The flhD operon is the master operon of the flagellar regulon and a global regulator of metabolism. The genome sequence of the Escherichia coli K-12 strain MG1655 contained an IS1 insertion sequence element in the regulatory region of the flhD promoter. Another stock of MG1655 was obtained from the E. coli Genetic Stock Center. This stock contained isolates which were poorly motile and had no IS1 element upstream of the flhD promoter. From these isolates, motile subpopulations were identified after extended incubation in motility agar. Purified motile derivatives contained an IS5 element insertion upstream of the flhD promoter, and swarm rates were sevenfold higher than that of the original isolate. For a motile derivative, levels of flhD transcript had increased 2.7-fold, leading to a 32-fold increase in fliA transcript and a 65-fold increase in flhB::luxCDABE expression from a promoter probe vector. A collection of commonly used lab strains was screened for IS element insertion and motility. Five strains (RP437, YK410, MC1000, W3110, and W2637) contained IS5 elements upstream of the flhD promoter at either of two locations. This correlated with high swarm rates. Four other strains (W1485, FB8, MM294, and RB791) did not contain IS elements in the flhD regulatory region and were poorly motile. Primer extension determined that the transcriptional start site of flhD was unaltered by the IS element insertions. We suggest that IS element insertion may activate transcription of the flhD operon by reducing transcriptional repression.An important source of genome plasticity is derived from transpositional events of insertion sequence (IS) elements (34, 35). They generally encode no functions other than those involved in their mobility (for a review, see reference 30) and display a nonrandom distribution in the chromosome of Escherichia coli (9, 16). Many IS elements have been shown to activate the expression of neighboring genes, for example, through the formation of hybrid promoters or disruption of transcriptional repression. This has also been seen with cryptic operons, which depend upon mutations for activation. Two examples in E. coli are the bgl and ade operons, which can be activated by IS element insertion upstream or downstream of the promoter (18,42,49,50,53). The chitobiose operon, chb (formerly cel), was thought to be cryptic but can be induced by chitobiose, as well as being activated by IS element insertion upstream of the structural genes under noninducing conditions (40, 44).Flagellar motility enables bacteria to escape from detrimental conditions and to reach more favorable environments. In E. coli, the flagellar regulon involves the expression of at least 14 operons in a regulated cascade to produce functional flagellar and chemotaxis machinery (for a review, see reference 14). The flhD operon at the apex of the flagellar regulon has been identified as the primary target of regulation by many environmental factors (for a review, see reference 61). It consists of two genes, flhD and flhC, whose product...

The bacterial flagellar hook is a tubular helical structure made by the polymerization of multiple copies of a protein, FlgE. Here we report the structure of the hook from Campylobacter jejuni by cryo-electron microscopy at a resolution of 3.5 Å. On the basis of this structure, we show that the hook is stabilized by intricate inter-molecular interactions between FlgE molecules. Extra domains in FlgE, found only in Campylobacter and in related bacteria, bring more stability and robustness to the hook. Functional experiments suggest that Campylobacter requires an unusually strong hook to swim without its flagella being torn off. This structure reveals details of the quaternary organization of the hook that consists of 11 protofilaments. Previous study of the flagellar filament of Campylobacter by electron microscopy showed its quaternary structure made of seven protofilaments. Therefore, this study puts in evidence the difference between the quaternary structures of a bacterial filament and its hook.

The killing of Listeria monocytogenes following exposure to low pH, organic acids, and osmotic stress was enhanced by the addition of 5% (vol/vol) ethanol. At pH 3, for example, the presence of this agent stimulated killing by more than 3 log units in 40 min of exposure. The rate of cell death at pH 3.0 was dependent on the concentration of ethanol. Thus, while the presence 10% (vol/vol) ethanol at pH 3.0 stimulated killing by more than 3 log units in just 5 min, addition of 1.25% (vol/vol) ethanol resulted in less than 1 log unit of killing in 10 min. The ability of 5% (vol/vol) ethanol to stimulate killing at low pH and at elevated osmolarity was also dependent on the amplitude of the imposed stress, and an increase in the pH from 3.0 to 4.0 or a decrease in the sodium chloride concentration from 25 to 2.5% led to a marked reduction in the effectiveness of 5% (vol/vol) ethanol as an augmentative agent. Combinations of organic acids, low pH, and ethanol proved to be particularly effective bactericidal treatments; the most potent combination was pH 3.0, 50 mM formate, and 5 % (vol/vol) ethanol, which resulted in 5 log units of killing in just 4 min. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane.The gram-positive bacterium Listeria monocytogenes is recognized as a food-borne pathogen with significance for humans (11), and major outbreaks of infection have been linked to the consumption of contaminated coleslaw (24), cheeses (14), and pasteurized milk (12). Today, L. monocytogenes is a major concern to manufacturers worldwide due to the high mortality rate of listeriosis in susceptible populations and to the resistance of the pathogen to a number of food preservation practices. In particular, the ability of the organism to grow at refrigeration temperatures (30) and on dry surfaces (31) and its ability to tolerate acidic conditions (1, 2, 7) make it well adapted to food environments which normally restrict bacterial growth. Consequently, control of this bacterium is a significant challenge for the food manufacturer.Acidification of foods with short-chain organic acids, either by fermentation or by deliberate addition, is an important and widespread mechanism for controlling food-borne pathogens in a variety of foods. However, a number of studies have demonstrated that L. monocytogenes is more acid tolerant than most food-borne pathogens, although the sensitivity of the organism to organic acids varies with the nature of the acidulant used (28). An additional consideration relevant to the survival of this pathogen in foods is that fact that acid tolerance can be enhanced by exposing the organism to moderately acidic conditions (8, 17), a factor which can further reduce the effectiveness of acid-based preservation systems against L. monocytogenes.The innate resistance of L. monocytogenes to many of the food preservation systems that are effective against other foodborne pathogens has prompted research aimed at developing combination systems for more effective control of this p...

The type III secretion system of the Salmonella flagellum consists of 6 integral membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, and FliR. However, in some other type III secretion systems, a homologue of FliO is apparently absent, suggesting it has a specialized role. Deleting the fliO gene from the chromosome of a motile strain of Salmonella resulted in a drastic decrease of motility. Incubation of the ΔfliO mutant strain in motility agar, gave rise to pseudorevertants containing extragenic bypass mutations in FliP at positions R143H or F190L. Using membrane topology prediction programs, and alkaline phosphatase or GFPuv chimeric protein fusions into the FliO protein, we demonstrated that FliO is bitopic with its N-terminus in the periplasm and C-terminus in the cytoplasm. Truncation analysis of FliO demonstrated that overexpression of FliO43–125 or FliO1–95 was able to rescue motility of the ΔfliO mutant. Further, residue leucine 91 in the cytoplasmic domain was identified to be important for function. Based on secondary structure prediction, the cytoplasmic domain, FliO43–125, should contain beta-structure and alpha-helices. FliO43–125-Ala was purified and studied using circular dichroism spectroscopy; however, this domain was disordered, and its structure was a mixture of beta-sheet and random coil. Coexpression of full-length FliO with FliP increased expression levels of FliP, but coexpression with the cytoplasmic domain of FliO did not enhance FliP expression levels. Overexpression of the cytoplasmic domain of FliO further rescued motility of strains deleted for the fliO gene expressing bypass mutations in FliP. These results suggest FliO maintains FliP stability through transmembrane domain interaction. The results also demonstrate that the cytoplasmic domain of FliO has functionality, and it presumably becomes structured while interacting with its binding partners.

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