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The collagenolytic mechanism of Vibrio collagenase, a virulence factor, remains unclear. Here, the authors report the structure of Vibrio collagenase VhaC and propose the mechanism for collagen recognition and degradation, providing new insight into bacterial collagenolysis.

Bacterial leaf blight caused by Gram-negative pathogen Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive bacterial diseases on rice. Due to the resistance, toxicity and environmental issues of chemical bactericides, new biological strategies are still in need. Although peptaibols produced by Trichoderma spp. can inhibit the growth of several Gram-positive bacteria and plant fungal pathogens, it still remains unclear whether peptaibols have anti-Xoo activity to control bacterial leaf blight on rice. In this study, we evaluated the antibacterial effects of Trichokonins A (TKA), peptaibols produced by Trichoderma longibrachiatum SMF2, against Xoo. The in vitro antibacterial activity analysis showed that the growth of Xoo was significantly inhibited by TKA, with a minimum inhibitory concentration of 54 μg/mL and that the three TKs in TKA all had remarkable anti-Xoo activity. Further inhibitory mechanism analyses revealed that TKA treatments resulted in the damage of Xoo cell morphology and the release of intracellular substances, such as proteins and nucleic acids, from Xoo cells, suggesting the damage of the permeability of Xoo cell membrane by TKA. Pathogenicity analyses showed that the lesion length on rice leaf was significantly reduced by 82.2% when treated with 27 μg/mL TKA. This study represents the first report of the antibacterial activity of peptaibols against a Gram-negative bacterium. Thus, TKA can be of a promising agent in controlling bacterial leaf blight on rice.

Predator-prey interactions play important roles in the cycling of marine organic matter. Here the authors show that a Gram-negative bacterium isolated from marine sediments can kill and feed on Gram-positive bacteria by secreting a peptidoglycan-degrading enzyme.

The ocean harbors a variety of bacteria that contain huge protease resources and offer a great potential for industrial and biotechnological applications. Here, we isolated a protease-secreting bacterium Vibrio pomeroyi strain 12613 from Atlantic seawater and purified a protease VP9 from strain 12613. VP9 was identified as a metalloprotease of the M4 family. VP9 could hydrolyze casein and gelatin but not elastin and collagen. With gelatin as the substrate, VP9 showed the highest activity at 40°C and pH 6.0–8.0. It was stable at temperatures of 50°C and less and in the range of pH 5.0–11.0. VP9 also had good tolerance to NaCl, non-ionic detergents, and organic solvent methanol. Unlike other M4 metalloproteases, VP9 has distinct collagen-swelling ability, and its collagen-swelling effect was concentration dependent. The relative expansion volume of collagen increased by approximately eightfold after treatment with 10 μM VP9 at 37°C for 12 h. The collagen-swelling mechanism of VP9 on bovine-insoluble type I collagen was further studied. Atomic force microscopy observation and biochemical analyses showed that VP9 can degrade proteoglycans in collagen fibers, resulting in the release of collagen fibrils from collagen fibers and the swelling of the latter. In addition, VP9 can degrade glycoproteins, a non-collagenous constituent interacting with collagen in the skin. The characteristics of VP9, such as sufficient specificity toward proteoglycans and glycoproteins but no activity toward collagen, suggest its promising potential in the unhairing and fiber-opening processing in leather industry.

Abstract Nitrogen is one of the most important nutrients needed for plants and algae to survive, and the photosynthetic ability of algae is related to nitrogen abundance. Red algae are unique photosynthetic eukaryotic organisms in the evolution of algae, as they contain phycobilisomes (PBSs) on their thylakoid membranes. In this report, the in vivo chlorophyll (Chl) a fluorescence kinetics of nitrogen-starved Porphyridium cruentum were analyzed to determine the effects of nitrogen deficiency on photosynthetic performance using a multi-color pulse amplitude modulation (PAM) chlorophyll fluorometer. Due to nitrogen starvation, the photochemical efficiency of PSII and the activity of PSII reaction centers (RCs) decreased, and photoinhibition of PSII occurred. The water-splitting system on the donor side of PSII was seriously impacted by nitrogen deficiency, leading to the inactivation of the oxygen-evolving complex (OEC) and decreased light energy conversion efficiency. In nitrogen-starved cells, a higher proportion of energy was used for photochemical reactions, and thermal dissipation was reduced, as shown by qP and qN. The ability of nitrogen-starved cells to tolerate and resist high photon flux densities was weakened. Our results showed that the photosynthetic performance of P. cruentum was severely impacted by nitrogen deficiency.

