Integration and intramolecular transposition of the TnBP3 Bordetella pertussis transposon in the Escherichia coli K-12 cells -- mutant for the phosphoenolpyruvate-dependent phosphotransferase system Hpr protein

Integration of a plasmid carrying the TnBP3 transposon of Bordetella pertussis into the chromosome of Escherichia coli and transpositions of the integrated structure within a chromosome in the wild-type and mutant cells ptsH devoid of the major Hpr protein of the phosphoenolpyruvate-dependent phosphotransferase system were studied. When transposed to a new chromosome site, the integrated structure was precisely (or almost precisely) excised from the metY gene sequence, which resulted in restoration of the Met+ phenotype. The integration and transposition events were only observed in the E. coli cells carrying the ptsH+ allele. The ptsH mutations inhibited integration and intramolecular transposition, which were restored after phenotypic or genetic suppression of the ptsH mutation. The intensity of the processes studied were suggested to depend on the integrity of a chain that ensures transferring of the phosphoryl residue by proteins of the phosphotransferase system in E. coli K12. The results obtained indicate that the ptsH mutants of E. coli can serve as the optimal host for cloning of fragments carrying repeated sequences of B. pertussis, which may apply to the repeated sequences of other microorganisms.     

Unique Regulation of Carbohydrate Chemotaxis in Bacillus subtilis by the Phosphoenolpyruvate-Dependent Phosphotransferase System and the Methyl-Accepting Chemotaxis Protein McpC

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a major role in the ability of Escherichia coli to migrate toward PTS carbohydrates. The present study establishes that chemotaxis toward PTS substrates in Bacillus subtilis is mediated by the PTS as well as by a methyl-accepting chemotaxis protein (MCP). As for E. coli, a B. subtilis ptsH null mutant is severely deficient in chemotaxis toward most PTS carbohydrates. Tethering analysis revealed that this mutant does respond normally to the stepwise addition of a PTS substrate (positive stimulus) but fails to respond normally to the stepwise removal of such a substrate (negative stimulus). An mcpC null mutant showed no response to the stepwise addition or removal of d-glucose or d-mannitol, both of which are PTS substrates. Therefore, in contrast to E. coli PTS carbohydrate chemotaxis, B. subtilis PTS carbohydrate chemotaxis is mediated by both MCPs and the PTS; the response to positive stimulus is primarily McpC mediated, while the duration or magnitude of the response to negative PTS carbohydrate stimulus is greatly influenced by components of the PTS and McpC. In the case of the PTS substrate d-glucose, the response to negative stimulus is also partially mediated by McpA. Finally, we show that B. subtilis EnzymeI-P has the ability to inhibit B. subtilis CheA autophosphorylation in vitro. We hypothesize that chemotaxis in the spatial gradient of the capillary assay may result from a combination of a transient increase in the intracellular concentration of EnzymeI-P and a decrease in the concentration of carbohydrate-associated McpC as the cell moves down the carbohydrate concentration gradient. Both events appear to contribute to inhibition of CheA activity that increases the tendency of the bacteria to tumble. In the case of d-glucose, a decrease in d-glucose-associated McpA may also contribute to the inhibition of CheA. This bias on the otherwise random walk allows net migration, or chemotaxis, to occur.  In enteric bacteria, chemotaxis toward many carbohydrate attractants is dependent upon components of the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) (1, 9, 15). This carbohydrate transport system consists of an autophosphorylating histidine kinase, EnzymeI, a common phosphocarrier protein, HPr, and a number of substrate-specific transporters, the EnzymeII complexes. At the expense of PEP, EnzymeI autophosphorylates on a histidine residue and transfers this phosphoryl group to a histidine residue on HPr. HPr-P then donates this phosphoryl group to a carbohydrate-specific EnzymeII complex. The carbohydrate substrate is the final phosphoryl group acceptor, as it is transported into the cell and is concomitantly phosphorylated by EnzymeII (13).Chemotaxis is also controlled by a phosphoryl transfer cascade. CheA, in response to an attractant- or repellent-bound receptor (methyl-accepting chemotaxis protein [MCP]), alters its rate of autophosphorylation appropriately to transiently increase or decrease the intracellular CheY-P pool and thereby modulate swimming behavior (4, 16). In enteric bacteria, increased CheY-P leads to tumbling (19). In Bacillus subtilis, increased CheY-P leads to smooth swimming (3). In enteric bacteria, chemotaxis toward PTS substrates requires CheA, CheY, EnzymeI, and HPr but does not depend on the presence of an MCP (12, 18). These observations have led investigators to suggest that the changes in the phosphorylation state of PTS components that accompany carbohydrate transport regulate CheA activity (10).Recent work has provided the following model for the role of the PTS in chemotaxis toward its substrates in Escherichia coli. As the bacteria encounter a PTS carbohydrate, HPr dephosphorylates EnzymeI faster than the latter protein can be rephosphorylated. The resulting increase in unphosphorylated EnzymeI and the resulting decrease in PEP both function to decrease the rate of CheA autophosphorylation. This is believed to lead to a transient decrease in the CheY-P pool that suppresses tumbling, allowing the bacteria to move up the carbohydrate gradient (10).This article describes studies on the process of carbohydrate chemotaxis in B. subtilis. In particular, we provide evidence that McpC is absolutely required for any response to all of the PTS carbohydrates tested. This is surprising considering the fact that McpC has previously been shown to also mediate chemotaxis toward eight different amino acids (11). McpA has previously been shown to partially mediate chemotaxis toward glucose (7). This result is confirmed in the present study with the use of direct behavioral assays. Our results suggest the existence of a multidimensional signaling mechanism involving both the PTS and specific MCPs, an unprecedented finding in the study of the molecular control of bacterial carbohydrate chemotaxis.     

