Filamentous structures in the cell envelope are associated with bacteroidetes gliding machinery.

Many bacteria belonging to the phylum Bacteroidetes move on solid surfaces, called gliding motility. In our previous study with the Bacteroidetes gliding bacterium Flavobacterium johnsoniae, we proposed a helical loop track model, where adhesive SprB filaments are propelled along a helical loop on the cell surface. In this study, we observed the gliding cell rotating counterclockwise about its axis when viewed from the rear to the advancing direction of the cell and revealed that one labeled SprB focus sometimes overtook and passed another SprB focus that was moving in the same direction. Several electron microscopic analyses revealed the presence of a possible multi-rail structure underneath the outer membrane, which was associated with SprB filaments and contained GldJ protein. These results provide insights into the mechanism of Bacteroidetes gliding motility, in which the SprB filaments are propelled along tracks that may form a multi-rail system underneath the outer membrane. The insights may give clues as to how the SprB filaments get their driving force.

[Application of ultrasound in treating postpartum pubis symphysis diastasis by bone setting manipulation].

OBJECTIVE: To explore application value of ultrasound in treating postpartum pubis symphysis diastasis by bone setting manipulation. METHODS: Retrospective analysis was performed on 30 patients (case group) with postpartum pubis symphysis diastasis diagnosed in Wangjing Hospital, China Academy of Chinese Medical Sciences from June 2017 to January 2021, aged from 21 to 43 years old, with an average of (33.0±3.5) years old. The main clinical manifestations were mobility disorders such as turning over and walking, and all patients were treated by bone setting manipulation. Before and after treatment, pain and degree of pubic symphysis separation were evaluated by visual analogue scale(VAS) and ultrasonography. In normal group, 30 menopausal women aged from 49 to 59 years old with an average of(54.0±2.9) years old who wanted to remove intra uterine device(IUD) and were underwent conventional pelvic plain radiographswere selected, and the width of pubic symphysis space was measured by ultrasound and plain radiographs. RESULTS: In normal group, the width of pubic symphysis was about (5.2±1.7) mm by ultrasonography, X-ray measurement was (5.0±2.1) mm, and showed no difference(P>0.05).In case group, the width of pubic symphysis measured by ultrasound before manipulation was about (9.5±1.8) mm, VAS was 6.05(5.27, 6.80) scores;while the width of pubic symphysis measured by ultrasound before manipulation was about (5.8±1.3) mm, VAS was 0(0, 0) scores, and there were statistical difference before and after manipulation (P<0.05). CONCLUSION: Ultrasound is examation method with safe, non radioactive, easy to repeat for many times, could clearly show cartilage, ligament and bone structure around pubic symphysis, and is more suitable for the imaging diagnosis of postpartum pubis symphysis diastasis, which provide quantitative imaging basis for clinical evaluation of the curative effect of bone setting manipulation in treating postpartum pubis symphysis diastasis.

The Value of Transrectal Ultrasound in the Preoperative Diagnosis of Complex Anal Fistula (CAF): Based on a Retrospective Cohort Study.

