DNA Microarrays for Identifying Fishes

In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a “Fish Chip” for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products.
Introduction
Compared with terrestrial ecosystems, little is known about marine biodiversity and changes in species richness and ecosystem function. This is mainly because of sampling difficulties and problems in taxonomy. Many marine organisms and especially their diverse developmental stages, such as (1) eggs and larvae of fishes and invertebrates, (2) zoo- and phytoplankton, and (3) benthic invertebrates, are cumbersome and difficult to identify by morphological characters. Classical microscopy methods are time-consuming and require a high degree of taxonomic expertise, which is currently falling short. In many cases identifying a species is the major bottleneck in marine biodiversity and ecosystem research, hampering the necessary monitoring of marine biodiversity. As an example, a review of 138 studies on invertebrate diversity in European seas showed that approximately one-third of the specimens could not have been identified to species level (Schander and Willassen 2005).
DNA-based identification methods are meanwhile established (Barlow and Tzotzos 1995) as powerful tools, exhibiting an unprecedented accuracy because of their inherently highest possible resolution, which can reach even the level of single base changes in a whole genome. Using these methods, the following marine animals have been investigated: eggs, larvae, and adults of fishes (Rocha-Olivares 1998; Noell et al. 2001; Fox et al. 2005; Ward et al. 2005), planktonic copepods (Bucklin et al. 1999), invertebrate larvae (Garland and Zimmer 2002; Barber and Boyce 2006), and prey in the gut content or feces of penguins, whales, and fishes (Jarman et al. 2002; Saitho et al. 2003).
Sequences of the small subunit of the rRNA gene are used as a standard method for identifying microbial organisms (Ludwig et al. 2004), and a fragment of the mitochondrial cytochrome oxidase I gene is in use as “DNA-barcode” for the identification of metazoans (Hebert et al. 2002; Ward et al. 2005). As shown above, a growing number of recently published studies are using molecular genetic identification methods. Nevertheless, their application is still restricted mainly because of methodical problems and because special knowledge and experience in molecular genetics is required. This is especially true if DNA-based identification is performed by using microarray platforms that are error-prone and difficult to quantify (Shi et al. 2006). Whereas most of the methods presently in use, such as PCR-based DNA amplification followed by sequencing techniques, allow to handle only single or a few species at the same time, DNA microarrays are believed to have the potential of identifying hundreds of species in parallel and to differentiate them against an even larger number of related species.
Commonly, microarrays are glass microscope slides on which oligonucleotide probes are spotted that are complementary to the DNA target sequences to be analysed (Relógio et al. 2002, Pirrung 2002, Dufva 2005). The DNA target, which is usually fluorophore-labelled during PCR amplification, hybridises with the oligonucleotide probe on the microarray and can be detected after washing steps by its label. Whereas applying of DNA microarrays for gene expression has already reached the routine level of high throughput systems (Blohm and Guiseppi-Elie 2001, Hoheisel 2006), they have been only recently applied for the identification of organisms, such as microbes (Wang et al. 2002; Call et al. 2003; Korimbocus et al. 2005; Loy and Bodrossy 2005), animals (Pfunder et al. 2004), and plants (Rønning et al. 2005). In terms of identifying marine organisms, microarrays have been used for bacteria (Peplies et al. 2003; Peplies et al. 2004) and phytoplankton (Metfies and Medlin 2004; Metfies et al. 2005; Godhe et al. 2007) Other DNA-hybridisation methods for the identification of higher marine organisms, such as invertebrate larvae (Goffredi et al. 2006), copepods (Kiesling et al. 2002), and larvae of fishes (Rosel and Kocher 2002), have been applied, but microarrays have not been used for this kind of studies. Other applications of microarrays in research on marine organisms are gene expression analysis (Williams et al. 2003; Lidie et al. 2005; Wang et al. 2006; Cohen et al. 2007; Jenny et al. 2007) and genotyping in population genetics (Moriya et al. 2004; Moriya et al. 2007).
One of the methodical limitations for using microarrays is the design of species specific probes. On the one hand, oligonucleotide probes designed in silico do not always exhibit the experimental hybridisation properties they were selected for and must be empirically tested. On the other hand, the molecular marker must have highly selective characteristics, such as low intraspecific and a high interspecific variation. One of the most frequently used markers in phylogenetics of fishes is the mitochondrial 16S rRNA gene. This gene has a well-characterized secondary structure (Meyer 1993; Ortí et al. 1996) and especially the loop regions exhibit many insertions, deletions, and substitutions forming highly variable molecular features, which usually allow the design of highly specific probes. This is underlined by a study on lionfishes (Kochzius et al. 2003), which showed that individuals of one species exhibit identical 16S rDNA haplotypes even though they were sampled at sites thousands of kilometres apart, but clear differences could be detected between closely related lionfish species.
In this study, the development of a DNA microarray is described, demonstrating the suitability of the 16S rRNA gene for designing oligonucleotides as microarray probes to differentiate at least 11 fish species from European seas. Based on these data, a “Fish Chip” for approximately 50 fish species is under construction to support the identification of eggs and larval stages from species that are otherwise difficult to identify, and of adult or processed fishes in fisheries industry.
Material and Methods
Sampling and DNA Extraction
To consider possible intraspecies sequence variations, fishes were collected in five different regions of the European seas: North Sea, Bay of Biscay, and Western, Central, as well as Eastern Mediterranean (Fig. 1, Table 1). All together 267 individual samples from 79 fish species were investigated. In addition sequences from the EMBL sequence data base were included. Voucher specimens and tissue samples have been preserved in absolute ethanol and stored at 4°C or were frozen at -20°C. DNA was extracted from gill filaments with the Agowa mag midi DNA isolation kit (AGOWA, Berlin, Germany) or from muscle tissue with the DNeasy tissue kit (Qiagen, Hilden, Germany) according to the instructions of the manufacturer.
Figure 1Map with the sampling areas; NS North Sea; BB Bay of Biscay; WM Western Mediterranean; CM Central Mediterranean; EM Eastern Mediterranean.Table 1Number of sequences per fish species and sampling regionSpeciesFamilyOrderNorth SeaBay of BiscayWestern MediterraneanCentral MediterraneanEastern MediterraneanEMBL sequence data base and other projectsTotalAccession number of EMBL data base sequencesTarget species Boops boopsSparidaePerciformes3418AF247396 Engraulis encrasicolusEngraulidaeClupeiformes3216 Helicolenus dactylopterusSebastidaeScorpaeniformes316111AY538975 Lophius budegassaLophiidaeLophiiformes369 Pagellus acarneSparidaePerciformes3339 Scomber scombrusScombridaePerciformes3126AB120717, AF055615 Scophthalmus rhombusScophthalmidaePleuronectiformes33118AY359665 Serranus cabrillaSerranidaePerciformes347 Sparus aurataSparidaePerciformes3216AF247432 Trachurus trachurusCarangidaePerciformes125210AB108498, AB096007 Trigla lyraTriglidaeScorpaeniformes336Additional species Chelidonichthys lucernusTriglidaeScorpaeniformes13610 Diplodus sargusSparidaePerciformes246 Gadus morhuaGadidaeGadiformes1236X99772, NC_002081, AY850363 Merluccius merlucciusMerlucciidaeGadiformes233614 Mullus surmuletusMullidaePerciformes252211 Mullus barbatusMullidaePerciformes257 Pagellus erythrinusSparidaePerciformes12710 Platichthys flesusPleuronectidaePleuronectiformes42410AY359670, AB125255, AY157320, AF113180 Pleuronectes platessaPleuronectidaePleuronectiformes426AY359673, AY157328 Psetta maximaScophthalmidaePleuronectiformes134 Sardina pilchardusClupeidaeClupeiformes33410 Scorpaena notataScorpaenidaeScorpaeniformes5611 Scorpaena porcusScorpaenidaeScorpaeniformes3317 Serranus hepatusSerranidaePerciformes2237 Solea soleaSoleidaePleuronectiformes332311AB125247, AF488442, AF112845 Zeus faberZeidaeZeiformes332614NC_003190, AF488474, AF221894-AF221896, AP002941 Σ 230Accession numbers are given for sequences obtained from EMBL sequence databaseTaxonomy according to FishBase (2007)
Polymerase Chain Reaction and Sequencing
A fragment of approximately 1380 bp length from the mitochondrial 16S rRNA gene was amplified with the primer 16fiF140 (5′-CGY AAG GGA AHG CTG AAA-3′), which has a single-base modification compared with Palumbi et al. (1991, unpublished manuscript) as well as with the newly designed primer 16fiR1524 (5′-CCG GTC TGA ACT CAG ATC ACG TAG-3′). Polymerase chain reaction (PCR) reactions with a total volume of 15 μl contained 1.5 μl 10 X reaction buffer, 1.5 μl dNTPs (10 mM), 0.05 μl of each primer (100 pmol/μl), 5 μl DNA-extract, 0.3 μl Teg polymerase (3 U/μl; Prokaria, Reykjavik, Iceland), and 6.6 μl deionized water. Thermal profile began at 94°C for 4 min, followed by 35 cycles of 94°C (30 s), 54°C (30 s), 72°C (90 s), with a final step of 7 min at 72°C.
