Lysogeny Broth Agar Research Articles

Luteibacter mycovicinus sp. nov., a yellow-pigmented gammaproteobacterium found as an endohyphal symbiont of endophytic Ascomycota.

We isolated and described a yellow-pigmented strain of bacteria (strain 9143T), originally characterized as an endohyphal inhabitant of an endophytic fungus in the Ascomycota. Although the full-length sequence of its 16S rRNA gene displays 99 % similarity to Luteibacter pinisoli, genomic hybridization demonstrated <30 % genomic similarity between 9143T and its closest named relatives, further supported by average nucleotide identity results. This and related endohyphal strains form a well-supported clade separate from L. pinisoli and other validly named species including the most closely related Luteibacter rhizovicinus. The name Luteibacter mycovicinus sp. nov. is proposed, with type strain 9143T (isolate DBL433), for which a genome has been sequenced and is publicly available from the American Type Culture Collection (ATCC TSD-257T) and from the Leibniz Institute DSMZ (DSM 112764T). The type strain reliably forms yellow colonies across diverse media and growth conditions (lysogeny broth agar, King's Medium B, potato dextrose agar, trypticase soy agar and Reasoner's 2A (R2A) agar). It forms colonies readily at 27 °C on agar with a pH of 6-8, and on salt (NaCl) concentrations up to 2 %. It lacks the ability to utilize sulphate as a sulphur source and thus only forms colonies on minimal media if supplemented with alternative sulphur sources. It is catalase-positive and oxidase-negative. Although it exhibits a single polar flagellum, motility was only clearly visible on R2A agar. Its host range and close relatives, which share the endohyphal lifestyle, are discussed.

In vitro analysis of various antibiotic and cement combinations against S. epidermidis and S. lugdunensis for treatment of periprosthetic shoulder infection

BackgroundPeriprosthetic joint infections (PJIs) are devastating potential complications after arthroplasty surgery with significant therapeutic challenges. Many authors advocate for two-stage revision arthroplasty for PJI of the shoulder which includes explanation with antibiotic spacer placement. The optimal antibiotic/cement combination for PJI in the shoulder is not known. The purpose of this study is to analyze various cement and antibiotic concentrations against Coagulase negative staphylococcus species, S. epidermidis and S. lugdunensis. MethodsFive strains of coagulase negative Staph were taken from clinically documented orthopedic infections Both Simplex and Palacos cement were used to create antibiotic laden beads and mixed with either ertapenem, vancomycin, gentamicin, or a combination of ertapenem + vancomycin or ertapenem + gentamicin. The bacteria were plated on lysogeny broth agar plates and 3 beads were added per plate. Samples were analyzed for zone of inhibition at 24 hours, 72 hours, and 1 week. After 1 week, beads were transferred to new plates with bacteria and the process was repeated for 6 weeks. ResultsResults showed that ertapenem beads with both Simplex and Palacos cement showed the largest zones of inhibition for all samples. Vancomycin in Palacos cement and vancomycin in combination with ertapenem in Simplex and Palacos cement showed consistent zones of inhibition for the duration of the experiments. Ertapenem in combination with either vancomycin or gentamicin may allow for a powerful initial burst of killing followed by consistent antibiotic elution as opposed to gentamicin alone. ConclusionWhile many premade spacers on the market are infused with gentamicin, our in vitro models demonstrate more efficacious bacterial eradication for antibiotics such as ertapenem and vancomycin specifically for certain low virulence organisms that are commonly found in PJI around the shoulder.

Sea hedgehog-inspired surface-enhanced Raman scattering biosensor probe for ultrasensitive determination of Staphylococcus aureus in food supplements

Staphylococcus aureus contamination in food supplements poses substantial challenges to public health and large-scale production but the sensitive detection in a timely manner remains a bottleneck. Drawing inspiration from the sea hedgehog, gold nanostars (AuNSs) were leveraged to design an ultrasensitive surface-enhanced Raman scattering (SERS) biosensor for the determination of Staphylococcus aureus in food supplements. Besides the surface enhancement furnished by the AuNSs, Raman reporter molecules and specific aptamers sequentially self-assembled onto these AuNSs to construct the “three-in-one” SERS biosensor probe for label-based quantitation of Staphylococcus aureus. Following incubation with contaminated health product samples, the gold nanostars@Raman reporter-aptamer specifically recognize and assemble around Staphylococcus aureus cells, forming a distinctive sea hedgehog structure. This unique configuration results in an amplified Raman signal at 1338 cm−1 and an enhancement factor of up to 6.71 × 107. The entire quantitative detection process can be completed within 30 min, boasting an exceptional limit of detection as low as 1.0 CFU mL−1. The method exhibits a broad working range for the determination of Staphylococcus aureus, with concentrations spanning 2.15 CFU mL−1 to 2.15 × 105 CFU mL−1. Furthermore, it demonstrates outstanding precision, with relative standard deviation values consistently below 5.0%. As a showcase to validate the practicality of the SERS method, we conducted tests on determining Staphylococcus aureus in a herbal food supplement, i.e., Ginkgo Biloba extract (GBE); the results align closely with those obtained through the conventional lysogeny broth agar plate method, pointing to the potential applicability in real-world scenarios.

