RESUMO
Receptor-binding proteins (RBPs) allow phages to dock onto their host and initiate infection through the recognition of proteinaceous or saccharidic receptors located on the cell surface. FhuA is the ferrichrome hydroxamate transporter in Escherichia coli and serves as a receptor for the well-characterized phages T1, T5, and phi80. To further characterize how other FhuA-dependent phages attach to FhuA, we isolated and published the genomes of three new FhuA-dependent coliphages: JLBYU37, JLBYU41, and JLBYU60. We identified the egions of FhuA involved in phage attachment by testing the effect of mutant fhuA alleles containing single-loop deletions of extracellular loops (L3, L4, L5, L8, L10, and L11) on phage infectivity. Deletion of loop 8 resulted in complete resistance to SO1-like phages JLBYU37 and JLBYU60 and the previously isolated vB_EcoD_Teewinot phage, but no single-loop deletions significantly altered the infection of T1-like JLBYU41. Additionally, lipopolysaccharide (LPS) truncation coupled with the L5 mutant significantly impaired the infectivity of JLBYU37 and JLBYU60. Moreover, significant reductions in the infectivity of JLBYU41 were observed upon LPS truncation in the L8 mutant strain. Analysis of the evolutionary relationships among FhuA-dependent phage RBPs highlights the conservation of L8 dependence in JLBYU37, JLBYU60, Teewinot, T5, and phi80, but also showcases how positive selective pressure and/or homologous recombination also selected for L4 dependence in T1 and even the lack of complete loop dependence in JLBYU41. IMPORTANCE Phage attachment is the first step of phage infection and plays a role in governing host specificity. Characterizing the interactions taking place between phage tail fibers and bacterial receptors that better equip bacteria to survive within the human body may provide insights to aid the development of phage therapeutics.
Assuntos
Bacteriófagos , Proteínas de Escherichia coli , Humanos , Proteínas de Escherichia coli/química , Proteínas de Bactérias/metabolismo , Ferricromo/metabolismo , Ferricromo/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Lipopolissacarídeos/metabolismo , Receptores Virais/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Colífagos/genética , Colífagos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismoRESUMO
BACKGROUND: Phosphate is an essential compound for life. Escherichia coli employs a signal transduction pathway that controls the expression of genes that are required for the high-affinity acquisition of phosphate and the utilization of alternate sources of phosphorous. These genes are only expressed when environmental phosphate is limiting. The seven genes for this signaling pathway encode the two-component regulatory proteins PhoB and PhoR, as well as the high-affinity phosphate transporter PstSCAB and an auxiliary protein called PhoU. As the sensor kinase PhoR has no periplasmic sensory domain, the mechanism by which these cells sense environmental phosphate is not known. This paper explores the hypothesis that it is the alternating conformations of the PstSCAB transporter which are formed as part of the normal phosphate transport cycle that signal phosphate sufficiency or phosphate limitation. RESULTS: We tested two variants of PstB that are predicted to lock the protein in either of two conformations for their signaling output. We observed that the pstBQ160K mutant, predicted to reside in an inward-facing, open conformation signaled phosphate sufficiency whereas the pstBE179Q mutant, predicted to reside in an outward-facing, closed conformation signaled phosphate starvation. Neither mutant showed phosphate transport. CONCLUSIONS: These results support the hypothesis that the alternating conformations of the PstSCAB transporter are sensed by PhoR and PhoU. This sensory mechanism thus controls the alternate autokinase and phospho-PhoB phosphatase activities of PhoR, which ultimately control the signaling state of the response regulator PhoB.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Transdução de Sinais/genética , Fosfatase Alcalina/análise , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/genética , Histidina Quinase/genética , Histidina Quinase/metabolismo , Homeostase/genética , Homeostase/fisiologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Proteínas Quinases , Alinhamento de Sequência , Inanição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Proper phosphate signaling is essential for robust growth of Escherichia coli and many other bacteria. The phosphate signal is mediated by a classic two component signal system composed of PhoR and PhoB. The PhoR histidine kinase is responsible for phosphorylating/dephosphorylating the response regulator, PhoB, which controls the expression of genes that aid growth in low phosphate conditions. The mechanism by which PhoR receives a signal of environmental phosphate levels has remained elusive. A transporter complex composed of the PstS, PstC, PstA, and PstB proteins as well as a negative regulator, PhoU, have been implicated in signaling environmental phosphate to PhoR. RESULTS: This work confirms that PhoU and the PstSCAB complex are necessary for proper signaling of high environmental phosphate. Also, we identify residues important in PhoU/PhoR interaction with genetic analysis. Using protein modeling and docking methods, we show an interaction model that points to a potential mechanism for PhoU mediated signaling to PhoR to modify its activity. This model is tested with direct coupling analysis. CONCLUSIONS: These bioinformatics tools, in combination with genetic and biochemical analysis, help to identify and test a model for phosphate signaling and may be applicable to several other systems.
