Small molecule inhibition of ATM kinase increases CRISPR-Cas9 1-bp insertion frequency.

Mutational outcomes following CRISPR-Cas9-nuclease cutting in mammalian cells have recently been shown to be predictable and, in certain cases, skewed toward single genotypes. However, the ability to control these outcomes remains limited, especially for 1-bp insertions, a common and therapeutically relevant class of repair outcomes. Here, through a small molecule screen, we identify the ATM kinase inhibitor KU-60019 as a compound capable of reproducibly increasing the fraction of 1-bp insertions relative to other Cas9 repair outcomes. Small molecule or genetic ATM inhibition increases 1-bp insertion outcome fraction across three human and mouse cell lines, two Cas9 species, and dozens of target sites, although concomitantly reducing the fraction of edited alleles. Notably, KU-60019 increases the relative frequency of 1-bp insertions to over 80% of edited alleles at several native human genomic loci and improves the efficiency of correction for pathogenic 1-bp deletion variants. The ability to increase 1-bp insertion frequency adds another dimension to precise template-free Cas9-nuclease genome editing.

Short chain fatty acids produced by colonizing intestinal commensal bacterial interaction with expressed breast milk are anti-inflammatory in human immature enterocytes.

Necrotizing enterocolitis (NEC) is a devastating intestinal emergency that affects ten percent of very low birth weight premature babies and costs society in both expense and heartache. It is probably caused by an inappropriate interaction of colonizing bacteria with an immature intestine. A possible preventative measure is to feed prematures their mother's expressed breast milk in conjunction with a probiotic. This synbiotic prevention reduces the severity and incidence of this condition. This study was designed to determine the mechanism of the synbiotic effect in human and mouse fetal intestine. Breast milk interacting with a NEC preventative probiotic such as Bifidobacterium infantis can produce increased levels of short chain fatty acids (acetate, propionate and butyrate) (SCFAs). SCFAs are known to be anti-inflammatory in mature enterocytes and immunocytes. Very little is known about their role in immature intestine. When exposed to a human fetal cell line, fetal intestinal organoids and fetal mouse intestine, these SCFAs were anti-inflammatory. Their mechanism of anti-inflammation differed from those reported for mature cells by involving the G-protein coupled receptor (GPR 109A) and inhibiting histone deacetylase 4 and 5. These bacterial metabolites may help explain the synbiotic anti-inflammatory effect of breast milk and probiotics given to premature infants at risk for NEC.

TraDIS-Xpress: a high-resolution whole-genome assay identifies novel mechanisms of triclosan action and resistance.

Understanding the genetic basis for a phenotype is a central goal in biological research. Much has been learnt about bacterial genomes by creating large mutant libraries and looking for conditionally important genes. However, current genome-wide methods are largely unable to assay essential genes which are not amenable to disruption. To overcome this limitation, we developed a new version of "TraDIS" (transposon directed insertion-site sequencing) that we term "TraDIS-Xpress" that combines an inducible promoter into the transposon cassette. This allows controlled overexpression and repression of all genes owing to saturation of inserts adjacent to all open reading frames as well as conventional inactivation. We applied TraDIS-Xpress to identify responses to the biocide triclosan across a range of concentrations. Triclosan is endemic in modern life, but there is uncertainty about its mode of action with a concentration-dependent switch from bacteriostatic to bactericidal action unexplained. Our results show a concentration-dependent response to triclosan with different genes important in survival between static and cidal exposures. These genes include those previously reported to have a role in triclosan resistance as well as a new set of genes, including essential genes. Novel genes identified as being sensitive to triclosan exposure include those involved in barrier function, small molecule uptake, and integrity of transcription and translation. We anticipate the approach we show here, by allowing comparisons across multiple experimental conditions of TraDIS data, and including essential genes, will be a starting point for future work examining how different drug conditions impact bacterial survival mechanisms.