Objective: A case-control study was employed to retrospectively analyze the value of transrectal ultrasound in the preoperative diagnosis of complex anal fistula (CAF). Methods: The clinical data of 128 patients with CAF treated in our hospital from March 2019 to June 2021 were analyzed retrospectively. All patients were examined by transrectal ultrasound and MRI with Hitachi HI Vision Ascendus ultrasound diagnostic apparatus and MRI. The general data of the patients (age, sex, course of disease, complications, and previous operation history) and ultrasonic image characteristics were recorded. The consistency of internal orifice, head, branch/abscess, and abscess detected by ultrasound, MRI, and ultrasound combined with MRI were compared, and the sensitivity, accuracy, and specificity of ultrasound, MRI, and the combination of ultrasound and MRI (ultrasound+MRI) in the diagnosis of different Parks classification of anal fistula (AF) were compared. Results: The ultrasound images of the rectal probe in typical cases were compared with the MRI images. The characteristics of the ultrasound images were as follows: the outer orifice of AF was a thin strip of mixed echo or low echo leading to the skin side, and the inner orifice showed local dilated low echo, mixed echo, or interruption of mucosal continuity. The following are the MRI image features: abnormal long bar signal shadow from the dorsal side of the end of the coccyx to the S5 plane, low signal on T1WI, high signal on T2WI, blurred boundary, uneven signal, bifurcation in the lower end of the tail for "Y" shape, one branch opening at the body surface at about 6 o'clock, the other walking horizontally, passing through the levator ani muscle to the right posterior position of the rectum at about 6:00 o'clock, and penetrating the inner mouth of the rectum at 6 o'clock. The detection of internal orifice, head, branch/abscess, and abscess were compared by three examination methods. There was significant difference in the detection rate of internal orifice and branch/purulent cavity among the three methods (P < 0.05). The detection rates of internal mouth and branch/abscess cavity by ultrasound and MRI (94.77% and 94.94%) were higher than those by single ultrasound (75.16% and 79.78%) and MRI (81.05% and 83.15%) (P < 0.05). There was no significant difference in the detection rate of ultrasound, MRI internal orifice, and branch/purulent cavity (P > 0.05). There was no significant difference in the detection rate of supervisor and abscess among the three methods (P > 0.05). The results of operation included transsphincter type (n = 53), intersphincter type (n = 45), and superior sphincter type (n = 30). Analysis of transsphincter type AF detected by three methods: 42 cases of transsphincter type AF and 86 cases of nonsphincter type AF were detected by ultrasound, 36 cases of transsphincter type AF and 92 cases of nontranssphincter type AF were detected by MRI, 57 cases of transsphincter type AF and 71 cases of nonsphincter type AF were detected by ultrasound and MRI. The comparison of the efficacy of the three methods in the diagnosis of transsphincter AF and the sensitivity of the three methods in the diagnosis of transsphincter AF showed significant difference (P < 0.05). The sensitivity of ultrasound and MRI in the diagnosis of transsphincter AF (96.23%) was higher than those of single ultrasound (67.92%) and MRI (64.15%) (P < 0.05). There was no significant difference in the accuracy and specificity of the three methods in the diagnosis of transsphincter AF (P > 0.05). There were 41 cases of intersphincter type AF and 87 cases of nonsphincter type AF detected by ultrasound, 38 cases of intersphincter type AF and 90 cases of nonsphincter intersphincter type AF detected by MRI, and 45 cases of intersphincter type AF and 83 cases of nonsphincter intersphincter type AF detected by ultrasound and MRI. The sensitivity and accuracy of the three methods in the diagnosis of intersphincter AF were statistically significant (P < 0.05). The sensitivity and accuracy (100.00% and 100.00%) of ultrasound and MRI in the diagnosis of intersphincter AF were higher than those of single ultrasound (66.67% and 79.69%) and MRI (71.11% and 85.16%) (P < 0.05). There was no significant difference in the specificity of the three methods in the diagnosis of intersphincter AF (P > 0.05). The results of three methods were compared, including 24 cases of superior sphincter type AF and 89 cases of nonsuperior sphincter type AF, 21 cases of superior sphincter type AF, and 107 cases of nonsuperior sphincter type AF detected by MRI and 93 cases of superior sphincter type AF and 128cases of nonsuperior sphincter type AF detected by ultrasound and MRI. There was no significant difference in the sensitivity, accuracy, and specificity of the three methods in the diagnosis of superior sphincter AF (P > 0.05). Conclusion: The sphincter, anorectal, and surrounding tissues were clearly demonstrated by transrectal ultrasound. The internal orifice, head, branch/abscess, abscess, and the relationship between abscess and sphincter in the diagnosis of CAF were in good agreement with the surgical results. Ultrasound+MRI can take into account the advantages of ultrasound and MRI, make up for each other, and improve the detection rate of internal orifice and branch/abscess. It can improve the sensitivity of diagnosis of transsphincter AF and the sensitivity and accuracy of intersphincter AF, which can provide intuitive and valuable imaging information for surgical intervention.

[Correlative analysis of cervical curvature and atlantoaxial instability].

