined as in Gibon et al. [6]. For the 100 inbred line panel each sample was divided and measured for enzymatic activity under two incubation temperatures (25uC and 35uC) to address the presence of G6E interaction. Measurements were performed in 96-well plates where each sample was measured for V max and V blank in the same plate. Each plate contained 40 experimental samples, as well as 4 common reference samples and 4 blank samples for normalization of the data. Raw enzyme activity data was standardized in two ways: 1) by plate mean; and 2) by common reference samples mean. Both methods yielded similar quality of data standardization.

Protein Quantification
Rabbit antibodies raised against IDH were generously provided by Dr. Michael Hodges (IBP Orsay, France). Protein extraction of IDH for blotting and procedures for gels were performed as in Hendriks et al. [43], except dithiothreitol was added to the sample buffer. Quantification was performed with a Li-Cor Odyssey.

Sequence Analyses
Sequencing and SNP discovery were conducted as previously described by Whitt and Buckler [44]. One or more amplicons (400-1600 bp each) was sequenced in each of the 17 selected CCM loci across 28 diverse maize inbred lines and 16 teosinte accessions (scheme of the corresponding enzymes of the selected genes and their pathway locations demonstrated in Figure S2 and EMBL/GenBank accession numbers of the amplicons listed in File S1). Four overlap regions, a, c, d, and f, covering approximately 3.5 kb of genomic sequence from the IDH gene were sequenced across the 288 inbred line set selected from the core 300-line association-panel [24], and an additional 0.75 kb region, e, was sequenced across the 100 lines subset ( Figure 1A). The 1.2 kb region, b, was first sequenced across four lines and then genotyped across the 288 inbred line set.
Pairwise LD for idh was calculated as r 2 between all SNPs pairs ( Figure 1B), and nucleotide diversity, p, the average number of nucleotide differences per site between two sequences, for CCM genes was estimated using the TASSEL software [45].

Statistical Analyses
In order to evaluate the proportion of the co-regulation factor in the observed enzymatic activity variation, we calculated the heritability of the average standardized activity across the nine enzymes. The genetic variation for this overall mean-activity was highly significant and explained 53% of the total phenotypic variation ( Table 1), indicating that co-regulation is a major component in the observed enzyme activity variation. In order to determine the remaining enzyme-specific variation (which is the relevant variation for the candidate-gene association mapping), we calculated the normalized, enzyme-specific activity for each enzyme as the difference between the standardized enzyme activity and the overall mean-activity.
All association tests were carried out using TASSEL software [45]. We used the mixed-linear model (MLM) function that accounts for different levels of relatedness and controls the type I error rates as described by Yu et al. [31]. Population structure was also included in the association analyses to eliminate nonfunctional, spurious associations resulting from population stratification [30]. Trait heritabilities, ANOVAs and Pearson correlations were calculated using JMP5.1 software (SAS institute).

Supporting Information
Table S1 Activities of nine CCM enzymes for eight different maize inbred lines in five replications.


text:
Comparative transcriptomics and proteomics of p-hydroxybenzoate producing Pseudomonas putida S12: novel responses and implications for strain improvement

A transcriptomics and proteomics approach was employed to study the expression changes associated with p-hydroxybenzoate production by the engineered Pseudomonas putida strain S12palB1. To establish p-hydroxybenzoate production, phenylalanine-tyrosine ammonia lyase (pal/tal) was introduced to connect the tyrosine biosynthetic and p-coumarate degradation pathways. In agreement with the efficient p-hydroxybenzoate production, the tyrosine biosynthetic and p-coumarate catabolic pathways were upregulated. Also many transporters were differentially expressed, one of which—a previously uncharacterized multidrug efflux transporter with locus tags PP1271-PP1273—was found to be associated with p-hydroxybenzoate export. In addition to tyrosine biosynthesis, also tyrosine degradative pathways were upregulated. Eliminating the most prominent of these resulted in a 22% p-hydroxybenzoate yield improvement. Remarkably, the upregulation of genes contributing to p-hydroxybenzoate formation was much higher in glucose than in glycerol-cultured cells. Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2626-z) contains supplementary material, which is available to authorized users.