Apparent Genetic Rescue of Adult Shank3 Exon 21 Insertion Mutation Mice Tempered by Appropriate Control Experiments.

SHANK3 (ProSAP2) is among the most common genes mutated in autism spectrum disorders (ASD) and is the causative gene in Phelan-McDermid syndrome (PMS). We performed genetic rescue of Shank3 mutant phenotypes in adult mice expressing a Shank3 exon 21 insertion mutation (Shank3G ). We used a tamoxifen-inducible Cre/loxP system (CreTam ) to revert Shank3G to wild-type (WT) Shank3+/+ We found that tamoxifen treatment in adult Shank3GCreTam+ mice resulted in complete rescue of SHANK3 protein expression in the brain and appeared to rescue synaptic transmission and some behavioral differences compared to Shank3+/+CreTam+ controls. However, follow-up comparisons between vehicle-treated, WT Cre-negative mice (Shank3+/+CreTam- and Shank3+/+CreTam+) demonstrated clear effects of CreTam on baseline synaptic transmission and some behaviors, making apparently positive genetic reversal effects difficult to interpret. Thus, while the CreTam tamoxifen-inducible system is a powerful tool that successfully rescues Shank3 expression in our Shank3G/G reversible mutants, one must exercise caution and use appropriate control comparisons to ensure sound interpretation.

Morphine and Fentanyl Citrate Induce Retrotransposition of Long Interspersed Element-1.

The retroelement long interspersed element-1 (LINE-1 or L1) comprises about 17% of the human genome. A single human cell has 80 to 100 copies of retrotransposition-competent L1, approximately 10% of which are 'hot' and actively 'jump' around the genome. Recent observations demonstrated that low-molecular weight compounds may induce L1 retrotransposition through unknown mechanisms. Herein, we demonstrated that the painkillers morphine and fentanyl citrate trigger L1 retrotransposition in neuronal cells without inducing DNA damage or up-regulating L1 mRNA expression. This effect was blocked by an antagonist of Toll-like receptor 4 (TLR4). Taken together, the data suggest that L1 retrotransposition due to morphine and fentanyl citrate is distinct from that triggered by DNA damage, requires TLR4, and is a novel type of genomic instability. Thus, we propose that L1 retrotransposition should be characterized as a component of the pharmacological activity of these analgesic agents.

Chemogenomic profiling of Plasmodium falciparum as a tool to aid antimalarial drug discovery.

The spread of Plasmodium falciparum multidrug resistance highlights the urgency to discover new targets and chemical scaffolds. Unfortunately, lack of experimentally validated functional information about most P. falciparum genes remains a strategic hurdle. Chemogenomic profiling is an established tool for classification of drugs with similar mechanisms of action by comparing drug fitness profiles in a collection of mutants. Inferences of drug mechanisms of action and targets can be obtained by associations between shifts in drug fitness and specific genetic changes in the mutants. In this screen, P. falciparum, piggyBac single insertion mutants were profiled for altered responses to antimalarial drugs and metabolic inhibitors to create chemogenomic profiles. Drugs targeting the same pathway shared similar response profiles and multiple pairwise correlations of the chemogenomic profiles revealed novel insights into drugs' mechanisms of action. A mutant of the artemisinin resistance candidate gene - "K13-propeller" gene (PF3D7_1343700) exhibited increased susceptibility to artemisinin drugs and identified a cluster of 7 mutants based on similar enhanced responses to the drugs tested. Our approach of chemogenomic profiling reveals artemisinin functional activity, linked by the unexpected drug-gene relationships of these mutants, to signal transduction and cell cycle regulation pathways.

Novel Insertion Mutation in KCNJ5 Channel Produces Constitutive Aldosterone Release From H295R Cells.