OBJECTIVE: To investigate the correlation between the changes of cervical curvature and atlantoaxial instability. METHODS: The correlation between the changes of cervical curvature and atlantoaxial instability was retrospectively studied in 50 outpatients with abnormal cervical curvature (abnormal cervical curvature group) from January 2018 to December 2019. There were 24 males and 26 females in abnormal cervical curvature group, aged from 18 to 42 years old with an average of(30.62±5.83) years. And 53 patients with normal cervical curvature (normal cervical curvature group) during the same period were matched, including 23 males and 30 females, aged from 21 to 44 years with an average of(31.98±6.11) years. Cervical spine X-ray films of 103 patients were taken in lateral position and open mouth position. Cervical curvature and variance of bilateral lateral atlanto-dental space(VBLADS) were measured and recorded, Pearson correlation coefficient analysis was used to study the correlation between the changes of cervical curvature and atlantoaxial instability. RESULTS: Atlantoaxial joint instability accounted for 39.6%(21/53) in normal cervical curvature group and 84.0%(42/50) in abnormal cervical curvature group. There was significant difference between two groups(P<0.01). VBLADS in abnormal cervical curvature group was (1.79±1.01) mm, which was significantly higher than that in normal cervical curvature group(0.55±0.75) mm(P<0.01). Pearson correlation coefficient analysis showed that the size of cervical curvature was negatively correlated with VBLADS. CONCLUSION: Cervical curvature straightening and inverse arch are the cause of atlantoaxial instability, the smaller the cervical curvature, the more serious the atlantoaxial instability.

Type IX Secretion System Effectors and Virulence of the Model Flavobacterium columnare Strain MS-FC-4.

Flavobacterium columnare causes columnaris disease in wild and cultured freshwater fish and is a major problem for sustainable aquaculture worldwide. The F. columnare type IX secretion system (T9SS) secretes many proteins and is required for virulence. The T9SS component GldN is required for secretion and gliding motility over surfaces. Genetic manipulation of F. columnare is inefficient, which has impeded identification of secreted proteins that are critical for virulence. Here, we identified a virulent wild-type F. columnare strain (MS-FC-4) that is highly amenable to genetic manipulation. This facilitated isolation and characterization of two deletion mutants lacking core components of the T9SS. Deletion of gldN disrupted protein secretion and gliding motility and eliminated virulence in zebrafish and rainbow trout. Deletion of porV disrupted secretion and virulence but not motility. Both mutants exhibited decreased extracellular proteolytic, hemolytic, and chondroitin sulfate lyase activities. They also exhibited decreased biofilm formation and decreased attachment to fish fins and other surfaces. Using genomic and proteomic approaches, we identified proteins secreted by the T9SS. We deleted 10 genes encoding secreted proteins and characterized the virulence of mutants lacking individual or multiple secreted proteins. A mutant lacking two genes encoding predicted peptidases exhibited reduced virulence in rainbow trout, and mutants lacking a predicted cytolysin showed reduced virulence in zebrafish and rainbow trout. The results establish F. columnare strain MS-FC-4 as a genetically amenable model to identify virulence factors. This may aid development of measures to control columnaris disease and impact fish health and sustainable aquaculture. IMPORTANCE Flavobacterium columnare causes columnaris disease in wild and aquaculture-reared freshwater fish and is a major problem for aquaculture. Little is known regarding the virulence factors involved in this disease, and control measures are inadequate. The type IX secretion system (T9SS) secretes many proteins and is required for virulence, but the secreted virulence factors are not known. We identified a strain of F. columnare (MS-FC-4) that is well suited for genetic manipulation. The components of the T9SS and the proteins secreted by this system were identified. Deletion of core T9SS genes eliminated virulence. Genes encoding 10 secreted proteins were deleted. Deletion of two peptidase-encoding genes resulted in decreased virulence in rainbow trout, and deletion of a cytolysin-encoding gene resulted in decreased virulence in rainbow trout and zebrafish. Secreted peptidases and cytolysins are likely virulence factors and are targets for the development of control measures.