PCR products were purified by using the ExoSAP-IT for PCR clean-up (GE Healthcare, Uppsala, Sweden). The newly designed sequencing primer 16fiseq1463 (5′-TGC ACC ATT AGG ATG TCC TGA TCC AAC-3′) was used to sequence one strand of the amplified fragment using the BigDye Terminator Cycle Sequencing Kit (ver. 3.1, PE Biosystems, Foster City, USA). The sequencing reactions were run in an ABI Prism 3730 automated DNA Analyzer (Applied Biosystems, Foster City, USA) according to the manufacturer’s instructions.
Sequence Analysis and Oligonucleotide Probe Design
In the framework of this project, 267 sequences have been acquired and 944 sequences have been obtained from EMBL sequence database and other projects, representing approximately 380 species of fish from European seas. Probes were designed based on 230 sequences obtained from 27 species (Table 1). A multiple alignment of these 230 sequences was performed with the programme Clustal W (Thompson et al. 1994) as implemented in BioEdit (version 7.0.4.1; Hall 1999) to ensure that all sequences represent a homologous fragment of the 16S rDNA. Before probe design gaps have been removed from each sequence. A computer program developed by the bioinformatics group of the Centre for Applied Gene Sensor Technology (CAG) and the Zentrum für Technomathematik (ZeTeM), both at University of Bremen, was used to design species-specific oligonucleotide probes, which ideally cover all sequences of one species and do not match any other species (Nölte 2002). The following criteria have been considered: (1) length of 23 to 27 bp, (2) melting temperature (Tm) of 81 to 85°C based on the unified model (SantaLucia 1998), (3) GC content of 52% to 54%, (4) secondary structure of the oligonucleotides and the target sequence, (5) possible dimer formation, and (6) the energy content of a bond between the probe and the target sequence. Minimal free energy (mfe) structures are computed by using RNAfold (Hofacker et al. 1994). Probes exhibiting strong secondary structures or binding to a region of the target with such a strong secondary structure were not used. If more than one probe qualified for a species, the one with the highest binding energy between probe and target was chosen. Already during the design-phase the selected oligonucleotide probes were tested in silico against 1211 background sequences from approximately 380 fish species.
Preparation of DNA Microarrays and Hybridisation Experiments
Aminosilane (3-aminopropyltrimethoxysilane)-coated glass slides were used with a PDITC-linker (1,4-phenylendiisothiocyanate) from Asper Biotech (Tartu, Estonia). Oligonucleotide probes (Thermo Hybaid, Ulm, Germany) with a 5′-amino-C6-modification were spotted in 150 mM Na3PO4 buffer (pH 8.5) at a concentration of 30 μM using a spotting robot based on a modified version of the contactless TopSpot® technology. The spotted volume of this oligonucleotides solution was 200 pl, producing a spot diameter of approximately 220 μm. Each probe was spotted in four replicates per block. An array contained five blocks and three arrays were spotted on one microarray slide (Fig. 2). After spotting, the microarrays were incubated for 16 h in a wet chamber to ensure efficient covalent binding of the oligonucleotides. Finally, the microarrays were shrink-wrapped under a nitrogen atmosphere and were storable at 4°C for up to 6 months.
Fig. 2Layout of the microarray
DNA for hybridisation experiments was amplified and labelled with 5′-Cy5-modified primers. The primers 16sar-L (3′-CGC CTG TTT AAC AAA AAC AT-3′) and 16sbr-H (5′-CCG GTT TGA ACT CAG ATC ACG T-3′) amplify a fragment of approximately 600 bp length from the mitochondrial 16S rRNA gene (Palumbi et al. 1991, unpublished manuscript). PCR reactions with a volume of 100 μl contained 10 μl 10 X reaction buffer, 4 μl dNTPs (5 mM), 2 μl of each primer (10 μM), 2 μl DNA-extract, 0.4 μl Taq polymerase (5 U/μl), 2 μl BSA (20 mg/ml), and 77.6 μl deionized water. The PCR thermal profile began at 95°C for 2 min, followed by 35 cycles of 95°C (30 s), 54°C (45 s), 72°C (60 s), followed by a final step of 10 min at 72°C. The Cy5-labelled PCR amplified DNA was purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany).
Hybridisation experiments were performed with 11 target and 14 nontarget fish species. As nontarget species fishes were chosen that are closely related to the target species and from which false-positive signals could be expected according to the in silico specificity tests performed during the design phase of the probes (Table 2).
Table 2Nontarget species tested in hybridisation experimentsSpeciesFamilyOrderDentex dentexSparidaePerciformesDiplodus vulgarisSparidaePerciformesGadus morhuaGadidaeGadiformesMelanogrammus aeglefinusGadidaeGadiformesMerlangius merlangusGadidaeGadiformesMerluccius merlucciusGadidaeGadiformesMicromesistius poutassouGadidaeGadiformesMullus surmuletusMullidaePerciformesPollachius pollachiusGadidaeGadiformesPollachius virensGadidaeGadiformesPsetta maximaScophthalmidaePleuronectiformesSerranus hepatusSerranidaePerciformesTrachurus mediterraneusCarangidaePerciformesTrachuru picturatusCarangidaePerciformesTaxonomy according to FishBase (2007)
A positive control at a concentration of 1 nM (5′-CGT GTG AGT CGA TGG ATC ATA-3′; 5′-Cy3-labelled) and 10 nM of the purified Cy5-labelled PCR product were hybridized to the microarray in a volume of 65 μl using GeneFrames® (ABgene House, Epsom, UK), which were applied to the microarray slides according to the manufacturer’s instruction (Fig. 2). Hybridisation was conducted at 50°C in a hybridisation oven. After 2 h hybridisation time the GeneFrames® were removed and the microarrays were washed 5 minutes each with 2 × SSC (sodium chloride trisodium citrate) buffer containing 0.05% SDS (sodium dodecyl sulphate), 1 × SSC containing 0.05% SDS, and 1 × SSC. Finally the microarrays were dried in a centrifuge at 2000 rpm for 3 minutes.
Measurement of Fluorescence Signals and Data Analysis
Hybridisation signals were measured using an Axon 4000B fluorescence microarray scanner at 635 nm (Cy5) as well as at 528 nm (Cy3). The fluorescence signal analysis was conducted with the software GenePix 4.1 (Axon, Union City, USA). The fluorescence signals of each probe were measured and the arithmetic mean was calculated. However, data were removed from the analysis if the spots showed artefacts caused during the spotting process (e.g., inhomogeneous spots documented by a monitoring camera during spotting) or experimental artefacts (e.g., air bubbles). Background noise was corrected by subtracting the arithmetic mean of the negative control measurement from the arithmetic mean of the spot measurements. Negative values were set to zero.
Results
Probes for 11 commercially important fish species have been designed (Table 3). Positions of the oligonucleotide probes in the 16S rDNA fragment used for probe design are given in Fig. 3. Following the nomenclature of Ortí et al. (1996), the binding sites of the microarray probes for Engraulis encrasicolus, Sparus aurata, and Trigla lyra are located in the variable region j, whereas the probes for Boops boops, Helicolenus dactylopterus, Lophius budegassa, Pagellus acarne, Scomber scombrus, Scophthalmus rhombus, Serranus cabrilla, and Trachurus trachurus bind to the variable region l.
Table 3Oligonucleotide probes for the identification of fish species from European seasSpecies nameProbe nameProbe sequence (5′-3′), 5′-amino-C6-modifiedLength (bp)Tm (°C)GC (%)Oligo mfeDimer mfeSpecificity (in silico)Boops boopsBooboo_315GCACCACACTCCTAAACCCAAGA2382.6452≥0−0.07speciesEngraulis encrasicolusEngenc_213CAAGTCCTAAATACCCGCAGCCT2382.4952≥0−0.17speciesHelicolenus dactylopterusHeldac_317ACCCCTCCTACAATTAAGAGCCG2381.8452≥0−0.22speciesLophius budegassaLopbud_312AACACCCTTCCTATCACCCAGAGCTAC2784.3952≥0−0.2genusPagellus acarnePagaca_317TACTACACTCCCACATCCGAGAGC2482.7754≥0−0.89speciesScomber scombrusScosco_321CAACTACTCCTACAGTCAAGAGCCACC2782.9152≥0−0.43speciesScophthalmus rhombusScorho_322CCCCTTAACTCCTCGAAGCAAGA2381.8852≥0−0.37speciesSerranus cabrillaSercab_313CCATTTTCCTACAACCCAGAGCGAC2582.7452≥0−0.18speciesSparus aurataSpaaur_201AGAACAGCTCACGTCAAACACCC2383.0252≥0−0.5speciesTrachurus trachurusTratra_333TTCCTCTCCTCCCACAAGCAAGA2383.6252≥0−0.15genusTrigla lyraTrilyr_232AAGACCGAACCAAATGAGCCCTG2383.1652≥0−0.17familyThe number in the probe name indicates the binding site in the 16S rDNA sequenceOligo mfe minimal free energy of the secondary structure of the oligonucleotide; Dimer mfe minimal free energy of the dimer of two identical oligonucleotide moleculesValues for mfe are given in kcal/molFig. 3Alignment (5′ > 3′) of representative 16S rDNA sequences from the target species with binding sites (light grey) of probes (5′ > 3′; probes hybridise to the reverse complementary target strand). Double stranded (dark grey) and single stranded regions (grey) of the secondary structure are indicated in the reference sequence of Pygoplites nattereri (Ortí et al. 1996; Accession number: U33590)
All single target hybridisations of the Cy5-labelled 16S rDNA fragment gave true-positive fluorescence signals for the corresponding probe (Fig. 4), but the hybridisation efficiency is very different. The signal intensity of the weakest probe-target pair gave approximately 1,000 arbitrary units and the strongest almost 30,000 (Table 4). Under the experimental conditions selected, single target hybridisations with B. boops, E. encrasicolus, H. dactylopterus, L. budegassa, S. rhombus, S. aurata, T. trachurus, and T. lyra did not show any false-positive signals. Eight probes gave under these conditions very weak false-positive signals with P. acarne, S. scombrus, and S. cabrilla, representing approximately 7% of all possible cross-hybridisation reactions. The values of these false-positive signals were several orders of magnitude lower than the true positive-signals.
