Recombinant Photorhabdus luminescens subsp. laumondii Zinc import ATP-binding protein ZnuC (znuC)
Description
Introduction to Photorhabdus luminescens and ZnuC
Photorhabdus luminescens is a bioluminescent, Gram-negative bacterium that belongs to the family Enterobacteriaceae. It is a nematode symbiont that is pathogenic to insects . Photorhabdus has the means to secrete virulence factors and establish microbial infections .
Zinc is an essential trace metal for all forms of life, playing crucial roles in various biological processes . It functions as a structural component in proteins and is critical for catalytic activity in many enzymes . Zinc homeostasis, involving zinc uptake, storage, and efflux, is tightly regulated within cells . ZnuC is an ATP-binding protein involved in zinc transport .
Zinc and Bacterial Pathogenesis
Zinc is vital for bacterial survival and pathogenesis . High-affinity zinc uptake systems are essential for pathogenic bacteria to counteract the host's nutritional immunity . Nutritional immunity is a strategy employed by hosts to limit the availability of essential nutrients, such as zinc, to invading pathogens .
ZnuABC Transporter
The ZnuABC transporter is a high-affinity zinc uptake system found in bacteria . It is composed of three proteins:
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ZnuA: A periplasmic zinc-binding protein.
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ZnuB: An integral membrane protein that forms the transmembrane channel.
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ZnuC: An ATP-binding protein that provides the energy for zinc transport across the membrane.
Role of ZnuC in Zinc Transport
ZnuC is a critical component of the ZnuABC transporter, essential for zinc import in bacteria . ATP-binding proteins like ZnuC utilize the energy from ATP hydrolysis to drive the translocation of zinc across the cytoplasmic membrane .
Zinc Finger Proteins
Zinc finger proteins are characterized by the coordination of one or more zinc ions (Zn2+), which stabilizes their structure . Zinc finger domains are relatively small protein motifs containing multiple finger-like protrusions that make tandem contacts with their target molecule . These domains may or may not bind zinc, and can bind other metals such as iron, or no metal at all . Zinc finger proteins are involved in various cellular processes, including gene transcription, translation, mRNA trafficking, and protein folding .
Regulation of Zinc Uptake
The Zur (zinc uptake regulator) protein regulates zinc homeostasis in bacteria . Zur is a transcriptional repressor that binds to specific DNA sequences in the promoter regions of zinc uptake genes, such as znuABC, under zinc-replete conditions . Under zinc-limiting conditions, Zur releases from the DNA, allowing transcription of the zinc uptake genes .
Research Findings
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Transcriptional Analysis: Studies have shown that the expression of znuA is regulated by Zur in response to zinc availability .
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Electrophoretic Mobility Shift Assay (EMSA): EMSA experiments have demonstrated that Zur directly binds to the promoter region of znuA in a zinc-dependent manner .
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Mutant Studies: Mutants lacking functional ZnuABC transporters exhibit defects in zinc uptake and symbiotic interactions .
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Complementation Studies: Symbiotic defects associated with znu-derived mutants can be overcome by adding zinc to the environment, indicating the importance of zinc uptake for bacterial symbiosis .
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Reporter Gene Fusions: Reporter constructs using gfpmut2 gene fusions have been used to study gene expression in Photorhabdus luminescens .
Table: Examples of Zinc Transporters and Regulators
Product Specs
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Target Background
This protein is a component of the ZnuABC ABC transporter complex, responsible for zinc import into the cell. It plays a crucial role in energy coupling for the transport system.
KEGG: plu:plu2114
STRING: 243265.plu2114
Q&A
Basic Research Questions
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What is the function of ZnuC in Photorhabdus luminescens and why is it important in bacterial physiology?
ZnuC functions as the ATP-binding component of the ZnuABC transporter system, which is essential for high-affinity zinc acquisition in Photorhabdus luminescens. This system is crucial because bacteria must acquire zinc from extremely limited environments, and ATP binding cassette (ABC) transporters perform this function . The ZnuABC system consists of a periplasmic solute binding protein (ZnuA), a membrane permease (ZnuB), and an ATP-binding protein (ZnuC). ZnuC provides the energy required for zinc transport through ATP hydrolysis, enabling the bacterium to maintain zinc homeostasis, which is essential for numerous cellular processes including protein synthesis, DNA replication, and defense against oxidative stress .
