Cuproptosis: Cellular and molecular mechanisms underlying copper-induced cell death.

Tsvetkov et al. (2022) discovered a new form of cell death triggered by targeted accumulation of Cu in mitochondria that drives lipoylated TCA cycle enzyme aggregation via direct Cu binding.

Complete functional analysis of type IV pilus components of a reemergent plant pathogen reveals neofunctionalization of paralog genes.

Type IV pilus (TFP) is a multifunctional bacterial structure involved in twitching motility, adhesion, biofilm formation, as well as natural competence. Here, by site-directed mutagenesis and functional analysis, we determined the phenotype conferred by each of the 38 genes known to be required for TFP biosynthesis and regulation in the reemergent plant pathogenic fastidious prokaryote Xylella fastidiosa. This pathogen infects > 650 plant species and causes devastating diseases worldwide in olives, grapes, blueberries, and almonds, among others. This xylem-limited, insect-transmitted pathogen lives constantly under flow conditions and therefore is highly dependent on TFP for host colonization. In addition, TFP-mediated natural transformation is a process that impacts genomic diversity and environmental fitness. Phenotypic characterization of the mutants showed that ten genes were essential for both movement and natural competence. Interestingly, seven sets of paralogs exist, and mutations showed opposing phenotypes, indicating evolutionary neofunctionalization of subunits within TFP. The minor pilin FimT3 was the only protein exclusively required for natural competence. By combining approaches of molecular microbiology, structural biology, and biochemistry, we determined that the minor pilin FimT3 (but not the other two FimT paralogs) is the DNA receptor in TFP of X. fastidiosa and constitutes an example of neofunctionalization. FimT3 is conserved among X. fastidiosa strains and binds DNA non-specifically via an electropositive surface identified by homolog modeling. This protein surface includes two arginine residues that were exchanged with alanine and shown to be involved in DNA binding. Among plant pathogens, fimT3 was found in ~ 10% of the available genomes of the plant associated Xanthomonadaceae family, which are yet to be assessed for natural competence (besides X. fastidiosa). Overall, we highlight here the complex regulation of TFP in X. fastidiosa, providing a blueprint to understand TFP in other bacteria living under flow conditions.

CYTOKININ RESPONSE FACTOR 2 is involved in modulating the salt stress response.

Cytokinin has strong connections to development and a growing role in the abiotic stress response. Here we show that CYTOKININ RESPONSE FACTOR 2 (CRF2) is additionally involved in the salt (NaCl) stress response. CRF2 promoter-GUS expression indicates CRF2 involvement in the response to salt stress as well as the previously known cytokinin response. Interestingly, CRF2 mutant seedlings are quite similar to the wild type (WT) under non-stressed conditions yet have many distinct changes in response to salt stress. Cytokinin levels measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) that increased in the WT after salt stress are decreased in crf2, potentially from CRF2 regulation of cytokinin biosynthesis genes. Ion content measured by inductively coupled plasma optical emission spectrometry (ICP-OES) was increased in the WT for Na, K, Mn, Ca and Mg after salt stress, whereas the corresponding Ca and Mg increases are lacking in crf2. Many genes examined by RNA-seq analysis were altered transcriptionally by salt stress in both the WT and crf2, yet interestingly approximately one-third of salt-modified crf2 transcripts (2655) showed unique regulation. Different transcript profiles for salt stress in crf2 compared with the WT background was further supported through an examination of co-expressed genes by weighted gene correlation network analysis (WGCMA) and principal component analysis (PCA). Additionally, Gene Ontology (GO) enrichment terms found from salt-treated transcripts revealed most photosynthesis-related terms as only being affected in crf2, leading to an examination of chlorophyll levels and the efficiency of photosystem II (via the ratio of variable fluorescence to maximum fluorescence, Fv /Fm ) as well as physiology after salt treatment. Salt stress-treated crf2 plants had both reduced chlorophyll levels and lower Fv /Fm values compared with the WT, suggesting that CRF2 plays a role in the modulation of salt stress responses linked to photosynthesis.