Figure 4Signals (background subtracted from absolute signal) of single target and nontarget hybridisations. White bars represent true-positive signals; false-positive signals are shown as grey bars. Numbers at the basis of the bars indicate the amount of measured spots. The number of hybridisations is given in brackets after target and nontarget names. Replication and absolute signal intensities (± standard deviation) of hybridized targets to the corresponding probe are given in Table 4.Table 4Target hybridisationsHybridized targetsNo. of hybridisationsMeasurements of specific probesMeasured probes/absolute no. of probesMean absolute fluorescence signal in arbitrary unitsStandard deviationBoops boops221/402991±1491Engraulis encrasicolus220/401659±962Helicolenus dactylopterus220/403502±912Lophius budegassa240/403450±1515Pagellus acarne227/403727±1270Scophthalmus rhombus115/201528±269Scomber scombrus240/4027827±5330Serranus cabrilla240/4010814±4396Sparus aurata240/40963±227Trachurus trachurus120/202015±880Trigla lyra235/402343±560
Single nontarget hybridisations showed 48 very weak false-positive signals, representing 31% of 154 possible cross-hybridisations. These false-positive signals were at least one order of magnitude weaker than the true-positive signal. Merlangius merlangus and Merluccius merluccius did not gave any false-positive signal, whereas Dentex dentex, Diplodus vulgaris, Gadus morhua, Melanogrammus aeglefinus, Micromesistius poutassou, Mullus surmuletus, Pollachius pollachius, Pollachius virens, Psetta maxima, Serranus hepatus, Trachurus mediterraneus, and Trachurus picturatus showed two to eight cross-hybridisations. Considering all possible cross-hybridisations, only 18% showed usually very weak false-positive signals.
Discussion
The results show that the “Fish Chip” described enables the identification of 11 commercially important fish species from European seas in certain experimental limits. These limits are primarily given by the fact that the fluorescence signal intensities of true-positive hybridisation signals were heterogeneous. This phenomenon is commonly encountered in DNA microarray experiments (Peplies et al. 2003; Warsen et al. 2004; Korimbocus et al. 2005; Rønning et al. 2005; Tobler et al. 2006) and can probably be overcome only by an extreme methodical effort (Shi et al. 2006). The problems encountered when single-colour microarray experiments need to be quantified are severe and in part not yet solved, because complex parameters are influencing the results. These are the sequence dependent hybridisation efficiency, specifically steric hindrance, secondary structures (Southern et al. 1999), and the relative position of the fluorescent label at the target (Zhang et al. 2005).
Sometimes the duplex formation can be favoured by using spacers, which obviously give the captures a greater degree of freedom from their neighbouring molecules and from the surface (Southern et al. 1999), leading to an enhancement of signal intensity with increasing spacer length (Peplies et al. 2003).
An important criterion of probe design was the base composition (Southern et al. 1999) and therefore all but one capture oligonucleotides were designed to have a GC content of 52% and 54% for the exception (Table 3). The different hybridisation efficiencies of the captures and the varying sensitivity for the different fishes is still a severe disadvantage, because it can hamper the estimation of a small amount of fishes of one species in the presence of a larger number of individuals of another species in a mixed sample. These limits are presently under investigation with fish eggs and other biological material.
Because all oligonucleotide probes bind to the variable regions j and l of the 16S rRNA gene, which represent large single-stranded loops, the secondary structure is unlikely to be a factor contributing to the partially low sensitivity. Also, the POL effect, a phenomenon decreasing the fluorescence signal with increasing distance between the probe binding site and the label on the target (Zhang et al. 2005) seems not to be important in this case. Most of the probes used in this study hybridise to the variable region l of the 16S rDNA and their distance from the fluorescence label is more or less identical.
Although cross-hybridisations occur, true-positive signals could clearly be differentiated from false-positive signals because of their generally higher signal. Most false-positive signals occurred when the 16S rDNA fragment of nontarget species was hybridised on the microarray.
Testing of nontarget species is seemingly important in studies using DNA microarrays for the identification of organisms, because even if a comprehensive sequence background is utilised for probe design and even if extensive in silico testing has been performed, the specificity of oligonucleotide probes has to be evaluated with closely related species and specifically those that show cross-hybridisations in silico.
This study shows that the 16S rRNA gene of fishes is suitable to design oligonucleotide probes that are able to differentiate eleven fish species from European seas by single target hybridisation on a microarray. Such a “Fish Chip” can hopefully be applied in marine environmental and fisheries research, as well as in fisheries and food control if the uneven hybridisation signal intensities of the different probe-target pairs can be improved or compensated.
The correct identification of fish eggs and larvae is crucial for fish stock assessment based on ichthyoplankton surveys. Genetic identification has shown that the majority of eggs in the Irish Sea, wrongly believed to be from cod, were actually from whiting, leading to an overestimation of cod stocks (Fox et al. 2005). A study on food fish in the United States revealed that three-quarters of fish sold as “red snapper” were mislabelled and belonged to other species (Marko et al. 2004), a situation that needs better analytical tools to be changed. The European Union (EU) also has strict regulations for seafood labelling, which must include, for example, the species name (EU Council Regulation No 104/2000; EU Commission Regulation No 2065/2001). Approximately 420 species of fish are sold in the German market alone, making a reliable identification method urgently necessary to protect the customer. DNA microarrays might have the potential to fulfill these requirements.
Recent efforts in compiling sequences of fishes, such as the European projects “FishTrace” http://www.fishtrace.org) and “Fish & Chips” (http://www.fish-and-chips.uni-bremen.de; Kochzius et al. 2007), as well as the international “Fish Barcode of Life Initiative” http://www.fishbol.org; Ward et al. 2005), will provide the necessary sequence background for the design of species specific oligonucleotide probes for the development of DNA microarrays for the identification of fishes.

capture oligonucleotide
dna chips
species identification
pisces


Increased Nitric Oxide Production and GFAP Expression in the Brains of Influenza A/NWS Virus Infected Mice

The cause of influenza to the brain was investigated using the A/NWS/33 influenza virus infected BALB/c mouse model. NOS-2 mRNA levels in the infected mouse brain was greater than in control mice in all brain regions examined, particularly in the olfactory bulb and hippocampus by 1 day p.i. On the contrary, no differences in NOS-1 or NOS-3 mRNA levels were found between infected and control mice. There was also a marked increase in the levels of metabolites of nitric oxide in the olfactory bulb and hippocampus. Immunohistochemistry showed positive staining for anti-NOS-2 primarily in the hippocampus of infected mice. Further, anti-NOS-2 and GFAP staining was mostly found around capillary blood vessels of the hippocampus starting early in the course of the disease. These results indicate that the NWS enhances the activation of astrocytes and NOS-2 expression which in turn enhances NO production and the expansion of capillary blood vessels.
Introduction
Influenza associated encephalopathy (IE) is one of the most severe neurological diseases amongst children, mostly under the age of 5. It is marked by rapid progression and the mortality rate is estimated to range from 27 to 44% [1]. Recently, we reported that production of the biological free radical gas, nitric oxide (NO), in the serum and cerebrospinal fluid (CSF) of patients with IE was increased in the initial stages and correlated with disease severity [2, 3].
Yokota et al. hypothesized that the influenza virus activates the limbic system followed by glial cell activation which in turn produces NO and inflammatory cytokines which flood the brain and cause a cytokine storm [4]. Recent in vitro studies by Imanishi et al. showed that a mouse lung-adapted strain of influenza virus could induce inducible NO synthase (NOS-2) mRNA expression and NOS-2-mediated NO production in murine macrophage cell [5]. Moreover, Ino et al. studied five autopsies cases of IE patients. They reported that four out of the five patients showed degenerated astrocytic spines in the cortex, by glial fibrillary acidic protein (GFAP) staining. They suspected that the IE was largely due to these altered glial cells (Ino et al. unpublished).
Taken together, it is likely that IE is related to both the influence of NO and glial cells changes. Nevertheless, there have been no reports on a relationship between NOS-2 and glial cells within the brain after infection with the influenza virus. Thus, the aim of these studies is to better understand the clinical state of IE by focusing on NO and astrocytes in the central nervous system (CNS) after influenza virus infection. By using the A/NWS/33 influenza virus infected BALB/c model mouse, we were able to demonstrate that influenza virus infection leads to an up regulation of NOS-2 and astrocytes, mostly around capillary blood vessels of the hippocampus and olfactory bulb, starting at an early stage of the disease.
Although IE has been reported for more than a decade, a therapy for IE has not yet been defined. We expect that our results will provide evidence which will aid in the development of a novel IE therapy.
Experimental procedures
Virus
Influenza A/NWS virus, which is a mouse brain adapted type of human influenza A/NWS/33 virus was used. (A kind gift from Dr. K. Hayashi, Toyama University, Japan.)