Introduction
The aromatic compound p-hydroxybenzoate is a building block of liquid crystal polymers, high performance plastics that are employed in electronic devices such as mobile phones. At present, such compounds are derived from fossil resources, but the increasing demand for 'green' polymers and resins requires other approaches to the synthesis of the building blocks that constitute these materials. Biological production from renewable feedstock is a very attractive option, although the hydrophobic nature of aromatic compounds makes specific demands on the production host. Therefore, we have developed a solvent-tolerant bacterium, Pseudomonas putida S12, as a host for the biological production of aromatic compounds from renewable resources (Verhoef et al. 2007;Nijkamp et al. 2005Nijkamp et al. , 2007Verhoef et al. 2009;Wierckx et al. 2005). This organism can tolerate a second phase of aromatic and aliphatic hydrophobic compounds such as toluene and 1octanol, which property makes this organism an excellent platform host for the production of toxic hydrophobic compounds.
For the production of the tyrosine-derived aromatics phenol, p-hydroxystyrene and p-hydroxybenzoate, we have exploited P. putida S12_427 as a platform strain ( Fig. 1; Verhoef et al. 2007Verhoef et al. , 2009Wierckx et al. 2008). Strain S12_427 was obtained by subjecting wild-type P. putida S12 to a series of random and targeted optimization steps, key elements in which were the overexpression of the aroF-1 encoded class-I DAHP synthase, random mutagenesis and antimetabolite screening ( Fig. 1; Wierckx et al. 2005). Transcriptomics and metabolic flux analyses on P. putida S12TPL3, a phenol producing strain based on P. putida S12_427, indicated that the metabolic flux towards tyrosine was significantly improved (Wierckx et al. 2008. For the production of p-hydroxybenzoate, tyrosine biosynthesis in P. putida S12_427 was connected to the endogenous p-coumarate catabolic pathway by introducing the pal/tal gene from Rhodosporidium toruloides ( Fig. 1; Verhoef et al. 2007). This gene encodes phenylalanine/ tyrosine ammonia lyase (Pal/Tal) that converts tyrosine into p-coumarate. The resulting synthetic pathway could be employed to accumulate the p-coumarate catabolic intermediate p-hydroxybenzoate, after eliminating the pobA gene encoding p-hydroxybenzoate hydroxylase. Figure 3 presents an overview of p-hydroxybenzoate biosynthesis in the resulting p-hydroxybenzoate producing strain, P. putida S12palB1 (Verhoef et al. 2007).
In the present study, we employed a transcriptomics and proteomics approach to study the expression changes associated with p-hydroxybenzoate production by P. putida S12palB1. Thus, insight could be provided into the effects brought about by p-hydroxybenzoate accumulation, in conjunction with the impact from the mostly non-targetedstrain improvement strategy used to obtain the parent strain, P. putida S12_427. Based on these insights, new strategies for further targeted-strain improvement may be designed. Special attention was focused on the pathways directly involved in p-hydroxybenzoate production, i.e., aromatic amino acid metabolism and p-coumarate degradation. Although previous research suggested no major differences between p-hydroxybenzoate production on glucose or glycerol (Verhoef et al. 2007), substrate specific effects at the transcript and proteome level cannot be excluded. Especially in view of the artificial connection that was made between an anabolic and a catabolic pathway to achieve p-hydroxybenzoate production, carbon source dependent regulatory effects were anticipated. The optimized p-hydroxybenzoate production strain P. putida S12palB1 was therefore compared to non-optimized control strains on glucose as well as on glycerol.