Arthrobacter vasquezii sp. nov., isolated from a soil sample from Union Glacier, Antarctica.

A Gram-stain-positive, catalase-positive, non-motile bacteria, with a rod-coccus cycle (designated as EH-1B-1T) was isolated from a soil sample from Union Glacier in Ellsworth Mountains, Antarctica. Strain EH-1B-1T had an optimal growth temperature of 28 °C and grew at pH 7-10. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, C16 : 0 and anteiso-C17 : 0. The G+C content based on the whole genome sequence was 63.1 mol%. Strain EH-1B-1T was most closely related to members of the genus Arthrobacter, namely Arthrobacter subterraneus and Arthrobacter tumbae. The strain grew on tryptic soy agar, Reasoner's 2A agar, lysogeny broth agar and nutrient agar. The average nucleotide identity and digital DNA-DNA hybridization values between strain EH-1B-1T and its closest reference type strains ranged from 78 to 88 % and from 20.9 to 36.3 %, respectively. Based on phenotypic, chemotypic and genotypic evidence, it is proposed that strain EH-1B-1T represents a novel species of Arthrobacter, for which the name Arthrobacter vasquezii sp. nov. is proposed, with strain EH-1B-1T (RGM 3386T=LMG 32961T) as the type strain.

OpaR Exerts a Dynamic Control over c-di-GMP Homeostasis and cpsA Expression in Vibrio parahaemolyticus through Its Regulation of ScrC and the Trigger Phosphodiesterase TpdA.

The second messenger cyclic dimeric GMP (c-di-GMP) plays a central role in controlling decision-making processes that are vitally important for the environmental survival of the human pathogen Vibrio parahaemolyticus. The mechanisms by which c-di-GMP levels and biofilm formation are dynamically controlled in V. parahaemolyticus are poorly understood. Here, we report the involvement of OpaR in controlling c-di-GMP metabolism and its effects on the expression of the trigger phosphodiesterase (PDE) TpdA and the biofilm-matrix related gene cpsA. Our results revealed that OpaR negatively modulates the expression of tpdA by maintaining a baseline level of c-di-GMP. The OpaR-regulated PDEs ScrC, ScrG, and VP0117 enable the upregulation of tpdA, to different degrees, in the absence of OpaR. We also found that TpdA plays the dominant role in c-di-GMP degradation under planktonic conditions compared to the other OpaR-regulated PDEs. In cells growing on solid medium, we observed that the role of the dominant c-di-GMP degrader alternates between ScrC and TpdA. We also report contrasting effects of the absence of OpaR on cpsA expression in cells growing on solid media compared to cells forming biofilms over glass. These results suggest that OpaR can act as a double-edged sword to control cpsA expression and perhaps biofilm development in response to poorly understood environmental factors. Finally, using an in-silico analysis, we indicate outlets of the OpaR regulatory module that can impact decision making during the motile-to-sessile transition in V. parahaemolyticus. IMPORTANCE The second messenger c-di-GMP is extensively used by bacterial cells to control crucial social adaptations such as biofilm formation. Here, we explore the role of the quorum-sensing regulator OpaR, from the human pathogen V. parahaemolyticus, on the dynamic control of c-di-GMP signaling and biofilm-matrix production. We found that OpaR is crucial to c-di-GMP homeostasis in cells growing on Lysogeny Broth agar and that the OpaR-regulated PDEs TpdA and ScrC alternate in the dominant role over time. Furthermore, OpaR plays contrasting roles in controlling the expression of the biofilm-related gene cpsA on different surfaces and growth conditions. This dual role has not been reported for orthologues of OpaR, such as HapR from Vibrio cholerae. It is important to investigate the origins and consequences of the differences in c-di-GMP signaling between closely and distantly related pathogens to better understand pathogenic bacterial behavior and its evolution.