Experimental animals
In total, 82 male BALB/c mice (5-week-old) were purchased from Japan Charles River Co. and given free access to food and water (Oriental Yeast, Co., Tokyo) and acclimatized for at least a week before the experiment.
Anesthetics
Each mouse was anesthetized by inhalation of ether before being inoculated intranasally with 50 μl NWS, 5.0 × 107 plaque-forming units (PFU)/ml (5.0 × 105 PFU/mouse). For negative control mice, the same amounts of 0.1 M phosphate-buffered saline (PBS) were intranasally inoculated. The mice were examined at 1, 3 and 6 days postinfection (p.i.).
Body weight
Body weight changes were monitored daily.
Euthanasia
All mice were decapitated whilst under ether inhalational anesthesia. Each brain was divided into eight regions (frontal cortex, occipital cortex, cerebellum, medullar oblongata, hippocampus, corpus striatum, thalamus/hypothalamus and olfactory bulb). Then each section was immediately frozen with liquid nitrogen.
NO2− and NO3− measurements by HPLC-UV method
Both NO2 and NO3 (NOx) levels in each brain region were measured by HPLC-UV system (ENO-10, EICOM) which was reported by us [3]. Briefly, thawed brain regions were deproteinized with a ten times volume of methanol and then centrifuged (12,000 r.p.m. for 20 min at 4°C) to avoid an occlusion in the polystyrene polymer column used in this system, by protein or other macromolecules. Detection was measured at 540 nm (absorption) and the concentrations were calculated from the area under the curve (AUC) of NO2 and NO3 (Power Chrome, EICOM, Kyoto) by Griess reaction.
RNA extraction
RNA was extracted from each part of the brain by using a QIA amp RNA extraction kit (QIAGEN K.K., Tokyo), according to the protocol suggested by the manufacturer.
cDNA synthesis
cDNA was synthesized from purified RNA by using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland). Random hexamer primer was used and the protocol was as follows; after annealing for 10 min at 25°C, and incubating 30 min at 55°C, the reaction was heated to 85°C for 5 min and chilled on ice.
Real time reverse transcriptase-polymerase chain reaction (RT-PCR)
Messenger RNA levels of NOS-1, NOS-2 and NOS-3 were quantified by using a Light Cycler Fast Start DNA Master HybProbe (Roche). This was done according to the following protocol; Pre-incubation was 95°C for 30 s. This was followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 62°C for 15 s, amplification at 72°C for 8 s for 45 cycles. Cycles were followed by 30 s 40°C cooling program. The primers and probes were designed by the Light Cycler primer and probe set (Roche Diagnostics Applied Science, Tokyo). NOS-2, NOS-3 and NOS-1 primer sequences were designed on GenBank database NM_0100927, NM_008713, and NM_008712 respectively. All data were normalized by dividing with the corresponding GAPDH mRNA from the same sample.
Immunohistochemistry
Anesthetized animals were perfused intracardially with 20 ml of 0.1 M PBS, followed by 30 ml of 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed, postfixed overnight and cryoprotected in 30% sucrose, processed through graded alcohols and embedded in paraffin. Then, they were cut as 6 μm thick coronal sections. They were incubated with 0.3% H2O2 for 20 min. The sections were then rinsed with tris-buffered saline, pH 7.6 (TBS). To identify reactive astrocytes, we stained with Anti-GFAP (rabbit polyclonal, DAKO, Denmark). Sections were incubated over night at a 1:2,000 dilution. The NOS-2 stains were incubated for 4 h at a 1:1,500 dilution (Anti-iNOS/NOSII, rabbit polyclonal IgG;Upstate Biotechnology, Lake Placid, NY). After the first staining, each section was incubated with the secondary antibody for 30 min (Nichirei Histofine Simple Stain, anti-rabbit IgG) at room temperature after TBS rinsing. Labeling was visualized by incubating with a solution containing 2%, 3,3′-diaminobenzidine, 0.1 M imidazole and 30% H2O2.
All sections were counterstained with Mayer’s hematoxylin (SIGMA, MI).
Statistical analysis
Fisher’s exact test was used when comparing body weights. All results obtained from the real time RT-PCR were subjected to one- or two-way analysis of variance (ANOVA) and differences among the means were analyzed by two-way ANOVA followed by a Newman–Keuls range test at the 0.05 significance level. Results are expressed as mean ± standard error.
Results
Body weight
Figure 1 shows the change in body weight of the non-infected (PBS) and infected (Flu) mice over day. The mice infected with virus showed a decrease in weight and ruffled hair from 3 days p.i. and by 6 days p.i. and there was a significant decrease in the body weight of the infected mice as compared to the non-infected mice (P < 0.05). Prominent neurological signs in the infected mice did not appear during the experimental period and none of the mice died during the experiment, although spontaneous locomotor activity was reduced in the infected mice.
Fig. 1Changes in body weight of A/NWS/33 influenza virus infected BALB/c model mice after virus infection. Each result shows the mean ± SE value of eight experiments. () non-infected (PBS treatment) mice group, () infected (Flu) mice group
Viral titers
At one day p.i., 120 PFU/50 mg virus was detected in the infected mouse brain and 136 × 104 PFU/100 mg virus was detected in the infected mouse lung.
Real-time RT-PCR for NOS-2, NOS-3 and NOS-1
Figure 2 shows the NOS-2/GAPDH and NOS-3/GAPDH ratios as measured in the olfactory bulb and hippocampus of infected mouse brains as compared to the non-infected mice on days 1, 3 and 6 p.i. Due to the scattered results obtained in both the infected and non-infected mice, data on the NOS-1/GAPDH ratio is not reported. The increases in the NOS-2/GAPDH ratio as seen in the olfactory bulb(OB) and hippocampus(HIP) at day 1 and 3 p.i., were obvious as shown in Fig. 2a and b. However, no changes were seen in the levels of NOS-3/GAPDH in the same regions (Fig. 2a’, b’).
Fig. 2NOS-2/GAPDH and NOS-3/GAPDH ratios in the olfactory bulb and hippocampus of non-infected and infected mice on days 1, 3 and 6 p.i. The open column shows the mean and S.E. value obtained from nine non-infected mice and the closed column shows the mean and S.E. value obtained from eight infected mice. *Significant difference between non-infected mice and infected mice. *P < 0.05. (a and a’): olfactory bulb, (b and b’): hippocampus
Changes in brain NO levels between non-infected and infected mice
Figure 3 shows that NO levels in the eight brain regions examined, either with or without NWS virus infection, changes in a day-by-day manner after virus infection. The levels of NO2 and NO3 were measured separately, as shown in the Materials and methods. In the isolated brain regions, NO3 levels were more than 40-fold higher than NO2 levels, and the final data are shown as the total value (NO) of NO2 plus NO3. At day 1 p.i., the NO levels detected in olfactory bulb (OB), hippocampus (HIP) and frontal cortex (FC) of the infected mice (Flu) were significantly increased as compared to the levels in the same brain regions of the non-infected (PBS) mice (P < 0.05). Further, at day 6 p.i., the increased levels of NO in the olfactory bulb (OB) and the frontal cortex (FC) returned to the levels seen in non-infected mice. However, the levels of NO in the hippocampus (HIP) were significantly increased (P < 0.05) at all the time points examined.
Fig. 3Day to day changes in NO levels in the eight brain regions either with or without NWS virus infection. The open column shows the mean and S.E. value obtained from eight non-infected mice and the closed column shows the mean and S.E. value obtained from seven infected mice. *Significant difference between non-infected mice and infected mice. *P < 0.05. OB: olfactory bulb, HIP: hippocampus, St: striatum, Hypo/Thal: hypothalamus and thalamus, MO: medulla oblongata, CE: cerebellum, FC: frontal cortex, and OC: occipital cortex
Immunohistochemistry
Figure 4 shows the results of anti-NOS-2 staining and GFAP staining in the hippocampus of non-infected (PBS) and infected mice (Flu) at 1 (Fig. 4a) and 6 (Fig. 4b) days p.i. in the same magnification each. The brown-yellow staining was recognized as positive staining for anti-NOS-2. We found positive staining in the hippocampus, especially in the area surrounding capillary blood vessels of the infected mice at 1 day p.i. (Fig. 4a, Day 1, Infected). By 6 days p.i., positive staining had spread to the striatum, hippocampus and cortex of the infected mice and positive staining were seen in the areas surrounding capillary vessels (Fig. 4b, Day 6, Infected). Add that infected mice showed an increase in GFAP staining on days 1 much more so than that seen in non-infected mice. Positive staining was most notable around hippocampal blood vessels in infected mice. Additionally, most of the blood vessels in hippocampi of infected mice were markedly expanded as compared to those seen in the non-infected mice.
Fig. 4Anti-NOS-2 immunohistochemistry and GFAP immunohistochemistry in the hippocampus at 1 (Fig. 4a) and 6 (Fig. 4b) days p.i
Discussion
Since our aim was to see the effect to the brain after influenza infection, this experiment was undertaken by using the A/NWS/33 virus because it is proven by Li et al that the NWS/33 strain can replicate in the brain of adult mice after intracerebral injection, add that the virus usually fails to spread beyond the respiratory epithelium after internasal instillation [6].
During the experiment, we observed that the NWS virus infected mice lose significantly more body weight at 6 days p.i. (Fig. 1) in agreement with previous studies [5, 7]. Since bodyweight changes are an indicator of the overall condition of the mice, this bear out that the mice was infected to the virus. More over, we have checked that RT-PCR for A/NWS/33 virus were positive in all brain regions expect medullar oblongata on day 1 p.i. (data not shown).