Bacterial strains, plasmids and culture conditions
The strains and plasmids used in this study are listed in Table 1. The media used were Luria broth (LB; Sambrook et al. 1982) and a phosphate buffered minimal medium adapted from a previously described medium (Verhoef et al. 2007) containing 3.88 g l −1 K 2 HPO 4 and 1.63 g l −1 NaH 2 PO 4 . In minimal media, 40 mM of glycerol (MMGly) or 20 mM of glucose (MMG) were used as the sole carbon source unless stated otherwise. Antibiotics were added as required to the media at the following concentrations: gentamicin, 10 µg ml −1 (MMG and MMGly) or 25 µg ml −1 (LB); tetracycline, 10 µg ml −1 (Escherichia coli) or 60 µg ml −1 (P. putida S12). The expression of the introduced copy of aroF-1 as well as the expression of P. putida S12 P. putida S12_427 P. putida S12palB1 P. putida S12palB2 P. putida S12palB3 P. putida S12TPL3 (Wierckx et al. 2005) ΔoprB-1  Fig. 1 Genealogy of aromatics producing mutants of P. putida S12. The right hand column shows how each successive mutant was obtained. oprB-1 porin B, aroF-1 class-I DAHP synthase, pobA p-hydroxybenzoate hydroxylase, hpd 4-hydroxyphenylpyruvate dioxygenase, PP1271-PP1273 multidrug efflux MFS transporter, pal/tal phenylalanine/tyrosine ammonia lyase, tpl tyrosine-phenol lyase, NTG N-methyl-N'-nitro-Nnitrosoguanidine, MFP r resistant to 100 mg/l m-fluoro-DL-phenylalanine, MFT r resistant to 100 mg/l m-fluoro-L-tyrosine the multidrug efflux major facilitator superfamily (MFS) transporter in the pBNTmfs vector was induced by addition of 0.1 mM of sodium salicylate. Shake flask experiments were performed in 250-ml Erlenmeyer flasks containing 50 ml of MMG or MMGly in a horizontal shaking incubator at 30°C. Cultures were inoculated to a starting optical density at 600 nm (OD 600 ) of 0.2 with cells from a preculture on MMG or MMGly.
Carbon-limited chemostat cultivations were performed as described previously (Verhoef et al. 2007) using minimal medium containing either 10 mM glucose or 20 mM glycerol, 10 mg l −1 gentamicin and 0.1 mM sodium salicylate, in 1-l fermentors with a BioFlo110 controller (New Brunswick Scientific, Nijmegen, The Netherlands). Chemostats were inoculated with a 35-ml inoculum of a late-log phase preculture on MMGly or MMG. The dilution rate (D) was set at 0.05 h −1 for 16 h, after which it was increased to 0.1 h −1 . The cultures were considered to be at steady state when no significant changes were measured in cell density, stirring speed and, when applicable, phydroxybenzoate concentration after at least five volume changes at D=0.1 h −1 .
Sampling for microarray and 2D-DIGE analysis Samples were drawn for microarray and two dimensional difference-in-gel-electrophoresis (2D-DIGE) analysis from chemostat cultures at steady state conditions. OD 600 was measured and aromatics and carbon source concentrations were analyzed in the sample supernatants. For each condition (Table 3), three separate chemostat cultivations were performed. For microarray analysis, samples were quenched in ice-cold methanol and centrifuged. The pellet was covered by 1 ml RNAlater (Ambion, Foster City, CA,  (Hartmans et al. 1990) P. putida S12_427 b P. putida S12 with an enhanced tyrosine production (Wierckx et al. 2008) P. putida S12_427 pJTTpal P. putida S12 427 containing plasmid pJTTpal This study P. putida S12palB1 pobA knockout strain derived from P. putida S12 427 containing plasmid pJTTpal (Verhoef et al. 2007) P. putida S12B2 pobA and hpd knockout strain derived from P. putida S12 427 This study P. putida S12palB2 P. putida S12B2 containing plasmid pJT'Tpal This study P. putida S12pal_mfsB2 P. putida S12B2 containing plasmids pJT'Tpal and pBNTmfs This study P. putida S12B3 Multidrug efflux MFS transporter (PP1271-PP1273) knockout strain derived from P. putida S12B2 This study P. putida S12palB3 P. putida S12B3 containing plasmid pJT'Tpal This study P. putida S12pal_mfsB3 P. putida S12palB3 containing plasmids pJT'Tpal and pBNTmfs This study P. putida S12 pJTTpal P. putida S12 containing plasmid pJTTpal (Nijkamp et al. 2005) P. putida S12 pJNNmcs(t) P. putida S12 containing plasmid pJNNmcs(t) (Wierckx et al. 2005 This study a Ap r , Gm r and Tc r , ampicillin, gentamicin and tetracycline resistance, respectively b Previously known as P. putida S12 TPL3c c Previously known as pTacpal USA) and incubated for at least 1 h at 4°C, after which the RNAlater was removed. The pellet was flash frozen in liquid nitrogen and stored at−80°C. Samples for proteomics analysis were centrifuged at 4,000 rpm for 20 min at 2°C. The pellet was flash frozen in liquid nitrogen and stored at−80°C.
Messenger RNA isolation and cDNA preparation for microarray analysis Messenger RNA was isolated from the frozen pellets as described by Wierckx et al. (Wierckx et al. 2008). Random priming, cDNA synthesis, purification, fragmentation, and labeling were performed according to the microarray manufacturer's instructions (Affymetrix, Santa Clara, CA, USA).

Microarray analysis
High-density custom microarrays based on the genome of P. putida KT2440 with additional probe sets were used for transcriptomics analysis (Wierckx et al. 2008;). The end-labeled cDNA fragments were hybridized to the microarray according to standard manufacturer's protocols. Hybridized microarrays were scanned by Serv-iceXS (Leiden, The Netherlands) on a high resolution Gene Chip Scanner 3000 7G system with autoloader (Affymetrix, Santa Clara, CA, USA) using standard default analysis settings (filter: 570 nm; pixel size: 2.5 μm). The resulting microarray data (.cel files) were imported into Genespring GX software package version 7.3.1 (Agilent Technologies, Santa Clara, CA, USA) using the GC RMA algorithm. After normalization (signals below 0.01 were taken as 0.01; per chip: normalize to 50th percentile; per gene: normalize to specific sample), probesets representing intergenic regions and control genes were removed, as well as nonchanging genes (between 0.667-and 1.334-fold change). On the resulting set of differentially expressed genes a one-way ANOVA test (parametric test, p value of 0.05) was applied. Genes that were differentially expressed by at least a factor 2.5 were selected for further analysis.