Primary aldosteronism accounts for 5%-10% of hypertension and in a third of cases is caused by autonomous aldosterone production by adenomas (APA). Somatic mutations in the potassium channel encoded by KCNJ5 have been detected in surgically removed APAs. To better understand the role of these mutations, we resequenced the KCNJ5 channel in a large Australian primary aldosteronism cohort. KCNJ5 mutations were detected in 37 APAs (45% of the cohort), including previously reported E145Q (n = 3), G151R (n = 20), and L168R (n = 13) mutations. In addition, we found a novel 12-bp in-frame insertion mutation (c.414-425dupGCTTTCCTGTTC, A139_F142dup) that duplicates the AFLF sequence in the pore helix upstream of the selectivity filter. Expressed in Xenopus oocytes, the A139_F142dup mutation depolarized the oocytes and produced a G-protein-sensitive Na(+) current with altered K(+) selectivity and loss of inward rectification but retained Ba(2+) sensitivity. Transfected into H295R cells, A139_F142dup increased basal aldosterone release 2.3-fold over the wild type. This was not increased further by incubation with angiotensin II. Although the A139_F142dup mutant trafficked to the plasma membrane of H295R cells, it showed reduced tetramer stability and surface expression compared with the wild-type channel. This study confirms the frequency of somatic KCNJ5 mutations in APAs and the novel mutation identified (A139_F142dup) extend the phenotypic range of the known KCNJ5 APA mutations. Being located in the pore helix, it is upstream of the previously reported mutations and shares some features in common with selectivity filter mutants but additionally demonstrates insensitivity to angiotensin II and decreased channel stability.

Instability of the Arabidopsis mutant csn5a-2 caused by epigenetic modification of intronic T-DNA.

T-DNA insertion mutants play a crucial role in elucidating Arabidopsis gene function. In some cases, two or more T-DNA mutants are combined to study genetic interactions between homologous genes or genes hypothesized to act in the same pathway. We studied the significance of protein-protein interactions between CSN5A and ROP11 by crossing three independent rop11 T-DNA insertion mutants with csn5a-2, a partial loss-of-function intronic T-DNA insertion mutant. The csn5a-2 single mutant is severely stunted, but double rop11 csn5a-2mutants were rescued and exhibited increased CSN5A transcript and protein levels. The rescued phenotype was maintained in non-Mendelian fashion when the csn5a-2 single mutant was re-isolated from the rop11-1 csn5a-2 double mutant, and was sensitive to two inhibitors of DNA methylation. Loss of kanamycin resistance was also observed in re-isolated csn5a-2. These findings indicate that the rescue of csn5a-2 resulted from a trans T-DNA-mediated epigenetic effect on the csn5a-2 intronic T-DNA, similar to recent reports involving the intronic T-DNA mutants ag-TD, ben1-1, and cob-6. Thus the work reported here provides further support for the recommendation that mutants created through novel combinations of T-DNA alleles should be carefully evaluated for evidence of epigenetic modification of T-DNA before final conclusions are drawn.

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis.

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.

Insertional hypermutation in mineral oil-induced plasmacytomas.

Unless stimulated by a chronic inflammatory agent, such as mineral oil, plasma cell tumors are rare in young BALB/c mice. This raises the questions: What do inflammatory tissues provide to promote mutagenesis? And what is the nature of mutagenesis? We determined that mineral oil-induced plasmacytomas produce large amounts of endogenous retroelements--ecotropic and polytropic murine leukemia virus and intracisternal A particles. Therefore, plasmacytoma formation might occur, in part, by de novo insertion of these retroelements, induced or helped by the inflammation. We recovered up to ten de novo insertions in a single plasmacytoma, mostly in genes with common retroviral integration sites. Additional integrations accompany tumor evolution from a solid tumor through several generations in cell culture. The high frequency of de novo integrations into cancer genes suggests that endogenous retroelements are coresponsible for plasmacytoma formation and progression in BALB/c mice.

Hygromycin B and apramycin antibiotic resistance cassettes for use in Campylobacter jejuni.