The reason why we determined the amounts of influenza virus in this experiment is as followed. During the past experiment, we had experienced that the mice die within 3 days p.i. when inoculating any more than the virus we used this time and when we use fewer viruses, there were hardly any difference in the non-infected and infected behavioral change.
Our results show that an enhancement NO biosynthesis was induced in the NWS virus infected mouse brain. Over-production of NO was substantiated by determining an increase in the expression of NOS-2 mRNA as shown in Fig. 2, and increased immuno-histochemical staining for anti-NOS-2 as shown in Fig. 4. At 1 day p.i., the amount of NOS-2 mRNA within the infected mouse brain was greater than that seen in control mice in all brain regions studied. Above all, the olfactory bulb showed the greatest increase. On the basis of these results, we compared NOS-2 mRNA expression in the olfactory bulb on days 1, 3, and 6 p.i., respectively. This showed that the NOS-2 mRNA ratio was greater in the infected mouse brain at all experimental times studied (Fig. 2). On the contrary, there was no difference in NOS-3 or NOS-1 mRNA levels between infected and control mice (data not shown). These results are important since these three isoforms of NOS, NOS-2 the inducible form of NOS, is capable of producing a continuous flux of NO and a large amount of NO generated by NOS-2 over a sustained period is implicated in the pathogenesis of various disorders and with the response to infectious organisms [8].
In regards to the immuno-histochemical analysis, positive staining for anti-NOS-2 was mostly detected in the hippocampus of infected mouse brains, whereas hardly any staining was seen in control brains (Fig. 4). Furthermore, positive staining had spread to the hippocampus and striatum by 6 days p.i., which correlated with the transition of NO levels.
NOS-2 has been shown to play a role in the defense against bacteria, parasites and viruses. However, it seems that NO induced by the influenza virus has a dual role. Rimmelzwaan et al. reported an inhibitory effect of NO on virus growth [9], whereas Akaike et al. reported that NO played a pathological role in the influenza virus-induced pneumonia in mice [10]. The fact that the influenza virus can induce NO has been reported previously [2, 3]. Yao et al. reported that the mRNA’s of both NOS-2 and NOS increased in the brain after the intranasal infection with influenza A virus of newborn mice [11]. Further, Imanishi et al. pointed out that a mouse lung-adapted strain of influenza virus could induce NOS-2 mRNA expression and NOS-2-mediated NO production in murine macrophage cells [5]. Our study has demonstrated that the NWS virus can induce NOS-2 expression in the infected mouse brain, especially in the olfactory bulb and hippocampus. These results may relate to the fact that intranasally administered NWS virus can invade the brain through the olfactory pathway as has been reported previously [12–16]. Recently, Yossi et al. reported that astrocytes are capable of rapid and massive NOS-2-dependent NO production in brains of normal mice in situ [17]. According to the report by Ino et al. on the brain autopsy of patients who had died of IE, four out of five IE patients showed degenerated astrocytic spines in the cortex by GFAP staining. They suspected that the IE was largely due to altered glial cells (Ino et al. unpublished). Takahashi et al. also made a statement on the autopsied case of a 2-year-old girl with encephalopathy associated with influenza A virus infection and according to their report; reactive astrocytes were seen in the vicinity of blood vessel in the brain but astrocytosis was not seen anywhere, including the areas where virus antigen-positive neurons were abundant [18]. Additionally, Yokota et al. hypothesized that the influenza virus activates the limbic system followed by the activation of glial cells which produce NO and other inflammatory cytokines which together flood the brain and cause a cytokine storm [4]. Astrocytes are the major glial cell in the CNS and have important physiological properties in terms of CNS homeostasis. Astrocytes also influence the formation and maintenance of the blood–brain barrier (BBB). The importance of astrocytes is demonstrated by a study from Bush et al. According to their report, an astrocyte ablation led to failure of BBB repair and vasogenic edema [19]. There have been several reports related to glial cells and NO [17, 20, 21]. However, there has not been a report considering the relationship between influenza virus infection, NOS-2 and astrocytes together. By determining an increase in GFAP staining as shown in Fig. 4, our results show that astrocytes are activated in the infected mouse brain and especially in hippocampus. Interestingly, we noticed that both the increased NOS-2 and GFAP staining were mostly found around capillary blood vessels of the hippocampus starting at an early stage. Our results suggest that since NOS-2 and astrocytes became activated around capillary blood vessels after viral infection, this may lead to brain capillary blood vessels break down. However, we were unable to determine whether the NOS-2 measured is derived from vascular endothelial cells or macrophages which will be an issue for future experiments.
Although IE has been mostly reported in Asian countries, influenza infection occurs in epidemics in both the northern and southern hemispheres and is a pandemic disease throughout the year. Recently, influenza virus which is resistant to anti-influenza drugs has been shown to emerge and there is real concern in regards to a new type of influenza pandemic [22]. However, no standardized therapy for the treatment of IE has yet been defined. In this paper, we suggest that the influenza virus causes the activation of astrocytes and NOS-2 in the brain. Thus it is likely that our results will add supportive evidence for the development of a novel IE therapy.

balb/c mouse
no
nos-2
olfactory bulb
hippocampus
astrocyte
influenza a/nws/33 virus
brain capillary blood vessels


Time sequence of the damage to the acceptor and donor sides of photosystem II by UV-B radiation as evaluated by chlorophyll a fluorescence

The effects of ultraviolet-B (UV-B) radiation on photosystem II (PS II) were studied in leaves of Chenopodium album. After the treatment with UV-B the damage was estimated using chlorophyll a fluorescence techniques. Measurements of modulated fluorescence using a pulse amplitude modulated fluorometer revealed that the efficiency of photosystem II decreased both with increasing time of UV-B radiation and with increasing intensity of the UV-B. Fluorescence induction rise curves were analyzed using a mechanistic model of energy trapping. It appears that the damage by UV-B radiation occurs first at the acceptor side of photosystem II, and only later at the donor side.
Introduction
The solar spectrum that reaches the earth’s surface includes wavelengths in the visible or photosynthetically active range (PAR, 400–700 nm), in the ultraviolet-A (UV-A, 320–400 nm), ultraviolet-B (UV-B, 280–320 nm) and in the ultraviolet-C (UV-C, 200–280 nm) ranges. While UV-C is totally absorbed by the atmosphere, ozone is the principal atmospheric attenuator of UV-B radiation (Cockell and Horneck 2001). The depletion of ozone by the emission into the atmosphere of man-made chlorine- and bromine-containing compounds has been correlated with an increase in the background level of UV-B radiation.
UV-B radiation can affect a multitude of physiological and morphological plant processes that ultimately can lead to inhibition of growth and reproduction (Jansen et al. 1998). Potential molecular targets for direct UV-B damage via photomodification or photosensitization include nucleotides, proteins, lipids and pigments (Jordan 2002). Besides direct macromolecular damage, UV-B can also induce specific signal transduction pathways and changes in gene expression that stimulates acclimation and repair processes (Hollósy 2002; Brosché and Strid 2003).
The photosynthetic process can be affected by UV-B radiation at different levels, including alterations in plant and leaf morphology that decreases light interception (Jansen 2002), changes in stomatal function that limit the availability of CO2, changes in photosynthetic pigments (Strid and Porra 1992), on the expression of photosynthetic genes and on enzymes of the carbon fixation pathway. However, it is the effects of UV-B radiation on light harvesting and primary photochemical reactions of photosynthetic membranes, particularly on the Photosystem II (PS II) reaction center, which has attracted much attention and study (Vass 1997; Vass et al. 2005).
PS II is one of the reaction centers that, together with the cytochrome b6f complex, the photosystem I (PS I) reaction center, and the ATP-synthase, forms the electron transport chain that drives energy transduction in the thylakoid membranes of oxygenic eukaryotes. The photochemical core of PS II is formed by the D1/D2 heterodimer, where the redox electron carriers and cofactors of electron transport are bound. Light absorbed by the antenna system of PS II induces the excitation of a special reaction center chlorophyll (P680), which is photo-oxidized on the first electron transfer reaction of PS II, with a pheophytin molecule (Pheo) acting as the primary electron acceptor and the formation of a radical pair state (P680+Pheo−). This radical pair state is “stabilized” at the reducing (acceptor) side of PS II by the electron transfer from Pheo− to a primary D2-bound quinone electron acceptor (QA) and then to the secondary D1-bound quinone acceptor (QB), which, upon accumulation of two reducing equivalents, and becoming protonated to plastoquinol, dissociates from the reaction center. At the oxidizing (donor) side of PS II, P680+ is reduced by a redox-active tyrosine residue of D1 (Yz) which acts as an electron transfer intermediate between P680+ and the oxygen-evolving complex (OEC), the metalloenzyme system composed of a cluster of four Mn ions and inorganic cofactors (Ca2+ and Cl−) that oxidizes water into molecular oxygen. Accordingly, the consecutive photochemical formation of the primary radical pair P680+Pheo− by light absorption and the reduction of P680+ forming the radical YZ+ drives the sequential four-step oxidation of the OEC inducing the S-state transitions and ultimately the splitting of the water molecule. For detailed reviews on oxygen evolution and PS II structure and function see, Diner and Babcock (1996), Xiong et al. (1998), and Van Rensen and Curwiel (2000).