Zinc Oxide-Based Nanoformulation Zinkicide Mitigates the Xylem-Limited Pathogen Xylella fastidiosa in Tobacco and Southern Highbush Blueberry.

The xylem-limited pathogen Xylella fastidiosa causes severe economic losses worldwide, and no effective antimicrobial disease management options are available. The goal of this study was to evaluate the efficacy of a novel ZnO-based nanoparticle formulation, Zinkicide TMN110 (ZnK), against X. fastidiosa in vitro and in planta. In vitro, minimum bactericidal concentration (MBC) of ZnK analyzed in Pierce's Disease 2 medium was estimated at approximately 60 ppm. Time-kill kinetics assay showed a 100% reduction of culturable X. fastidiosa in less than 1 h after ZnK treatment. Microfluidic chambers assays showed that ZnK also inhibits X. fastidiosa cell aggregation and growth under flow conditions. Phytotoxicity assessments in the greenhouse demonstrated that ZnK can be applied as a soil drench in 50 ml at 500 ppm/plant/week up to four times to tobacco and blueberry without causing visible damage. ZnK was also evaluated for disease control in the greenhouse using tobacco infected with X. fastidiosa subsp. fastidiosa strain TemeculaL. ZnK soil drench weekly applications at concentrations of 500 followed by 1,000 ppm (500/1,000) and 500/500/1,000 ppm (in 50 ml each), reduced X. fastidiosa populations by >2 to 3 log10 units and disease severity by approximately 57 and 76%, respectively, compared with the untreated control. Similarly, when blueberry plants infected with X. fastidiosa subsp. multiplex strain AlmaEm3 were soil drenched with ZnK at concentrations 1,000/1,000 ppm and 1,000/1,000/500 ppm (in 200 ml each), the bacterial population was reduced by approximately 1 to 2 log10 units, and disease severity decreased by approximately 39 and 43%, respectively. Overall, this study shows antibacterial activity of ZnK against X. fastidiosa and its effectiveness in plants to reduce disease symptoms under controlled conditions.

The Copper-Binding Protein CutC Is Involved in Copper Homeostasis and Affects Virulence in the Xylem-Limited Pathogen Xylella fastidiosa.

Copper (Cu) is an essential element that can be toxic if homeostasis is disrupted. Xylella fastidiosa, a xylem-limited plant pathogenic bacterium that causes disease in many economically important crops worldwide, has been exposed to Cu stress caused by wide application of Cu-containing antimicrobials used to control other diseases. However, X. fastidiosa Cu homeostasis mechanisms are still poorly understood. The potentially Cu-related protein CutC, which is involved in Cu tolerance in Escherichia coli and humans, has not been analyzed functionally in plant pathogenic bacteria. We demonstrate that recombinantly expressed X. fastidiosa CutC binds Cu and deletion of cutC gene (PD0586) in X. fastidiosa showed increased sensitivity to Cu-shock compared with wild type (WT) strain TemeculaL. When infecting plants in the greenhouse, cutC mutant showed decreased disease incidence and severity compared with WT but adding Cu exaggerated severity. Interestingly, the inoculation of cutC mutant caused reduced symptoms in the acropetal regions of plants. We hypothesize that X. fastidiosa cutC is involved in Cu homeostasis by binding Cu in cells, leading to Cu detoxification, which is crucial to withstand Cu-shock stress. Unveiling the role of cutC gene in X. fastidiosa facilitates further understanding of Cu homeostasis in bacterial pathogens.

The Influence of Copper Homeostasis Genes copA and copB on Xylella fastidiosa Virulence Is Affected by Sap Copper Concentration.