Assuntos
Escherichia coli/metabolismo , Fosfatos/metabolismo , Transdução de Sinais , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Robust growth in many bacteria is dependent upon proper regulation of the adaptive response to phosphate (Pi) limitation. This response enables cells to acquire Pi with high affinity and utilize alternate phosphorous sources. The molecular mechanisms of Pi signal transduction are not completely understood. PhoU, along with the high-affinity, Pi-specific ATP-binding cassette transporter PstSCAB and the two-component proteins PhoR and PhoB, is absolutely required for Pi signaling in Escherichia coli. Little is known about the role of PhoU and its function in regulation. We have demonstrated using bacterial two-hybrid analysis and confirmatory coelution experiments that PhoU interacts with PhoR through its PAS (Per-ARNT-Sim) domain and that it also interacts with PstB, the cytoplasmic component of the transporter. We have also shown that the soluble form of PhoU is a dimer that binds manganese and magnesium. Alteration of highly conserved residues in PhoU by site-directed mutagenesis shows that these sites play a role in binding metals. Analysis of these phoU mutants suggests that metal binding may be important for PhoU membrane interactions. Taken together, these results support the hypothesis that PhoU is involved in the formation of a signaling complex at the cytoplasmic membrane that responds to environmental Pi levels.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Magnésio/metabolismo , Manganês/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fosfatos/metabolismo , Fatores de Transcrição/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Membrana Celular/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Ligação Proteica , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
Expression of the Pho regulon in Escherichia coli is induced in response to low levels of environmental phosphate (P(i)). Under these conditions, the high-affinity PstSCAB(2) protein (i.e., with two PstB proteins) is the primary P(i) transporter. Expression from the pstSCAB-phoU operon is regulated by the PhoB/PhoR two-component regulatory system. PhoU is a negative regulator of the Pho regulon; however, the mechanism by which PhoU accomplishes this is currently unknown. Genetic studies of phoU have proven to be difficult because deletion of the phoU gene leads to a severe growth defect and creates strong selection for compensatory mutations resulting in confounding data. To overcome the instability of phoU deletions, we employed a promoter-swapping technique that places expression of the phoBR two-component system under control of the P(tac) promoter and the lacO(ID) regulatory module. This technique may be generally applicable for controlling expression of other chromosomal genes in E. coli. Here we utilized P(phoB)::P(tac) and P(pstS)::P(tac) strains to characterize phenotypes resulting from various DeltaphoU mutations. Our results indicate that PhoU controls the activity of the PstSCAB(2) transporter, as well as its abundance within the cell. In addition, we used the P(phoB)::P(tac) DeltaphoU strain as a platform to begin characterizing new phoU mutations in plasmids.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Fatores de Transcrição/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Deleção de Genes , Genes Essenciais , Proteínas de Membrana Transportadoras/genética , Fosfatos/metabolismo , Regiões Promotoras Genéticas , Recombinação Genética , Supressão Genética , Fatores de Transcrição/genéticaRESUMO
The ability to control the expression of chromosomal genes is important for many applications, including metabolic engineering and the functional analysis of cellular processes. This mini-review presents recent work on the application of techniques that allow researchers to replace a chromosomal promoter with one designed for a specific level of activity, thereby exerting precise transcriptional control while retaining the natural genetic context of a gene or operon. This technique, termed promoter swapping, involves the creation of a PCR product that encodes a removable antibiotic resistance cassette and an engineered promoter. Short homology sequences on the ends of the PCR fragment target it for homologous recombination with the chromosome catalyzed by phage-derived recombination proteins. After the PCR product is introduced by electroporation into an appropriate acceptor strain, antibiotic resistance selects the desired recombination products. The antibiotic resistance cassette is then removed from the strain by site-specific recombination leaving the engineered promoter precisely positioned upstream of a target gene but downstream of a short scar consisting of a single site-specific recombination site.
Assuntos
Bactérias/enzimologia , Bactérias/genética , Regulação da Expressão Gênica , Engenharia Genética/métodos , Mutagênese Insercional/métodos , Regiões Promotoras Genéticas , Recombinação Genética , Bactérias/metabolismo , Biotecnologia/métodos , Redes e Vias Metabólicas/genéticaRESUMO
Phosphorus is required for many biological molecules and essential functions, including DNA replication, transcription of RNA, protein translation, posttranslational modifications, and numerous facets of metabolism. In order to maintain the proper level of phosphate for these processes, many bacteria adapt to changes in environmental phosphate levels. The mechanisms for sensing phosphate levels and adapting to changes have been extensively studied for multiple organisms. The phosphate response of Escherichia coli alters the expression of numerous genes, many of which are involved in the acquisition and scavenging of phosphate more efficiently. This review shares findings on the mechanisms by which E. coli cells sense and respond to changes in environmental inorganic phosphate concentrations by reviewing the genes and proteins that regulate this response. The PhoR/PhoB two-component signal transduction system is central to this process and works in association with the high-affinity phosphate transporter encoded by the pstSCAB genes and the PhoU protein. Multiple models to explain how this process is regulated are discussed.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fosfatos/metabolismo , Regulon , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Transdução de Sinais , Fatores de TranscriçãoRESUMO
Structures of the inactive and activated forms of the receiver domain of PhoB reported in this issue of Structure (Bachhawat et al., 2005) suggest that the OmpR/PhoB subclass of transcription factors becomes active by dimerization about a symmetric axis utilizing the alpha4-beta5-alpha5 surface.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Estrutura Terciária de ProteínaRESUMO
In addition to the Pho regulon, phosphate starvation also stimulates the accumulation of RpoS. Several deletion mutations within the pstSCAB-phoU operon were tested for the accumulation of RpoS during exponential growth. Our data suggest that the processed 3' end of the pstA message stimulates translation of rpoS.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , DNA Intergênico/genética , Proteínas de Escherichia coli/genética , Fosfatos/deficiência , Biossíntese de Proteínas , Fator sigma/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Intergênico/fisiologia , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Modelos Genéticos , Mutação , Fator sigma/metabolismo , Transcrição GênicaRESUMO
In Escherichia coli, PhoR is the histidine kinase of the phosphate regulon. It has been postulated that PhoR may function as a phospho-PhoB phosphatase. Experiments with four precise phoR deletion mutants supported this hypothesis and suggested that this activity resides within the histidine phosphorylation domain. This biochemical activity was confirmed by using a separately expressed histidine phosphorylation domain.