From the above brief description of the PS II components and the potential molecular targets of UV-B radiation, it is clear that several components of PS II could be directly affected, as actually has been observed (Vass et al. 1996). Several studies, most of them in vitro, have shown that UV-B can inhibit PS II electron transport by damage to the quinone electron acceptors’ redox function (Rodrigues et al. 2006), to Yz function, to the OEC, and to the D1 protein. It should be noted that the time-sequence of events that ultimately leads to inactivation of PS II function is not completely clear, as it might involve a direct destruction or impairment of the absorbing molecule, modification of the protein environment to which these redox components are bound as well as a process of energy transfer by the sensitizer species to the damaged site (Vass 1997).
The time sequence of damage to acceptor and donor sides of PS II, respectively has never been measured in a single experiment. In this work, we have studied the effect of UV-B radiation on the function of PS II in leaves of Chenopodium album using both steady-state (light-adapted) fluorescence and the time-resolved fast Chl-a fluorescence induction curve. For a review on Chl-a fluorescence, see Govindjee (1995). The results were analyzed based on a mechanistic model of energy trapping and electron transport of PS II (Vredenberg 2000). It appears that damage to the acceptor side of PS II occurs first, and that only later the donor side becomes affected.
Materials and methods
Plant growth
Plants of Chenopodium album L. were grown as described earlier (Rodrigues et al. 2006). After around 28 days the plants were transferred to a growth cabinet with a constant temperature of 20°C, 70% relative humidity and a PAR level of 60 μmol m−2 s−1. The plants were watered daily and after 10–14 days the sampling of leaves for the UV exposure treatments started. Several different batches of plants were used and no significant difference could be detected when comparing results from experiments replicated with plants with similar ages but from different batches.
UV-B radiation treatments
The plant material used for the exposure to UV light was either fully developed detached leaves or leaf discs (0.6 cm2) floating on petri-dishes filled with demineralized water with the abaxial leaf surface exposed to the radiation. The control samples were either kept in the darkness or exposed to white light (PAR). In some experiments leaves were vacuum infiltrated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in 0.5% ethanol before being exposed to UV-B, with the controls infiltrated with a 0.5% ethanol solution.
UV-B irradiations were performed using a Vilber Lourmat lamp with filter (VL-115M, Marne-la-Vallée, France). This lamp has a peak emission at 312 nm. UV-B irradiance was measured with an optometer (United Detector Technology Inc, Baltimore, USA) equipped with a probe specific for UV radiation.
Chlorophyll a fluorescence measurements
Measurements of Chl-a fluorescence were performed at room temperature on leaf samples that had been dark adapted for 1 h after the exposure to UV-B radiation, unless otherwise stated. Steady-state Chl-a fluorescence was measured with a pulse-amplitude fluorometer (PAM 101–103, Walz Inc., Effeltrich, Germany). A weak modulated measuring light (1.6 kHz, 650 nm) was used for the determination of the minimal fluorescence level Fo, followed by a 1 s pulse of saturating light (5,500 μmol m−2 s−1) for the measurement of the maximum fluorescence level Fm. The leaf was then irradiated with a fixed or increasing level of actinic PAR and, at each level, we measured the steady state light-adapted fluorescence (Fo′), the maximum fluorescence of a light-adapted state (Fm′) induced by a saturating light pulse superimposed on the actinic light, and after removing the actinic light and the application during 10 s of far-red light (6 μmol m−2 s−1, 720 nm), the minimum fluorescence of a light-adapted state (Fo′). During the saturating pulses the modulation of the measuring light was automatically changed to 100 kHz. Several fluorescence parameters were calculated from the measured fluorescence levels (Schreiber et al. 1986; Genty et al. 1989; Van Kooten and Snel 1990): the potential photochemical efficiency (quantum yield) of PS II, expressed as the ratio Fv/Fm with Fv = Fm − Fo; the coefficient of photochemical quenching, qP, given by (Fm′ − Fs)/Fv′ with Fv′ = Fm′ − Fo′; the efficiency of excitation energy capture by open PS II reaction centers, Fv′/Fm′; and non-photochemical quenching, qN, given by 1 − (Fm′ − Fo′)/(Fm − Fo) (Walters and Horton 1991).
The fast Chl-a fluorescence rise was measured with the Plant Efficiency Analyser (PEA, Hansatech Ltd, Norfolk, UK). Leaf samples were excited with a saturating pulse of red light (peak at 650 nm, 3,500 μmol m−2 s−1) and the fluorescence signal recorded at an acquisition interval of 10 μs in the first 2 ms of excitation (for more details on this instrument see Strasser et al. (1995, 2000): a model describing discrete steps of excitation energy and electron transfer associated with PS II was published by Zhu et al. (2005). Due to the limiting response time of the fluorescence detector in the time region below 50 μs, the Fo level was taken as the model-calculated (see below) fluorescence level at 10 μs. This procedure gave similar results to the method of extrapolation as proposed by Vredenberg (2000). The fast Chl-a fluorescence rise is plotted in a log-time scale either as the absolute measured values or as the relative variable fluorescence (rFv), calculated as (Fm − Ft)/(Fm − Fo), with Ft as the fluorescence level at any time.
Modeling and interpretation of fast Chlorophyll a fluorescence rise
The fast Chl-a fluorescence rise curve was modeled according to Vredenberg et al. (2001). The mathematical formulation is based on a three-state model of energy trapping and fluorescence (Vredenberg 2000) and equates the polyphasic fluorescence rise upon a saturating pulse of light to a multi-state transfer pattern of the PS II reaction center.
Results and discussion
Exposure of the leaves to UV-B radiation caused an increase of Fo and a decrease of Fm (Fig. 1B), resulting in a decrease of the Fv/Fm value (Fig. 1A). Using a lower intensity of UV-B radiation on grape leaves, Pfündel (2003) also found a decrease of Fm. However, in his experiments, the Fo first decreased with time, and only increased after about 3 h. The increase of the Fo indicates an increase in the amount of QB-nonreducing centers; the decrease of the Fv/Fm value indicates that UV-B has a strong damaging effect on the functioning of PS II.
Fig. 1Effect of UV-B radiation of variable duration on Fo, Fm, and the Fv/Fm ratio in leaf discs. The UV-B radiation level was 720 (■), or 150 (◯) μmol m−2 s−1, respectively (PAR level was zero); control samples were kept in darkness. Each value is the mean of four measurements and the bars indicate ±S.D.; bars not shown are within the symbols
Illumination with white light during 6 h decreased the photochemical quenching qP from 1.0 at the lowest intensity to a value of 0.58 at 656 μmol m−2 s−1 (Fig. 2). Curwiel et al. (1993), using the same plant material, reported a decrease to about 0.80 after illumination at about 500 μmol m−2 s−1. Up to 100 μmol m−2 s−1, irradiation with UV-B has a stronger effect on qP than white light; at 200 μmol m−2 s−1 and higher, the effect of UV-B is smaller. The efficiency of excitation energy capture by open PS II reaction centers, Fv′/Fm′, was already decreased strongly after 6 h at 5.4 μmol m−2 s−1 of UV-B, while the effect of white light is much smaller; comparable results were found for the quantum yield of PS II given by (Fm′ − Fs)/Fm′. The non-photochemical qN increased from 0 at the lowest intensities to 0.51 (white light) and 0.41 (UV-B) at the highest irradiation levels. Curwiel et al. (1993) reported a value of about 0.6 after illumination with white light at about 550 μmol m−2 s−1.
Fig. 2Effects of 6 h irradiation, at various levels, of detached leaves on some fluorescence parameters. CO is control; control is white light (PAR); UV-B is given by the Vilber lamp (zero PAR). Each value is the average of 4 measurements and the bars indicate ±S.D.; bars not shown are within the symbols
Measurements of Chl-a fluorescence transients of intact leaves with the PEA fluorometer are presented in Fig. 3. In Fig. 3A the effects of 0, 1, 3, and 6 h irradiation with the Vilber UV-B lamp at 150 μmol m−2 s−1 are illustrated. Also in these measurements, the most obvious effects of UV-B are an increase of the Fo level and a decrease in Fm. After infiltration with water (plus 0.5% ethanol) the effects of UV-B appear the same (compare in Fig. 3B, □ with ■). Compared with infiltration with water, infiltration with DCMU (●) causes a much higher Fo level, and a faster increase to the J-P level; irradiation with UV-B after DCMU-infiltration (◯) causes a still higher Fo level and a slower increase to the J-P level. Comparable results were found using another PS II-inhibiting herbicide, atrazine (not shown). The higher Fo level indicates an increased amount of QB-nonreducing centers. Apart from the Fo level, the differences between the curves before and after UV-B treatment are much smaller in the presence of DCMU. This may be related with the finding by Jansen et al. (1993) that PS II-inhibitors like DCMU, which replace QB from its niche on the D1 protein, inhibit UV-B-driven D1 degradation.