Inactivation of the Phosphoenolpyruvate-Dependent Phosphotransferase System in Various Species of Bacteria by Vinylglycolic Acid

The alkenoic hydroxyacid 2-hydroxy-3-butenoic acid (vinylglycolate) specifically inhibited the phosphotransferase system in a variety of bacteria while not affecting respiration-coupled transport systems.     

Isolation and Genetic Study of Erwinia Mutants Devoid of Common Components of the Phosphoenolpyruvate-Dependent Phosphotransferase System

Mutants of bacteria belonging the genus Erwinia(Erwinia chrysanthemi andErwinia carotovora) with pleiotropic disturbances in the utilization of many substrates were obtained through chemical and transposon mutagenesis. Genetic studies revealed that these mutants had defective ptsI or ptsH genes responsible for the synthesis of common components of the phosphoenolpyruvate-dependent phosphotransferase system, enzyme I and the HPr protein, respectively. The ptsI ⁺ allele in both Erwinia species was cloned in vivo. Mapping of obtained mutations indicated that theptsIand ptsH genes ofE. chrysanthemi do not constitute a linkage group. The ptsI gene is located at 100 min of the chromosomal map, whereas theptsH gene is located at 175 min. Sequencing of a portion of theE. chrysanthemi ptsI gene showed that a product of the cloned DNA region had up to 68% homology with the N terminus of Escherichia coli enzyme I.     

Effects of Insert Size on Transposition Efficiency of the Sleeping Beauty Transposon in Mouse Cells

Transposon vectors are widely used in prokaryotic and lower eukaryotic systems. However, they were not available for use in vertebrate animals until the recent reconstitution of a synthetic fish transposon, Sleeping Beauty (SB). The reacquisition of transposability of the SB transposase fostered great enthusiasm for using transposon vectors as tools in vertebrate animals, particularly for gene transfer to facilitate accelerated integration of transgenes into chromosomes. Here, we report the effects of insert sizes on transposition efficiency of SB. A significant effect of insert size on efficiency of transposition by SB was found. The SB transposase enhanced the integration efficiency effectively for SB transposon up to approximately 5.6 kb, but lost its ability to enhance the integration efficiency when the transposon size was increased to 9.1 kb. This result indicates that the SB transposon system is highly applicable for transferring small genes, but may not be applicable for transferring very large genes. Received October 20, 2000; accepted December 15, 2000.     

