Biofilm formation by Vibrio cholerae facilitates environmental persistence, and hyperinfectivity within the host. Biofilm formation is regulated by 3',5'-cyclic diguanylate (c-di-GMP) and requires production of the type IV mannose-sensitive hemagglutinin (MSHA) pilus. Here, we show that the MSHA pilus is a dynamic extendable and retractable system, and its activity is directly controlled by c-di-GMP. The interaction between c-di-GMP and the ATPase MshE promotes pilus extension, whereas low levels of c-di-GMP correlate with enhanced retraction. Loss of retraction facilitated by the ATPase PilT increases near-surface roaming motility, and impairs initial surface attachment. However, prolonged retraction upon surface attachment results in reduced MSHA-mediated surface anchoring and increased levels of detachment. Our results indicate that c-di-GMP directly controls MshE activity, thus regulating MSHA pilus extension and retraction dynamics, and modulating V. cholerae surface attachment and colonization.

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.

Many bacteria use population density to control gene expression via quorum sensing. In Vibrio cholerae, quorum sensing coordinates virulence, biofilm formation, and DNA uptake by natural competence. The transcription factors AphA and HapR, expressed at low and high cell density respectively, play a key role. In particular, AphA triggers the entire virulence cascade upon host colonisation. In this work we have mapped genome-wide DNA binding by AphA. We show that AphA is versatile, exhibiting distinct modes of DNA binding and promoter regulation. Unexpectedly, whilst HapR is known to induce natural competence, we demonstrate that AphA also intervenes. Most notably, AphA is a direct repressor of tfoX, the master activator of competence. Hence, production of AphA markedly suppressed DNA uptake; an effect largely circumvented by ectopic expression of tfoX. Our observations suggest dual regulation of competence. At low cell density AphA is a master repressor whilst HapR activates the process at high cell density. Thus, we provide deep mechanistic insight into the role of AphA and highlight how V. cholerae utilises this regulator for diverse purposes.

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 ...

16Molecular motors convert chemical energy directly into mechanical work 1 and are found in all 17domains of life 2 . These motors are critical to intracellular transport 3 , motility 4,5 , macromolecular 18protein assembly 3,6 , and many essential processes 7 . A wide-spread class of related bacterial 19 motors drive the dynamic activity of extracellular fibers, such as type IV pili (T4P), that are 20 extended and retracted using so-called secretion motor ATPases. Among these, the tight 21 adherence (tad) pili are critical for surface sensing, surface attachment, and biofilm formation 8-22 10