Peptidoglycan is the fundamental structural constituent of the bacterial cell wall. Despite many years of research, the architecture of peptidoglycan is still largely elusive. Here, we report the high-resolution architecture of peptidoglycan from the model Gram-positive bacterium Bacillus subtilis. We provide high-resolution evidence of peptidoglycan architecture remodeling at different growth stages. Side wall peptidoglycan from B. subtilis strain AS1.398 changed from an irregular architecture in exponential growth phase to an ordered cable-like architecture in stationary phase. Thickness of side wall peptidoglycan was found to be related with growth stages, with a slight increase after transition to stationary phase. Septal disks were synthesized progressively toward the center, while the surface features were less clear than those imaged with side walls. Compared with previous studies, our results revealed slight differences in architecture of peptidoglycan from different B. subtilis strains, expanding our knowledge about the architectural features of B. subtilis peptidoglycan.

Elastases have been widely studied because of their important uses as medicine and meat tenderizers. However, there are relatively few studies on marine elastases. Myroilysin, secreted by Myroides profundi D25 from deep-sea sediment, is a novel elastase. In this study, we examined the elastin degradation mechanism of myroilysin. When mixed with insoluble bovine elastin, myroilysin bound hydrophobically, suggesting that this elastase may interact with the hydrophobic domains of elastin. Consistent with this, analysis of the cleavage pattern of myroilysin on bovine elastin and recombinant tropoelastin revealed that myroilysin preferentially cleaves peptide bonds with hydrophobic residues at the P1 and/or P1′ positions. Scanning electron microscopy (SEM) of cross-linked recombinant tropoelastin degraded by myroilysin showed preferential damages of spherules over cross-links, as expected for a hydrophobic preference. The degradation process of myroilysin on bovine elastin fibres was followed by light microscopy and SEM, revealing that degradation begins with the formation of crevices and cavities at the fibre surface, with these openings increasing in number and size until the fibre breaks into small pieces, which are subsequently fragmented. Our results are helpful for developing biotechnological applications for myroilysin.

Proteases in the M23 family have specific activities toward elastin and bacterial peptidoglycan. The peptidoglycan-degrading property makes these proteases have potential as novel antimicrobials. Because M23 proteases cannot be maturely expressed in Escherichia coli, it is significant to improve the production of these enzymes in their wild strains. Pseudoalterin is a new M23 protease secreted by the deep-sea bacterium Pseudoalteromonas sp. CF6-2. In this study, the fermentation conditions of strain CF6-2 for pseudoalterin production were optimized using single factor experiments and response surface methodology to improve the enzyme yield. To reduce the fermentation cost, bovine artery powder instead of elastin was determined as a cheap and efficient inducer. Based on single factor experiments, artery powder content, culture temperature and culture time were determined as the main factors influencing pseudoalterin production and were further optimized by the central composite design. The optimal values of these factors were determined as: artery powder of 1.2%, culture temperature of 20.17 °C and culture time of 28.04 h. Under the optimized conditions, pseudoalterin production reached 100.02 ± 9.0 U/mL, more than twice of that before optimization. These results lay a good foundation for developing the biotechnological potential of pseudoalterin.