Xylella fastidiosa is a xylem-limited plant pathogenic bacterium that causes diseases worldwide in crops such as grape, citrus, and olive. Although copper (Cu)-containing compounds are not used for management of X. fastidiosa-caused diseases, they are widely used in X. fastidiosa hosts in vineyards and orchards. The accumulation of Cu in soils and, therefore, plant saps, could be a challenge for X. fastidiosa survival. Here, the molecular basis of Cu homeostasis was studied in relation to virulence. Although homologous Cu-related genes copA (X. fastidiosa loci PD0100) and copB (PD0101) have been characterized in other bacteria, their functions differ among bacterial species. In vitro, both copA and copB mutants were more sensitive to Cu than the wild-type (WT) strain. Interestingly, the copA mutant was more sensitive to Cu shock, while the copB mutant was more sensitive to chronic Cu treatments. In tobacco greenhouse experiments with normal watering, both mutants reduced virulence compared with WT. But when Cu was added as a drench treatment, both copA and copB mutants had increased disease severity approximately 20 and 50% compared with mutants without Cu added, respectively, which were significantly higher than the approximately 5% observed for WT under the same conditions. These results indicate that the pathogen's Cu homeostasis affects virulence and is influenced by Cu concentration in the environment. Understanding Cu homeostasis in X. fastidiosa will help discern the outcome of Cu treatments and the adaptation of this pathogen to the xylem of plants that have been exposed to high Cu concentrations because of agricultural practices.

Getting Everyone to the Fair: Supporting Teachers in Broadening Participation in Science and Engineering Fairs.

Science and Engineering (S&E) fairs are a valuable educational activity and are believed to increase students' engagement and learning in science and engineering. However, due to differences in resources, many schools do not implement fairs to achieve these benefits for their students. This study reports the findings of a program intended to increase the participation of students from low-achieving and under-resourced schools in a regional fair program that feeds into the international fair competition. We found that the number of schools and projects participating in our regional fair increased dramatically since the start of the program. Teachers had mostly positive expectations for the project and expressed buy-in for the effort the project would take. They recruited a diverse pool of students to participate in the school fairs. Quasi-experimental methods allowed us to explore the impact of completing S&E fairs on student gains on science self-efficacy, interest and value perceptions. Controlling for pre-existing differences in these attitudes, we found that students not completing projects showed declines in their science attitudes during the year. Students who completed projects maintained similar attitudes, while those whose projects advanced to the regional fair had substantial gains on all three variables. It is unknown whether this gain can be attributed to the experience of engaging with a quality project, from being the kind of student who completes a quality project, or some other factor. Future research with greater experimental control could address these questions.

Zinkicide Is a ZnO-Based Nanoformulation with Bactericidal Activity against Liberibacter crescens in Batch Cultures and in Microfluidic Chambers Simulating Plant Vascular Systems.

Phloem-limited bacterial "Candidatus Liberibacter" species are associated with incurable plant diseases worldwide. Antimicrobial treatments for these pathogens are challenging due to the difficulty of reaching the vascular tissue they occupy at bactericidal concentrations. Here, in vitro antimicrobial mechanisms of Zinkicide TMN110 (ZnK), a nonphytotoxic zinc oxide (ZnO)-based nanoformulation, were compared to those of bulk ZnO (b-ZnO) using as a model the only culturable species of the genus, Liberibacter crescens Minimum bactericidal concentration (MBC) determination and time-kill assays showed that ZnK has a bactericidal effect against L. crescens, whereas b-ZnO is bacteriostatic. When ZnK was used at the MBC (150 ppm), its antimicrobial mechanisms included an increase in Zn solubility, generation of intracellular reactive oxygen species, lipid peroxidation, and cell membrane disruption; all of these were of greater intensity than those of b-ZnO. Inhibition of biofilms, which are important during insect vector colonization, was stronger by ZnK than by b-ZnO at concentrations between 2.5 and 10 ppm in batch cultures; however, neither ZnK nor b-ZnO removed L. crescens preformed biofilms when applied between 100 and 400 ppm. In microfluidic chambers simulating source-to-sink phloem movement, ZnK significantly outperformed b-ZnO in Zn mobilization and bactericidal activity against L. crescens planktonic cells in sink reservoirs. In microfluidic chamber assays assessing antibiofilm activity, ZnK displayed a significantly enhanced bactericidal activity against L. crescens individual attached cells as well as preformed biofilms compared to that of b-ZnO. The superior mobility and antimicrobial activity of ZnK in microenvironments make this formulation a promising product to control plant diseases caused by "Candidatus Liberibacter" species and other plant vascular pathogens.IMPORTANCE "Candidatus Liberibacter" species are associated with incurable plant diseases that have caused billions of dollars of losses for United States and world agriculture. Chemical control of these pathogens is complicated, because their life cycle combines intracellular vascular stages in plant hosts with transmission by highly mobile insect vectors. To date, "Candidatus Liberibacter" species are mostly unculturable, except for Liberibacter crescens, a member of the genus that has been used as a model for in vitro assays. Here, we evaluated the potential of Zinkicide (ZnK) as an antimicrobial against "Candidatus Liberibacter" species in batch cultures and under flow conditions, using L. crescens as a biological model. ZnK displayed bactericidal activity against L. crescens in batch cultures and showed increased mobility and bactericidal activity in microfluidic devices resembling "Candidatus Liberibacter" species natural habitats. ZnK performance observed here against L. crescens makes this compound a promising candidate to control plant diseases caused by vascular pathogens.