Genetic Engineering of the Phosphocarrier Protein NPr of the Escherichia coli Phosphotransferase System Selectively Improves Sugar Uptake Activity

The Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS) in prokaryotes mediates the uptake and phosphorylation of its numerous substrates through a phosphoryl transfer chain where a phosphoryl transfer protein, HPr, transfers its phosphoryl group to any of several sugar-specific Enzyme IIA proteins in preparation for sugar transport. A phosphoryl transfer protein of the PTS, NPr, homologous to HPr, functions to regulate nitrogen metabolism and shows virtually no enzymatic cross-reactivity with HPr. Here we describe the genetic engineering of a "chimeric" HPr/NPr protein, termed CPr14 because 14 amino acid residues of the interface were replaced. CPr14 shows decreased activity with most PTS permeases relative to HPr, but increases activity with the broad specificity mannose permease. The results lead to the proposal that HPr is not optimal for most PTS permeases but instead represents a compromise with suboptimal activity for most PTS permeases. The evolutionary implications are discussed.     

Transposition and inheritance of Bordetella Tn-element in Escherichia coli K12

B. pertussis genetically mobile element TnBp3 integrates the plasmid in E. coli chromosome. During culturing under nonselective conditions the majority of cells of some E. coli strains lose the kanamycin resistance marker, which indicates the instability of TnBp3 inheriting. The stability of inheriting the integrated structure is higher in E. coli cells with recB-21 recC-12 sbcB-2 mutations. The role of RecBC recombination system in extrusion of TnBp3 is discussed.     

Bacteriophage Lambda Receptor Protein in Escherichia coli K-12: Lowered Affinity of Some Mutant Proteins for Maltose-Binding Protein In Vitro

Mutant and wild-type LamB proteins (phage λ receptor proteins) were purified by affinity chromatography with immobilized maltose-binding protein, and their transport functions were tested in reconstituted liposomes. Two mutant proteins exhibited a marked decrease in affinity for immobilized maltose-binding protein, as well as altered transport rates.     

Lactobacillus casei 64H Contains a Phosphoenolpyruvate-Dependent Phosphotransferase System for Uptake of Galactose, as Confirmed by Analysis of ptsH and Different gal Mutants

Galactose metabolism in Lactobacillus casei 64H was analyzed by genetic and biochemical methods. Mutants with defects in ptsH, galK, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin. ptsH mutations abolish growth on lactose, cellobiose, N-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in galK mutants. A mutation in galK in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a galK mutation in combination with a mutation in ptsH completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different.     

Modulation of Escherichia coli Adenylyl Cyclase Activity by Catalytic-Site Mutants of Protein IIAGlc of the Phosphoenolpyruvate:Sugar Phosphotransferase System

It is demonstrated here that in Escherichia coli, the phosphorylated form of the glucose-specific phosphocarrier protein IIA^Glc of the phosphoenolpyruvate:sugar phosphotransferase system is an activator of adenylyl cyclase and that unphosphorylated IIA^Glc has no effect on the basal activity of adenylyl cyclase. To elucidate the specific role of IIA^Glc phosphorylation in the regulation of adenylyl cyclase activity, both the phosphorylatable histidine (H90) and the interactive histidine (H75) of IIA^Glc were mutated by site-directed mutagenesis to glutamine and glutamate. Wild-type IIA^Glc and the H75Q mutant, in which the histidine in position 75 has been replaced by glutamine, were phosphorylated by the phosphohistidine-containing phosphocarrier protein (HPr~P) and were equally potent activators of adenylyl cyclase. Neither the H90Q nor the H90E mutant of IIA^Glc was phosphorylated by HPr~P, and both failed to activate adenylyl cyclase. Furthermore, replacement of H75 by glutamate inhibited the appearance of a steady-state level of phosphorylation of H90 of this mutant protein by HPr~P, yet the H75E mutant of IIA^Glc was a partial activator of adenylyl cyclase. The H75E H90A double mutant, which cannot be phosphorylated, did not activate adenylyl cyclase. This suggests that the H75E mutant was transiently phosphorylated by HPr~P but the steady-state level of the phosphorylated form of the mutant protein was decreased due to the repulsive forces of the negatively charged glutamate at position 75 in the catalytic pocket. These results are discussed in the context of the proximity of H75 and H90 in the IIA^Glc structure and the disposition of the negative charge in the modeled glutamate mutants.     

