Bacterial type IV pili are critical for diverse biological processes including horizontal gene transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic appendages extend and retract from the cell surface. In many type IVa pilus systems, extension occurs through the action of an extension ATPase, often called PilB, while optimal retraction requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog of PilT called PilU. However, the function of this protein has remained unclear because pilU mutants exhibit inconsistent phenotypes among type IV pilus systems and because it is relatively understudied compared to PilT. Here, we study the type IVa competence pilus of Vibrio cholerae as a model system to define the role of PilU. We show that the ATPase activity of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, however, contribute to pilus retraction in ΔpilT strains. Thus, these data suggest that PilU is a bona fide retraction ATPase that supports pilus retraction in a PilT-dependent manner. We also found that a ΔpilU mutant exhibited a reduction in the force of retraction suggesting that PilU is important for generating maximal retraction forces. Additional in vitro and in vivo data show that PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase is conserved in Acinetobacter baylyi, suggesting that the role of PilU described here may be broadly applicable to other type IVa pilus systems.

19Bacterial type IV pili are critical for diverse biological processes including horizontal gene 20 transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic 21 appendages extend and retract from the cell surface. In many type IVa pilus systems, extension 22 occurs through the action of an extension ATPase, often called PilB, while optimal retraction 23 requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog 24 of PilT called PilU. However, the function of this protein has remained unclear because pilU 25 mutants exhibit inconsistent phenotypes among type IV pilus systems and because it is 26 relatively understudied compared to PilT. Here, we study the type IVa competence pilus of 27Vibrio cholerae as a model system to define the role of PilU. We show that the ATPase activity 28 of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, 29 however, contribute to pilus retraction in ΔpilT strains. Thus, these data suggest that PilU is a 30 bona fide retraction ATPase that supports pilus retraction in a PilT-dependent manner. We also 31 found that a ΔpilU mutant exhibited a reduction in the force of retraction suggesting that PilU is 32 important for generating maximal retraction forces. Additional in vitro and in vivo data show that 33PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus 34 retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase 35 is conserved in Acinetobacter baylyi, suggesting that the role of PilU described here may be 36 broadly applicable to other type IVa pilus systems. 37 38Author Summary 39 Almost all bacterial species use thin surface appendages called pili to interact with their 40 environments. These structures are critical for the virulence of many pathogens and represent 41 one major way that bacteria share DNA with one another, which contributes to the spread of 42 antibiotic resistance. To carry out their function, pili dynamically extend and retract from the 43 bacterial surface. Here, we show that retraction of pili in some systems is determined by the 44 combined activity of two motor ATPase proteins. 45 46Type IV pili are ubiquitous surface appendages in Gram-negative bacteria that promote diverse 48 activities including attachment, virulence, biofilm formation, horizontal gene transfer, and 49 twitching motility [1][2][3][4][5]. These structures can dynamically extend and retract from the cell 50 surface, which is often critical for their function. A detailed mechanistic understanding of pilus 51 dynamic activity, however, remains lacking. 52Type IV pili are composed almost exclusively of a single protein called the major pilin, which 53 forms a helical fiber that extends from the cell surface [6]. Extension and retraction of the pilus is 54 hypothesized to occur through the interaction of cytoplasmic hexameric ATPases with the inner 55 membrane platform of the pilus machine, whereby ATP ...

A collection of recombinant rotaviruses that express the fluorescent markers UnaG, mKate, mRuby, TagBFP, CFP, or YFP as separate proteins was generated. Genes for the fluorescent proteins were inserted into genome segment 7 without compromising expression of the protein NSP3. These recombinant rotaviruses are valuable for analyzing rotavirus biology by fluorescence-based live-cell imaging.

Noroviruses were found to cause one-third of the diarrhea cases that previously had no identified etiology. Future work should attempt to ascertain etiologic agents in the approximately one-fifth of cases without a plausible microbial cause, understand the significance of multiple agents in stools, and guide interpretation of nonculture diagnostics.

Microorganisms encounter toxicities inside the host. Many pathogens exist as subpopulations to maximize survivability. Subpopulations of Staphylococcus aureus include antibiotic-tolerant small colony variants (SCVs). These mutants often emerge following antibiotic treatment but can be present in infections prior to antibiotic exposure. We hypothesize that haem toxicity in the host selects for respiration-deficient S. aureus SCVs in the absence of antibiotics. We demonstrate that some but not all respiration-deficient SCV phenotypes are more protective than the haem detoxification system against transient haem exposure, indicating that haem toxicity in the host may contribute to the dominance of menaquinone-deficient and haem-deficient SCVs prior to antibiotic treatment.