The assembly of large and complex organelles, such as the bacterial flagellum, poses the formidable problem of coupling temporal gene expression to specific stages of the organelle-assembly process. The discovery that levels of the bacterial flagellar regulatory protein FlgM are controlled by its secretion from the cell in response to the completion of an intermediate flagellar structure (the hook-basal body) was only the first of several discoveries of unique mechanisms that coordinate flagellar gene expression with assembly. In this Review, we discuss this mechanism, together with others that also coordinate gene regulation and flagellar assembly in Gram-negative bacteria.

We developed a bacterial genetic system based on translation of the his operon leader peptide gene to determine the relative speed at which the ribosome reads single or multiple codons in vivo. Low frequency effects of so-called “silent” codon changes and codon neighbor (context) effects could be measured using this assay. An advantage of this system is that translation speed is unaffected by the primary sequence of the His leader peptide. We show that the apparent speed at which ribosomes translate synonymous codons can vary substantially even for synonymous codons read by the same tRNA species. Assaying translation through codon pairs for the 5′- and 3′- side positioning of the 64 codons relative to a specific codon revealed that the codon-pair orientation significantly affected in vivo translation speed. Codon pairs with rare arginine codons and successive proline codons were among the slowest codon pairs translated in vivo. This system allowed us to determine the effects of different factors on in vivo translation speed including Shine-Dalgarno sequence, rate of dipeptide bond formation, codon context, and charged tRNA levels.

SummaryThe mechanism of length control of the flagellar hook is under debate between two theories. One claims that the FliK directly measures the hook length as a molecular ruler, while the other claims that the cytoplasmic substructure measures the amount of hook subunits to determine the hook length. Both agree that the FliK C-terminal domain catalyses the substrate-specificity switch to terminate hook elongation. In this study, we systematically created fliK mutants with deletions and insertions at various sites within the FliK N-terminal domain and analysed their effects on the final hook length. Insertions of peptide fragments from the Yersinia YscP into FliK gave rise to hooks with defined lengths, which was proportional to the molecular size of the FliK-YscP chimeras. Among fliK deletion mutants, only those with small truncations in three specific sites of FliK produced hooks of a defined, shortened length. For the majority of deletion mutants, FliK was secreted, but hook length was not controlled. On the other hand, for some deletion mutants FliK was not secreted, but the hook length was controlled, indicating that FliK secretion is not necessary for hooklength control. We conclude that FliK regulates hook length as an internal molecular ruler.

The rod component of the bacterial flagellum polymerizes from the inner membrane across the periplasmic space and stops at a length of 25 nm at the outer membrane. Bushing structures, the P-and L-rings, polymerize around the distal rod and form a pore in the outer membrane. The flagellar hook structure is then added to the distal rod growing outside the cell. Hook polymerization stops after the rod-hook structure reaches ∼80 nm in length. This study describes mutants in the distal rod protein FlgG that fail to terminate rod growth. The mutant FlgG subunits continue to polymerize close to the length of the normal rod-hook structure of 80 nm. These filamentous rod structures have multiple P-rings and fail to form the L-ring pore at the outer membrane. The flagella grow within the periplasm similar to spirochete flagella. This provides a simple method to evolve intracellular flagella as in spirochetes. The mechanism that couples rod growth termination to the ring assembly and outer membrane penetration exemplifies the importance of stopping points in the construction of a complex macromolecular machine that facilitate efficient coupling to the next step in the assembly pathway.

Flavor quality is of major importance to the consumer, but the flavor characteristics of beer appear to deteriorate greatly with time, at a rate depending on the composition of the beer and its storage conditions (notably pH). Prior to identifying the influence of pH on the development of the most intense staling flavors found in aged lager beers, the corresponding key flavor compounds were determined by aroma extract dilution analysis. In addition to trans-2-nonenal, beta-damascenone seems at least as important in the flavor of aged beer. Ethyl butyrate, dimethyl trisulfide, 2-acetylpyrazine, 3-(methylthio)propionaldehyde, 2-methoxypyrazine, maltol, gamma-nonalactone, and ethyl cinnamate are also relevant to the sensory profile of aged beer. Upon aging, a beer having a higher pH produces less beta-damascenone, because acid-catalyzed glycoside hydrolysis is decreased. On the other hand, it produces more 3-(methylthio)propionaldehyde, owing to Strecker degradation of methionine. Raising the beer pH additionally causes the release of 3-(methylthio)propionaldehyde from sulfitic adducts. These adducts, more stable at a lower pH, protect the aldehyde against premature oxidation to 3-(methylthio)propionic acid, thus making it available for dimethyl trisulfide formation during aging.