Many marine bacteria secrete exopolysaccharides (EPSs) that have important ecological and physiological functions. Numerous nutritional and environmental factors influence bacterial EPS production. However, the regulatory mechanisms of EPS production are poorly understood. The deep-sea Bacteroidetes bacterium Zunongwangia profunda SM-A87 can produce high quantities of EPS, and its EPS production is enhanced significantly by lactose. Here, we studied the reasons behind the significant advantage that lactose has over other carbon sources in EPS production in SM-A87. RNA-seq technologies were used to study lactose-regulated genes in SM-A87. The expression level of genes within the EPS gene cluster was up-regulated when lactose was added. Supplement of lactose also influenced the expression of genes located outside the EPS gene cluster that are also involved in EPS biosynthesis. The major glycosyl components of SM-A87 EPS are mannose, glucose and galactose. Genomic metabolic pathway analyses showed that the EPS precursor GDP-mannose can be synthesized from glucose, while the precursor UDP-glucose must be synthesized from galactose. Lactose can provide glucose and galactose simultaneously and prevent glucose inhibition. Lactose can also greatly stimulate the growth of SM-A87. Taken together, lactose acts not only as an inducer but also as a carbohydrate source for EPS production. This research broadens our knowledge of the regulation of EPS production in marine bacteria.

Antimicrobial peptides are promising alternative antimicrobial agents compared to conventional antibiotics. Understanding the mode of action is important for their further application. We examined the interaction between trichokonin VI, a peptaibol isolated from Trichoderma pseudokoningii, and Bacillus subtilis, a representative Gram-positive bacterium. Trichokonin VI was effective against B. subtilis with a minimal inhibitory concentration of 25 µM. Trichokonin VI exhibited a concentration- and time-dependent effect against B. subtilis, which was studied using atomic force microscopy. The cell wall of B. subtilis collapsed and the roughness increased upon treatment with trichokonin VI. Nanoindentation experiments revealed a progressive decrease in the stiffness of the cells. Furthermore, the membrane permeabilization effect of trichokonin VI on B. subtilis was monitored, and the results suggest that the leakage of intracellular materials is a possible mechanism of action for trichokonin VI, which led to alterations in the morphological and nanomechanical properties of B. subtilis.

Most marine bacteria secrete exopolysaccharide (EPS), which is important for bacterial survival in the marine environment. However, it is still unclear whether the self-secreted EPS is involved in marine bacterial motility. Here we studied the role of EPS in the lateral flagella-driven swarming motility of benthic bacterium Pseudoalteromonas sp. SM9913 (SM9913) by a comparison of wild SM9913 and ΔepsT, an EPS synthesis defective mutant. Reduction of EPS production in ΔepsT did not affect the growth rate or the swimming motility, but significantly decreased the swarming motility on a swarming plate, suggesting that the EPS may play a role in SM9913 swarming. However, the expression and assembly of lateral flagella in 1epsT were not affected. Instead, 1epsT had a different swarming behavior from wild SM9913. The swarming of ΔepsT did not have an obvious rapid swarming period, and its rate became much lower than that of wild SM9913 after 35 h incubation. An addition of surfactin or SM9913 EPS on the surface of the swarming plate could rescue the swarming level. These results indicate that the self-secreted EPS is required for the swarming of SM9913. This study widens our understanding of the function of the EPS of benthic bacteria. [ABSTRACT FROM AUTHOR]

Collagen is an insoluble protein that widely distributes in the extracellular matrix of marine animals. Collagen degradation is an important step in the marine nitrogen cycle. However, the mechanism of marine collagen degradation is still largely unknown. Here, a novel subtilisin-like collagenolytic protease, myroicolsin, which is secreted by the deep sea bacterium Myroides profundi D25, was purified and characterized, and its collagenolytic mechanism was studied. Myroicolsin displays low identity (<30%) to previously characterized subtilisin-like proteases, and it contains a novel domain structure. Protein truncation indicated that the Pro secretion system C-terminal sorting domain in the precursor protein is involved in the cleavage of the N-propeptide, and the linker is required for protein folding during myroicolsin maturation. The C-terminal β-jelly roll domain did not bind insoluble collagen fiber, suggesting that myroicolsin may degrade collagen without the assistance of a collagen-binding domain. Myroicolsin had broad specificity for various collagens, especially fish-insoluble collagen. The favored residue at the P1 site was basic arginine. Scanning electron microscopy and atomic force microscopy, together with biochemical analyses, confirmed that collagen fiber degradation by myroicolsin begins with the hydrolysis of proteoglycans and telopeptides in collagen fibers and fibrils. Myroicolsin showed strikingly different cleavage patterns between native and denatured collagens. A collagen degradation model of myroicolsin was proposed based on our results. Our study provides molecular insight into the collagen degradation mechanism and structural characterization of a subtilisin-like collagenolytic protease secreted by a deep sea bacterium, shedding light on the degradation mechanism of deep sea sedimentary organic nitrogen. [ABSTRACT FROM AUTHOR]

