Dataset Information

Growth of flagellar filaments of Escherichia coli is independent of filament length.

ABSTRACT: Bacterial flagellar filaments grow at their distal ends, from flagellin that travels through a central channel ?2 nm in diameter. The flagellin is extruded from the cytoplasm by a pump powered by a proton motive force (PMF). We measured filament growth in cells near the mid-exponential-phase with flagellin bearing a specific cysteine-for-serine substitution, allowing filaments to be labeled with sulfhydryl-specific fluorescent dyes. We labeled filaments first with a green maleimide dye and then, following an additional period of growth, with a red maleimide dye. The contour lengths of the green and red segments were measured. The average lengths of red segments (?2.3 ?m) were the same regardless of the lengths of the green segments from which they grew (ranging from less than 1 to more than 9 ?m in length). Thus, flagellar filaments do not grow at a rate that decreases exponentially with length, as formerly supposed. If flagellar filaments were broken by viscous shear, the broken filaments continued to grow. Identical results were obtained whether flagellin was expressed from fliC on the chromosome under the control of its native promoter or on a plasmid under the control of the arabinose promoter.

SUBMITTER: Turner L

PROVIDER: S-EPMC3347194 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Growth of flagellar filaments of Escherichia coli is independent of filament length.

Turner Linda L Stern Alan S AS Berg Howard C HC

Journal of bacteriology 20120323 10

Bacterial flagellar filaments grow at their distal ends, from flagellin that travels through a central channel ∼2 nm in diameter. The flagellin is extruded from the cytoplasm by a pump powered by a proton motive force (PMF). We measured filament growth in cells near the mid-exponential-phase with flagellin bearing a specific cysteine-for-serine substitution, allowing filaments to be labeled with sulfhydryl-specific fluorescent dyes. We labeled filaments first with a green maleimide dye and then, ...[more]

PMID: 22447900

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Project description:When deprived of FtsZ, Escherichia coli cells (VIP205) grown in liquid form long nonseptated filaments due to their inability to assemble an FtsZ ring and their failure to recruit subsequent divisome components. These filaments fail to produce colonies on solid medium, in which synthesis of FtsZ is induced, upon being diluted by a factor greater than 4. However, once the initial FtsZ levels are recovered in liquid culture, they resume division, and their plating efficiency returns to normal. The potential septation sites generated in the FtsZ-deprived filaments are not annihilated, and once sufficient FtsZ is accumulated, they all become active and divide to produce cells of normal length. FtsZ-deprived cells accumulate defects in their physiology, including an abnormally high number of unsegregated nucleoids that may result from the misplacement of FtsK. Their membrane integrity becomes compromised and the amount of membrane proteins, such as FtsK and ZipA, increases. FtsZ-deprived cells also show an altered expression pattern, namely, transcription of several genes responding to DNA damage increases, whereas transcription of some ribosomal or global transcriptional regulators decreases. We propose that the changes caused by the depletion of FtsZ, besides stopping division, weaken the cell, diminishing its resiliency to minor challenges, such as dilution stress.ImportanceOur results suggest a role for FtsZ, in addition to its already known effect in the constriction of E. coli, in protecting the nondividing cells against minor stress. This protection can even be exerted when an inactive FtsZ is produced, but it is lost when the protein is altogether absent. These results have implications in fields like synthetic biology or antimicrobial discovery. The construction of synthetic divisomes in the test tube may need to preserve unsuspected roles, such as this newly found FtsZ property, to guarantee the stability of artificial containers. Whereas the effects on viability caused by inhibiting the activity of FtsZ may be partly overcome by filamentation, the absence of FtsZ is not tolerated by E. coli, an observation that may help in the design of effective antimicrobial compounds.

Project description:Escherichia coli morphotype E flagellar filaments have a characteristic surface pattern of short-pitch loops when examined by electron microscopy. Seven of the 50 known E. coli H (flagellar antigen) serotypes (H1, H7, H12, H23, H45, H49, and H51) produce morphotype E filaments. Polymerase chain reaction was used to amplify flagellin structural (fliC) genes from E. coli strains producing morphotype E flagellar filaments and from strains with flagellar filaments representing other morphotypes. A single DNA fragment was obtained from each strain, and the size of the amplified DNA correlated with the molecular mass of the corresponding flagellin protein. This finding and hybridization data suggest that these bacteria are monophasic. fliC genes from three E. coli serotypes (H1, H7, and H12) possessing morphotype E flagellar filaments were sequenced in order to assess the contribution of conserved flagellin primary sequence to the characteristic filament architecture. The H1 and H12 fliC sequences were identical in length (1,788 bp), while the H7 fliC sequence was shorter (1,755 bp). The deduced molecular masses of the FliC proteins were 60,857 Da (H1), 59,722 Da (H7), and 60,978 Da (H12). The H1, H7, and H12 flagellins demonstrated 98 to 99% identity over the amino-terminal region (190 amino acid residues) and 89% (H7) to 99% (H1 and H12) identity in the carboxy-terminal region (100 amino acid residues). The complete primary amino acid sequences for H1 and H12 flagellins differed by only 10 amino acids, accounting for previously reported serological cross-reactions. However, the central region of H7 flagellin had only 38% identity with H1 and H12 flagellins. The characteristic morphology of morphotype E flagellar filaments is therefore not dependent on a highly conserved primary sequence within the exposed central region. Comparison of morphotype E E. coli flagellins with those from E. coli K-12, Serratia marcescens, and several Salmonella serovars supported the established concept of highly conserved terminal regions flanking a variable central region.

Dataset Information

Growth of flagellar filaments of Escherichia coli is independent of filament length.

Publications

Growth of flagellar filaments of Escherichia coli is independent of filament length.

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