The mechanochemical origins of the microtubule sliding motility within the kinesin-5 domain organization

The conserved kinesin-5 bipolar tetrameric motors slide apart microtubules during mitotic spindle assembly and elongation. Kinesin-5 bipolar organization originates from its conserved tetrameric helical minifilament, which position the C-terminal tail domains of two subunits near the N-terminal motor domains of two anti-parallel subunits (Scholey et al, 2014). This unique tetrameric structure enables kinesin-5 to simultaneously engage two microtubules and transmit forces between them, and for multiple kinesin-5 motors to organize via tail to motor interactions during microtubule sliding (Bodrug et al, 2020). Here, we show how these two structural adaptations, the kinesin-5 tail-motor domain interactions and the length of the tetrameric minifilament, determine critical aspects of kinesin-5 motility and sliding mechanisms. An x-ray structure of the 34-nm kinesin-5 minifilament reveals how the dual dimeric N-terminal coiled-coils emerge from the tetrameric central bundle. Using this structure, we generated active bipolar mini-tetrameric motors from Drosophila and human orthologs, which are half the length of native kinesin-5. Using single-molecule motility assays, we show that kinesin-5 tail domains promote mini-tetramers static pauses that punctuate processive motility. During such pauses, kinesin-5 mini-tetramers form multi-motor clusters mediated via tail to motor domain cross-interactions. These clusters undergo slow and highly processive motility and accumulate at microtubule plus-ends. In contrast to native kinesin-5, mini-tetramers require tail domains to initiate microtubule crosslinking. Although mini-tetramers are highly strained in initially aligning microtubules, they slide microtubules more efficiently than native kinesin-5, due to their decreased minifilament flexibility. Our studies reveal that the conserved kinesin-5 motor-tail mediated clustering and the length of the tetrameric minifilament are key features for sliding motility and are critical in organizing microtubules during mitotic spindle assembly and elongation.

Mab_3083c Is a Homologue of RNase J and Plays a Role in Colony Morphotype, Aggregation, and Sliding Motility of Mycobacterium abscessus

Mycobacterium abscessus is an opportunistic pathogen causing human diseases, especially in immunocompromised patients. M. abscessus strains with a rough morphotype are more virulent than those with a smooth morphotype. Morphotype switch may occur during a clinical infection. To investigate the genes involved in colony morphotype switching, we performed transposon mutagenesis in a rough clinical strain of M. abscessus. A morphotype switching mutant (smooth) named mab_3083c::Tn was obtained. This mutant was found to have a lower aggregative ability and a higher sliding motility than the wild type strain. However, its glycopeptidolipid (GPL) content remained the same as those of the wild type. Complementation of the mutant with a functional mab_3083c gene reverted its morphotype back to rough, indicating that mab_3083c is associated with colony morphology of M. abscessus. Bioinformatic analyses showed that mab_3083c has a 75.4% identity in amino acid sequence with the well-characterized ribonuclease J (RNase J) of M. smegmatis (RNase JMsmeg). Complementation of the mutant with the RNase J gene of M. smegmatis also switched its colony morphology from smooth back to rough. These results suggest that Mab_3083c is a homologue of RNase J and involved in regulating M. abscessus colony morphotype switching.

Diguanylate Cyclase GdpX6 with c-di-GMP Binding Activity Involved in the Regulation of Virulence Expression in Xanthomonas oryzae pv. oryzae

Cyclic diguanylate monophosphate (c-di-GMP) is a secondary messenger present in bacteria. The GGDEF-domain proteins can participate in the synthesis of c-di-GMP as diguanylate cyclase (DGC) or bind with c-di-GMP to function as a c-di-GMP receptor. In the genome of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, there are 11 genes that encode single GGDEF domain proteins. The GGDEF domain protein, PXO_02019 (here GdpX6 [GGDEF-domain protein of Xoo6]) was characterized in the present study. Firstly, the DGC and c-di-GMP binding activity of GdpX6 was confirmed in vitro. Mutation of the crucial residues D403 residue of the I site in GGDEF motif and E411 residue of A site in GGDEF motif of GdpX6 abolished c-di-GMP binding activity and DGC activity of GdpX6, respectively. Additionally, deletion of gdpX6 significantly increased the virulence, swimming motility, and decreased sliding motility and biofilm formation. In contrast, overexpression of GdpX6 in wild-type PXO99A strain decreased the virulence and swimming motility, and increased sliding motility and biofilm formation. Mutation of the E411 residue but not D403 residue of the GGDEF domain in GdpX6 abolished its biological functions, indicating the DGC activity to be imperative for its biological functions. Furthermore, GdpX6 exhibited multiple subcellular localization in bacterial cells, and D403 or E411 did not contribute to the localization of GdpX6. Thus, we concluded that GdpX6 exhibits DGC activity to control the virulence, swimming and sliding motility, and biofilm formation in Xoo.

