Flagellar assembly mutants in Escherichia coli

Genetic and biochemical analysis of mutants defective in the synthesis of flagella in Escherichia coli revealed an unusual class of mutants. These mutants were found to produce short, curly, flagella-like filaments with low amplitude ( approximately 0.06 mum). The filaments were connected to characteristic flagellar basal caps and extended for 1 to 2 mum from the bacterial surface. The mutations in these strains were all members of one complementation group, group E, which is located between his and uvrC. The structural, serological, and chemical properties of the filament derived from the mutants closely resemble those of the flagellar hook structure. On the basis of these properties, it is suggested that these filaments are "polyhooks", i.e., repeated end-to-end polymers of the hook portion of the flagellum. Polyhooks are presumed to be the result of a defective cistron which normally functions to control the length of the hook region of the flagellum.     

Regulatory Network of the Initiation of Chromosomal Replication in Escherichia coli

ABSTRACT

The bacterial chromosome is replicated once during the division cycle, a process ensured by the tight regulation of initiation at oriC. In prokaryotes, the initiator protein DnaA plays an essential role at the initiation step, and feedback control is critical in regulating initiation. Three systems have been identified that exert feedback control in Escherichia coli, all of which are necessary for tight strict regulation of the initiation step. In particular, the ATP-dependent control of DnaA activity is essential. A missing link in initiator activity regulation has been identified, facilitating analysis of the reaction mechanism. Furthermore, key components of this regulatory network have also been described. Because the eukaryotic initiator complex, ORC, is also regulated by ATP, the bacterial system provides an important model for understanding initiation in eukaryotes. This review summarizes recent studies on the regulation of initiator activity.     

Genetic Analysis of Flagellar Mutants in Escherichia coli

Flagellar mutants in Escherichia coli were obtained by selection for resistance to the flagellotropic phage chi. F elements covering various regions of the E. coli genome were then constructed, and, on the basis of the ability of these elements to restore flagellar function, the mutations were assigned to three regions of the E. coli chromosome. Region I is between trp and gal; region II is between uvrC and aroD; and region III is between his and uvrC. F elements carrying flagellar mutations were constructed. Stable merodiploid strains with a flagellar defect on the exogenote and another on the endogenote were then prepared. These merodiploids yielded information on the complementation behavior of mutations in a given region. Region III was shown to include at least six cistrons, A, B, C, D, E, and F. Region II was shown to include at least four cistrons, G, H, I, and J. Examination of the phenotypes of the mutants revealed that those with lesions in cistron E of region III produce "polyhooks" and lesions in cistron F of region III result in loss of ability to produce flagellin. Mutants with lesions in cistron J of region II were entirely paralyzed (mot) mutants. Genetic analysis of flagellar mutations in region III suggested that the mutations located in cistrons A, B, C, and E are closely linked and mutations in cistrons D and F are closely linked.     

Flagellar formation in Escherichia coli electron transport mutants.

Mutants of Escherichia coli lacking ubiquinone or heme have been tested for motility and found to be essentially immotile. The loss of motility is identified with the loss of flagellum synthesis.     

Flagellar cross-repolymerization among different Salmonella serotypes and Escherichia coli

Flagellar preparations from several serotypes of Salmonella and one strain of Escherichia coli were tested for their ability to cross-repolymerize. Repolymerization was found not to be dependent on the antigenic determinants of the flagella.     

Subunit Organization and Reversal-associated Movements in the Flagellar Switch of Escherichia coli

Bacterial flagella contain a rotor-mounted protein complex termed the switch complex that functions in flagellar assembly, rotation, and clockwise/counterclockwise direction control. In Escherichia coli and Salmonella, the switch complex contains the proteins FliG, FliM, and FliN and corresponds structurally with the C-ring in the flagellar basal body. Certain features of subunit organization in the switch complex have been deduced previously, but details of subunit organization in the lower part of the C-ring and the molecular movements responsible for motor switching remain unclear. In this study, we use cross-linking, binding, and mutational experiments to examine subunit organization in the bottom of the C-ring and to probe movements that occur upon switching. The results show that FliN tetramers alternate with FliM C-terminal domains to form the bottom of the C-ring in an arrangement that closely reproduces the major features observed in electron microscopic reconstructions. When motors were switched to clockwise rotation by a repellent stimulus, cross-link yields were altered in a pattern indicating relative movement of FliN and FliM_C. These results are discussed in the framework of a structurally grounded hypothesis for the switching mechanism.     