Protein extraction
Protein extracts were prepared as described by Wijte et al. (Wijte et al. 2006). In brief, cells were boiled for 10 min in a Tris-SDS solution (0.2% SDS, 0.028 M Tris-HCl, 0.022 Tris-base, and 0.2 M DTT) and subsequently a DNase/ RNase treatment was given. Proteins were then extracted by the methanol/chloroform method. The protein pellet was solubilized in a solution with 8-M urea, 4% CHAPS and 30 mM Tris and sonicated on ice (4 ×5 s). Protein concentrations were determined with the RC/DC assay (BioRad, Veenendaal, The Netherlands).

2D gel electrophoresis
Proteomics analysis was performed using the 2D-DIGE method, according to Volkers et al. (Volkers et al. 2006) and Wijte et al. (Wijte et al. 2006). Protein samples were labeled according to the manufacturers' protocol (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) with CyDyes Cy3 and Cy5. The standard sample, consisting of a mixture of equal amounts of all samples in the experiment, was labeled with Cy2. First dimension electrophoresis was performed according to the manufacturers' protocol (GE Healthcare). In short, the labeled proteins were applied to a 24-cm Immobiline Dry-Strip pH 4-7 and rehydrated overnight or applied with cup-loading to a pre-hydrated 18-cm Dry-Strip pH 6-11. Iso-electric focusing was carried out using an IPGphor for a total of 60,750 Vh for the pH 4-7 strip and 30,750 Vh for the pH 6-11 strip. Prior to the second dimension electrophoresis, the strips were refocused for a total of 2,500 Vh. After that, the strips were incubated in equilibration buffer containing 1% (w/v) DTT for 15 min, followed by a 15-min incubation in equilibration buffer containing 2.5% (w/v) iodoacetamide. After equilibration, the strips were placed on top of 12.5% polyacrylamide labcast gels (pH 4-7) or 15% polyacrylamide lab-cast gels (pH 6-11) and sealed with a 1% agarose solution containing a trace of bromophenol blue. Gels were run for 1 h at 1 W per gel followed by 13 W per gel until the bromophenol blue had migrated to the bottom of the gel.

Image acquisition and data analysis
The gels were scanned using a Typhoon 9400 Imager at 100 µm resolution, according to the manufacturers' protocol (GE Healthcare). Data analysis was performed using DeCyder 2D Software version 6.5 with the DeCyder Extended Data Analysis module version 1.0 (both GE Healthcare). After one-way analysis of variance (student's t test with a p value of 0.05), significantly changed proteins were selected that had an average fold-change difference of at least 1.5 between the conditions tested.

In-gel digestion and protein identification
Preparative polyacrylamide gels were prepared following the protocol for analytical gels described above, with the following changes: only the Cy2-labeled standard was applied and after scanning the gels were stained with ProteomIQ Blue according to the manufacturer's instructions (Proteome Systems, Woburn, MA, USA). After excision, the proteins spots were digested in-gel with trypsin, using the protocol described by Havlis et al. (Havlis et al. 2003). Protein identification was performed by matrix-assisted laser desorption ionization mass spec-trometry and microliquid chromatography electrospray tandem mass spectrometry, as described previously (Volkers et al. 2006).

Analytical methods
Cell densities were determined at 600 nm with an Ultrospec 10 cell density meter (GE Healthcare). An OD 600 of 1 corresponds to 0.49 g l −1 of cell dry weight (CDW). The aromatic compounds were analyzed by HPLC (Agilent 1100 system) using a Zorbax 3.5 µm SB-C18 column (4.6× 50 mm) and a diode-array detector. As the eluent, a linear gradient of acetonitrile in KH 2 PO 4 -buffer (50 mM, pH 2) with 1% acetonitrile was used, increasing from 0-25% in 4.9 min at a flow of 1.5 ml min −1 . Glucose, gluconic acid and 2-ketogluconic acid were analyzed as described by Meijnen et al. (Meijnen et al. 2008). Glycerol was analyzed by ion chromatography (Dionex ICS3000 system), using an IonPac ICE AS1 column with 100 mM methyl sulphonic acid as the eluent at a flow of 1.2 ml min −1 .

Targeted gene disruption
The previously employed gene replacement vectors pJQpobA::tetA (Verhoef et al. 2007) and pJQhpd::tetA (Wierckx et al. 2008) were modified by replacing the tetA marker by the same gene flanked by loxP recombination sites (Sauer and Henderson 1988;Sternberg and Hamilton 1981) yielding pJQpobA::tetAloxP and pJQhpd::tetAloxP (Table 1). For construction of the gene replacement vector pJQmfs::tetAloxP, two parts of the multidrug efflux MFS transporter genes (locus tag PP1271-PP1273) were amplified by polymerase chain reaction (PCR) using primers NW23-NW26 (Table 2). The resulting PCR products mfs1 (5′ end) and mfs2 (3′ end) were digested with NotI/XbaI and XhoI/XbaI, respectively. Suicide plasmid pJQ200SK (Quandt and Hynes 1993) was digested with NotI and XhoI. The three resulting DNA fragments were ligated to yield pJQmfs. pJQmfs was linearized with XbaI and treated with bacterial alkaline phosphatase. The XbaI fragment from pSKtetlox (unpublished data) containing the loxP flanked tetA marker (Sauer and Henderson 1988;Sternberg and Hamilton 1981) was ligated into XbaI-digested pJQmfs yielding pJQmfs::tetAloxP. Targeted gene disruptions were performed as described by Verhoef et al. (Verhoef et al. 2007). The loxP-flanked tetA marker was removed by site-specific recombination after introducing pJNTcre (unpublished data) and induction of the Cre recombinase with 0.1 mM Na-salicylate. Colonies that were tetracycline sensitive were selected and plasmid pJNTcre was removed by overnight culturing in non-selective Luria broth. Replacement of the native gene by the disrupted copy was confirmed by a colony PCR.