Copper Supplementation in Watering Solution Reaches the Xylem But Does Not Protect Tobacco Plants Against Xylella fastidiosa Infection.

Xylella fastidiosa is a xylem-limited plant pathogenic bacterium that causes disease in many crops worldwide. Copper (Cu) is an antimicrobial agent widely used on X. fastidiosa hosts to control other diseases. Although the effects of Cu for control of foliar pathogens are well known, it is less studied on xylem-colonizing pathogens. Previous results from our group showed that low concentrations of CuSO4 increased biofilm formation, whereas high concentrations inhibited biofilm formation and growth in vitro. In this study, we conducted in planta experiments to determine the influence of Cu in X. fastidiosa infection using tobacco as a model. X. fastidiosa-infected and noninfected plants were watered with tap water or with water supplemented with 4 mM or 8 mM of CuSO4. Symptom progression was assessed, and sap and leaf ionome analysis was performed by inductively coupled plasma with optical emission spectroscopy. Cu uptake was confirmed by increased concentrations of Cu in the sap of plants treated with CuSO4-amended water. Leaf scorch symptoms in Cu-supplemented plants showed a trend toward more severe at later time points. Quantification of total and viable X. fastidiosa in planta indicated that CuSO4-amended treatments did not inhibit but slightly increased the growth of X. fastidiosa. Cu in sap was in the range of concentrations that promote X. fastidiosa biofilm formation according to our previous in vitro study. Based on these results, we proposed that the plant Cu homeostasis machinery controls the level of Cu in the xylem, preventing it from becoming elevated to a level that would lead to bacterial inhibition.

The mammalian phosphate carrier SLC25A3 is a mitochondrial copper transporter required for cytochrome c oxidase biogenesis.

Copper is required for the activity of cytochrome c oxidase (COX), the terminal electron-accepting complex of the mitochondrial respiratory chain. The likely source of copper used for COX biogenesis is a labile pool found in the mitochondrial matrix. In mammals, the proteins that transport copper across the inner mitochondrial membrane remain unknown. We previously reported that the mitochondrial carrier family protein Pic2 in budding yeast is a copper importer. The closest Pic2 ortholog in mammalian cells is the mitochondrial phosphate carrier SLC25A3. Here, to investigate whether SLC25A3 also transports copper, we manipulated its expression in several murine and human cell lines. SLC25A3 knockdown or deletion consistently resulted in an isolated COX deficiency in these cells, and copper addition to the culture medium suppressed these biochemical defects. Consistent with a conserved role for SLC25A3 in copper transport, its heterologous expression in yeast complemented copper-specific defects observed upon deletion of PIC2 Additionally, assays in Lactococcus lactis and in reconstituted liposomes directly demonstrated that SLC25A3 functions as a copper transporter. Taken together, these data indicate that SLC25A3 can transport copper both in vitro and in vivo.

