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Bacteria under the light microscope. (A) B. subtilis HH2 was cultured in glucose medium until OD600~1. (B) B. subtilis HH2 was cultured in cellulose medium until OD600~1.
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In the giant panda, adaptation to a high-fiber environment is a first step for the adequate functioning of intestinal bacteria, as the high cellulose content of the gut due to the panda's vegetarian appetite results in a harsh environment. As an excellent producer of several enzymes and vitamins, Bacillus subtilis imparts various advantages to anim...
Citations
... Studies have demonstrated the efficacy of probiotics, including Lactobacillus, Bifidobacterium, and Bacillus, in preventing bacterial infections in the gut [37][38][39]. In our previous research, we isolated B. subtilis strain HH2 from the feces of a healthy giant panda, which showed promising results in acting as a probiotic for pandas on a high-fiber diet, inhibiting E. coli and Staphylococcus aureus in vitro, and ameliorating TNBS-induced colitis [23][24][25]. The objective of this study was to evaluate the protective effects of B. subtilis HH2 in beagles challenged with ETEC. ...
This study evaluated the protective effect of Bacillus subtilis HH2 on beagles orally challenged with enterotoxigenic Escherichia coli (ETEC). We assessed the physiological parameters and the severity of diarrhea, as well as the changes in three serum immunoglobulins (IgG, IgA, and IgM), plasma diamine oxidase (DAO), D-lactate (D-LA), and the fecal microbiome. Feeding B. subtilis HH2 significantly reduced the severity of diarrhea after the ETEC challenge (p < 0.05) and increased serum levels of IgG, IgA, and IgM (p < 0.01). B. subtilis HH2 administration also reduced serum levels of DAO at 48 h after the ETEC challenge (p < 0.05), but no significant changes were observed in D-LA (p > 0.05). Oral ETEC challenge significantly reduced the richness and diversity of gut microbiota in beagles not pre-fed with B. subtilis HH2 (p < 0.05), while B. subtilis HH2 feeding and oral ETEC challenge significantly altered the gut microbiota structure of beagles (p < 0.01). Moreover, 14 days of B. subtilis HH2 feeding reduced the relative abundance of Deinococcus-Thermus in feces. This study reveals that B. subtilis HH2 alleviates diarrhea caused by ETEC, enhances non-specific immunity, reduces ETEC-induced damage to the intestinal mucosa, and regulates gut microbiota composition.
... Generally, the presence of glucose in the fermentation medium is reported to enhance gene expression and can encourage the growth and division of bacteria (Zhou et al., 2015). Likewise, earlier studies have also shown that in the presence of fibers, fever, and high salt in fermentation medium results in selective up-regulation of certain genes to resist exposure to elements in an exigent environment by secretion of some proteins for protection of cells as a defense mechanism (Zhou et al., 2015). ...
... Generally, the presence of glucose in the fermentation medium is reported to enhance gene expression and can encourage the growth and division of bacteria (Zhou et al., 2015). Likewise, earlier studies have also shown that in the presence of fibers, fever, and high salt in fermentation medium results in selective up-regulation of certain genes to resist exposure to elements in an exigent environment by secretion of some proteins for protection of cells as a defense mechanism (Zhou et al., 2015). A recent q-RTPCR study by Zhou et al. (2018) and Choub et al. (2021) reported lowest fold gene expression (surfactin sfp gene) in 1% glucose and highest expression in the presence of a combination of 0.67% glucose and 0.33% cellulose. ...