Characterization of Escherichia coli Flagellar Mutants That are Insensitive to Catabolite Repression

In Escherichia coli, the synthesis of the flagellar organelle is sensitive to catabolite repression. Synthesis requires the presence of the cyclic adenosine monophosphate receptor protein (Crp) and 3',5'-cyclic adenosine monophosphate (cAMP); i.e., mutants that lack Crp or adenylcyclase (Cya) synthesize no flagella. We isolated and characterized a series of mutants (cfs) that restored flagella-forming ability in a Crp strain of E. coli. The mutations in these strains were transferred onto episomes and they were then introduced into a variety of other strains. The presence of the mutation resulted in flagella synthesis in Cya and Crp strains as well as in the wild type grown under conditions of catabolite repression. Deletion analysis and other genetic studies indicated that: (i) the cfs mutations had a dominant effect when they were in the transconfiguration in merodiploids: (ii) they occurred in or very close to the flaI gene: and (iii) their expression required the presence of an intact flaI gene adjacent to the cfs mutation. Biochemical studies showed that the synthesis of at least two flagellar polypeptides, the hook subunit and an amber fragment of flagellin, were absent in strains that carried a cya mutation. Their synthesis was depressed in strains grown under conditions of catabolite repression. The presence of the cfs mutation restored the specific synthesis of these two polypeptides. We suggest that the formation of the flaI gene product is the step in flagellar synthesis that is catabolite sensitive and requires cAMP. We propose a regulatory function for the product of the flaI gene.     

Regulatory Mutations Affecting the Gluconate System in Escherichia coli

A spontaneously arising regulatory mutant of the gluconate system in Escherichia coli was isolated. This mutant became constitutive, probably in one step, for gluconate high-affinity transport, gluconokinase, and gluconate-6-P dehydrase. The mutation involved (gntR18) is cotransducible with asd. Pseudorevertants, derived from a mutant (M2) that shows a long lag for growth on gluconate mineral medium, were also isolated and characterized. They give constitutive levels of gluconokinase and gluconate-6-P dehydrase but lack high-affinity transport function. Genetic experiments performed with one of these pseudorevertants (M4) indicate that it carries a secondary mutation in the gntR gene. The M4 phenotype is thus the result of the interaction of expression of a constitutive mutation (gntR4) with the mutation of strain M2 (gntM2).     

Regulatory properties of phosphofructokinase 2 from Escherichia coli

Escherichia coli K12 contains two phosphofructokinases: phosphofructokinase 1, the most studied one, appears to behave as an allosteric enzyme, while phosphofructokinase 2 presents the features of a Michaelian enzyme. We show the present paper that, in fact, phosphofructokinase 2 also presents some regulatory properties in vitro: at high concentrations, ATP is an inhibitor of phosphofructokinase 2 and it provokes the tetramerization of the dimeric native enzyme. The binding of the two substrates to phosphofructokinase 2 is sequential and ordered as for phosphofructokinase 1, but in the former case fructose 6-phosphate is the first substrate to be bound and ADP the first product to be released. Each dimer of phosphofructokinase 2 binds two molecules of fructose 6-phosphate but only one molecule of the product fructose 1,6-phosphate. Although both phosphofructokinases of E. coli K12 present regulatory properties in vitro, the mechanism of regulation of the activity of the two enzymes is strikingly different. It can be asked whether or not these mechanisms operate in vivo.