Length of the flagellar hook and the capacity of the type III export apparatus.

Length determination in biology generally uses molecular rulers. The hook, a part of the flagellum of motile bacteria, has an invariant length. Here, we examined hook length and found that it was determined not by molecular rulers but probably by the amount of subunit protein secreted by the flagellar export apparatus. The export apparatus shares common features with the type III virulence-factor secretion machinery and thus may be used more widely in length determination of structures other than flagella.

Effect of cellular level of FliK on flagellar hook and filament assembly in Salmonella typhimurium.

Frameshift mutations in the fliK gene of Salmonella result in abnormal elongation of the hook and the failure to assemble filament (polyhook phenotype). Second-site suppressor mutations restore filament assembly, but the cells often remain defective in hook-length control (polyhook-filament phenotype). Where the suppressor mutations are intragenic, the second mutation restores the original frame, generating a region of frameshifted sequence, but restoring the natural C terminus. Some of these frameshifted sequences contain a UGA (opal) termination codon. These cells have few flagella and swarm poorly. We suspected that readthrough of UGA by tRNATrp might be the reason for the partial function. When the UGA codon was changed to the Trp codon UGG, flagellar assembly and function were restored to wild-type levels. Conversely, underexpression of the wild-type fliK gene, achieved by changing the sole Trp codon in the sequence (Trp271) to UGA, decreased both the number of flagella and the ability to swarm. These results validate the readthrough hypothesis and indicate that low levels of FliK sustain some degree of flagellation and motility. At low levels of FliK, most flagella had polyhooks. With increasing amounts, the morphology progressively changed to polyhook-filament, and eventually to wild-type hook-filament. When FliK was overproduced, the hook length was slightly shorter (46(+/-7) nm) than that of the wild-type strain (55(+/-9) nm). FliK levels were measured by immunoblotting. Wild-type levels were about 40 to 80 molecules/cell. FliK synthesized by UGA readthrough could be detected when overproduced from plasmid fliK-W271opal, and the levels indicated a probability of readthrough of 0.002 to 0.01. This value was used to estimate the cellular level of underexpressed FliK, which could partly restore function to a fliK mutant, at about 0.07 to 0.8 molecule/cell. These results suggest that FliK does not form a large structure in the cytoplasm and may function as a regulatory protein for protein export. A model for hook-length control is presented that involves feedback from the assembly point to the export apparatus.

Substrate specificities of exo- and endo-type cellulases in the hydrolysis of beta-(1----3)- and beta-(1----4)-mixed D-glucans.

An exo-type cellulase (Ex-1) was extracted from Irpex lacteus (Polyporus tulipiferae) and purified essentially to homogeneity. This cellulase attacked cellulosic substrates in an exo-wise fashion to produce almost exclusively cellobiose. In contrast, Ex-1 was found to attack beta-glucans having beta-(1----3)- and beta-(1----4)-mixed linkages in a way similar to an endo-type cellulase. The products formed from barley glucan by Ex-1 were 3(2)-O-beta-D-cellobiosyl-cellobiose much greater than 3(2)-O-beta-D-glucosyl-cellobiose greater than cellobiose much greater than or equal to cellotriose much greater than glucose in the early stage, but no laminaribiose was produced. An endo-type cellulase (En-1) obtained from the same fungus also hydrolyzed beta-glucans but in a typical endo-wise fashion and the products from barley glucan were 3(2)-O-beta-D-glucosyl-cellobiose much greater than 3(2)-O-beta-D-cellobiosyl-cellobiose greater than cellobiose much greater than laminaribiose; no glucose or cellotriose was produced. Thus, it seems likely that En-1 can attack any intramolecular linkage of beta-glucan, while Ex-1 requires the presence of at least cellobiosyl residues adjacent to a beta-(1----3)-D-linked glucosyl residue. This finding, together with the mode of hydrolysis of cellulosic substrates by Ex-1, suggests that the stereochemical structure of successive beta-(1----4)-cellobiosyl residues inserted by beta-(1----3)-D-glucosidic linkage is permissible in the action of Ex-1, although this enzyme prefers the beta-(1----4)-linked cellobiosyl sequence.

Effect of hook subunit concentration on assembly and control of length of the flagellar hook of Salmonella.

The flagellar hook of Salmonella is a filamentous polymer made up of subunits of the protein FlgE. Hook assembly is terminated when the length reaches about 55 nm. After our recent study of the effect of cellular levels of the hook length control protein FliK, we have now analyzed the effect of cellular levels of FlgE itself. When FlgE was overproduced in a wild-type strain, a fliC (flagellin) mutant, or a fliD (hook-associated protein 2 [HAP2], filament capping protein) mutant, the hooks remained at the wild-type length. In a fliK (hook length control protein) mutant, which produces long hooks (polyhooks), the overproduction of FlgE resulted in extraordinarily long hooks (superpolyhooks). In a flgK (HAP1, first hook-filament junction protein) mutant or a flgL (HAP3, second hook-filament junction protein) mutant, the overproduction of FlgE also resulted in longer than normal hooks. Thus, at elevated hook protein levels not only FliK but also FlgK and FlgL are necessary for the proper termination of hook elongation. When FlgE was severely underproduced, basal bodies without hooks were often observed. However, those hooks that were seen were of wild-type length, demonstrating that FlgE underproduction decreases the probability of the initiation of hook assembly but not the extent of hook elongation.

Effect of epidural analgesia with ambulation on labor duration.

BACKGROUND: Ambulatory epidural analgesia (AEA) is a popular choice for labor analgesia because ambulation reportedly increases maternal comfort, increases the intensity of uterine contractions, avoids inferior vena cava compression, facilitates fetal head descent, and relaxes the pelvic musculature, all of which can shorten labor. However, the preponderance of evidence suggests that ambulation during labor is not associated with these benefits. The purpose of this study is to determine whether ambulation with AEA decreases labor duration from the time of epidural insertion to complete cervical dilatation. METHODS: In this prospective, randomized study, 160 nulliparous women with AFA were randomly assigned to one of two groups: AEA with ambulation and AEA without ambulation. AEA blocks were initiated with 15-20 ml ropivacaine (0.07%) plus 100 microg fentanyl, followed by a continuous infusion of 0.07% ropivacaine plus 2 microg/ml fentanyl at 15-20 ml/h. Maternal measured variables included ambulation time, time from epidural insertion to complete dilatation, stage II duration, pain Visual Analogue Scale scores, and mode of delivery. APGAR scores were recorded at 1 and 5 min. Results are expressed as mean +/- SD or median and analyzed using the t test, chi-square, or the Mann-Whitney test at P < or = 0.05. RESULTS: The ambulatory group walked 25.0 +/- 23.3 min, sat upright 40.3 +/- 29.7 min, or both. Time from epidural insertion to complete dilatation was 240.9 +/- 146.1 min in the ambulatory group and 211.9 +/- 133.9 min in the nonambulatory group (P = 0.206). CONCLUSION: Ambulatory epidural analgesia with walking or sitting does not shorten labor duration from the time of epidural insertion to complete cervical dilatation.