Surfactin lipopeptide is an eco-friendly microbially synthesized bioproduct that holds considerable potential in therapeutics (antibiofilm) as well as in agriculture (antifungal). In the present study, production of surfactin by a marine strain Bacillus velezensis MS20 was carried out, followed by physico-chemical characterization, anti-biofilm activity, plant growth promotion, and quantitative Reverse Transcriptase—Polymerase Chain Reaction (q RT-PCR) studies. From the results, it was inferred that MS20 was found to produce biosurfactant (3,300 mg L –1 ) under optimized conditions. From the physicochemical characterization [Thin layer chromatography (TLC), Fourier Transform Infrared (FTIR) Spectroscopy, Liquid Chromatography/Mass Spectroscopy (LC/MS), and Polymerase Chain Reaction (PCR) amplification] it was revealed to be surfactin. From bio-assay and scanning electron microscope (SEM) images, it was observed that surfactin (MIC 50 μg Ml –1 ) has appreciable bacterial aggregation against clinical pathogens Pseudomonas aeruginosa MTCC424, Escherichia coli MTCC43, Klebsiella pneumoniae MTCC9751, and Methicillin resistant Staphylococcus aureus (MRSA) and mycelial condensation property against a fungal phytopathogen Rhizoctonia solani . In addition, the q-RTPCR studies revealed 8-fold upregulation (9.34 ± 0.11-fold) of srf A-A gene compared to controls. Further, treatment of maize crop (infected with R. solani ) with surfactin and MS20 led to the production of defense enzymes. In conclusion, concentration and synergy of a carbon source with inorganic/mineral salts can ameliorate surfactin yield and, application wise, it has antibiofilm and antifungal activities. In addition, it induced systemic resistance in maize crop, which makes it a good candidate to be employed in sustainable agricultural practices.
... Although B. subtilis can produce cellulase such as endoglucanase (encoded by eglS) and b-glucosidase (encoded by bglA and bglC), cellulose is not the preferred carbon source of B. subtilis, and high-fiber environment is even a kind of stress (Fujjta 2009;Robson and Chambliss 1987;Zhou et al. 2015). However, B. subtilis can also survive this cellulose stress by hydrolyzing cellulose and finally achieve stress adaptation to a certain extent, which may benefit from the strong transcriptional regulatory network in vivo (Zhou et al. 2015). ...
... Although B. subtilis can produce cellulase such as endoglucanase (encoded by eglS) and b-glucosidase (encoded by bglA and bglC), cellulose is not the preferred carbon source of B. subtilis, and high-fiber environment is even a kind of stress (Fujjta 2009;Robson and Chambliss 1987;Zhou et al. 2015). However, B. subtilis can also survive this cellulose stress by hydrolyzing cellulose and finally achieve stress adaptation to a certain extent, which may benefit from the strong transcriptional regulatory network in vivo (Zhou et al. 2015). RNA-binding protein Hfq is an important bacterial post-transcriptional regulator, which can interact directly or indirectly with a variety of RNA (sRNA, mRNA, tRNA and rRNA), proteins and DNA (Kavita et al. 2018;Santos et al. 2019). ...
Objective
To study the function of the RNA-binding protein Hfq in Bacillus subtilis cellulose decomposition.ResultsIn the medium with sodium carboxymethylcellulose (Na-CMC) as the sole carbon source, the knockout of Hfq resulted in a 38.0% ± 2.1% and 76.6% ± 7.1% decrease in cellulose hydrolysis ability and cellulase activity, respectively. The results of real-time quantitative PCR revealed that several cellulase genes (eglS, bglA, and bglC) were significantly downregulated in the Hfq knockout strain. The isogenic Δhfq complemented strain recovered the cellulose hydrolysis ability, cellulase activity, and expression level of cellulase genes. In addition, the survival of Hfq mutant in stationary phase was significantly affected.ConclusionRNA-binding protein Hfq is involved in the regulation of cellulose hydrolysis ability, cellulase activity, cellulase gene expression, and stationary phase survival.
... The positive regulation of cellulase activity by Hfq further explains the advantages given by this protein in cellulose hydrolysis ability and stationary phase survival under cellulose stress, since the cellulase activity of B. subtilis is closely related to the rate of obtaining nutrients regardless of being in Na-CMC medium or on Congo red plates (Figure 1-Figure 3 and Table S3). High-ber conditions constitute a stress environment for B. subtilis (Ziyao et al., 2015). The possible role of Hfq BS in stress adaptation has been emphasized by this study; nevertheless, it remains to be con rmed. ...
Objective
To study the function of the RNA-binding protein Hfq in Bacillus subtilis cellulose decomposition.