Background Pseudoalteromonas species are a group of marine gammaproteobacteria frequently found in deep-sea sediments, which may play important roles in deep-sea sediment ecosystem. Although genome sequence analysis of Pseudoalteromonas has revealed some specific features associated with adaptation to the extreme deep-sea environment, it is still difficult to study how Pseudoalteromonas adapt to the deep-sea environment due to the lack of a genetic manipulation system. The aim of this study is to develop a genetic system in the deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913, making it possible to perform gene mutation by homologous recombination. Results The sensitivity of Pseudoalteromonas sp. SM9913 to antibiotic was investigated and the erythromycin resistance gene was chosen as the selective marker. A shuttle vector pOriT- 4Em was constructed and transferred into Pseudoalteromonas sp. SM9913 through intergeneric conjugation with an efficiency of 1.8 × 10-3, which is high enough to perform the gene knockout assay. A suicide vector pMT was constructed using pOriT-4Em as the bone vector and sacB gene as the counterselective marker. The epsT gene encoding the UDPglucose lipid carrier transferase was selected as the target gene for inactivation by in-frame deletion. The epsT was in-frame deleted using a two-step integration-segregation strategy after transferring the suicide vector pMT into Pseudoalteromonas sp. SM9913. The ΔepsT mutant showed approximately 73% decrease in the yield of exopolysaccharides, indicating that epsT is an important gene involved in the EPS production of SM9913. Conclusions A conjugal transfer system was constructed in Pseudoalteromonas sp. SM9913 with a wide temperature range for selection and a high transfer efficiency, which will lay the foundation of genetic manipulation in this strain. The epsT gene of SM9913 was successfully deleted with no selective marker left in the chromosome of the host, which thus make it possible to knock out other genes in the same host. The construction of a gene knockout system for Pseudoalteromonas sp. SM9913 will contribute to the understanding of the molecular mechanism of how Pseudoalteromonas adapt to the deep-sea environment. [ABSTRACT FROM AUTHOR]

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile DMS through the action of DMSP lyases and is important in the global sulfur and carbon cycles. When released into the atmosphere from the oceans, DMS is oxidized, forming cloud condensation nuclei that may influence weather and climate. Six different DMSP lyase genes are found in taxonomically diverse microorganisms, and dddQ is among the most abundant in marine metagenomes. Here, we examine the molecular mechanism of DMSP cleavage by the DMSP lyase, DddQ, from Ruegeria lacuscaerulensis ITI_1157. The structures of DddQ bound to an inhibitory molecule 2-(N-morpholino)ethanesulfonic acid and of DddQ inactivated by a Tyr131Ala mutation and bound to DMSP were solved. DddQ adopts a β-barrel fold structure and contains a Zn2+ ion and six highly conserved hydrophilic residues (Tyr120, His123, His125, Glu129, Tyr131, and His163) in the active site. Mutational and biochemical analyses indicate that these hydrophilic residues are essential to catalysis. In particular, Tyr131 undergoes a conformational change during catalysis, acting as a base to initiate the β-elimination reaction in DMSP lysis. Moreover, structural analyses and molecular dynamics simulations indicate that two loops over the substrate-binding pocket of DddQ can alternate between "open" and "closed" states, serving as a gate for DMSP entry. We also propose a molecular mechanism for DMS production through DMSP cleavage. Our study provides important insight into the mechanism involved in the conversion of DMSP into DMS, which should lead to a better understanding of this globally important biogeochemical reaction. [ABSTRACT FROM AUTHOR]

We detected polysaccharide capsules from Zunongwangia profunda SM-A87 with atomic force microscopy (AFM). The molecular organization of the capsules at the single-polysaccharide-chain level was reported. Furthermore, we found that with ScanAsyst mode the polysaccharide capsules could be detected even in the presence of deliquescent water covering the capsule. [ABSTRACT FROM AUTHOR]