In situ imaging of bacterial outer membrane projections and associated protein complexes using electron cryo-tomography.

The ability to produce outer membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among diderm bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of ~90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified outer MEs and MVs in 13 diderm bacterial species and classified several major ultrastructures: (1) tubes with a uniform diameter (with or without an internal scaffold), (2) tubes with irregular diameter, (3) tubes with a vesicular dilation at their tip, (4) pearling tubes, (5) connected chains of vesicles (with or without neck-like connectors), (6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.

The Monoheme c Subunit of Respiratory Alternative Complex III Is Not Essential for Electron Transfer to Cytochrome aa3 in Flavobacterium johnsoniae.

Bacterial alternative complex III (ACIII) catalyzes menaquinol (MKH2) oxidation, presumably fulfilling the role of cytochromes bc1/b6f in organisms that lack these enzymes. The molecular mechanism of ACIII is unknown and so far the complex has remained inaccessible for genetic modifications. The recently solved cryo-electron microscopy (cryo-EM) structures of ACIII from Flavobacterium johnsoniae, Rhodothermus marinus, and Roseiflexus castenholzii revealed no structural similarity to cytochrome bc1/b6f and there were variations in the heme-containing subunits ActA and ActE. These data implicated intriguing alternative electron transfer paths connecting ACIII with its redox partner, and left the contributions of ActE and the terminal domain of ActA to the catalytic mechanism unclear. Here, we report genetic deletion and complementation of F. johnsoniae actA and actE and the functional implications of such modifications. Deletion of actA led to the loss of activity of cytochrome aa3 (a redox partner of ACIII in this bacterium), which confirmed that ACIII is the sole source of electrons for this complex. Deletion of actE did not impair the activity of cytochrome aa3, revealing that ActE is not required for electron transfer between ACIII and cytochrome aa3. Nevertheless, absence of ActE negatively impacted the cell growth rate, pointing toward another, yet unidentified, function of this subunit. Possible explanations for these observations, including a proposal of a split in electron paths at the ActA/ActE interface, are discussed. The described system for genetic manipulations in F. johnsoniae ACIII offers new tools for studying the molecular mechanism of operation of this enzyme. IMPORTANCE Energy conversion is a fundamental process of all organisms, realized by specialized protein complexes, one of which is alternative complex III (ACIII). ACIII is a functional analogue of well-known mitochondrial complex III, but operates according to a different, still unknown mechanism. To understand how ACIII interacts functionally with its protein partners, we developed a genetic system to mutate the Flavobacterium johnsoniae genes encoding ACIII subunits. Deletion and complementation of heme-containing subunits revealed that ACIII is the sole source of electrons for cytochrome aa3 and that one of the redox-active subunits (ActE) is dispensable for electron transfer between these complexes. This study sheds light on the operation of the supercomplex of ACIII and cytochrome aa3 and suggests a division in the electron path within ACIII. It also shows a way to manipulate protein expression levels for application in other members of the Bacteroidetes phylum.

The Carboxy-Terminal Region of Flavobacterium johnsoniae SprB Facilitates Its Secretion by the Type IX Secretion System and Propulsion by the Gliding Motility Machinery.

