Actinobacterial species are mostly thought to be nonmotile. Recent studies have revealed the degenerate evolution of flagella in this phylum and different flagellar rod compositions from the classical model. Moreover, flagella-independent motility by various means has been reported in Streptomyces spp. and Mycobacterium spp., but the underlying mechanisms remain elusive.

Bacteria can propel themselves by rotating a flagellum or a flagellar bundle. To image this thin structure in motile bacteria, the flagella can be vitally stained with fluorophores. This chapter describes a flagellar staining protocol with the additional possibility of visualizing the cell body. It offers the opportunity to track conformational changes of flagella and simultaneously track the positions of the cell bodies. The additional use of a filter increases the number of motile cells and improves the signal-to-noise ratio of images. The flagellar staining requires a prior introduction of a surface-exposed cysteine, which is not covered in this chapter.

The first steps of vision take place in the ciliary outer segment compartment of photoreceptor cells. The protein composition of outer segments is uniquely suited to perform this function. The most abundant among these proteins is the visual pigment, rhodopsin, whose outer segment trafficking involves intraflagellar transport (IFT). Here, we report three major findings from the analysis of mice in which ciliary transport was acutely impaired by conditional knockouts of IFT-B subunits. First, we demonstrate the existence of a sorting mechanism whereby mislocalized rhodopsin is recruited to and concentrated in extracellular vesicles prior to their release, presumably to protect the cell from adverse effects of protein mislocalization. Second, reducing rhodopsin expression significantly delays photoreceptor degeneration caused by IFT disruption, suggesting that controlling rhodopsin levels may be an effective therapy for some cases of retinal degenerative disease. Last, the loss of IFT-B subunits does not recapitulate a phenotype observed in mutants of the BBSome (another ciliary transport protein complex relying on IFT) in which non-ciliary proteins accumulate in the outer segment. Whereas it is widely thought that the role of the BBSome is to primarily participate in ciliary transport, our data suggest that the BBSome has another major function independent of IFT and possibly related to maintaining the diffusion barrier of the ciliary transition zone.

Leishmania species, members of the kinetoplastid parasites, cause leishmaniasis, a neglected tropical disease, in millions of people worldwide. Leishmania has a complex life cycle with multiple developmental forms, as it cycles between a sand fly vector and a mammalian host; understanding their life cycle is critical to understanding disease spread. One of the key life cycle stages is the haptomonad form, which attaches to insect tissues through its flagellum. This adhesion, conserved across kinetoplastid parasites, is implicated in having an important function within their life cycles and hence in disease transmission. Here, we discover the kinetoplastid-insect adhesion proteins (KIAPs), which localise in the attached Leishmania flagellum. Deletion of these KIAPs impairs cell adhesion in vitro and prevents Leishmania from colonising the stomodeal valve in the sand fly, without affecting cell growth. Additionally, loss of parasite adhesion in the sand fly results in reduced physiological changes to the fly, with no observable damage of the stomodeal valve and reduced midgut swelling. These results provide important insights into a comprehensive understanding of the Leishmania life cycle, which will be critical for developing transmission-blocking strategies.

Biofilms aid bacterial adhesion to surfaces via direct and indirect mechanisms, and formation of biofilms is considered as an important strategy for adaptation and survival in sub-optimal environmental conditions. However, the molecular underpinnings of biofilm formation in subsurface sediment/groundwater ecosystems where microorganisms often experience fluctuations in nutrient input, pH, nitrate or metal concentrations is underexplored. We examined biofilm formation under different nutrient, pH, metal, and nitrate regimes of 16 Rhodanobacter strains isolated from subsurface groundwater wells spanning diverse pH (3.5 to 5) and nitrate levels (13.7 to 146 mM). Eight Rhodanobacter strains demonstrated significant biofilm growth under low pH, suggesting adaptation to survive and grow at low pH. Biofilms intensified under aluminum stress, particularly in strains possessing fewer genetic traits associated with biofilm formation warranting further investigation. Through RB-TnSeq, proteomics, use of specific mutants and transmission electron microscopy analysis, we discovered flagellar loss under aluminum stress, indicating a potential relationship between motility, metal tolerance, and biofilm growth. Comparative genomic analyses revealed absence of flagella and chemotaxis genes, and presence of putative Type VI secretion system in the high biofilm-forming strain FW021-MT20. This study identifies genetic determinants associated with biofilm growth in a predominant environmental genus, Rhodanobacter, under metal stress and identifies traits aiding survival and adaptation to contaminated subsurface environments.