Construction of expression plasmids
Plasmid pBNTmcs(Gm r ) was constructed as follows. The chloramphenicol (Cm) marker from pBBR1mcs (Kovach et al. 1995) was amplified by PCR using the primers Cm_F-AvaI and Cm_R_MluI (Table 2). The resulting fragment was ligated into the pJNTmcs (Meijnen et al. 2008) vector using the restriction sites MluI and Kpn2I (compatible with AvaI). From this vector, the salicylate-inducible NagR/ pNagAa promoter, tac ribosomal binding site and Cm marker were amplified using primers RV165 and Cm_R_MluI (Table 2) and digested with Kpn2I and XmajI. The resulting fragment was ligated in a Kpn2I and XbaI (compatible with XmajI) digested pBBR1mcs vector, yielding pBNTmcs(Cm r ). The Cm marker was replaced by a gentamicin (Gm) marker. The Gm marker of pJNNmcs (previously known as pTN-1 (Wierckx et al. 2005)) was obtained by PCR using primers KN34 and KN30 (Table 2) and cloned into the PagI and NcoI restriction sites of pBNTmcs(Cm r ), yielding pBNTmcs(Gm r ).
For constructing the MFS-transporter expression plasmid pBNTmfs(Gm r ), the multidrug efflux MFS transporter (PP1271-PP1273) was amplified by PCR on genomic DNA of P. putida S12 using oligonucleotide primers KN48 and KN49 ( Table 2). The resulting fragment was digested with KpnI and NotI and ligated into KpnI-NotIdigested pBNTmcs(Gm r ).
Plasmid pJT'Tmcs was constructed by digesting pJTTmcs (Meijnen et al. 2008) with HindIII and BamHI and the resulting fragment was treated with S1 nuclease, according to manufacturer's instructions. The nuclease treated vector was ligated, yielding pJT'Tmcs. For constructing pJT'Tpal, the pal gene was obtained from pJTTpal as a KpnI-NotI fragment and purified from agarose gel. The purified fragment was ligated into KpnI-NotIdigested pJT'Tmcs, yielding pJT'Tpal.