Dominant, Heritable Resistance to Stewart's Wilt in Maize Is Associated with an Enhanced Vascular Defense Response to Infection with Pantoea stewartii.

Vascular wilt bacteria such as Pantoea stewartii, the causal agent of Stewart's bacterial wilt of maize (SW), are destructive pathogens that are difficult to control. These bacteria colonize the xylem, where they form biofilms that block sap flow leading to characteristic wilting symptoms. Heritable forms of SW resistance exist and are used in maize breeding programs but the underlying genes and mechanisms are mostly unknown. Here, we show that seedlings of maize inbred lines with pan1 mutations are highly resistant to SW. However, current evidence suggests that other genes introgressed along with pan1 are responsible for resistance. Genomic analyses of pan1 lines were used to identify candidate resistance genes. In-depth comparison of P. stewartii interaction with susceptible and resistant maize lines revealed an enhanced vascular defense response in pan1 lines characterized by accumulation of electron-dense materials in xylem conduits visible by electron microscopy. We propose that this vascular defense response restricts P. stewartii spread through the vasculature, reducing both systemic bacterial colonization of the xylem network and consequent wilting. Though apparently unrelated to the resistance phenotype of pan1 lines, we also demonstrate that the effector WtsE is essential for P. stewartii xylem dissemination, show evidence for a nutritional immunity response to P. stewartii that alters xylem sap composition, and present the first analysis of maize transcriptional responses to P. stewartii infection.

The mitochondrial metallochaperone SCO1 maintains CTR1 at the plasma membrane to preserve copper homeostasis in the murine heart.

SCO1 is a ubiquitously expressed, mitochondrial protein with essential roles in cytochrome c oxidase (COX) assembly and the regulation of copper homeostasis. SCO1 patients present with severe forms of early onset disease, and ultimately succumb from liver, heart or brain failure. However, the inherent susceptibility of these tissues to SCO1 mutations and the clinical heterogeneity observed across SCO1 pedigrees remain poorly understood phenomena. To further address this issue, we generated Sco1hrt/hrt and Sco1stm/stm mice in which Sco1 was specifically deleted in heart and striated muscle, respectively. Lethality was observed in both models due to a combined COX and copper deficiency that resulted in a dilated cardiomyopathy. Left ventricular dilation and loss of heart function was preceded by a temporal decrease in COX activity and copper levels in the longer-lived Sco1stm/stm mice. Interestingly, the reduction in copper content of Sco1stm/stm cardiomyocytes was due to the mislocalisation of CTR1, the high affinity transporter that imports copper into the cell. CTR1 was similarly mislocalized to the cytosol in the heart of knockin mice carrying a homozygous G115S substitution in Sco1, which in humans causes a hypertrophic cardiomyopathy. Our current findings in the heart are in marked contrast to our prior observations in the liver, where Sco1 deletion results in a near complete absence of CTR1 protein. These data collectively argue that mutations perturbing SCO1 function have tissue-specific consequences for the machinery that ultimately governs copper homeostasis, and further establish the importance of aberrant mitochondrial signaling to the etiology of copper handling disorders.

Efficient genome editing in Fusarium oxysporum based on CRISPR/Cas9 ribonucleoprotein complexes.

The Fusarium oxysporum species complex (FOSC) is an economically important group of pathogenic filamentous fungi that are able to infect both animals and plants. Reverse genetic techniques, including gene disruption/deletion methods, to study these fungi are available although limitations exist resulting in decreased efficiency. Herein we describe a gene editing system developed using a F. oxysporum-optimized Cas9 ribonucleoprotein (RNP) and protoplast transformation method. The Cas9 protein and sgRNA were assembled to form a stable RNP in vitro and this complex was transferred into fungal protoplasts for gene editing with PEG-mediated transformation. In order to determine if the Cas9 RNP system is functional in the FOSC protoplasts and assess the efficacy of the system, two genes, URA5 and URA3, were selected for targeted disruption generating uracil auxotroph mutants that are resistant to 5-fluoroorotic acid, 5-FOA. In addition, a gene in a secondary metabolite biosynthetic cluster, the ortholog of BIK1, was mutated using this system and the maximum efficiency of this gene disruption was about 50%. Further analysis of the bik1 mutant confirmed that this polyketide synthase was involved in the synthesis of the red pigment, bikaverin. The mutants generated in this study displayed the strong expected phenotypes, demonstrating this F. oxysporum-optimized CRISPR/Cas9 system is stable and can efficiently disrupt the genes of interest.