An Optimized Transformation Protocol for Escherichia coli BW3KD with Supreme DNA Assembly Efficiency.

DNA cloning requires two steps: the assembly of recombinant DNA molecules and the transformation of the product into a host organism for replication. High efficiencies in both processes can increase the success rate. Recently, we developed an Escherichia coli BW3KD strain with higher transformation efficiency than commonly used cloning strains. Here, we further developed a simple method named TSS-HI (transformation storage solution optimized by Hannahan and Inoue method) for competent cell preparation, which combined the advantages of three common methods for operational simplicity and high transformation efficiency. When competent BW3KD cells were prepared using this developed method, the transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/μg DNA, which exceeded the levels of commercial chemically competent cells and homemade electrocompetent cells. BW3KD cells formed colonies within 7 h on lysogeny broth agar plates, quicker than the well-known fast-growing E. coli cloning strain Mach1. The competent cells worked effectively for the transformation of assembled DNA of 1 to 7 fragments in one step and promoted efficiencies of transformation or cloning with large plasmids. The cloning efficiency of BW3KD cells prepared by this method increased up to 828-fold over that of E. coli XL1-Blue MRF' cells prepared by a common method. Thus, competent cells are suitable for different cloning jobs and should help with the increased demand for DNA assembly in biological studies and biotechnology. IMPORTANCE DNA transformation is commonly used in cloning; however, high transformation efficiency becomes a limiting factor in many applications, such as the construction of CRISPR and DNA libraries, the assembly of multiple fragments, and the transformation of large plasmids. We developed a new competent cell preparation method with unmatched transformation efficiency. When the BW3KD strain, derived from Escherichia coli BW25113 cells, was prepared by this method, its transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/μg DNA, which broke the record for chemically prepared competent cells. Routine cloning could be completed in 1 day due to the high growth rate of this strain. The competent cells were shown to be highly efficient for transformation or cloning with large plasmids and for the assembly of multiple fragments. The results highlight the effectiveness of the new protocol and the usefulness of the BW3KD strain as the host.

SPORULATION OF SPOROSARCENA PASTEURII (BACILLUS PASTEURII) ON DIFFERENT NUTRIENT MEDIUM IN LABORATORY CONDITIONS

The bacterium Sporosarcina pasteurii can produce spore at the adverse environmental condition. The sporulation is different in different nutrients medium. This study helps to search out the better nutrient medium that can use in laboratory experiments for the sporulation of Sporosarcina pasteurii. As a result, the seven to ten days of observation showed that the Sporulation of Sporosarcina pasteurii is good in Nutrient agar medium than tryptic soy agar and lysogeny broth agar. In this study, used a hemocytometer for counting the spore on the surface of agar medium with the help of an optical microscope. SEM is used to analyze the morphology of the Sporosarcina pasteurii and its spores. It helps the research work related to Sporosarcina pasteurii in the microbiological and environmental research area.

Phage-based forensic tool for spatial visualization of bacterial contaminants in cheese

Traditional procedures for microbial testing typically involve a homogenizing step. These methods give valuable information on the presence or enumeration of a bacterial contaminant, but not where the contaminant was in the original sample. Spatial information could be useful in troubleshooting sources of bacterial contamination in a processing plant. For example, if the contaminant was localized on the top of a food such as cheese, this might indicate dripping condensate along a specific processing line as its source. The objective of this proof-of-concept study was to evaluate the use of a genetically engineered phage to detect bacterial contaminants on cheese to be able to visualize the contaminants without the use of magnification. In this study, a T7 bacteriophage engineered to overexpress the luciferase NanoLuc (Promega, Madison, WI) was utilized to reveal the spatial location of Escherichia coli on lysogeny broth (LB) agar and queso fresco (QF). Four scenarios were tested to explore how phage may be applied, with a blue bioluminescent signal revealing the spatial location of contaminants: (1) phage applied topically via molten soft agar to E. coli-inoculated (a) LB agar or (b) QF; and (2) phage incorporated within (a) LB agar or (b) QF and then inoculated with E. coli. Each was tested in triplicate. Cultures of E. coli BL21 grown for 18 h were serially diluted in phosphate-buffered saline and inoculated onto 8 ± 0.5 g of LB agar or QF in 6-well plates. Plates were incubated at 37°C for 8 h for condition 1a, 24 h for 1b and 2b, and 22 h for 2a. For 1a and 1b, stock phage was added to molten soft agar, applied topically, and incubated for 2 additional hours to allow for E. coli infection. After incubation, the substrate NanoGlo (Promega) was added to cover the surface of the agar or cheese and imaged immediately in a dark box using a digital camera and long exposure to capture the bioluminescent signal. Photographs captured small blue spots where the incubated colony-forming units were located. The lowest inoculum level of E. coli detected for each scenario was 1.43 × 101 ± 9.94, 1.18 × 101 ± 7.07, 5.48 × 101 ± 1.19 × 101, and 2.37 × 101 ± 1.40 × 101 cfu/well, for 1a, 1b, 2a, and 2b, respectively. These data demonstrate that the reporter phage proof-of-concept could be used as a forensic tool to visualize the spatial location of bacteria in a cheese matrix. Future work will translate this concept to dairy-relevant phage-pathogen systems.

