Fabienne F. V. Chevance

Coordinating assembly of a bacterial macromolecular machine

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.

FliK regulates flagellar hook length as an internal ruler

The 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 hook‐length control. We conclude that FliK regulates hook length as an internal molecular ruler.

The Effects of Codon Context on In Vivo Translation Speed

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.

Flk prevents premature secretion of the anti-σ factor FlgM into the periplasm

The flk locus of Salmonella typhimurium was identified as a regulator of flagellar gene expression in strains defective in P‐ and l‐ring formation. Flk acts as a regulator of flagellar gene expression by modulating the protein levels of the anti‐σ28 factor FlgM. Evidence is presented which suggests that Flk is a cytoplasmic‐facing protein anchored to the inner membrane by a single, C‐terminal transmembrane‐spanning domain (TMS). The specific amino acid sequence of the TMS is not essential for Flk activity, but membrane anchoring is essential. Membrane fractionation and visualization of protein fusions of green fluorescent protein derivatives to Flk suggested that the Flk protein is present in the membrane as punctate spots in number that are much greater than the number of flagellar basal structures. The turnover of the anti‐σ28 factor FlgM was increased in flk mutant strains. Using FlgM–β‐lactamase fusions we show the increased turnover of FlgM in flk null mutations is due to FlgM secretion into the periplasm where it is degraded. Our data suggest that Flk inhibits FlgM secretion by acting as a braking system for the flagellar‐associated type III secretion system. A model is presented to explain a role for Flk in flagellar assembly and gene regulatory processes.

Identical folds used for distinct mechanical functions of the bacterial flagellar rod and hook

The bacterial flagellum is a motile organelle driven by a rotary motor, and its axial portions function as a drive shaft (rod), a universal joint (hook) and a helical propeller (filament). The rod and hook are directly connected to each other, with their subunit proteins FlgG and FlgE having 39% sequence identity, but show distinct mechanical properties; the rod is straight and rigid as a drive shaft whereas the hook is flexible in bending as a universal joint. Here we report the structure of the rod and comparison with that of the hook. While these two structures have the same helical symmetry and repeat distance and nearly identical folds of corresponding domains, the domain orientations differ by ∼7°, resulting in tight and loose axial subunit packing in the rod and hook, respectively, conferring the rigidity on the rod and flexibility on the hook. This provides a good example of versatile use of a protein structure in biological organisms.

Autonomous and FliK-Dependent Length Control of the Flagellar Rod in Salmonella enterica

Salmonella flgG point mutations produce filamentous rod structures whose lengths are determined by FliK. FliK length variants produce rods with lengths proportional to the corresponding FliK molecular size, suggesting that FliK controls the length of not only the hook but also the rod by the same molecular mechanism.

Mlc of Thermus thermophilus: a Glucose-Specific Regulator for a Glucose/Mannose ABC Transporter in the Absence of the Phosphotransferase System

We report the presence of Mlc in a thermophilic bacterium. Mlc is known as a global regulator of sugar metabolism in gram-negative enteric bacteria that is controlled by sequestration to a glucose-transporting EII(Glc) of the phosphotransferase system (PTS). Since thermophilic bacteria do not possess PTS, Mlc in Thermus thermophilus must be differently controlled. DNA sequence alignments between Mlc from T. thermophilus (Mlc(Tth)) and Mlc from E. coli (Mlc(Eco)) revealed that Mlc(Tth) conserved five residues of the glucose-binding motif of glucokinases. Here we show that Mlc(Tth) is not a glucokinase but is indeed able to bind glucose (K(D) = 20 microM), unlike Mlc(Eco). We found that mlc of T. thermophilus is the first gene within an operon encoding an ABC transporter for glucose and mannose, including a glucose/mannose-binding protein and two permeases. malK1, encoding the cognate ATP-hydrolyzing subunit, is located elsewhere on the chromosome. The system transports glucose at 70 degrees C with a K(m) of 0.15 microM and a V(max) of 4.22 nmol per min per ml at an optical density (OD) of 1. Mlc(Tth) negatively regulates itself and the entire glucose/mannose ABC transport system operon but not malK1, with glucose acting as an inducer. MalK1 is shared with the ABC transporter for trehalose, maltose, sucrose, and palatinose (TMSP). Mutants lacking malK1 do not transport either glucose or maltose. The TMSP transporter is also able to transport glucose with a K(m) of 1.4 microM and a V(max) of 7.6 nmol per min per ml at an OD of 1, but it does not transport mannose.