An in vivo test of the biologically relevant roles of carotenoids as antioxidants in animals.

Carotenoids are well known for their contribution to the vibrant coloration of many animals and have been hypothesized to be important antioxidants. Surprisingly few examples of carotenoids acting as biologically relevant antioxidants in vivo exist, in part because experimental designs often employ carotenoid doses at levels that are rarely observed in nature. Here, we used an approach that reduces carotenoid content from wild-type levels to test for the effect of carotenoids as protectants against an oxidative challenge. We used the marine copepod Tigriopus californicus reared on a carotenoid-free or a carotenoid-restored diet of nutritional yeast and then exposed them to a pro-oxidant. We found that carotenoid-deficient copepods not only accumulated more damage but also were more likely to die during an oxidative challenge than carotenoid-restored copepods. We suggest that carotenoid reduction, and not supplementation, better tests the proposed roles of carotenoids in other physiological functions in animals.

Chloramphenicol Derivatives with Antibacterial Activity Identified by Functional Metagenomics.

A functional metagenomic approach identified novel and diverse soil-derived DNAs encoding inhibitors to methicillin-resistant Staphylococcus aureus (MRSA). A metagenomic DNA soil library containing 19 200 recombinant Escherichia coli BAC clones with 100 Kb average insert size was screened for antibiotic activity. Twenty-seven clones inhibited MRSA, seven of which were found by LC-MS to possess modified chloramphenicol ( Cm) derivatives, including three new compounds whose structures were established as 1-acetyl-3-propanoylchloramphenicol, 1-acetyl-3-butanoylchloramphenicol, and 3-butanoyl-1-propanoylchloramphenicol. Cm was used as the selectable antibiotic for cloning, suggesting that heterologously expressed enzymes resulted in derivatization of Cm into new chemical entities with biological activity. An esterase was found to be responsible for the enzymatic regeneration of Cm, and the gene trfA responsible for plasmid copy induction was found to be responsible for inducing antibacterial activity in some clones. Six additional acylchloramphenicols were synthesized for structure and antibacterial activity relationship studies, with 1- p-nitrobenzoylchloramphenicol the most active against Mycobacterium intracellulare and Mycobacterium tuberculosis, with MICs of 12.5 and 50.0 µg/mL, respectively.

Natural Competence Rates Are Variable Among Xylella fastidiosa Strains and Homologous Recombination Occurs In Vitro Between Subspecies fastidiosa and multiplex.

Xylella fastidiosa, an etiological agent of emerging crop diseases around the world, is naturally competent for the uptake of DNA from the environment that is incorporated into its genome by homologous recombination. Homologous recombination between subspecies of X. fastidiosa was inferred by in silico studies and was hypothesized to cause disease emergence. However, no experimental data are available on the degree to which X. fastidiosa strains are capable of competence and whether recombination can be experimentally demonstrated between subspecies. Here, using X. fastidiosa strains from different subspecies, natural competence in 11 of 13 strains was confirmed with plasmids containing antibiotic markers flanked by homologous regions and, in three of five strains, with dead bacterial cells used as source of donor DNA. Recombination frequency differed among strains and was correlated to growth rate and twitching motility. Moreover, intersubspecific recombination occurred readily between strains of subsp. fastidiosa and multiplex, as demonstrated by movement of antibiotic resistance and green fluorescent protein from donor to recipient cells and confirmed by DNA sequencing of the flanking arms of recombinant strains. Results demonstrate that natural competence is widespread among X. fastidiosa strains and could have an impact in pathogen adaptation and disease development.