Campylobacter jejuni genetic manipulation is restricted by the limited number of antibiotic resistance cassettes available for use in this diarrheal pathogen. In this study, two antibiotic resistance cassettes were developed, encoding for hygromycin B and apramycin resistance, for use in mutagenesis or for selection of gene expression and complementation constructs in C. jejuni. First, the marker genes were successfully modified to allow for insertional mutagenesis or deletion of a gene-of-interest, and were bracketed with restriction sites for the facilitation of site-specific cloning. These hygromycin B and apramycin markers are encoded by plasmids pAC1H and pAC1A, respectively. We also modified an insertional gene-delivery vector to create pRRH and pRRA, containing the hygromycin B and apramycin resistance genes, and 3 unique restriction sites for the directional introduction of genes into the conserved multi-copy rRNA gene clusters of the C. jejuni chromosome. We determined the effective antibiotic concentrations required for selection, and established that no harmful effects or fitness costs were associated with carrying hygromycin B or apramycin resistance under standard C. jejuni laboratory conditions. Using these markers, the arylsulfatase reporter gene astA was deleted, and the ability to genetically complement the astA deletion using pRRH and pRRA for astA gene insertion was demonstrated. Furthermore, the relative levels of expression from the endogenous astA promoter were compared to that of polycistronic mRNA expression from the constitutive promoter upstream of the resistance gene. The development of additional antibiotic resistance cassettes for use in Campylobacter will enable multiple gene deletion and expression combinations as well as more in-depth study of multi-gene systems important for the survival and pathogenesis of this important bacterium.

High-level expression of sugar inducible gene2 (HSI2) is a negative regulator of drought stress tolerance in Arabidopsis.

BACKGROUND: HIGH-LEVEL EXPRESSION OF SUGAR INDUCIBLE GENE2 (HSI2), also known as VAL1, is a B3 domain transcriptional repressor that acts redundantly with its closest relative, HSI2-LIKE1 (HSL1), to suppress the seed maturation program following germination. Mutant hsi2 hsl1 seedlings are arrested early in development and differentially express a number of abiotic stress-related genes. To test the potential requirement for HSI2 during abiotic stress, hsi2 single mutants and plants overexpressing HSI2 were subjected to simulated drought stress by withholding watering, and characterized through physiological, metabolic and gene expression studies. RESULTS: The hsi2 mutants demonstrated reduced wilting and maintained higher relative water content than wild-type after withholding watering, while the overexpressing lines displayed the opposite phenotype. The hsi2 mutant displayed lower constitutive and ABA-induced stomatal conductance than wild-type and accumulated lower levels of ABA metabolites and several osmolytes and osmoprotectants following water withdrawal. Microarray comparisons between wild-type and the hsi2 mutant revealed that steady-state levels of numerous stress-induced genes were up-regulated in the mutant in the absence of stress but down-regulated at visible wilting. Plants with altered levels of HSI2 responded to exogenous application of ABA and a long-lived ABA analog, but the hsi2 mutant did not show altered expression of several ABA-responsive or ABA signalling genes 4 hr after application. CONCLUSIONS: These results implicate HSI2 as a negative regulator of drought stress response in Arabidopsis, acting, at least in part, by regulating transpirational water loss. Metabolic and global transcript profiling comparisons of the hsi2 mutant and wild-type plants do not support a model whereby the greater drought tolerance observed in the hsi2 mutant is conferred by the accumulation of known osmolytes and osmoprotectants. Instead, data are consistent with mutants experiencing a relatively milder dehydration stress following water withdrawal.

Transposon mutagenesis in Bifidobacterium breve: construction and characterization of a Tn5 transposon mutant library for Bifidobacterium breve UCC2003.