The cyanobacterium Spirulina ( Arthrospira) platensis is a good source of phycobiliprotein purification. C-phycocyanin (C-PC) is the major phycobiliprotein, while allophycocyanin (APC) is less abundant in S. platensis. Previously reported methods for C-PC purification are only able to offer either high purity or high efficiency. This paper describes one-step anion exchange chromatography method with continuous pH gradient elution for simultaneous purification of C-PC and APC with high purity and high recovery. Crude C-PC and APC were extracted and concentrated by ammonium sulfate fractionation at saturation of 25% and 60%, then purified on a DEAE-Sepharose Fast Flow chromatography column with continuous pH gradient elution from pH 5.0 to 3.6. After this single-step chromatography, C-PC and APC with high purity and recovery were simultaneously obtained. The purity ratios of C-PC and APC reached 5.59 (A/A) and 5.19 (A/A), respectively. Their purity was further demonstrated by electrophoresis and fluorescence emission spectroscopy. Moreover, the total recovery yield of pure C-PC and APC were 67.04% and 80.0%, representing 111.83 and 29.28 mg·g lyophilized weight, respectively. The obtained C-PC and APC remained stable over a pH range of 4-9. This purification method for high purity and recovery of C-PC and APC proved to be fairly efficient compared with previously reported methods. [ABSTRACT FROM AUTHOR]

Red algae are a group of eukaryotic photosynthetic organisms. Phycobilisomes (PBSs), which are composed of various types of phycobiliproteins and linker polypeptides, are the main light-harvesting antennae in red algae, as in cyanobacteria. Two morphological types of PBSs, hemispherical- and hemidiscoidal-shaped, are found in different red algae species. PBSs harvest solar energy and efficiently transfer it to photosystem II (PS II) and finally to photosystem I (PS I). The PS I of red algae uses light-harvesting complex of PS I (LHC I) as a light-harvesting antennae, which is phylogenetically related to the LHC I found in higher plants. PBSs, PS II, and PS I are all distributed throughout the entire thylakoid membrane, a pattern that is different from the one found in higher plants. Photosynthesis processes, especially those of the light reactions, are carried out by the supramolecular complexes located in/on the thylakoid membranes. Here, the supramolecular architecture, function and regulation of thylakoid membranes in red algal are reviewed. [ABSTRACT FROM AUTHOR]

The article offers information on the oxidation oftrimethylamine to trimethylamine N-oxide facilitates high hydrostatic pressure tolerance in a generalist bacterial lineage. It mentions the improved High hydrostatic pressure (HHP) tolerance in the presence of TMA, implying the universality of this HHP tolerance strategy in marine Bacteroidetes.

Effects of salt stress on Artemisia scoparia and A. vulgaris “Variegate” were examined. A. scoparia leaves became withered under NaCl treatment, whereas A. vulgaris “Variegate” leaves were not remarkably affected. Chlorophyll content decreased in both species, with a higher reduction in A. scoparia. Contents of proline, MDA, soluble carbohydrate, and Na+ increased in both species under salt stress, but A. vulgaris “Variegate” had higher level of proline and soluble carbohydrate and lower level of MDA and Na+. The ratios of K+/Na+, Ca2+/Na+, and Mg2+/Na+ in A. vulgaris “Variegate” under NaCl stress were higher. Moreover, A. vulgaris “Variegate” had higher transport selectivity of K+/Na+ from root to stem, stem to middle mature leaves, and upper newly developed leaves than A. scoparia under NaCl stress. A. vulgaris “Variegate” chloroplast maintained its morphological integrity under NaCl stress, whereas A. scoparia chloroplast lost integrity. The results indicated that A. scoparia is more sensitive to salt stress than A. vulgaris “Variegate.” Salt tolerance is mainly related to the ability of regulating osmotic pressure through the accumulation of soluble carbohydrates and proline, and the gradient distribution of K+ between roots and leaves was also contributed to osmotic pressure adjustment and improvement of plant salt tolerance.