Results
General expression features of p-hydroxybenzoate producing P. putida S12palB1 P. putida S12palB1 was cultured in carbon limited chemostats with either glycerol or glucose as the carbon source. In agreement with previous observations (Verhoef et al. 2007), the carbon source had no major effect on product formation: on either substrate comparable concentrations of p-hydroxybenzoate and t-cinnamate were produced, at a similar product-to-substrate yield (Y ps ; Table 3). The biomass density as well as the biomass-to-substrate yield was slightly higher on glycerol (Table 3; data not shown). As control cultures, wild-type P. putida S12 variant strains were cultivated in chemostats and harbored either an empty expression plasmid (pJNNmcs(t); (Wierckx et al. 2008)), or the Pal/Tal expression plasmid (pJTTpal; Nijkamp et al. 2007; Table 3). Samples were drawn from the chemostat cultivations for transcriptomics and proteomics analysis. Comparisons were made between the optimized p-hydroxybenzoate producing strain P. putida S12palB1 and the non-optimized control strains, on either glucose or glycerol as the carbon source. Thus, the responses underlying the improved performance of the platform strain P. putida S12_427, as well as the impact of metabolic engineering on strain S12_427 to generate the p-hydroxybenzoate producing strain P. putida S12palB1 (Fig. 1), could be assessed for either carbon source.
Remarkably, the carbon source employed appeared to have a major effect on global transcript levels in the p-hydroxybenzoate producing P. putida S12palB1. With glucose as the carbon source, 215 genes were differentially expressed (fold change >2.5) in P. putida S12palB1 compared to the control strain, whereas only 88 genes were differentially expressed on glycerol. Proteome analysis showed a similar trend with 285 differentially expressed spots (fold change >1.5) in glucose-grown P. putida S12palB1 compared to the control strain, vs. 110 spots in the glycerol-grown cultures. From the glucose cultures, 93 out of the 285 spots were analyzed, representing 60 different proteins. Of the 110 differentially expressed spots of the glycerol cultures, 39 were analyzed representing 26 unique proteins.
The distribution of the differentially expressed genes and proteins over the different Clusters of Orthologous Groups (COG) categories (based on the NCBI-COG database of P. putida KT2440 (http://www.ncbi.nlm.nih.gov/sutils/coxik. cgi?gi=266)) was very similar for glucose and glycerolgrown P. putida S12palB1 (Fig. 2a, b). Nearly all COG groups were represented. About one quarter of the differentially expressed genes and proteins were annotated as having a hypothetical (including unknowns) or general function only. Another 25% could be assigned to amino acid transport and metabolism. A similar response had been observed previously in the phenol-producing P. putida S12TPL3. This was proposed to relate to the selection procedure by which the parent strain P. putida S12_427  CDW cell dry weight, Y ps p-hydroxybenzoate to substrate yield in C-mol p-hydroxybenzoate per C-mol substrate (C-mol%) a Average of three chemostat cultures, errors represent the standard deviation was obtained, i.e., resistance to fluoroanalogs of the amino acids phenylalanine and tyrosine (Wierckx et al. 2005). In addition, the COG groups of transcription and translation were well-represented, indicative of a substantial overall phenotypic difference between the p-hydroxybenzoate production strain and the control strain. The slight overrepresentation of the COG groups 'energy production and conversion' and 'signal transduction' were likely to relate to a solvent stress response, relating to the accumulation of phydroxybenzoate ( Fig. 2; Park et al. 1998Park et al. , 2001).
Specific expression features of P. putida S12palB1 relating to p-hydroxybenzoate production A number of differentially expressed genes and proteins in P. putida S12palB1 could be clearly linked to improved p-hydroxybenzoate production. These were classified into three functional groups: (1) aromatic amino acid biosynthesis and p-coumarate catabolism (i.e., the synthetic pathway of p-hydroxybenzoate biosynthesis), (2) aromatic amino acid catabolism, and (3) transport systems (Table 4; Fig. 3). A complete overview of differentially expressed genes and proteins is provided in Table 1 of the supplemental data.

Aromatic amino acid biosynthesis and p-coumarate degradation
In P. putida S12palB1, five genes of the tyrosine biosynthetic pathway were upregulated, among which the aroF-1 gene that was present in an additional copy in this strain (Table 4). Moreover, the tryptophan biosynthetic pathway was found to be upregulated, both at the transcript level (trpCDGE) and at the protein level (TrpCDGE as well as TrpA; Table 4). These findings are in good agreement with previous results in phenol producing P. putida S12TPL3 (Wierckx et al. 2008) as well as with the improved phydroxybenzoate biosynthesis. The upregulation of the aromatic amino acid biosynthetic genes in general appeared to be somewhat lower on glycerol compared to glucose (by a factor 1.5-3). An exception was the dramatic effect observed for phhAB, the upregulation of which was lower by a factor 20-30 on glycerol-grown P. putida S12palB1 compared to glucose-grown cells.
In addition to the aromatic amino acid biosynthetic genes, all genes involved in the conversion of pcoumarate into p-hydroxybenzoate (Mitra et al. 1999;Jimenez et al. 2002;Venturi et al. 1998) were upregulated compared to the control strains (Table 4). Again, the level of upregulation of these genes was apparently carbonsource dependent, being lower by an order of magnitude in the glycerol-grown cells compared to the glucose-grown cultures.

Tyrosine degradation pathways
In addition to the tyrosine biosynthetic genes, genes involved in the degradation of tyrosine were upregulated (Table 4, Fig. 3), although exclusively in glucose-grown cultures. The tyrosine decarboxylase-encoding gene PP2552 was upregulated 48-fold in glucose-grown P. putida S12palB1. However, no accumulation of tyramine was observed. Considering that tyramine is a dead-end product, the contribution of tyrosine decarboxylase to tyrosine catabolism appeared to be limited. As suggested earlier for phenol-producing P. putida S12TPL3 (Wierckx et al. 2008 Fig. 2 Global response on transcriptome and proteome level between the control strain and P. putida S12palB1 on glucose (a) and glycerol (b). Percentages of differentially expressed genes (fold changes of at least 2.5) and proteins (fold change of at least 1.5) are given per COG group 0.65 2.4±0.1 c a Fold change in expression level of P. putida S12palB1 compared to P. putida S12 pJTTpal on glycerol as substrate; values below 1 represent down regulation and values above 1 represent upregulation in P. putida S12palB1 b Fold change in expression level of P. putida S12palB1 compared to P. putida S12 pJNNmcs(t) on glucose as substrate; values below 1 represent down regulation and values above 1 represent upregulation in P. putida S12palB1 c Average of all spots per protein, errors represent the maximum deviation from the mean. See supplemental data for values per spot d Below threshold homogentisate pathway of which the upregulated hpd gene encodes the first enzyme, i.e., 4-hydroxyphenylpyruvate dioxygenase.