Intraflagellar transport has traditionally been studied in immobilized flagella. In this issue, Gray et al. (https://doi.org/10.1083/jcb.202401154) introduced a novel methodology for fast imaging in free-swimming Leishmania, revealing the impacts of flagellum immobilization on intraflagellar transport and its inverse correlation with cell swimming speed.

FliL is a bacterial flagellar protein demonstrated to associate with, and regulate ion flow through, the stator complex in a diverse array of bacterial species. FliL is also implicated in additional functions such as stabilizing the flagellar rod, modulating rotor bias, sensing the surface, and regulating gene expression. How can one protein do so many things? Its location is paramount to understanding its numerous functions. This review will look at the evidence, attempt to resolve some conflicting findings, and offer new thoughts on FliL.

Astaxanthin biosynthesis in Haematococcus pluvialis is driven by energy. However, the effect of the flagella-mediated energy-consuming movement process on astaxanthin accumulation has not been well studied. In this study, the profiles of astaxanthin and NADPH contents in combination with the photosynthetic parameters with or without flagella enabled by pH shock were characterized. The results demonstrated that there was no significant alteration in cell morphology, with the exception of the loss of flagella observed in the pH shock treatment group. In contrast, the astaxanthin content in the flagella removal groups was 62.9%, 62.8% and 91.1% higher than that of the control at 4, 8 and 12 h, respectively. Simultaneously, the increased Y(II) and decreased Y(NO) suggest that cells lacking the flagellar movement process may allocate more energy towards astaxanthin biosynthesis. This finding was verified by NADPH analysis, which revealed higher levels in flagella removal cells. These results provide preliminary insights into the underlying mechanism of astaxanthin accumulation enabled by energy reassignment in movement-lacking cells.

Serratia marcescens is an opportunistic human pathogen that produces a vibrant red pigment called prodigiosin. Prodigiosin has implications in virulence of S. marcescens and promising clinical applications. We discovered that addition of the virulent flagellotropic bacteriophage χ (Chi) to a culture of S. marcescens stimulates a greater than fivefold overproduction of prodigiosin. Active phage infection is required for the effect, as a χ-resistant strain lacking flagella does not respond to phage presence. Via a reporter fusion assay, we have determined that the addition of a χ-induced S. marcescens cell lysate to an uninfected culture causes a threefold increase in transcription of the pig operon, containing genes essential for pigment biosynthesis. Replacement of the pig promoter with a constitutive promoter abolished the pigmentation increase, indicating that regulatory elements present in the pig promoter likely mediate the phenomenon. We hypothesize that S. marcescens detects the threat of phage-mediated cell death and reacts by producing prodigiosin as a stress response. Our findings are of clinical significance for two main reasons: (i) elucidating complex phage-host interactions is crucial for development of therapeutic phage treatments, and (ii) overproduction of prodigiosin in response to phage could be exploited for its biosynthesis and use as a pharmaceutical.

Trypanosoma cruzi uses various mechanisms to cope with osmotic fluctuations during infection, including the remodeling of organelles such as the contractile vacuole complex (CVC). Little is known about the morphological changes of the CVC during pulsation cycles occurring upon osmotic stress. Here, we investigated the structure-function relationship between the CVC and the flagellar pocket domain where fluid discharge takes place-the adhesion plaque-during the CVC pulsation cycle. Using TcrPDEC2 and TcVps34 overexpressing mutants, known to have low and high efficiency for osmotic responses, we described a structural phenotype for the CVC that matches their corresponding physiological responses. Quantitative tomography provided data on the volume of the CVC and spongiome connections. Changes in the adhesion plaque during the pulsation cycle were also quantified and a dense filamentous network was observed. Together, the results suggest that the adhesion plaque mediates fluid discharge from the central vacuole, revealing new aspects of the osmoregulatory system in T. cruzi.