The Major Outer Membrane Protein MopB Is Required for Twitching Movement and Affects Biofilm Formation and Virulence in Two Xylella fastidiosa strains.

MopB is a major outer membrane protein (OMP) in Xylella fastidiosa, a bacterial plant pathogen that causes losses on many economically important crops. Based on in silico analysis, the uncharacterized MopB protein of X. fastidiosa contains a ß-barrel structure with an OmpA-like domain and a predicted calcium-binding motif. Here, MopB function was studied by mutational analysis taking advantage of the natural competence of X. fastidiosa. Mutants of mopB were constructed in two different X. fastidiosa strains, the type strain Temecula and the more virulent WM1-1. Deletion of the mopB gene impaired cell-to-cell aggregation, surface attachment, and biofilm formation in both strains. Interestingly, mopB deletion completely abolished twitching motility. Electron microscopy of the bacterial cell surface revealed that mopB deletion eliminated type IV and type I pili formation, potentially caused by destabilization of the outer membrane. Both mopB mutants showed reduced virulence using tobacco (Nicotiana tabacum) as a host under greenhouse conditions. These results suggest that MopB has pleiotropic functions in biofilm formation and twitching motility and is important for virulence of X. fastidiosa.

Copper import into the mitochondrial matrix in Saccharomyces cerevisiae is mediated by Pic2, a mitochondrial carrier family protein.

Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria.

Calcium-Enhanced Twitching Motility in Xylella fastidiosa Is Linked to a Single PilY1 Homolog.

The plant-pathogenic bacterium Xylella fastidiosa is restricted to the xylem vessel environment, where mineral nutrients are transported through the plant host; therefore, changes in the concentrations of these elements likely impact the growth and virulence of this bacterium. Twitching motility, dependent on type IV pili (TFP), is required for movement against the transpiration stream that results in basipetal colonization. We previously demonstrated that calcium (Ca) increases the motility of X. fastidiosa, although the mechanism was unknown. PilY1 is a TFP structural protein recently shown to bind Ca and to regulate twitching and adhesion in bacterial pathogens of humans. Sequence analysis identified three pilY1 homologs in X. fastidiosa (PD0023, PD0502, and PD1611), one of which (PD1611) contains a Ca-binding motif. Separate deletions of PD0023 and PD1611 resulted in mutants that still showed twitching motility and were not impaired in attachment or biofilm formation. However, the response of increased twitching at higher Ca concentrations was lost in the pilY1-1611 mutant. Ca does not modulate the expression of any of the X. fastidiosa PilY1 homologs, although it increases the expression of the retraction ATPase pilT during active movement. The evidence presented here suggests functional differences between the PilY1 homologs, which may provide X. fastidiosa with an adaptive advantage in environments with high Ca concentrations, such as xylem sap.

Metal Uptake by Mitochondrial Carrier Family Proteins Using Lactococcus lactis.

Metal ion homeostasis in mitochondria is essential to maintaining proper cellular physiology. However, the ability of metals to bind off target or form complexes with multiple metabolites presents major challenges to understanding the mechanisms that govern this homeostasis. Adding further to the complexity, some of the major mitochondrial transporters have shown substrate promiscuity. In many cases, mitochondrial metals are found in the matrix compartment that is surrounded by the impermeable inner membrane. Four major classes of transporters facilitate the movement of solute across the inner membrane. These are mitochondrial carrier family, ATP-binding cassette transporters, mitochondrial pyruvate carriers, and sideroflexins. For iron, the matrix is the site of iron-sulfur clusters and heme synthesis and therefore transport must occur in a coordinated fashion with the cellular needs for these critical cofactors. Iron could be transported in numerous forms as it has been shown to form complexes with abundant metabolites such as citrate, nucleotides, or glutathione. Here, we describe assays to study iron (or any metal) transport by mitochondrial carrier family proteins expressed in Lactococcus lactis using a nisin-controlled expression system.