Transport systems
The extracellular accumulation of p-hydroxybenzoate in cultures of P. putida S12palB1 is likely to involve cellular transport systems. This is supported by the upregulation of numerous transport systems in P. putida S12palB1 compared to the control strains. Among these were a multidrug efflux MFS transporter (PP1271-PP1273) and a wide range of (aromatic) amino acid transporters ( Table 4). The nature of the carbon source employed apparently also affected the expression of these transporters. The upregulation of the MFS transporter genes PP1271-1273 showed little carbon-substrate dependency, being slightly lower on glycerol than on glucose. For the amino acid transporter-encoding genes, much larger upregulation was observed depending on the carbon source used. Remarkably at the protein level, even downregulation of some amino acid transporters was observed in glycerol-grown P. putida S12palB1 compared to the control strain, as opposed to the glucose-grown cultures ( Table 4).
Improvement of p-hydroxybenzoate production by targeted deletion of the hpd gene We previously demonstrated that the addition of tyrosine to chemostat-cultivated P. putida S12palB1 resulted in improved p-hydroxybenzoate titers, indicating that product formation was limited by the availability of tyrosine.  Fig. 3 Schematic overview of differentially expressed genes involved in relevant pathways of p-hydroxybenzoate biosynthesis, as derived from transcriptomics analysis of P. putida S12palB1. Gene names are indicated in italic or when absent, indicated by locus tags from P. putida KT2440. Red lines indicate upregulation and green lines indicate downregulation in P. putida S12palB1. The text boxes describe the proposed role in the enhanced p-hydroxybenzoate production of the differentially expressed genes However, part of the added tyrosine was consumed to generate biomass indicating that p-hydroxybenzoate production competed for tyrosine with other pathways (Verhoef et al. 2007). The upregulation of genes involved in tyrosine degradation as observed in the present study is in agreement with these observations. As argued above, tyrosine is probably mainly degraded via the homogentisate pathway. Therefore, the hpd gene was disrupted in P. putida S12palB1 to improve tyrosine availability.
The resulting strain, P. putida S12palB2, showed a marked improvement of p-hydroxybenzoate production characteristics in shake flask cultures with glucose as the carbon source. Product titers increased from 1.8 to 2.3 mM compared to strain S12palB1, at a similar final biomass content (Fig. 4a, b). The Y ps improved from 10.5 C-mol% for strain S12palB1 to 13.4 C-mol% for stain S12palB2, i.e., a relative yield improvement of 22%. Also, the productivity showed considerable improvement: the overall specific production rate q p increased from 1.6 to 2.3 μmol p-hydroxybenzoate (g CDW min) −1 , while the maximum specific production rate (q p,max ) increased from 3.8 to 4.4 μmol p-hydroxybenzoate (g CDW min) −1 . Maximal q p was achieved between 10 and 12 h of cultivation, in line with the observed transient accumulation of p-coumarate (Fig. 4a, b). The latter is likely to relate to transient glucose repression of the fcs gene, which immediately affected the p-coumarate pool (Fig. 3).
Interestingly, when P. putida S12palB2 was cultured on glycerol, p-hydroxybenzoate production was also improved. The product-to-substrate yield showed a relative increase of 21%, even though hpd was only slightly upregulated (below threshold of 2.5-fold) in glycerolgrown P. putida S12palB1. Transient accumulation of p-coumarate did not occur on glycerol since fcs was not repressed (unpublished data). Thus, disrupting the hpd gene clearly had a stimulating effect on p-hydroxybenzoate production. Fig. 4 Growth and production of p-hydroxybenzoate of P. putida S12palB1 (a), P. putida S12palB2 (b), P. putida S12palB3 (c), P. putida S12B3 (d), P. putida S12pal_mfsB3 (e) and P. putida S12pal_mfsB2 (f) in mineral medium with 20 mM glucose as carbon source in shake flask cultures. p-hydroxybenzoate (square), p-coumarate (triangle) and CDW (cell dry weight; diamond). The data are averages of triplicate experiments. The maximum variation between the triplicates was less than 10% Export of p-hydroxybenzoate in P. putida S12palB1 Transcriptome and proteome analysis in the present study showed extensive differential expression of cellular transport systems in P. putida S12palB1. Among the transporters identified, the multidrug efflux MFS transporter (P. putida KT2440 locus tags PP1271-PP1273) was upregulated on both glucose and glycerol, with only minor variation between the two carbon substrates. Since p-hydroxybenzoate accumulation was comparable in glucose and glycerolgrown cultures, the MFS transporter was considered as a putative p-hydroxybenzoate exporter in P. putida S12. To assess the role of this transporter in p-hydroxybenzoate transport, all three ORFs were deleted in P. putida S12palB2, yielding strain P. putida S12palB3. The inactivation of the MFS transporter resulted in slightly impaired growth and significantly lower p-hydroxybenzoate titers, although production was not completely eliminated (Fig. 4c). When the Pal/Tal expression plasmid pJTTpal was removed from P. putida S12palB3, resulting in the non-producing strain P. putida S12B3, normal growth was restored (Fig. 4d). Thus, the growth impairment observed in P. putida S12palB3 must be attributed to a combined effect of the transporter knock-out and intracellular p-hydroxybenzoate production.
Complementation of the MFS transporter in P. putida S12palB3 restored normal growth as well as production of p-hydroxybenzoate (Fig. 4e). These results suggest that the MFS transporter PP1271-1273 plays an important role in p-hydroxybenzoate export. Overexpression of the MFS transporter in P. putida S12palB2 did not result in improved p-hydroxybenzoate production (Fig. 4f). Since the complementation experiment indicated (Fig. 4e) that the MFS transporter was functionally expressed the MFS transporter clearly is not a limiting factor for p-hydroxybenzoate production.