Flavobacterium johnsoniae SprB moves rapidly along the cell surface, resulting in gliding motility. SprB secretion requires the type IX secretion system (T9SS). Proteins secreted by the T9SS typically have conserved C-terminal domains (CTDs) belonging to the type A CTD or type B CTD family. Attachment of 70- to 100-amino-acid type A CTDs to a foreign protein allows its secretion. Type B CTDs are common but have received little attention. Secretion of the foreign protein superfolder green fluorescent protein (sfGFP) fused to regions spanning the SprB type B CTD (sfGFP-CTDSprB) was analyzed. CTDs of 218 amino acids or longer resulted in secretion of sfGFP, whereas a 149-amino-acid region did not. Some sfGFP was secreted in soluble form, whereas the rest was attached on the cell surface. Surface-attached sfGFP was rapidly propelled along the cell, suggesting productive interaction with the motility machinery. This did not result in rapid cell movement, which apparently requires additional regions of SprB. Secretion of sfGFP-CTDSprB required coexpression with sprF, which lies downstream of sprB SprF is similar in sequence to Porphyromonas gingivalis PorP. Most F. johnsoniae genes encoding proteins with type B CTDs lie immediately upstream of porP/sprF-like genes. sfGFP was fused to the type B CTD from one such protein (Fjoh_3952). This resulted in secretion of sfGFP only when it was coexpressed with its cognate PorP/SprF-like protein. These results highlight the need for extended regions of type B CTDs and for coexpression with the appropriate PorP/SprF-like protein for efficient secretion and cell surface localization of cargo proteins.IMPORTANCE The F. johnsoniae gliding motility adhesin SprB is delivered to the cell surface by the type IX secretion system (T9SS) and is rapidly propelled along the cell by the motility machinery. How this 6,497-amino-acid protein interacts with the secretion and motility machines is not known. Fusion of the C-terminal 218 amino acids of SprB to a foreign cargo protein resulted in its secretion, attachment to the cell surface, and rapid movement by the motility machinery. Efficient secretion of SprB required coexpression with the outer membrane protein SprF. Secreted proteins that have sequence similarity to SprB in their C-terminal regions are common in the phylum Bacteroidetes and may have roles in adhesion, motility, and virulence.

Design and Implementation of a Novel Polarimetric Active Radar Calibrator for Gaofen-3 SAR.

The Chinese first fully polarimetric space-borne synthetic aperture radar (SAR)-Gaofen-3 (GF-3) was launched in August 2016, which operates at the C-band and the resolution can reach 1 m. Polarimetric SAR calibration is a procedure that corrects the polarization distortion of a measured scattering matrix by referring to the scattering matrix of a known target. The present paper describes the principle, design, manufacture, and measurement results of a novel polarimetric active radar calibrator (PARC) designed for GF-3. A new design method for PARC was presented and two dual-polarized antennas with very high polarization purity were used. The internal calibration technique was introduced to ensure balance in the amplitude and phase, which ensures the precision of the PARC's scattering matrices. The results we obtained through measurement in the microwave anechoic chamber and experiments in in-orbit calibration agree well with the theoretical predictions, and the novel PARC presented is proved to be well suited for polarization and radiometric calibration of GF-3.

Carrageenan catabolism is encoded by a complex regulon in marine heterotrophic bacteria.

Macroalgae contribute substantially to primary production in coastal ecosystems. Their biomass, mainly consisting of polysaccharides, is cycled into the environment by marine heterotrophic bacteria using largely uncharacterized mechanisms. Here we describe the complete catabolic pathway for carrageenans, major cell wall polysaccharides of red macroalgae, in the marine heterotrophic bacterium Zobellia galactanivorans. Carrageenan catabolism relies on a multifaceted carrageenan-induced regulon, including a non-canonical polysaccharide utilization locus (PUL) and genes distal to the PUL, including a susCD-like pair. The carrageenan utilization system is well conserved in marine Bacteroidetes but modified in other phyla of marine heterotrophic bacteria. The core system is completed by additional functions that might be assumed by non-orthologous genes in different species. This complex genetic structure may be the result of multiple evolutionary events including gene duplications and horizontal gene transfers. These results allow for an extension on the definition of bacterial PUL-mediated polysaccharide digestion.

The Type IX Secretion System Is Required for Virulence of the Fish Pathogen Flavobacterium columnare.