An Alternative Route for Recycling of N-Acetylglucosamine from Peptidoglycan Involves the N-Acetylglucosamine Phosphotransferase System in Escherichia coli

A set of enzymes dedicated to recycling of the amino sugar components of peptidoglycan has previously been identified in Escherichia coli. The complete pathway includes the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase, of the catabolic pathway for use of N-acetylglucosamine (GlcNAc). Mutations in nagA result in accumulation of millimolar concentrations of GlcNAc6P, presumably by preventing peptidoglycan recycling. Mutations in the genes encoding the key enzymes upstream of nagA in the dedicated recycling pathway (ampG, nagZ, nagK, murQ, and anmK), which were expected to interrupt the recycling process, reduced but did not eliminate accumulation of GlcNAc6P. A mutation in the nagE gene of the GlcNAc phosphotransferase system (PTS) was found to reduce by 50% the amount of GlcNAc6P which accumulated in a nagA strain and, together with mutations in the dedicated recycling pathway, eliminated all the GlcNAc6P accumulation. This shows that the nagE-encoded PTS transporter makes an important contribution to the recycling of peptidoglycan. The manXYZ-encoded PTS transporter makes a minor contribution to the formation of cytoplasmic GlcNAc6P but appears to have a more important role in secretion of GlcNAc and/or GlcNAc6P from the cytoplasm.Peptidoglycan (PG) or murein, the rigid shape-forming layer of the bacterial cell envelope, undergoes extensive degradation and resynthesis during normal bacterial growth. It is estimated that 40 to 50% of the PG is broken down and reused each generation (for a review, see reference 22). PG is a matrix of chains of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) sugars cross-linked by peptide bridges. Over the last 20 years the pathways for recycling both the peptide and amino sugar portions of the PG have been elucidated, and a number of genes involved in this process have been identified. Most of the genes involved encode dedicated enzymes whose only function seems to be to recover the material produced during PG turnover and to reuse it to synthesize more PG or as a source of energy. However, some of the enzymes shown to be involved have apparently been recruited from another metabolic pathway (e.g., murQ- and nagA-encoded enzymes [see below]), while other specialized PG-recycling enzymes have a subsidiary function (e.g., ampG- and ampD-encoded enzymes in β-lactamase induction [20]).The pathway for recycling the amino sugar part of PG in Escherichia coli is shown in Fig. ​Fig.11 (for a review, see reference 22). Periplasmic hydrolases (lytic transglycosylases, Slt) and endopeptidases break the PG backbone, liberating anhydro-muropeptides (principally GlcNAc-anhydro-MurNAc [anhMurNAc]-tetrapeptide), which are transported into the cytoplasm by the ampG-encoded transporter (10). The peptide portion is cleaved off either by the membrane-associated amiD-encoded amidase (28) or by the ampD-encoded cytoplasmic amidase (11), liberating the disaccharide. The tetrapeptide is converted to a tripeptide and free d-Ala, both of which are reused to produce UDP-MurNAc-pentapeptide (11). The GlcNAc-anhMurNAc disaccharide is cleaved by the nagZ-encoded β-N-acetylglucosaminidase (2, 32), and then both sugars are converted to their 6-phosphate forms by the specific kinases NagK (29) and AnmK (31). The latter produces MurNAc-6-phosphate (MurNAc6P), which is converted to GlcNAc6P by the murQ-encoded etherase (12, 30). MurNAc6P is