Dexamethasone improves therapeutic outcomes in a preclinical bacterial epididymitis mouse model.

Can dexamethasone improve infertility-related cauda epididymidal tissue damage caused by bacterial epididymitis? Dexamethasone in addition to anti-microbial treatment effectively reduces long-term deleterious epididymal tissue damage by dampening the host's adaptive immune response. Despite effective anti-microbial treatment, ~40% of patients with epididymitis experience subsequent sub- or infertility. An epididymitis mouse model has shown that the host immune response is mainly responsible for the magnitude of epididymal tissue damage that is fundamentally causative of the subsequent fertility issues. Bacterial epididymitis was induced in male mice by using uropathogenic Escherichia coli (UPEC). From Day 3 after infection onwards, mice were treated with daily doses of levofloxacin (20mg/kg, total n= 12 mice), dexamethasone (0.5mg/kg, total n= 9) or both in combination (total n= 11) for seven consecutive days. Control animals were left untreated, i.e. given no interventional treatment following UPEC infection (total n= 11). Half of the animals from each group were killed either at 10 or 31days post-infection. A mouse model of induced bacterial epididymitis was applied to adult male C57BL/6J mice. At the respective endpoints (10 or 31days post-infection), epididymides were collected. Effectiveness of antibiotic treatment was assessed by plating of epididymal homogenates onto lysogeny broth agar plates. Overall tissue morphology and the degree and nature of tissue damage were assessed histologically. Quantitative RT-PCR was used to assess local cytokine transcript levels. Blood was drawn and serum analysed for systemic IgG and IgM levels by ELISA. In addition, correlation analyses of clinical data and serum-analyses of IgG and IgM levels in patients with epididymitis were performed. The addition of dexamethasone to the standard anti-microbial treatment did not further worsen epididymal tissue integrity. In fact, an obviously dampened immune response and reduced tissue reaction/damage was observed at both 10 and 31days post-infection following combined treatment. More specifically, epididymal duct continuity was preserved, enabling sperm transit. In contrast, in untreated or antibiotic-treated animals, damage of the epididymal duct and duct constrictions were observed, associated with a lack of cauda spermatozoa. In line with the bacteriostatic/bactericidal effect of levofloxacin (alone as well as in combination), local cytokine transcript levels were significantly and similarly reduced in animals treated with levofloxacin alone (P< 0.01) or in combination with dexamethasone (P< 0.05) compared to UPEC-infected untreated animals. Interestingly, the addition of dexamethasone to the anti-microbial treatment induced a unique dampening effect on adaptive immunity, since systemic IgG and IgM levels as well as the pan-T cell marker CD3 were reduced at both 10 and 31days post-infection. Breeding studies to address the fertility-protecting effect of the combined treatment were not possible in the experimental animals because the vas deferens was ligated (model specific). Whereas innate immunity is necessary and involved in acute bacterial clearance, adaptive immunity seems to be responsible for long-term, subclinical immunological activities that may negatively affect the pathogenesis of bacterial epididymitis even after effective bacterial eradication. These effects can be reduced in mice by the additional treatment with dexamethasone. This immunological characteristic of bacterial epididymitis shows similarities to the Jarisch-Herxheimer reaction known from other types of bacterial infection. The study was supported by grants from the Deutsche Forschungsgemeinschaft, Monash University and the Medical Faculty of Justus-Liebig University to the International Research Training Group on 'Molecular pathogenesis of male reproductive disorders' (GRK 1871). R.W., K.L.L. and M.P.H. were supported by grants from the National Health and Medical Research Council of Australia (ID1079646, ID1081987, ID1020269 and ID1063843) and by the Victorian Government's Operational Infrastructure Support Program. The authors have no conflicts of interest to declare. No clinical trial involved.