Results
In the medium with sodium carboxymethylcellulose (Na-CMC) as the sole carbon source, the knockout of Hfq resulted in a 38.0% ± 2.1% and 76.6% ± 7.1% decrease in cellulose hydrolysis ability and cellulase activity, respectively. The results of real-time quantitative PCR revealed that several cellulase genes (eglS, bglA, and bglC) were significantly downregulated in the Hfq knockout strain. The isogenic ΔHfq complemented strain recovered the cellulose hydrolysis ability, cellulase activity, and expression level of cellulase genes. In addition, the survival of Hfq mutant in stationary phase was significantly affected.
Conclusion
RNA-binding protein Hfq is involved in the regulation of cellulose hydrolysis ability, cellulase activity, cellulase gene expression, and stationary phase survival.
... 2018;Xue et al., 2015;Yang et al., 2018;Zhang et al., 2018). Although the main diet of adult giant pandas is bamboo, their gut microbiota have an extremely low abundance of typical cellulolytic bacteria, such as Ruminococcus and Bacteroides (M. Chen et al., 2015;Li et al., 2010;Xue et al., 2015). Instead, Bacillus and Clostridia (Xue et al., 2015;Z. Zhou et al., 2015) with limited cellulolytic activity are abundant (Xue et al., 2015;Zhang et al., 2018;Zhu et al., 2011), which would account for the tiny loss of cellulose (~8%) in fecal bamboos (Dierenfeld et al., 1982). ...
... The transcription of Bacillus subtilis HH2 cultured in the presence of various carbon sources was studied using transcriptome sequencing techniques. The different carbon sources were observed to activate specific transcriptional controls (such as elevated expression of cellulose and the reduction of non-essential protein synthesis to save energy) in the giant panda [39]. These studies reported Bacillus as a suitable candidate for micro-ecological preparations. ...
With increasing age, the rumen microbiota of new-born ruminants become central in the translation of fibrous feed substances into essential nutrients. However, the colonization process of the microbial community (especially fungal community) remains poorly understood in ruminants at pre-weaning stages. In this study, the rumen bacterial and fungal colonization processes were investigated in goats at eight stages using amplicon sequencing. For bacteria, we found 36 common core genera at D0, D3, D14, D28, and D56, including mainly Bacillus, Alloprevotella, Bacteroides, Prevotella_1, Lactococcus, and Ruminococcaceae_NK4A214. Firmicutes was the dominant phylum among the total microbiota in newborn goat kids (prior to nursing), while Bacillus, Lactococcus, and Pseudomonas were predominant genera. Interestingly, the proportion of Bacillus was as high as 55% in newborn animals. After milk nursing, the predominant phylum changed to Bacteroidetes, while the proportion of Bacillus and Lactobacillus was very low. CowPi was used to predict the functional gene pathways and we found increases in the abundance of genes associated with amino acid related enzymes, DNA repair and recombination proteins, aminoacyl tRNA biosynthesis, and peptidases after D3. With regard to fungi, we found that there were 51 common genera at day 0 (D0), D3, D14, D28, and D56, including mainly Cryptococcus, Aspergillus, and Caecomyces. Aspergillus occupied approximately 47% at day 0, but then it decreased from day 3 to day 14. This study indicates that the core microbes of rumen emerged shortly after birth, but the abundance was very different from the core genus of the adult rumen. In addition, we also report a detailed scheme of the bacterial and fungal colonization process in rumens and propose three distinct stages during the rumen colonization process in pre-weaning goats, which will offer a reference for the development of milk substitutes for small ruminants.
... are quite common in the gut of different animals, including the ones with high-fiber feeding habits, such as soil invertebrates or the giant panda 53,54 , few studies have focused on their carbohydrolytic potential 39 . For example, predominant B. subtilis strains from the intestinal microbial community of the giant panda, seem to have the capacity to growth in a higher fiber environment 55 , opening the possibility that also in fish, Bacillus spp. may have a decisive role in shaping their host digestive capacity towards the efficient utilization of PF-diets. ...