also the product of transport of MurNAc by the MurNAc-specific phosphotransferase system (PTS) transporter MurP. The murP and murQ genes form an operon for use of MurNAc as a carbon source (4). Thus, the MurQ protein has both catabolic and recycling functions (12, 30). Similarly, further use of the GlcNAc6P involves an enzyme normally involved in the catabolism of GlcNAc, the nagA-encoded GlcNAc6P deacetylase of the GlcNAc degradation pathway (21). The deacetylase converts GlcNAc6P to glucosamine-6-phosphate (GlcN6P), which can be converted to UDP-GlcNAc, the first dedicated compound for the synthesis of the cell wall components, by the glmM- and glmU-encoded enzymes (16, 17).Open in a separate windowFIG. 1.Scheme for recycling of PG in E. coli. The enzymes and substrates are described in the text. Slt is the major soluble lytic transglycosylase. OM, outer membrane; PP, periplasm; IM, inner membrane. The enzymes involved in converting UDP-GlcNAc into the components of the PG and outer membrane are not shown. Arrows with a question mark indicate the pathways postulated to exist based on the results described in this work.It has been known for many years that mutations in nagA lead to very high levels of GlcNAc6P (33). Strains carrying nagA mutations are Nag^Sensitive (i.e., they do not grow in medium containing GlcNAc and another carbon source). The toxicity of the accumulated sugar phosphates means that secondary mutations that alleviate this toxicity arise spontaneously in vivo (33). GlcNAc6P is the inducing signal for the NagC repressor of the nag regulon, and the accumulation of GlcNAc6P in the nagA strain results in derepression (endogenous induction) of the nag regulon (25). One class of suppressor mutations result in noninducible versions of NagC that are not sensitive to GlcNAc6P, so that the nag genes stay repressed (23), implying that overexpression of the nag regulon genes is one cause of the toxicity. Amino sugars are essential constituents of the bacterial PG and lipopolysaccharide (LPS) in gram-negative bacteria. In the absence of an exogenous supply of amino sugars, glmS, encoding GlcN6P synthase, is an essential gene (for a review, see reference 7). As GlcNAc6P accumulates in nagA cells growing in medium devoid of amino sugars, it must ultimately be derived from the de novo synthesis of GlcN6P by GlmS, which is destined for synthesis of PG and the LPSs of the outer membrane. As no acetyltransferase for GlcN6P has been characterized, the most likely origin of the GlcNAc6P in nagA strains is recycling of the PG. The LPS of the outer membrane of gram-negative bacteria also contains GlcN, but it is not known to undergo any turnover and the work of Park (21) showed that radioactive GlcN was stably incorporated into the LPS fraction, whereas radioactivity was slowly lost from the PG of isolated sacculi.In this work the effect of mutations in the recycling pathway on the accumulation of GlcNAc6P in vivo was investigated. The results show that mutations in one or more genes of the recycling pathway reduce but do not eliminate GlcNAc6P accumulation in nagA strains. However, when these mutations are present in the same strain with a mutation in the nagE gene encoding the GlcNAc6P-specific transporter of the GlcNAc PTS, GlcNAc6P levels decrease to the background level. This shows that the GlcNAc PTS is another pathway that is involved in recycling the GlcNAc component of PG. The manXYZ-encoded PTS transporter is also capable of GlcNAc uptake, and its effect on the recycling process was also examined.     