Mycobacterium smegmatis expands across surfaces using hydraulic sliding

Mycobacterium smegmatis spreads over soft agar surfaces by sliding motility, a form of passive motility in which growth and reduction of surface adhesion enable the bacteria to push each other outwards. Hence, sliding motility is mostly associated with round colonies. However, M. smegmatis sliding colonies can also produce long, pointed dendrites. Round sliding colonies were readily reproduced, but our non-round colonies were different from those seen previously. The latter (named digitate colonies) had centimetre-long linear protrusions, containing a central channel filled with a free-flowing suspension of M. smegmatis and solid aggregates. Digitate colonies had both a surface pellicle and an inner biofilm component surrounding a central channel, which sat in a cleft in the agar. Time-lapse microscopy showed that the expansion of the fluid-filled channel enabled the lengthwise extension of the protrusions without any perceptible growth of the bacteria taking place. These observations represent a novel type of sliding motility, named hydraulic sliding, associated with a specialised colony structure and the apparent generation of force by expansion of a liquid core. As this structure requires pellicle formation without an initial liquid culture it implies the presence of an unstudied mycobacterial behaviour that may be important for colonisation and virulence.Originality-Significance StatementThis study is the first to identify a new form of passive motility in the mycobacteria; hydraulic sliding, in which liquid expansion is the cause of motility. This form of motility has so far never been described in bacteria. The study also reveals new ways mycobacteria can form biofilms and colonize complex three-dimensional substrates, aspects of mycobacterial biology that are important for infection, pathogenesis and vaccine development.Author SummaryMycobacterium smegmatis is used as a non-pathogenic model organism for pathogenic mycobacteria. During growth, M. smegmatis can move passively over soft agar surfaces by a process called sliding motility, whereby colony growth directly pushes cells outwards. Although passive, sliding motility is believed to be important in allowing bacteria to colonise surfaces. Sliding motility however does not fully account for how M. smegmatis produces dendritic colonies. We attempted to generate dendritic colonies but found instead that the cells produced colonies that had larger protrusions radiating from them (digitate colonies). Digitate colonies are a previously unobserved phenomenon, in that the bacteria create a biofilm-lined, fluid-filled, pellicle-covered, deep cleft in the agar and move across the surface by the expansion of the contained liquid core of the protrusions. Given the new structure and the new mechanism of expansion we have termed this set of behaviours hydraulic sliding. These observations are important as it is a new variation in the way bacteria can move, generate biofilms (notably mycobacterial pellicle) and colonize complex three-dimensional substrates.

Myosin 1b is an actin depolymerase

The regulation of actin dynamics is essential for various cellular processes. Former evidence suggests a correlation between the function of non-conventional myosin motors and actin dynamics. Here we investigate the contribution of myosin 1b to actin dynamics using sliding motility assays. We observe that sliding on myosin 1b immobilized or bound to a fluid bilayer enhances actin depolymerization at the barbed end, while sliding on myosin II, although 5 times faster, has no effect. This work reveals a non-conventional myosin motor as another type of depolymerase and points to its singular interactions with the actin barbed end.

A thin-film extensional flow model for biofilm expansion by sliding motility

In the presence of glycoproteins, bacterial and yeast biofilms are hypothesized to expand by sliding motility. This involves a sheet of cells spreading as a unit, facilitated by cell proliferation and weak adhesion to the substratum. In this paper, we derive an extensional flow model for biofilm expansion by sliding motility to test this hypothesis. We model the biofilm as a two-phase (living cells and an extracellular matrix) viscous fluid mixture, and model nutrient depletion and uptake from the substratum. Applying the thin-film approximation simplifies the model, and reduces it to one-dimensional axisymmetric form. Comparison with Saccharomyces cerevisiae mat formation experiments reveals good agreement between experimental expansion speed and numerical solutions to the model with O ( 1 ) parameters estimated from experiments. This confirms that sliding motility is a possible mechanism for yeast biofilm expansion. Having established the biological relevance of the model, we then demonstrate how the model parameters affect expansion speed, enabling us to predict biofilm expansion for different experimental conditions. Finally, we show that our model can explain the ridge formation observed in some biofilms. This is especially true if surface tension is low, as hypothesized for sliding motility.