Bacterial polar flagella, comprised of flagellin, are essential for bacterial motility. Pseudoalteromonas sp. strain SM9913 is a bacterium isolated from deep-sea sediments. Unlike other Pseudoalteromonas strains that have a long polar flagellum, strain SM9913 has an abnormally short polar flagellum. Here, we investigated the underlying reason for the short flagellum and found that a single-base mutation was responsible for the altered flagellar assembly. This mutation leads to the fragmentation of the flagellin gene into two genes, PSM_A2281, encoding the core segment and the C-terminal segment, and PSM_A2282, encoding the N-terminal segment, and only gene PSM_A2281 is involved in the production of the short polar flagellum. When a chimeric gene of PSM_A2281 and PSM_A2282 encoding an intact flagellin, A2281::82, was expressed, a long polar flagellum was produced, indicating that the N-terminal segment of flagellin contributes to the production of a polar flagellum of a normal length. Analyses of the simulated structures of A2281 and A2281::82 and that of the flagellar filament assembled with A2281::82 indicate that due to the lack of two a-helices, the core of the flagellar filament assembled with A2281 is incomplete and is likely too weak to support the stability and movement of a long flagellum. This mutation in strain SM9913 had little effect on its growth and only a small effect on its swimming motility, implying that strain SM9913 can live well with this mutation in natural sedimentary environments. This study provides a better understanding of the assembly and production of bacterial flagella. IMPORTANCE Polar flagella, which are essential organelles for bacterial motility, are comprised of multiple flagellin subunits. A flagellin molecule contains an N-terminal segment, a core segment, and a C-terminal segment. The results of this investigation of the deep-sea sedimentary bacterium Pseudoalteromonas sp. strain SM9913 demonstrate that a single-base mutation in the flagellin gene leads to the production of an incomplete flagellin without the N-terminal segment and that the loss of the N-terminal segment of the flagellin protein results in the production of a shortened polar flagellar filament. Our results shed light on the important function of the N-terminal segment of flagellin in the assembly and stability of bacterial flagellar filament. [ABSTRACT FROM AUTHOR]

Halotolerant enzymes are beneficial for industrial processes requiring high salt concentrations and low water activity. Most halophilic proteins are evolved to have reduced hydrophobic interactions on the surface and in the hydrophobic cores for their haloadaptation. However, in this study, we improved the halotolerance of a thermolabile esterase, E40, by increasing intraprotein hydrophobic interactions. E40 was quite unstable in buffers containing more than 0.3 M NaCl, and its kcat and substrate affinity were both significantly reduced in 0.5 M NaCl. By introducing hydrophobic residues in loop 1 of the CAP domain and/or α7 of the catalytic domain in E40, we obtained several mutants with improved halotolerance, and the M3 S202W I203F mutant was the most halotolerant. ("M3" represents a mutation in loop 1 of the CAP domain in which residues R22-K23-T24 of E40 are replaced by residues Y22-K23-H24-L25-S26 of Est2.) Then we solved the crystal structures of the S202W I203F and M3 S202W I203F mutants to reveal the structural basis for their improved halotolerance. Structural analysis revealed that the introduction of hydrophobic residues W202 and F203 in α7 significantly improved E40 halotolerance by strengthening intradomain hydrophobic interactions of F203 with W202 and other residues in the catalytic domain. By further introducing hydrophobic residues in loop 1, the M3 S202W I203F mutant became more rigid and halotolerant due to the formation of additional interdomain hydrophobic interactions between the introduced Y22 in loop 1 and W204 in α7. These results indicate that increasing intraprotein hydrophobic interactions is also a way to improve the halotolerance of enzymes with industrial potential under high-salt conditions. [ABSTRACT FROM AUTHOR]