Flavobacterium columnare, a member of the phylum Bacteroidetes, causes columnaris disease in wild and aquaculture-reared freshwater fish. The mechanisms responsible for columnaris disease are not known. Many members of the phylum Bacteroidetes use type IX secretion systems (T9SSs) to secrete enzymes, adhesins, and proteins involved in gliding motility. The F. columnare genome has all of the genes needed to encode a T9SS. gldN, which encodes a core component of the T9SS, was deleted in wild-type strains of F. columnare The F. columnare ΔgldN mutants were deficient in the secretion of several extracellular proteins and lacked gliding motility. The ΔgldN mutants exhibited reduced virulence in zebrafish, channel catfish, and rainbow trout, and complementation restored virulence. PorV is required for the secretion of a subset of proteins targeted to the T9SS. An F. columnare ΔporV mutant retained gliding motility but exhibited reduced virulence. Cell-free spent media from exponentially growing cultures of wild-type and complemented strains caused rapid mortality, but spent media from ΔgldN and ΔporV mutants did not, suggesting that soluble toxins are secreted by the T9SS.IMPORTANCE Columnaris disease, caused by F. columnare, is a major problem for freshwater aquaculture. Little is known regarding the virulence factors produced by F. columnare, and control measures are limited. Analysis of targeted gene deletion mutants revealed the importance of the type IX protein secretion system (T9SS) and of secreted toxins in F. columnare virulence. T9SSs are common in members of the phylum Bacteroidetes and likely contribute to the virulence of other animal and human pathogens.

The unusual cellulose utilization system of the aerobic soil bacterium Cytophaga hutchinsonii.

Cellulolytic microorganisms play important roles in global carbon cycling and have evolved diverse strategies to digest cellulose. Some are 'generous,' releasing soluble sugars from cellulose extracellularly to feed both themselves and their neighbors. The gliding soil bacterium Cytophaga hutchinsonii exhibits a more 'selfish' strategy. It digests crystalline cellulose using cell-associated cellulases and releases little soluble sugar outside of the cell. The mechanism of C. hutchinsonii cellulose utilization is still poorly understood. In this review, we discuss novel aspects of the C. hutchinsonii cellulolytic system. Recently developed genetic manipulation tools allowed the identification of proteins involved in C. hutchinsonii cellulose utilization. These include periplasmic and cell-surface endoglucanases and novel cellulose-binding proteins. The recently discovered type IX secretion system is needed for cellulose utilization and appears to deliver some of the cellulolytic enzymes and other proteins to the cell surface. The requirement for periplasmic endoglucanases for cellulose utilization is unusual and suggests that cello-oligomers must be imported across the outer membrane before being further digested. Cellobiohydrolases or other predicted processive cellulases that play important roles in many other cellulolytic bacteria appear to be absent in C. hutchinsonii. Cells of C. hutchinsonii attach to and glide along cellulose fibers, which may allow them to find sites most amenable to attack. A model of C. hutchinsonii cellulose utilization summarizing recent progress is proposed.

Demonstration of a microwave photonic synthetic aperture radar based on photonic-assisted signal generation and stretch processing.

A microwave photonic synthetic aperture radar (MWP SAR) is developed and experimentally demonstrated. In the transmitter, microwave photonic frequency doubling is used to generate a linearly-frequency-modulated (LFM) radar signal; while in the receiver, photonic stretch processing is employed to receive the reflection signal. The presented MWP SAR operates in Ku band with a bandwidth of 600MHz, and is evaluated through a series of inverse SAR imaging tests both in a microwave anechoic chamber and in a field trial. Its imaging performance verifies that the proposed MWP SAR works perfect and shows the potential of overcoming the conventional radar bandwidth bottleneck.

Diverse C-Terminal Sequences Involved in Flavobacterium johnsoniae Protein Secretion.