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How does zinc transport via ZnuC differ between Photorhabdus luminescens and other bacterial species like Escherichia coli?
While the general mechanism of zinc transport via ZnuC is conserved across bacterial species, there are notable differences between P. luminescens and E. coli systems. In E. coli, zinc homeostasis involves a balance between the import system ZnuABC and export system ZntA, both regulated by zinc-responsive transcription factors . P. luminescens ZnuC contains distinct motifs with a high concentration of histidine residues (HHHHDHHQ) in its sequence that may play specific roles in its function . Additionally, P. luminescens inhabits both insect and nematode hosts, suggesting its zinc acquisition systems may be adapted to these unique host environments. The expression patterns of znuC in P. luminescens also differ from E. coli, as P. luminescens likely has specific adaptations for environmental transitions between its hosts .
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What are the structural characteristics of P. luminescens ZnuC protein?
The P. luminescens ZnuC protein consists of 271 amino acids with a molecular sequence starting with MSTLITLKNVAVNFGDRRVLNNISLHLQRGNILTLLGPNGAGKSTLVRVVLGLIEPSSGTIEQTDGLKIGYVPQKLHLDPTLPLTVKRFMMLKPGVKSGDILPALERVNAAHLLQQPMQKLSGGESQRVLLARALLNQPQLLVLDEPTQGVDVNGQLALYDLINQIRTELHCAVLMVSHDLHLVMAKTDEVLCLNQHICCSGTPEVVSTHPEFIAMFGHHGAEQLAIYRHQHDHHQHNHHHDLKGKIILENNRECHS . The protein contains the characteristic nucleotide-binding domain (NBD) typical of ABC transporters, with the Walker A motif (GPNGAGKST) for ATP binding, Walker B motif for ATP hydrolysis, and signature C motif. Notably, it contains histidine-rich regions that likely participate in zinc coordination. The protein adopts a structure similar to other ABC transporter NBDs, with two lobes that sandwich the ATP molecule during the transport cycle .
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What expression systems are commonly used for producing recombinant P. luminescens ZnuC?
Escherichia coli is the most commonly used expression system for producing recombinant P. luminescens ZnuC due to its simplicity, rapid growth, and high protein yields . The expression typically involves cloning the znuC gene into an expression vector with an inducible promoter (such as T7 or arabinose-inducible systems) and optimal codon usage for E. coli. Other expression systems that can be used include yeast, mammalian cells, and baculovirus-insect cell systems, particularly when post-translational modifications or specific folding environments are required . For functional studies, the pSEVA-based vectors have been successfully used for expression in Photorhabdus and related bacteria, allowing for native regulation and function assessment . When high purity is required, the protein is typically expressed with affinity tags such as His-tag for simplified purification and can achieve >85% purity through optimized protocols .
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What methods are used to assess the function of ZnuC in zinc transport?
Several methodologies are employed to assess ZnuC function in zinc transport:
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Genetic approaches: Constructing znuC knockout strains and complementation studies to evaluate growth under zinc-limited conditions .
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Transport assays: Using radioactive 65Zn or fluorescent zinc probes to measure zinc uptake in cells expressing wild-type or mutant ZnuC .
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ATPase activity assays: Measuring ATP hydrolysis rates in purified ZnuC to assess its enzymatic function, typically using colorimetric phosphate detection methods .
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Expression analysis: Quantifying znuC expression using reporter systems (like Lux) under various zinc concentrations to understand regulation .
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Growth experiments: Comparing growth curves of wild-type and znuC mutant strains under normal and zinc-limited conditions to assess physiological impact .
Method Measured Parameters Advantages Limitations Gene knockouts Growth in zinc-limited media Direct assessment of physiological relevance May have compensatory mechanisms 65Zn uptake Transport rate, Km, Vmax Quantitative measurement of transport Requires radioactive materials ATPase assays ATP hydrolysis rate Direct measure of enzyme activity May not reflect in vivo activity Reporter systems Transcriptional response Real-time monitoring possible Indirect measure of transport Bacterial growth Growth rate, lag phase Physiologically relevant Non-specific, affected by multiple factors -