Fig. 3Fluorescence induction curves after various treatments of leaves. (A) Fluorescence transients after 0, 60, 180, and 360 min irradiation with UV-B light at 150 μmol m−2 s−1. ( PAR is zero). (B) ■, After infiltration with 0.5% ethanol (CO is control), no UV-B; ●, after infiltration with 10−5 M DCMU in 0.5% ethanol, no UV-B; □, after infiltration with 0.5% ethanol and 6 h irradiation with 150 μmol m−2 s−1 UV-B light; ◯, after infiltration with 10−5 M DCMU in 0.5% ethanol and 6 h irradiation with UV-B. Each curve is the average of three measurements
To study the kinetics of the various parts of the transients, induction curves were normalized (Fig. 4). While the level of the J transient increases a little after 60 min UV-B, it decreases clearly after 180 and 360 min UV-B; the kinetics of the O-J increase become lower after UV-B irradiation. In all cases the level of the I transient decreases and the kinetics of the J-(I)P rise decreases. From curves like those in Fig. 4, rate constants of reactions involved in the photochemical trapping in the reaction center of PS II were calculated using the Three-State Trapping Model of PS II (Vredenberg 2000; Vredenberg et al. 2001). The rate constants for QA− oxidation by QB (kAB1) decreased very fast, both at 150 and 720 μmol m−2 s−1; it is down to about 50% of control already after 15 min UV-B irradiation (Fig. 5A). The rate constant for QA− oxidation by QB− (kAB2) was down to 5% of control, already after 15 min UV-B (not shown). This indicates that UV-B irradiation has a fast action on the reducing side of PS II. The rate constants for Yz+ reduction by the OEC in S1 and S2 (k1 and k2 in Fig. 5B and C, respectively), were also affected, but much slower. From this we conclude that the damage by UV-B starts at the reducing side, and affects the donor side later. In Fig. 5D it is illustrated that kL (excitation rate) increases by UV-B irradiation. This indicates that UV-B radiation decreases the antenna size, or that a lower number of open reaction centers is available after UV-B treatment.
Fig. 4Relative variable fluorescence measured after illumination of leaves during various times (T, in min) with UV-B light at 150 (A) or 720 μmol m−2 s−1 (B); PAR is zero. CO is control. Each curve is the average of three (A) or four (B) measurementsFig. 5Rate constants of some reactions involved in the photochemical trapping in the reaction center of PS II, calculated using the Three-State Trapping Model of PS II (Vredenberg 2000). Curves like those presented in Fig. 5 were taken as a source for the calculations; these curves were measured after various times of irradiation with the Vilber lamp at 150 μmol m−2 s−1 (○) or 720 μmol m−2 s−1 (■); PAR was zero. Average of four measurements and the bars indicate ±S.D.; bars not shown are within the symbols
As may be concluded from the results presented in Fig. 5, UV-B radiation affects rate constants of reactions at both the acceptor and donor sides of PS II. With respect to the acceptor side, Rodrigues et al. (2006) reported that QA− is a photosensitizer for UV-B radiation; absorption of UV-B by this semiquinone radical initiates reactions leading to damage to PS II. The group of Vass, most often using isolated plant preparations, has reported several times on the effect of UV-B radiation on the donor side of PS II. In a recent paper (Szilard et al. 2006), they describe a model where UV-B-induced inhibition of water oxidation is caused either by direct absorption within the catalytic manganese cluster or by damaging intermediates of the water oxidation process.
Vass et al. (1996) measured the effects of UV-B radiation on the OEC, the QAFe2+ complex, the redox-active tyrosines, and the D1 protein in isolated spinach PS II membrane particles. While all these functions were affected, the OEC appeared to be the most sensitive; the authors concluded that the primary damage by UV-B occurs at the OEC, and that modification and/or inactivation of tyrosine and the quinone acceptor complex are subsequent events. Our work confirms that UV-B radiation affects both acceptor and donor sides of PS II. However, our analysis leads to the conclusion that the acceptor side of PS II is affected first and the donor side later. This different conclusion may be caused by the different material and methods of the two works. Vass et al. (1996) used PS II particle membranes, while in our work intact leaves were measured. Furthermore, Vass et al. (1996) measured the effect of UV-B separately on the various PS II functions, while we calculated the effects of UV-B from one single measurement.

photosystem ii
uv-b radiation
chlorophyll a fluorescence
chenopodium album
photodamage
photosynthesis


The role of GlnD in ammonia assimilation in Mycobacterium tuberculosis

Summary
Introduction
Mycobacterium tuberculosis is responsible for the largest number of human deaths from a single bacterial agent. Nearly two million people die from tuberculosis each year and more than eight million are newly infected.1 A better understanding of the basic metabolism of this pathogen could lead to new strategies for eradication. Although progress has been made in understanding some of the nutritional requirements of this organism both in vitro and in vivo, in particular its carbon source acquisition,2,3 little is known about nitrogen metabolism.
M. tuberculosis has four enzymes with glutamine synthetase (GS) activity (GlnA1-4).4,5 Of these, GlnA1, GlnA3 and GlnA4 synthesise l-glutamine, whereas GlnA2 synthesises the d-glutamine and d-isoglutamine required for cell wall biosynthesis.5 The major GS, GlnA1, is expressed to a high level and is exported.6 The role of the secreted enzyme is not clear, but it has been suggested that it may play a role in pH modulation, although the biosynthetic reaction does require a source of ATP.
GS catalyses the production of glutamine from glutamate and ammonia. Since this reaction requires ATP, it needs to be strictly regulated in the presence of excess ammonia to conserve both energy and glutamate pools in the cell. GS activity can be controlled by several mechanisms, including feedback inhibition, transcriptional control of gene expression and by post-translational modification. In Escherichia coli a regulatory cascade of three proteins, GlnD, PII and GlnE, is involved in the latter mechanism (Fig. 1). GlnD is a uridylyl transferase which modifies the PII protein. PII in turn controls the activity of GlnE. GlnE is an adenylyl transferase which controls the interconversion of GS and GS-AMP. Transfer of the AMP moiety to the GS enzyme reduces its glutamine synthetic activity. GlnE is also able to deadenylylate GS with the predominant reaction being determined by interaction with the PII protein; PII promotes the adenylylation reaction, whereas PII-UMP promotes the deadenylylation reaction. In this way the cells can rapidly control GS biosynthetic activity in response to ammonia availability. An M. tuberculosis GlnA1 mutant is auxotrophic for glutamine, and attenuated in macrophages and guinea pigs,7 suggesting that the assimilation of ammonia via this pathway is required in vivo.
We have previously shown that, in contrast to other bacteria including closely related organisms such as Streptomyces coelicolor, GlnE is an essential gene in M. tuberculosis.8 Thus the control of GS activity seems critical to normal growth. Here we show that glnD is not essential and that although it has an effect on GS activity in the cells, it is not required for virulence.
Materials and methods
Culture
M. tuberculosis H37Rv (ATCC25618) was grown in Middlebrook 7H9 plus 10% v/v OADC supplement (Becton Dickinson) and 0.05% w/v Tween 80, Middlebrook 7H10 agar with 10% v/v OADC supplement or TSM media (1.5 g/L K2HPO4, 0.5 g/L KH2PO4, 0.5 g/L MgSO4, 0.5 mg/L CaCl2, 0.1 mg/L ZnSO4, 0.1 mg/L CuSO4 and 50 mg/L ferric chloride) supplemented with 10% v/v OADC and 0.05% w/v Tween 80.9 Working pH 7.2. For TSM-high ammonia, 30 mM (NH4)2SO4 was added; for TSM-low ammonia, 0.1 mM (NH4)2SO4 was added, l-amino acids (alanine asparagine, glutamine and glutamate) were added to 3 mM. Growth curves were obtained in 12 mm diameter borosilicate tubes with 4–5 ml media and stirring at 250 rpm with an 8 mm flea. Hygromycin was used at 100 μg/ml and kanamycin at 20 μg/ml.
Quantitative RT-PCR
Probes and primers were designed for quantitative PCR for sigA (endogenous control), amt, glnB and glnD using the software Primer Express (Table 1). cDNA was synthesised from RNA using RT and random hexamer primers using AMV reverse transcriptase. PCR was carried out in a Taqman 7900 using a standard PCR master mix. For sigA, amt, glnB and glnD, the primer pairs were SigA-R and SigA-F, Amt-R and Amt-F, GlnB-R and GlnB-F, and GlnD-F and GlnD-R, respectively, and the probes used were SigA-T, Amt-T, GlnB-T and GlnD-T. The primer and probe concentrations were first optimised. The optimal primer concentration was 300 nM for all four genes, the probe concentration was 100 nM for sigA and glnB, 125 nm for glnD and 200 nm for amt. In order to measure relative gene expression levels, standard curves for each primer-probe set were generated using genomic DNA. CT values were converted into the equivalent of ng using the standard curve. Control reactions without RT were used to confirm that there was no significant contaminating genomic DNA present. CT values for genomic DNA were converted to ng and subtracted from the plus RT values. In order to standardise the samples to ensure that equal amounts of cDNA were used, each value was standardised to sigA to generate unit-less values. At least three independent RNA samples were assayed in triplicate for each gene.
Construction of glnD mutant
We used our previous method for generating delivery vectors with a marker cassette.10 The delivery vector was constructed by amplifying two regions flanking glnD such that an in-frame deletion was engineered and cloning them into p1NIL. Primer pairs gap7 CACAACGGATACCACAAC and gap8 CGTCAATGCTGTTGCTGC, and gap5 CAAGACCTGGGGAGACGC and gap6 CAGTTTGTCGGTGCCCTC were used to amplify the upstream and downstream regions and the PCR products were cloned into pGEM EasyT (Promega). The two regions were then excised as KpnI-EcoRI and EcoRI–HindIII fragments respectively and cloned into the KpnI-HindIII sites of p1NIL thereby deleting 1.7 kbp of the glnD gene. The marker cassette from pGOAL19 (hyg, lacZ, sacB) was introduced as a PacI fragment to generate the final delivery vector pKOD3.