Flavobacteriumjohnsoniae and many related bacteria secrete proteins across the outer membrane using the type IX secretion system (T9SS). Proteins secreted by T9SSs have amino-terminal signal peptides for export across the cytoplasmic membrane by the Sec system and carboxy-terminal domains (CTDs) targeting them for secretion across the outer membrane by the T9SS. Most but not all T9SS CTDs belong to the family TIGR04183 (type A CTDs). We functionally characterized diverse CTDs for secretion by the F. johnsoniae T9SS. Attachment of the CTDs from F. johnsoniae RemA, AmyB, and ChiA to the foreign superfolder green fluorescent protein (sfGFP) that had a signal peptide at the amino terminus resulted in secretion across the outer membrane. In each case, approximately 80 to 100 amino acids from the extreme carboxy termini were needed for efficient secretion. Several type A CTDs from distantly related members of the phylum Bacteroidetes functioned in F. johnsoniae, supporting the secretion of sfGFP by the F. johnsoniae T9SS. F. johnsoniae SprB requires the T9SS for secretion but lacks a type A CTD. It has a conserved C-terminal domain belonging to the family TIGR04131, which we refer to as a type B CTD. The CTD of SprB was required for its secretion, but attachment of C-terminal regions of SprB of up to 1,182 amino acids to sfGFP failed to result in secretion. Additional features outside the C-terminal region of SprB may be required for its secretion.IMPORTANCE Type IX protein secretion systems (T9SSs) are common in but limited to members of the phylum Bacteroidetes Most proteins that are secreted by T9SSs have conserved carboxy-terminal domains that belong to the protein domain family TIGR04183 (type A CTDs) or TIGR04131 (type B CTDs). Here, we identify features of T9SS CTDs of F. johnsoniae that are required for protein secretion and demonstrate that type A CTDs from distantly related members of the phylum function with the F. johnsoniae T9SS to secrete the foreign protein sfGFP. In contrast, type B CTDs failed to target sfGFP for secretion, suggesting a more complex association with the T9SS.

Genetic analyses unravel the crucial role of a horizontally acquired alginate lyase for brown algal biomass degradation by Zobellia galactanivorans.

Comprehension of the degradation of macroalgal polysaccharides suffers from the lack of genetic tools for model marine bacteria, despite their importance for coastal ecosystem functions. We developed such tools for Zobellia galactanivorans, an algae-associated flavobacterium that digests many polysaccharides, including alginate. These tools were used to investigate the biological role of AlyA1, the only Z. galactanivorans alginate lyase known to be secreted in soluble form and to have a recognizable carbohydrate-binding domain. A deletion mutant, ΔalyA1, grew as well as the wild type on soluble alginate but was deficient in soluble secreted alginate lyase activity and in digestion of and growth on alginate gels and algal tissues. Thus, AlyA1 appears to be essential for optimal attack of alginate in intact cell walls. alyA1 appears to have been recently acquired via horizontal transfer from marine Actinobacteria, conferring an adaptive advantage that might benefit other algae-associated bacteria by exposing new substrate niches. The genetic tools described here function in diverse members of the phylum Bacteroidetes and should facilitate analyses of polysaccharide degradation systems and many other processes in these common but understudied bacteria.

Development of Timolol-Loaded Galactosylated Chitosan Nanoparticles and Evaluation of Their Potential for Ocular Drug Delivery.

This study was conducted to develop timolol maleate (TM)-loaded galactosylated chitosan (GC) nanoparticles (NPs) (TM-GC-NPs) followed by optimization via a four-level and three-factor Box-Behnken statistical experimental design. The optimized nanoparticles showed a particle size of 213.3 ± 6.83 nm with entrapment efficiency of 38.58 ± 1.31% and drug loading of 17.72 ± 0.28%. The NPs were characterized with respect to zeta potential, pH, surface morphology, and differential scanning calorimetry (DSC). The determination of the oil-water partition coefficient demonstrated that the TM-GC-NPs had a high liposolubility at pH 6 as compared to timolol-loaded chitosan nanoparticles (TM-CS-NPs) and commercial TM eye drops. The in vitro release study indicated that TM-GC-NPs had a sustained release effect compared with the commercial TM eye drops. Ocular tolerance was studied by the hen's egg chorioallantoic membrane (HET-CAM) assay and the formulation was non-irritant and could be used for ophthalmic drug delivery. The in vitro transcorneal permeation study and confocal microscopy showed enhanced penetration, and retention in the cornea was achieved with TM-GC-NPs compared with the TM-CS-NPs and TM eye drops. Preocular retention study indicated that the retention of TM-GC-NPs was significantly longer than that of TM eye drops. The in vivo pharmacodynamic study suggested TM-GC-NPs had a better intraocular pressure (IOP) lowering efficacy and a prolonged working time compared to commercial TM eye drops (P ≤ 0.05). The optimized TM-GC-NPs could be prepared successfully promising their use as an ocular delivery system.