Sterile Capsule-Egg Cocoon Covering Constitutes an Antibacterial Barrier for Spider Parasteatoda tepidariorum Embryos.

Coexistence of organisms and pathogens has resulted in the evolution of efficient antimicrobial defense, especially at the embryonic stage. This investigation aimed to substantiate the hypothesis that the layers of silk in a spider cocoon play a role in the immunity of the embryos against microorganisms present in the external environment. A two-step interdisciplinary attempt has been made. First, the eggs and empty cocoons of the spider Parasteatoda tepidariorum were incubated on lysogeny broth agar media for 3 d. In the samples of eggs, no growth of bacteria was detected. This indicated that the eggs inside cocoons were sterile. Therefore, in the second step, the cocoons and egg surface were analyzed using SEM, TEM, and LM. The obtained images demonstrated that both inner and outer layers of the silk are built of threads of the same diameter, set in an irregular manner, and randomly clustered into groups. The threads in the outer layer were packed more densely than in the inner one. TEM analysis revealed threads of two types of fibrils and their arrangement. The resultant thread tangle of the cocoon, possibly correlated with the ultrastructure of the fibers, seems to be an example of a structure-function relationship playing a crucial ecoimmunological role in spider embryonic development.

Nitrate amendment reduces biofilm biomass and shifts microbial communities in remote, oligotrophic ponds

AbstractHumans have increased the amount of reactive N available in the environment by over an order of magnitude since the industrial revolution. Most studies have been conducted in ecosystems with pervasive anthropogenic nutrient inputs, so little is understood about how naive biofilm communities respond to elevated nutrients. Our nutrient-diffusing substrate (NDS) experiments, which were conducted in Alaskan freshwater ponds with very little anthropogenic nutrient inputs, suggest that P limits biofilm photoautotrophs. However, despite low water-column nutrient concentrations, overall biofilm biomass was not enhanced by the addition of N or P. Rather, we observed an ~60% biomass reduction with NO3– amendment in 15 oligotrophic ponds across 2 y. This widespread biomass reduction was accompanied by changes in microbial communities, but these trends were not observed with NH4+ or P amendment. Nonamended communities (i.e., no nutrient amendment other than lysogeny broth agar) were characterized by anaerobic...

Development of a Three-dimensional Hydrogel System for the Maintenance of Porcine Spermatogonial Stem Cell Self-renewal

Porcine spermatogonial stem cells (SSCs) prefer three-dimensional (3D) culture systems to 2D ones for the maintenance of self-renewal. Of the many 3D culture systems, agar-based hydrogels are candidates for supporting porcine SSC self-renewal, and there are various types of agar powder that can be used. In this study, we sought to identify an agar-based 3D hydrogel system that exhibited strong efficacy in the maintenance of porcine SSC self-renewal. First, 3D hydrogels with different mechanics were prepared with various concentrations of Bacto agar, lysogeny broth (LB) agar, and agarose powder, and the 3D hydrogel with the strongest alkaline phosphatase (AP) activity and greatest increase in colony size was identified for the different types of agar powder. Second, among the porcine SSCs cultured in the different 3D hydrogels, we analyzed the colony formation, morphology, and size; AP activity; and transcription and translation of porcine SSC-related genes, and these were compared to determine the optimal 3D hydrogel system for the maintenance of porcine SSC self-renewal. We found that 0.6% (w/v) Bacto agar-, 1% (w/v) LB agar-, and 0.2% (w/v) agarose-based 3D hydrogels showed the strongest maintenance of AP activity and the most pronounced increase in colony size in the culture of porcine SSCs. Moreover, among these hydrogels, the strongest transcription and translation of porcine SSC-related genes and largest colony size were detected in porcine SSCs cultured in the 0.2% (w/v) agarose-based 3D hydrogel, whereas there were no significant differences in colony formation and morphology. These results demonstrate that the 0.2% (w/v) agarose-based 3D hydrogel can be effectively used for the maintenance of porcine SSC self-renewal.

Impact of Polymer Colonization on the Fate of Organic Contaminants in Sediment.