STUDI PENDAHULUAN NONTUBERCULOUS MYCOBACTERIA (NTM): PEMBENTUKAN BIOFILM, MOTILITAS GESER, DAN POLA KEPEKAAN ANTIBIOTIK

Nontuberculous mycobacteria (NTM) adalah mikrorganisme yang banyak dijumpai di lingkungan, namun, baru-baru ini dianggap patogen karena kejadian infeksinya meningkat secara signifikan. Penelitian ini bertujuan untuk mengetahui kemampuan pembentukan biofilm isolat NTM, korelasinya dengan sifat motilitas geser, dan untuk menganalisis pola kepekaan antibiotik. Strain NTM yang dipakai dalam penelitian ini adalah 10 isolat klinis NTM yang diperoleh dari laboratorium TB, Departemen Mikrobiologi, Fakultas Kedokteran UGM Yogyakarta. Kemampuan pembentukan biofilm dideteksi dengan menggunakan uji mikrotiter dan pewarnaan dengan kristal violet 1%. Uji motilitas geser dilakukan pada medium motilitas, terdiri dari 0,3% Middlebrook 7H9-agar tanpa suplemen. Pola kepekaan antibiotik diteliti dengan teknik dilusi sesuai metode CLSI. Dari penelitian ini menunjukkan bahwa 7 dari 10 isolat NTM merupakan penghasil biofilm kuat, sementara 1 isolat sebagai strain penghasil biofilm moderat, dan 2 isolat tidak menghasilkan biofilm. Sementara itu, strain pembentuk biofilm mampu melakukan motilitas geser pada agar semisolid, dan 2 isolat NTM yang tidak memiliki kemampuan pembentukan biofilm tidak dapat melakukan motilitas geser. Sifat pembentukan biofilm berkorelasi dengan kemampuan isolat NTM untuk melakukan motilitas geser pada media agar semisolid. Klaritromisin merupakan antibiotik yang paling efektif terhadap isolat NTM yang diuji (poten terhadap 50% isolat uji), diikuti oleh gentamisin (40%), sedangkan kanamisin, levofloxacin, dan ofloxacin menunjukkan tingkat potensi yang sama (30%). Ceftriaxone hanya mampu menghambat pertumbuhan isolat NTM sekitar 20%. Selanjutnya, kotrimoksazol dan amoksisilin memiliki aktivitas in vitro yang buruk terhadap isolat NTM karena tidak ada isolat NTM yang sensitif terhadap kedua antibiotik ini. Nontuberculous mycobacteria (NTM) are ubiquitous organisms commonly found in the environment. However, recently it is considered as emerging global interest since the incidence increase significantly. This study aimed to investigate the biofilm forming ability of NTM isolates, correlated with the sliding motility properties, and to analyze their antibiotic susceptibility pattern. NTM strain included in this study were 10 NTM clinical isolates obtained from TB laboratory, Microbiology Departement, Faculty of Medicine UGM Yogyakarta. Biofilm forming capability was detected by using biofilm development assay in microtiter plate and staining with 1% crystal violet. Sliding motility assay was performed on motility medium, consisting of Middlebrook 7H9- 0.3% agar without supplements. Antibiotic susceptibility pattern was investigated by macrobroth dilution technique according to CLSI methods. Our study revealed that 7 out of 10 NTM isolates produced biofilm strongly, while 1 isolate demontrated as moderate biofilm former strain, and the remaining 2 isolates did not produce biofilm on polysterene substrate. Meanwhile, biofilm-former strain are able to slide on semisolid agar, and 2 non-adherent NTM isolates did not have ability to perform sliding motility. A good correlation was found between mycobacterial sliding and biofilm assembly of NTM isolates. Clarithromycin has been shown as the most effective antibiotic against NTM isolates tested, which was active against 50% of all isolates, followed by gentamycin (40%), while kanamycin, levofloxacin, and ofloxacin showed the same level of potency (30%). Ceftriaxone was only able to inhibit the growth of NTM isolates about 20%. Furthermore, cotrimoxazole and amoxicillin had poor in vitro activity against NTM species.

Peptidyl-Prolyl-cis/trans-Isomerases Mip and PpiB ofLegionella pneumophilaContribute to Surface Translocation, Growth at Suboptimal Temperature, and Infection

ABSTRACTThe gammaproteobacteriumLegionella pneumophilais the causative agent of Legionnaires’ disease, an atypical pneumonia that manifests itself with severe lung damage.L. pneumophila, a common inhabitant of freshwater environments, replicates in free-living amoebae and persists in biofilms in natural and man-made water systems. Its environmental versatility is reflected in its ability to survive and grow within a broad temperature range as well as its capability to colonize and infect a wide range of hosts, including protozoa and humans. Peptidyl-prolyl-cis/trans-isomerases (PPIases) are multifunctional proteins that are mainly involved in protein folding and secretion in bacteria. InL. pneumophilathe surface-associated PPIase Mip was shown to facilitate the establishment of the intracellular infection cycle in its early stages. The cytoplasmic PpiB was shown to promote cold tolerance. Here, we set out to analyze the interrelationship of these two relevant PPIases in the context of environmental fitness and infection. We demonstrate that the PPIases Mip and PpiB are important for surfactant-dependent sliding motility and adaptation to suboptimal temperatures, features that contribute to the environmental fitness ofL. pneumophila. Furthermore, they contribute to infection of the natural hostAcanthamoeba castellaniias well as human macrophages and human explanted lung tissue. These effects were additive in the case of sliding motility or synergistic in the case of temperature tolerance and infection, as assessed by the behavior of the double mutant. Accordingly, we propose that Mip and PpiB are virulence modulators ofL. pneumophilawith compensatory action and pleiotropic effects.

A new actin depolymerase: a Myosin 1 motor

The regulation of actin dynamics is essential for various cellular processes. Former evidence suggests a correlation between the function of non-conventional myosin motors and actin dynamics. We investigate the contribution of myosin1b to actin dynamics using sliding motility assays. We observe that sliding on myosin1b immobilized or bound to a fluid bilayer enhances actin depolymerization at the barbed end, while sliding on myosin II, although 5 times faster, has no effect. This work reveals a non-conventional myosin motor as a new type of depolymerase and points to its singular interactions with the actin barbed end.