Bifidobacteria are claimed to contribute positively to human health through a range of beneficial or probiotic activities, including amelioration of gastrointestinal and metabolic disorders, and therefore this particular group of gastrointestinal commensals has enjoyed increasing industrial and scientific attention in recent years. However, the molecular mechanisms underlying these probiotic mechanisms are still largely unknown, mainly due to the fact that molecular tools for bifidobacteria are rather poorly developed, with many strains lacking genetic accessibility. In this work, we describe the generation of transposon insertion mutants in two bifidobacterial strains, B. breve UCC2003 and B. breve NCFB2258. We also report the creation of the first transposon mutant library in a bifidobacterial strain, employing B. breve UCC2003 and a Tn5-based transposome strategy. The library was found to be composed of clones containing single transposon insertions which appear to be randomly distributed along the genome. The usefulness of the library to perform phenotypic screenings was confirmed through identification and analysis of mutants defective in D-galactose, D-lactose or pullulan utilization abilities.

Linking genes of unknown function with abiotic stress responses by high-throughput phenotype screening.

Over 13% of all genes in the Arabidopsis thaliana genome encode for proteins classified as having a completely unknown function, with the function of >30% of the Arabidopsis proteome poorly characterized. Although empirical data in the form of mRNA and proteome profiling experiments suggest that many of these proteins play an important role in different biological processes, their functional characterization remains one of the major challenges in modern biology. To expand the annotation of genes with unknown function involved in the response of Arabidopsis to different environmental stress conditions, we selected 1007 such genes and tested the response of their corresponding homozygous T-DNA insertional mutants to salinity, oxidative, osmotic, heat, cold and hypoxia stresses. Depending on the specific abiotic stresses tested, 12-31% of mutants had an altered stress-response phenotype. Interestingly, 832 out of 1007 mutants showed tolerance or sensitivity to more than one abiotic stress treatment, suggesting that genes of unknown function could play an important role in abiotic stress-response signaling, or general acclimation mechanisms. Further analysis of multiple stress-response phenotypes within different populations of mutants revealed interesting links between acclimation to heat, cold and oxidative stresses, as well as between sensitivity to ABA, osmotic, salinity, oxidative and hypoxia stresses. Our findings provide a significant contribution to the biological characterization of genes with unknown function in Arabidopsis and demonstrate that many of these genes play a key role in the response of plants to abiotic stresses.

Transport properties of members of the ZIP family in plants and their role in Zn and Mn homeostasis.

A better understanding of the role of the Arabidopsis ZIP family of micronutrient transporters is necessary in order to advance our understanding of plant Zn, Fe, Mn, and Cu homeostasis. In the current study, the 11 Arabidopsis ZIP family members not yet well characterized were first screened for their ability to complement four yeast mutants defective in Zn, Fe, Mn, or Cu uptake. Six of the Arabidopsis ZIP genes complemented a yeast Zn uptake-deficient mutant, one was able partially to complement a yeast Fe uptake-deficient mutant, six ZIP family members complemented an Mn uptake-deficient mutant, and none complemented the Cu uptake-deficient mutant. AtZIP1 and AtZIP2 were then chosen for further study, as the preliminary yeast and in planta analysis suggested they both may be root Zn and Mn transporters. In yeast, AtZIP1 and AtZIP2 both complemented the Zn and Mn uptake mutants, suggesting that they both may transport Zn and/or Mn. Expression of both genes is localized to the root stele, and AtZIP1 expression was also found in the leaf vasculature. It was also found that AtZIP1 is a vacuolar transporter, while AtZIP2 is localized to the plasma membrane. Functional studies with Arabidopsis AtZIP1 and AtZIP2 T-DNA knockout lines suggest that both transporters play a role in Mn (and possibly Zn) translocation from the root to the shoot. AtZIP1 may play a role in remobilizing Mn from the vacuole to the cytoplasm in root stellar cells, and may contribute to radial movement to the xylem parenchyma. AtZIP2, on the other hand, may mediate Mn (and possibly Zn) uptake into root stellar cells, and thus also may contribute to Mn/Zn movement in the stele to the xylem parenchyma, for subsequent xylem loading and transport to the shoot.

Knocking out ACR2 does not affect arsenic redox status in Arabidopsis thaliana: implications for as detoxification and accumulation in plants.