Plastic pellets and microbes are important constitutes in sediment, but the significance of microbes colonizing on plastic pellets to the environmental fate and transport of organic contaminants has not been adequately recognized and assessed. To address this issue, low-density polyethylene (LDPE), polyoxymethylene (POM) and polypropylene (PP) slices were preloaded with dichlorodiphenyltrichloroethanes (DDTs), polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) and incubated in abiotic and biotic sediment microcosms. Images from scanning electron microscope, Lysogeny Broth agar plates and confocal laser scanning microscope indicated that all polymer slices incubated in biotic sediments were colonized by microorganisms, particularly the LDPE slices. The occurrence of biofilms induced higher dissipation rates of DDTs and PAHs from the LDPE slice surfaces incubated in the biotic sediments than in the abiotic sediments. Plastic colonization on LDPE slice surfaces enhanced the biotransformation of DDT and some PAHs in both marine and river sediments, but had little impact on PCBs. By comparison, PP and POM with unique properties were shown to exert different impacts on the physical and microbial activities as compared to LDPE. These results clearly demonstrated that the significance of polymer surface affiliated microbes to the environmental fate and behavior of organic contaminants should be recognized.

Cranberry derivatives enhance biofilm formation and transiently impair swarming motility of the uropathogen Proteus mirabilis HI4320.

Proteus mirabilis is a major cause of catheter-associated urinary tract infection (CAUTI), emphasizing that novel strategies for targeting this bacterium are needed. Potential targets are P. mirabilis surface-associated swarming motility and the propensity of these bacteria to form biofilms that may lead to catheter blockage. We previously showed that the addition of cranberry powder (CP) to lysogeny broth (LB) medium resulted in impaired P. mirabilis swarming motility over short time periods (up to 16 h). Herein, we significantly expanded on those findings by exploring (i) the effects of cranberry derivatives on biofilm formation of P. mirabilis, (ii) whether swarming inhibition occurred transiently or over longer periods more relevant to real infections (∼3 days), (iii) whether swarming was also blocked by commercially available cranberry juices, (iv) whether CP or cranberry juices exhibited effects under natural urine conditions, and (v) the effects of cranberry on medium pH, which is an indirect indicator of urease activity. At short time scales (24 h), CP and commercially available pure cranberry juice impaired swarming motility and repelled actively swarming bacteria in LB medium. Over longer time periods more representative of infections (∼3 days), the capacity of the cranberry material to impair swarming diminished and bacteria would start to migrate across the surface, albeit by exhibiting a different motility phenotype to the regular "bull's-eye" swarming phenotype of P. mirabilis. This bacterium did not swarm on urine agar or LB agar supplemented with urea, suggesting that any potential application of anti-swarming compounds may be better suited to settings external to the urine environment. Anti-swarming effects were confounded by the ability of cranberry products to enhance biofilm formation in both LB and urine conditions. These findings provide key insights into the long-term strategy of targeting P. mirabilis CAUTIs.

The porcine gut microbial metagenomic library for mining novel cellulases established from growing pigs fed cellulose-supplemented high-fat diets1

The porcine gut microbiome is a novel genomic resource for screening cellulose-degrading enzymes. A plasmid metagenomic expression library was constructed from the hindgut microbiota of 6 Yorkshire growing pigs (25 to 40 kg) fed a high-fat basal diet supplemented with 10% Solka-Floc for 28 d. Fresh cecal and colonic digesta samples were collected, flash-frozen in liquid N, and stored under -80°C. Metagenomic DNA was extracted, mechanically sheared, and cleaned to remove small DNA fragments (<1.0 kb). The resulting DNA fragments were subjected to blunt-end polishing, fractionation, and purification by using commercial kits. The end-modified DNA fragments were ligated to pCR4Blunt-TOPO vector and transformed into competent Escherichia coli TOPO10 cells. Metagenomic plasmid libraries were screened for carboxymethyl cellulolytic activities by using lysogeny broth agar plates. The average insert size of the resulting library was approximately 4.2 kb. Screening for the ability to hydrolyze carboxymethyl cellulose yielded 14 positive colonies, giving an estimated 430 Mb of metagenomic DNA in the approximately 102,000 E. coli clones with an overall hit rate of 0.14%. The 11 assembled insert sequences included 4 function-related gene clusters, and a total of 18 putative carbohydrate active enzyme genes were identified. This included genes encoding 11 cellulases, 4 hemicellulases, 1 polygalacturonas, 1 glycoside hydrolase family 26 mannanase-family 5 cellulase chimeric enzyme gene, and 1 cellobiose phosphorylase. In conclusion, the coupling of functional metagenomic mining with biochemical characterization of fiber-degrading enzymes is a powerful strategy for exploring the enzymological underpinnings of the anaerobic fermentation of dietary fiber in the complex animal gut environment.