A Little Gene with Big Effects: a serT Mutant Is Defective in flgM Gene Translation

A conditional-lethal mutant was isolated as having a flagellar regulatory phenotype at 30 degrees C and being unable to grow at 42 degrees C. Chromosomal mapping localized the mutation to the serT gene, which encodes an essential serine tRNA species (tRNA((cmo)5UGA)(Ser)). DNA sequence analysis revealed the mutation to be a single base change in G:A at position 10 of the serT gene that lies within the D-stem of the essential tRNA((cmo)5)UGA(Ser) species. tRNA((cmo)5)UGA(Ser) recognizes UCA, UCG, and UCU codons, but UCU is also recognized by tRNA(GGA)(Ser) and UCG by tRNA(CGA)(Ser). No other tRNAs are known to read the UCA codon. Thus, the UCA codon is specifically recognized by tRNA((cmo)5)UGA(Ser). We show that the anti-sigma(28) activity of FlgM is defective in the serT mutant strain. The serT allele causes a 10-fold increase in sigma(28)-dependent fliC promoter transcription, indicating a defect in FlgM anti-sigma(28) activity in the presence of the serT mutation. The flgM gene contains only one UCA codon. Changing the UCA of flgM to ACG reversed the effect of the serT allele. Implications for context effects in regulation of gene expression are discussed.

Case for the genetic code as a triplet of triplets

Significance The genetic code for life is a triplet base code. It is known that adjacent codons can influence translation of a given codon and that codon pair biases occur throughout nature. We show that mRNA translation at a given codon can be affected by the two previous codons. Data presented here support a model in which the evolutionary selection pressure on a single codon is over five successive codons, including synonymous codons. This work provides a foundation for the interpretation of how single DNA base changes might affect translation over multiple codons and should be considered in the characterization of the effects of DNA base changes on human disease. The efficiency of codon translation in vivo is controlled by many factors, including codon context. At a site early in the Salmonella flgM gene, the effects on translation of replacing codons Thr6 and Pro8 of flgM with synonymous alternates produced a 600-fold range in FlgM activity. Synonymous changes at Thr6 and Leu9 resulted in a twofold range in FlgM activity. The level of FlgM activity produced by any codon arrangement was directly proportional to the degree of in vivo ribosome stalling at synonymous codons. Synonymous codon suppressors that corrected the effect of a translation-defective synonymous flgM allele were restricted to two codons flanking the translation-defective codon. The various codon arrangements had no apparent effects on flgM mRNA stability or predicted mRNA secondary structures. Our data suggest that efficient mRNA translation is determined by a triplet-of-triplet genetic code. That is, the efficiency of translating a particular codon is influenced by the nature of the immediately adjacent flanking codons. A model explains these codon-context effects by suggesting that codon recognition by elongation factor-bound aminoacyl-tRNA is initiated by hydrogen bond interactions between the first two nucleotides of the codon and anticodon and then is stabilized by base-stacking energy over three successive codons.

Analysis of Factors That Affect FlgM-Dependent Type III Secretion for Protein Purification with Salmonella enterica Serovar Typhimurium

The FlgM protein is secreted in response to flagellar hook-basal body secretion and can be used as a secretion signal to direct selected protein secretion via the flagellar type III secretion (T3S) system [H. M. Singer, M. Erhardt, A. M. Steiner, M. M. Zhang, D. Yoshikami, G. Bulaj, B. M. Olivera, and K. T. Hughes, mBio 3(3):e00115-12, 2012, http://dx.doi.org/10.1128/mBio.00115-12]. Conditions known to affect flagellar gene expression, FlgM stability, and flagellar T3S were tested either alone or in combination to determine their effects on levels of secreted FlgM. These conditions included mutations that affect activity of the flagellar FlhD4C2 master regulatory protein complex or the FlgM T3S chaperone σ(28), the removal of Salmonella pathogenicity island 1 (Spi1), the removal of flagellar late secretion substrates that could compete with FlgM for secretion, and changes in the ionic strength of the growth medium. Conditions that enhanced FlgM secretion were combined in order to maximize levels of secreted FlgM. An optimized FlgM secretion strain was used to secrete and isolate otherwise difficult-to-produce proteins and peptides fused to the C terminus of FlgM. These include cysteine-rich, hydrophobic peptides (conotoxins δ-SVIE and MrVIA), nodule-specific, cysteine-rich antimicrobial peptides (NCR), and a malaria surface antigen domain of apical membrane antigen AMA-1.