A deletion mutant was constructed according to our previous method.10 Briefly, we treated the vector DNA with UV and electroporated M. tuberculosis to generate a single cross-over strain.11 This strain was streaked out without antibiotics and double cross-overs selected and screened on 2% w/v sucrose and 50 μg/ml X-gal. White colonies were patch tested for hygromycin and kanamycin sensitivity and then screened by PCR for the deletion gene. Potential mutants were confirmed by Southern blotting using XhoI digested genomic DNA and hybridising to a probe derived from the upstream flanking region.
GS assays
Cell-free extracts were generated using the MiniBeadBeater.12 Culture filtrates were prepared using 0.2 μM filters and the filtrates were concentrated using Centricon 20 units (Amicon). Total GS activity was assayed using the transferase assay.13 GS activity is given in nmol per minute per mg of total protein.
Virulence assays
THP-1 cells were maintained in culture, treated with PMA to induce differentiation, washed and then infected as described.14 A total of 5×105 macrophages were infected at MOIs of 1:50 and 1:5 bacteria to macrophage. Extra-cellular bacteria were removed by washing several times. Determination of the initial inoculum was assessed by plating serial dilutions and the number of intra-cellular bacteria was monitored over 7 days. Mice were infected with approximately 106 viable mycobacteria in 200 μl of pyrogen-free saline via a lateral tail vein. Where appropriate, infected mice were killed by cervical dislocation in accordance with humane endpoint protocols under the Animals Scientific Procedures Act, 1986 (UK).
Results
Expression of glnD and nitrogen regulation
We are interested in the regulatory cascade that controls the activity of GS by post-translational modification. We previously demonstrated that GlnE, an adenylyl transferase which modifies GS, is essential in M. tuberculosis.8,15 The activity of GlnE is modified by the PII protein (encoded by GlnK or GlnB) and PII is controlled by the GlnD protein (Fig. 1). In order to gain a better understanding of this pathway, we extended our work to look at the other members of the cascade.
The glnD gene is arranged in an apparent operon with two other genes, amt and glnB (Fig. 3A). The start and stop codons of amt and glnB overlap, but there is a 60 bp gap between glnB and glnD which could theoretically contain a promoter. We wanted to determine if the genes in this region are controlled by ammonia availability. We used RT-quantitative PCR to look at the expression levels of the three genes relative to sigA (Fig. 2). Amt and glnB were up-regulated three-fold and glnD was up-regulated two-fold in low-ammonia medium as compared to high-ammonia medium. The lesser induction of glnD is not unexpected as it is often seen with genes that are at the 3′ end of the operon. Alternatively, glnD could be independently expressed from a promoter located in the intergenic region.
Construction of glnD mutant
In order to characterise the role of GlnD in the regulatory cascade which controls GS activity, we constructed a deletion mutant. An in-frame deletion of the gene was made in the vector p1NIL and the gene cassette from pGOAL19 containing the hyg, lacZ, sacB genes was inserted. A two-step homologous recombination process was used to generate the mutant.10 The in-frame deletion and expected genotype was confirmed by Southern blotting (Fig. 3). Out of 32 double cross-overs screened, 12 were mutants. One strain (Tame 69) was selected for further study.
We analysed the ability of the glnDΔ strain to grow in various nitrogen sources, since it has been shown that mutations in the control of GS activity can lead to deleterious effects on growth. We measured growth of the deletion mutant Tame 69 with various nitrogen sources. The mutant was able to use all the nitrogen sources and there was no difference in the growth rates from the wild-type strain (data not shown).9 Thus the mutant is not compromised in its ability to utilise any of these substrates.
Control of total GS activity
In other bacteria, GlnD is involved in the post-translational modification cascade which ultimately controls GS activity by modulating its adenylylation state. It has also been shown to play a role in regulation of nitrogen-controlled genes in Corynebacteria.16 Therefore, we investigated total GS activity of the mutant grown with ammonia or glutamate as nitrogen sources (Fig. 4) to determine if deletion of glnD affected the activity of GS. The majority of GS is found in the culture filtrate in M. tuberculosis6 and our data confirmed that we had higher activity in this fraction than in the cell-free extracts for the wild-type strain. Interestingly, we found that total GS activity was higher in ammonia-rich conditions (30 mM ammonium sulphate) as compared to either low ammonia (0.1 mM) or 7H9 (glutamate) in the wild-type strain. This was in agreement with RT-qPCR data for glnA1 which showed increased expression in high-ammonia medium (Pashley and Parish, unpublished data), although glnA2 expression was unchanged.9 There was no significant difference in the total GS activity between the mutant and wild-type in the cell-free extracts. However, in culture filtrates the situation was very different. We found a large reduction in total GS activity in the mutant strain grown in all media except low ammonia (where the level of activity was at its lowest). This was particularly pronounced in ammonia-rich conditions, where the wild-type activity was at its highest.
Virulence in macrophages and SCID mice
M. tuberculosis glnA1 mutants are glutamine auxotrophs and showed a reduced ability to multiply in macrophages and guinea pigs.7 Since extra-cellular GS activity was reduced in the glnD mutant, we determined whether GlnD played any role in intra-cellular survival. We used two measures of this: (1) the ability of the mutant to grow in macrophages and (2) its ability to cause disease in mice. For the macrophage infection assay we used the human macrophage-like THP-1 cell line. Infections were carried out at two different MOI (Fig. 5). The results showed that there was no difference between the wild-type and mutant strain, indicating that disruption of GlnD function had no effect on intra-cellular survival. We also tested virulence in the SCID mouse model to detect any strong attenuation profile (Fig. 5). Again, the mutant strain behaved just as the wild-type and was not attenuated.
Discussion
We have shown that expression of glnB and amt is controlled in response to ammonia levels. The up-regulation of these genes in low-ammonia conditions is in agreement with previous findings in other related bacteria. For example, in Corynebacterium glutamicum, these genes are transcribed as an operon under the control of nitrogen availability; the operon is switched off in nitrogen-rich media and is turned on during nitrogen starvation.17,18
Our previous results indicate that at least one member of the GS regulatory cascade is essential, as we have been unable to construct GlnE mutants in M. tuberculosis.8,15 Our current hypothesis is that GlnE is required to inactivate GlnA1 because the latter is expressed at such a high level. If it were all enzymatically active, the intra-cellular levels of glutamate and/or ATP would quickly be depleted. In agreement with this we have shown that the adenylylation function is essential, but the deadenylylation function is not (Pashley et al., unpublished). Deletion of GlnD would result in a lack of uridylyation of the PII protein. Since unmodified PII promotes the adenylylation activity of GlnE, inactivation of GS should still occur in the mutant and therefore deletion of GlnD is possible. However, our hypothesis predicts that PII is likely to be essential since it directly modulates GlnE activity. This concurs with our preliminary evidence that we are unable to make a glnB deletion mutant, in contrast to the relative ease of constructing a glnD mutant (Pashley and Parish, unpublished data).
Although the glnD mutant was viable, there were substantial changes in the level of GS activity. Normally, a large amount of GS is exported from the cell during growth.6 Export of GS has been linked to high levels of expression, rather than any specific export mechanism.19 If glnD deletion leads to reduced expression of glnA1, this would explain why only extra-cellular levels are depleted. The observation that total GS activity in the culture filtrates was markedly reduced in the glnD mutant is an intriguing one. It is possible that overall expression of GlnA1 is markedly reduced and therefore little of it will be exported to the outside. Alternatively, GlnA1 production could be completely abolished. The enzyme assay we used does not distinguish between the four M. tuberculosis GS enzymes, so GS activity measured could arise from GlnA2-4. However, this seems unlikely, since it has already been demonstrated that deletion of the glnA1 gene leads to glutamine auxotrophy, implying that all the biosynthetic activity in the cell is GlnA1.7
It is likely that the GlnD regulatory cascade only controls activity of GlnA1, although we cannot rule out the possibility that GlnA3 activity would also be modulated. However, GlnA2 and GlnA4 will not be controlled by this cascade (since they cannot be modified by GlnE). Thus GS activity arising from enzymes other than GlnA1 cannot be rule out. Further work to determine the levels of expression of each GS would help to further elucidate the mechanism of control of GS activity in the whole cell. However, the fact that GlnA1 is the major GS in M. tuberculosis confirms the importance of this post-transcriptional regulatory control system.
Although extra-cellular GS was markedly reduced in the glnD mutant, the strain was still fully virulent in macrophages and SCID mice. This was a surprising observation, since it has already been demonstrated that GlnA1 is required for virulence.7 Our data indicate that GS is not required at high levels for virulence in the SCID mouse and that the presence of the lower level of intra-cellular GS is sufficient for bacterial survival. It is possible that a defect may be seen in the glnD mutant under conditions in which intra-cellular multiplication is seen or in activated, rather than resting macrophages. However, since we have not measured extra-cellular GS directly during infection, we cannot exclude the possibility that extra-cellular GlnA1 is found in the glnD mutant in vivo. Therefore, the attenuation of GlnA1 mutants most likely results from their auxotrophic nature and the lack of availability of glutamine in the host environment.
In conclusion, we have shown that glnD is not an essential gene, but that it is required for the normal expression and activity of GS in culture filtrates of M. tuberculosis. Future work to address the question of how deletion of glnD results in altered expression of GS by determining the mechanism of regulation for ammonia-regulated genes is underway.

nitrogen metabolism
glutamine synthetase
gene regulation