Discussion
In the present study, the changes in transcript and protein levels underlying and affecting efficient p-hydroxybenzoate biosynthesis by P. putida S12palB1 have been studied. Many of the extensive general expression changes were found to relate to system-wide alterations originating from the optimization of the original platform strain (Verhoef et al. 2007;Wierckx et al. 2005;Wierckx et al. 2008). In addition, also p-hydroxybenzoate production appeared to have a substantial general impact. Part of the responses could be attributed to p-hydroxybenzoate stress, whereas others may be related to the 'short-circuiting' of an anabolic pathway (aromatic amino acid biosynthesis) and a catabolic pathway (p-coumarate degradation).
The pathways directly involved in p-hydroxybenzoate formation were considerably upregulated in P. putida S12palB1, in line with the elevated metabolic flux towards the product. These responses were comparable to those observed in P. putida S12TPL3 (Wierckx et al. 2008). The upregulation of the p-coumarate pathway is obviously unique for strain S12palB1 as this pathway requires the presence of p-coumaroyl-CoA for induction (Calisti et al. 2008). By contrast, no responses of the protocatechuate pathway were observed which is explained by the disruption of the first gene of this pathway, pobA, in P. putida S12palB1. The homogentisate pathway was shown to account for substantial loss of tyrosine, thus compromising p-hydroxybenzoate synthesis. Considerable improvement of both yield and productivity was achieved by targeted inactivation of hpd, the first gene of the homogentisate pathway.
Remarkably, the nature of the carbon source employed exerted major effects in P. putida S12palB1. Effects were observed both at the global expression level and specifically for genes and proteins that are directly associated with phydroxybenzoate biosynthesis and production. The effect was most prominent for the genes of the p-coumarate degradation pathway and the genes encoding phenylalanine hydroxylase, phhA and phhB. No such differences were observed between the control cultures on glucose and glycerol. Therefore, the carbon source-dependency of the expression of these genes must be considered a characteristic of P. putida S12palB1. Nonetheless, the transcript levels in glycerol-grown P. putida S12palB1 apparently sufficed to accommodate efficient p-hydroxybenzoate biosynthesis, since similar amounts were produced in glucose-grown and glycerol-grown cultures. Moreover, none of the upregulated 'p-hydroxybenzoate biosynthetic' gene products were detected in the proteomics analysis, which supported the notion that different transcript levels do not necessarily affect actual protein synthesis. It cannot be excluded, however, that the bottleneck for p-hydroxybenzoate production on either substrate is located upstream of the tyrosine biosynthetic pathway, e.g., at the level of the key aromatic precursors phosphoenolpyruvate and erythrose-4-phosphate.
In addition to metabolic genes, various transporter systems were differentially expressed in P. putida S12palB1. Although the multidrug efflux MFS transporter PP1271-PP1273 showed no similarity to known p-hydroxybenzoate transporters such as AaeAB of E. coli (Van Dyk et al. 2004), it was shown to be involved in p-hydroxybenzoate export. Overexpression did not improve production, however, suggesting that the innate transport capacity was sufficient to cope with the amount of p-hydroxybenzoate generated. The MFS transporter was furthermore not essential, and unlikely to be specific, for p-hydroxybenzoate export, since it was also upregulated in response to phenol (unpublished data), toluene (Volkers et al. 2008) and urea (in P. putida KT2440; (Reva et al. 2006)).
In addition to contributing to the general understanding of the genetic background underlying the improved p-hydroxybenzoate production characteristics of P. putida S12palB1, the transcriptomics and proteomics results provided leads that resulted in further strain improvement. Moreover, the fact that the available carbon source directly impacts the expression of key p-hydroxybenzoate biosynthetic pathways indicates that the p-hydroxybenzoate production capacity has not been exploited to its full potential. The precise nature of this upstream bottleneck preventing further yield improvement is currently under investigation.