Tris(S,S-dioxide)-trithiasumanene: strong fluorescence and cocrystal with 1,2,6,7,10,11-hexabutoxytriphenylene.

Thiophene rings in trithiasumanene (1) are oxidized regioselectively to form tris(S,S-dioxide)-trithiasumanene (3). Compound 3 displays strong indigo fluorescence in both solution and the solid state, and forms a 1 : 1 cocrystal with HBT to give a yellow emission in crystalline form.

A polysaccharide utilization locus from Flavobacterium johnsoniae enables conversion of recalcitrant chitin.

BACKGROUND: Chitin is the second most abundant polysaccharide on earth and as such a great target for bioconversion applications. The phylum Bacteroidetes is one of nature's most ubiquitous bacterial lineages and is essential in the global carbon cycle with many members being highly efficient degraders of complex carbohydrates. However, despite their specialist reputation in carbohydrate conversion, mechanisms for degrading recalcitrant crystalline polysaccharides such as chitin and cellulose are hitherto unknown. RESULTS: Here we describe a complete functional analysis of a novel polysaccharide utilization locus (PUL) in the soil Bacteroidete Flavobacterium johnsoniae, tailored for conversion of chitin. The F. johnsoniae chitin utilization locus (ChiUL) consists of eleven contiguous genes encoding carbohydrate capture and transport proteins, enzymes, and a two-component sensor-regulator system. The key chitinase (ChiA) encoded by ChiUL is atypical in terms of known Bacteroidetes-affiliated PUL mechanisms as it is not anchored to the outer cell membrane and consists of multiple catalytic domains. We demonstrate how the extraordinary hydrolytic efficiency of ChiA derives from synergy between its multiple chitinolytic (endo- and exo-acting) and previously unidentified chitin-binding domains. Reverse genetics show that ChiA and PUL-encoded proteins involved in sugar binding, import, and chitin sensing are essential for efficient chitin utilization. Surprisingly, the ChiUL encodes two pairs of SusC/D-like outer membrane proteins. Ligand-binding and structural studies revealed functional differences between the two SusD-like proteins that enhance scavenging of chitin from the environment. The combined results from this study provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and reveal important novel aspects of the PUL paradigm. CONCLUSIONS: By combining reverse genetics to map essential PUL genes, structural studies on outer membrane chitin-binding proteins, and enzymology, we provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and introduce a new saccharolytic mechanism used by the phylum Bacteroidetes. The presented discovery and analysis of the ChiUL will greatly benefit future enzyme discovery efforts as well as studies regarding enzymatic intramolecular synergism.

Developments in Methods for Measuring the Intestinal Absorption of Nanoparticle-Bound Drugs.

With the rapid development of nanotechnology, novel drug delivery systems comprising orally administered nanoparticles (NPs) have been paid increasing attention in recent years. The bioavailability of orally administered drugs has significant influence on drug efficacy and therapeutic dosage, and it is therefore imperative that the intestinal absorption of oral NPs be investigated. This review examines the various literature on the oral absorption of polymeric NPs, and provides an overview of the intestinal absorption models that have been developed for the study of oral nanoparticles. Three major categories of models including a total of eight measurement methods are described in detail (in vitro: dialysis bag, rat gut sac, Ussing chamber, cell culture model; in situ: intestinal perfusion, intestinal loops, intestinal vascular cannulation; in vivo: the blood/urine drug concentration method), and the advantages and disadvantages of each method are contrasted and elucidated. In general, in vitro and in situ methods are relatively convenient but lack accuracy, while the in vivo method is troublesome but can provide a true reflection of drug absorption in vivo. This review summarizes the development of intestinal absorption experiments in recent years and provides a reference for the systematic study of the intestinal absorption of nanoparticle-bound drugs.

Complete Genome Sequence of the Fish Pathogen Flavobacterium columnare Strain C#2.

Flavobacterium columnare is a Gram-negative bacterial pathogen that causes columnaris disease of freshwater fish. Flavobacterium columnare strain C#2 was isolated from a diseased warm-water fish and is typed as genomovar II. The genome consists of a single 3.33-Mb circular chromosome with 2,689 predicted coding genes.