Regulation of Lactose Transport by the Phosphoenolpyruvate-Sugar Phosphotransferase System in Membrane Vesicles of Escherichia coli

Regulation of lactose uptake by the phosphoenolpyruvate-sugar phosphotransferase system (PTS) has been demonstrated in membrane vesicles of Escherichia coli strain ML308-225. Substrates of the phosphotransferase system inhibited D-lactate energized uptake of lactose but did not inhibit uptake of either L-alanine or L-proline. This inhibition was reversed by intravesicular (but not extravesicular) phosphoenolpyruvate. Lactose uptake was also inhibited by enzyme III^glc preparations that were shocked into the vesicles, and this inhibition was reversed by phosphoenolpyruvate. Intravesicular HPr and enzyme I stimulated methyl α-glucoside uptake but did not inhibit or stimulate lactose accumulation. Vesicles maintained at 0°C for several days partially lost 1) the ability to take up lactose, 2) the ability to accumulate PTS substrates, and 3) PTS-mediated regulation. Phosphoenolpyruvate addition restored all of these activities. These results support a mechanism in which the relative proportions of phosphorylated and nonphosphorylated forms of a phosphotransferase constituent regulate the activity of the lactose permcase.     

反转录病毒MSCV介导汉坦病毒核蛋白基因在NIH3T3细胞中整合和表达

汉坦病毒是引起肾综合征出血热(HFRS)和汉坦病毒型肺炎综合征(HPS)的主要病原体.由S基因编码的核蛋白(NP)主要与机体的细胞免疫有关,并调节病毒的复制及诱导细胞程序性死亡.构建了汉坦病毒Z10株核蛋白cDNA与含有pac基因的反转录病毒鼠干细胞病毒(MSCV)重组体MSCV-FlagNP,通过磷酸钙转录法导入产病毒的包装细胞系BOSC23中,产生完整的重组MSCV-FlagNP病毒.然后以重组病毒感染NIH 3T3细胞,利用Puromycin的选择特性(pac基因)对感染细胞进行连续压力筛选,获得了转核蛋白抗性细胞.利用Southern blot和PCR方法分别对核蛋白基因在抗性细胞染色体整合情况及其完整性进行了鉴定.并且用Western blot在抗性细胞中可检测到核蛋白的表达.进一步以Flag单克隆抗体介导的免疫荧光染色联合共聚焦激光扫描荧光显微镜,分析了内源性Flag融合核蛋白在抗性细胞内分布,发现核蛋白主要分布于胞浆及胞核周围区,并且部分核蛋白可聚集形成胞浆包涵体.转核蛋白基因细胞模型的建立,对进一步研究汉坦病毒核蛋白功能以及病毒复制机制有重要意义.     

Mutant of Escherichia coli with Thermosensitive Protein in the Process of Cellular Division

A new thermosensitive mutant of Escherichia coli deficient in cell division was isolated by means of membrane filtration after nitrosoguanidine mutagenesis. The mutant cells grow normally at 30 C but stop dividing immediately after shift to 42 C, resulting in multinucleated filaments lacking septa. The number of colony-forming units does not decrease for at least 6 hr at 42 C. The maximum length of the filaments is 10 to 16 times that of normal cells. Addition of a high concentration of NaCl fails to stimulate cell division at 42 C. The filaments formed at 42 C divide abruptly 30 min after shift to 30 C, and synchronous increase of cell number is shown for 3 hr. The macromolecular synthesis of protein and nucleic acids at 42 C is normal on the whole. The cell division shown after the shift from 42 to 30 C is observed in the absence of thymine, but not in the presence of chloramphenicol or in a medium deficient in amino acids. However, the filament can divide to some extent in the presence of chloramphenicol if some protein synthesis is allowed to proceed at 30 C before the addition of the antibiotic. The elongated cells divide at 42 C provided that they are exposed to 30 C before being shifted to high temperature.     

Transposition of the Tn5 and Tn10 elements and duplications in Escherichia coli K12 genome

The kinetics of accumulation of resident transposon copies in a dividing population has been defined using a special experimental system. Analysis of the kinetics made it possible to estimate the probability of transposition for Tn5 as 2.5 X 10(-4) and for Tn10 as 2.3 X 10(-6) per cell per generation. Transposition of the composite elements does not depend on RecBC or RecF pathways of recombination. The fraction of the bacterial population with tandem duplications in the proA region of the genome is permanent for Escherichia coli. It is independent of the recombination pathways (RecBC of RecF) and the integrity of DNA polymerase I.     

Molecular Interactions of the Min Protein System Reproduce Spatiotemporal Patterning in Growing and Dividing Escherichia coli Cells

Oscillations of the Min protein system are involved in the correct midcell placement of the divisome during Escherichia coli cell division. Based on molecular interactions of the Min system, we formulated a mathematical model that reproduces Min patterning during cell growth and division. Specifically, the increase in the residence time of MinD attached to the membrane as its own concentration increases, is accounted for by dimerisation of membrane-bound MinD and its interaction with MinE. Simulation of this system generates unparalleled correlation between the waveshape of experimental and theoretical MinD distributions, suggesting that the dominant interactions of the physical system have been successfully incorporated into the model. For cells where MinD is fully-labelled with GFP, the model reproduces the stationary localization of MinD-GFP for short cells, followed by oscillations from pole to pole in larger cells, and the transition to the symmetric distribution during cell filamentation. Cells containing a secondary, GFP-labelled MinD display a contrasting pattern. The model is able to account for these differences, including temporary midcell localization just prior to division, by increasing the rate constant controlling MinD ATPase and heterotetramer dissociation. For both experimental conditions, the model can explain how cell division results in an equal distribution of MinD and MinE in the two daughter cells, and accounts for the temperature dependence of the period of Min oscillations. Thus, we show that while other interactions may be present, they are not needed to reproduce the main characteristics of the Min system in vivo.