Many plant species are able to reduce arsenate to arsenite efficiently, which is an important step allowing detoxification of As through either efflux of arsenite or complexation with thiol compounds. It has been suggested that this reduction is catalyzed by ACR2, a plant homologue of the yeast arsenate reductase ScACR2. Silencing of AtACR2 was reported to result in As hyperaccumulation in the shoots of Arabidopsis thaliana. However, no information of the in vivo As speciation has been reported. Here, we investigated the effect of AtACR2 knockout or overexpression on As speciation, arsenite efflux from roots and As accumulation in shoots. T-DNA insertion lines, overexpression lines and wild-type (WT) plants were exposed to different concentrations of arsenate for different periods, and As speciation in plants and arsenite efflux were determined using HPLC-ICP-MS. There were no significant differences in As speciation between different lines, with arsenite accounting for >90% of the total extractable As in both roots and shoots. Arsenite efflux to the external medium represented on average 77% of the arsenate taken up during 6 h exposure, but there were no significant differences between WT and mutants or overexpression lines. Accumulation of As in the shoots was also unaffected by AtACR2 knockout or overexpression. Additionally, after exposure to arsenate, the yeast (Saccharomyces cerevisiae) strain with ScACR2 deleted showed similar As speciation as the WT with arsenite-thiol complexes being the predominant species. Our results suggest the existence of multiple pathways of arsenate reduction in plants and yeast.

Genetic knockdown of a single organic anion transporter alters the expression of functionally related genes in Malpighian tubules of Drosophila melanogaster.

Insects excrete a wide variety of toxins via the Malpighian (renal) tubules. Previous studies have implicated three transporters in the secretion of the organic anion (OA) methotrexate (MTX) by the Drosophila Malpighian tubule: Drosophila multidrug resistance-associated protein (dMRP, CG6214), a multidrug efflux transporter (MET, CG30344), and an organic anion transporting polypeptide 58Dc (OATP58Dc, CG3380). RNA interference (RNAi) knockdown and P-element insertion mutation of single OA transporter genes were used to evaluate the importance of these three putative transporters in the secretion of MTX by the Malpighian tubules of Drosophila melanogaster. A major finding is that genetic knockdown of a single OA transporter gene leads to reductions in the expression of at least one other OA transporter gene and in secretion of MTX by Malpighian tubules isolated from flies reared on a standard diet. The pattern of changes indicates that decreases in MTX secretion do not correspond to decreases in dMRP expression in all of the RNAi lines. Genetic knockdown of a single OA transporter gene also alters the extent of upregulation of multiple OA transporter genes in the tubules in response to dietary MTX. Knockdown of dMRP is associated with a decrease in MET expression but an increase in OATP expression when flies are reared on MTX-enriched diet. Our results indicate that dMRP and MET are not the dominant MTX transporters in the tubules when flies are reared on MTX-enriched diets. At least one additional transporter, and possibly OATP, are required for MTX secretion. The implications of our results for studies using genetic knockdown techniques to identify OA transporters in whole tissues such as Malpighian tubules are discussed.

Arabidopsis RAP2.2 plays an important role in plant resistance to Botrytis cinerea and ethylene responses.