Microbial hormone-sensitive lipases (HSLs) contain a CAP domain and a catalytic domain. However, it remains unclear how the CAP domain interacts with the catalytic domain to maintain the stability of microbial HSLs. Here, we isolated an HSL esterase, E40, from a marine sedimental metagenomic library. E40 exhibited the maximal activity at 45 °C and was quite thermolabile, with a half-life of only 2 min at 40 °C, which may be an adaptation of E40 to the permanently cold sediment environment. The structure of E40 was solved to study its thermolability. Structural analysis showed that E40 lacks the interdomain hydrophobic interactions between loop 1 of the CAP domain and α7 of the catalytic domain compared with its thermostable homologs. Mutational analysis showed that the introduction of hydrophobic residues Trp202 and Phe203 inα7 significantly improved E40 stability and that a further introduction of hydrophobic residues in loop 1 made E40 more thermostable because of the formation of interdomain hydrophobic interactions. Altogether, the results indicate that the absence of interdomain hydrophobic interactions between loop 1 and α7 leads to the thermolability of E40. In addition, a comparative analysis of the structures of E40 and other thermolabile and thermostable HSLs suggests that the interdomain hydrophobic interactions between loop 1 and α7 are a key element for the thermostability of microbial HSLs. Therefore, this study not only illustrates the structural element leading to the thermolability of E40 but also reveals a structural determinant for HSL thermostability. [ABSTRACT FROM AUTHOR]

Peptide uptake is important for nutrition supply for marine bacteria. It is also an important step in marine nitrogen cycling. However, how marine bacteria absorb peptides is still not fully understood. DppA is the periplasmic dipeptide binding protein of dipeptide permease (Dpp; an important peptide transporter in bacteria) and exclusively controls the substrate specificity of Dpp. Here, the substrate binding specificity of deep-sea Pseudoalteromonas sp. strain SM9913 DppA (PsDppA) was analyzed for 25 different dipeptides with various properties by using isothermal titration calorimetry measurements. PsDppA showed binding affinities for 8 dipeptides. To explain the multispecific substrate recognition mechanism of PsDppA, we solved the crystal structures of unliganded PsDppA and of PsDppA in complex with 4 different types of dipeptides (Ala-Phe, Met-Leu, Gly-Glu, and Val-Thr). PsDppA alternates between an "open" and a "closed" form during substrate binding. Structural analyses of the 4 PsDppA-substrate complexes combined with mutational assays indicate that PsDppA binds to different substrates through a precise mechanism: dipeptides are bound mainly by the interactions between their backbones and PsDppA, in particular by anchoring their N and C termini through ion-pair interactions; hydrophobic interactions are important in binding hydrophobic dipeptides; and Lys457 is necessary for the binding of dipeptides with a C-terminal glutamic acid or glutamine. Additionally, sequence alignment suggests that the substrate recognition mechanism of PsDppA may be common in Gram-negative bacteria. All together, our results provide structural insights into the multispecific substrate recognition mechanism of marine Gram-negative bacterial DppA, which provides a better understanding of the mechanisms of marine bacterial peptide uptake. [ABSTRACT FROM AUTHOR]

Deseasin MCP-01 is a bacterial collagenolytic serine protease. Its catalytic domain alone can degrade collagen, and its C-terminal PKD domain is a collagen-binding domain (CBD) that can improve the collagenolytic efficiency of the catalytic domain by an unknown mechanism. Here, scanning electron microscopy (SEM), atomic force microscopy (AFM), zeta potential, and circular dichroism spectroscopy were used to clarify the functional mechanism of the PKD domain in MCP-01 collagenolysis. The PKD domain observably swelled insoluble collagen. Its collagenswelling ability and its improvement to the collagenolysis of the catalytic domain are both temperature-dependent. SEM observation showed the PKD domain swelled collagen fascicles with an increase of their diameter from 5.3 μm to 8.8 μm after 1 h of treatment, and the fibrils forming the fascicles were dispersed. AFM observation directly showed that the PKD domain bound collagen, swelled the microfibrils, and exposed the monomers. The PKD mutant W36A neither bound collagen nor disturbed its structure. Zeta potential results demonstrated that PKD treatment increased the net positive charges of the collagen surface. PKD treatment caused no change in the content or the thermostability of the collagen triple helix. Furthermore, the PKD-treated collagen could not be degraded by gelatinase. Therefore, though the triple helix monomers were exposed, the PKD domain could not unwind the collagen triple helix. Our study reveals the functional mechanism of the PKD domain of the collagenolytic serine protease MCP-01 in collagen degradation, which is distinct from that of the CBDs of mammalian matrix metalloproteases. [ABSTRACT FROM AUTHOR]