• Ethylene plays a crucial role in plant resistance to necrotrophic pathogens, in which ETHYLENE RESPONSE FACTORs (ERFs) are often involved. • Here, we evaluated the role of an ERF transcription factor, RELATED TO AP2 2 (RAP2.2), in Botrytis resistance and ethylene responses in Arabidopsis. We analyzed the resistance of transgenic plants overexpressing RAP2.2 and the T-DNA insertion mutant to Botrytis cinerea. We assessed its role in the ethylene signaling pathway by molecular and genetic approaches. • RAP2.2-overexpressing transgenic plants showed increased resistance to B. cinerea, whereas its T-DNA insertion mutant rap2.2-3 showed decreased resistance. Overexpression of RAP2.2 in ethylene insensitive 2 (ein2) and ein3 ein3-like 1 (eil1) mutants restored their resistance to B. cinerea. Both ethylene and Botrytis infection induced the expression of RAP2.2 and the induction was disrupted in ein2 and ein3 eil1 mutants. We identified rap2.12-1 as a T-DNA insertion mutant of RAP2.12, the closest homolog of RAP2.2. The hypocotyls of rap2.2-3 rap2.12-1 double mutants showed ethylene insensitivity. The constitutive triple response in constitutive triple response1 (ctr1) was partially released in the rap2.2-3 rap2.12-1 ctr1 triple mutants. • Our findings demonstrate that RAP2.2 functions as an important regulator in Botrytis resistance and ethylene responses.

An Arabidopsis T-DNA insertion mutant for galactokinase (AtGALK, At3g06580) hyperaccumulates free galactose and is insensitive to exogenous galactose.

Galactokinase (GALK, EC 2.7.1.6) is a cytosolic enzyme with a wide occurrence across the taxonomic kingdoms. It catalyzes the phosphorylation of α-d-galactose (Gal) to α-d-Gal-1-P. The cytotoxicity of free (unphosphorylated) Gal is well documented in plants and causes marked defects. An Arabidopsis GALK (AtGALK, At3g06580) was previously identified, cloned and functionally characterized in Escherichia coli and was suggested to occur as a single copy gene in Arabidopsis. We identified an AtGALK T-DNA insertion mutant (atgalk) that (i) is AtGALK transcript deficient; (ii) displays no GALK activity in vegetative tissues; and (iii) accumulates Gal up to 6.8 mg g(-1) FW in vegetative tissues, in contrast to wild-type plants. By constitutively overexpressing the AtGALK cDNA, atgalk was functionally rescued. Three independent transformed lines showed restored AtGALK transcripts and GALK activity and had low leaf Gal concentrations comparable with those observed in wild-type plants. Surprisingly, in vitro grown atgalk plants were largely insensitive to the exogenous application of up to 100 mM free Gal, while wild-type plants exhibited sensitivity to low Gal concentrations (10 mM). Furthermore, atgalk seedlings retained the capacity for uptake of exogenously supplied Gal (100 mM), accumulating up to 57 mg g(-1) FW in leaves. Leaves from soil-grown atgalk plants that exhibited no growth or morphological defects were used to demonstrate that the accumulating Gal occurred exclusively in the vacuoles of mesophyll protoplasts. Collectively, these findings suggest a novel Gal detoxification pathway that targets free Gal to the vacuole and is active in the atgalk mutant background.

A putative P-type ATPase required for virulence and resistance to haem toxicity in Listeria monocytogenes.

Regulation of iron homeostasis in many pathogens is principally mediated by the ferric uptake regulator, Fur. Since acquisition of iron from the host is essential for the intracellular pathogen Listeria monocytogenes, we predicted the existence of Fur-regulated systems that support infection. We examined the contribution of nine Fur-regulated loci to the pathogenicity of L. monocytogenes in a murine model of infection. While mutating the majority of the genes failed to affect virulence, three mutants exhibited a significantly compromised virulence potential. Most striking was the role of the membrane protein we designate FrvA (Fur regulated virulence factor A; encoded by frvA [lmo0641]), which is absolutely required for the systemic phase of infection in mice and also for virulence in an alternative infection model, the Wax Moth Galleria mellonella. Further analysis of the ΔfrvA mutant revealed poor growth in iron deficient media and inhibition of growth by micromolar concentrations of haem or haemoglobin, a phenotype which may contribute to the attenuated growth of this mutant during infection. Uptake studies indicated that the ΔfrvA mutant is unaffected in the uptake of ferric citrate but demonstrates a significant increase in uptake of haem and haemin. The data suggest a potential role for FrvA as a haem exporter that functions, at least in part, to protect the cell against the potential toxicity of free haem.