The gastrointestinal microbiota plays a critical role on host health and metabolism. This is particularly important in teleost nutrition, because fish do not possess some of the necessary enzymes to cope with the dietary challenges of aquaculture production. A main difficulty within fish nutrition is its dependence on fish meal, an unsustainable commodity and a source of organic pollutants. The most obvious sustainable alternatives to fish meal are plant feedstuffs, but their nutritive value is limited by the presence of high levels of non-starch polysaccharides (NSP), which are not metabolized by fish. The composition of fish-gut microbial communities have been demonstrated to adapt when the host is fed different ingredients. Thus, we hypothesized that a selective pressure of plant-based diets on fish gut microbiota, could be a beneficial strategy for an enrichment of bacteria with a secretome able to mobilize dietary NSP. By targeting bacterial sporulating isolates with diverse carbohydrase activities from the gut of European sea bass, we have obtained isolates with high probiotic potential. By inferring the adaptive fitness to the fish gut and the amenability to industrial processing, we identified the best two candidates to become industrially valuable probiotics. This potential was confirmed in vivo, since one of the select isolates lead to a better growth and feed utilization efficiency in fish fed probiotic-supplemented plant-based diets, thus contributing for sustainable and more cost-effective aquaculture practices.
... However, despite their high fiber diet, cellulolytic species typically present in herbivore guts, e.g., members of Ruminococcaceae or Bacteroides ( Zhang et al., 2018), are conspicuously absent or present at extremely low abundances in the gut of giant panda. Instead, Bacillus ( Zhou et al., 2015) and Clostridia, which have limited cellulolytic activity and thus cannot fully utilize bamboo ( Zhu et al., 2011), are widely observed ( Dierenfeld et al., 1982). Furthermore, the gut microbiota compositions of adult giant pandas exhibited extensive seasonal variation ( Xue et al., 2015;Wu et al., 2017). ...
Adult giant pandas (Ailuropoda melanoleuca) express transitional characteristics in that they consume bamboos, despite their carnivore-like digestive tracts. Their genome contains no cellulolytic enzymes; therefore, understanding the development of the giant panda gut microbiome, especially in early life, is important for decoding the rules underlying gut microbial formation, inheritance and dietary transitions. With deep metagenomic sequencing, we investigated the gut microbiomes of two newborn giant panda brothers and their parents living in Macao, China, from 2016 to 2017. Both giant panda cubs exhibited progressive increases in gut microbial richness during growth, particularly from the 6th month after birth. Enterobacteriaceae dominated the gut microbial compositions in both adult giant pandas and cubs. A total of 583 co-abundance genes (CAGs) and about 79 metagenomic species (MGS) from bacteria or viruses displayed significant changes with age. Seven genera (Shewanella, Oblitimonas, Helicobacter, Haemophilus, Aeromonas, Listeria, and Fusobacterium) showed great importance with respect to gut microbial structural determination in the nursing stage of giant panda cubs. Furthermore, 10 orthologous gene functions and 44 pathways showed significant changes with age. Of the significant pathways, 16 from Escherichia, Klebsiella, Propionibacterium, Lactobacillus, and Lactococcus displayed marked differences between parents and their cubs at birth, while 29 pathways from Escherichia, Campylobacter and Lactobacillus exhibited significant increase in cubs from 6 to 9 months of age. In addition, oxidoreductases, transferases, and hydrolases dominated the significantly changed gut microbial enzymes during the growth of giant panda cubs, while few of them were involved in cellulose degradation. The findings indicated diet-stimulated gut microbiome transitions and the important role of Enterobacteriaceae in the guts of giant panda in early life.
... It has thus been hypothesized that the degradation of cellulose, a key component of its bamboo diet, should be dependent on its gut microbiota. Some researchers have found some cellulose-degrading bacteria in the feces from giant pandas (such as, [6]). Based on metagenomic technology to detect bacteria diversity in gut microbiome of the giant panda, Zhu et al. [7] detected 13 operational taxonomic units closely related to Clostridium groups I and XIVa, both of which contain taxa known to digest cellulose, and putative genes coding for two cellulose-digesting enzymes. ...
The giant panda feeds almost exclusively on bamboo, a diet highly enriched in lignin and cellulose, but is characterized by a digestive tract similar to carnivores. It is still large unknown if and how the giant panda gut microbiota contributes to lignin and cellulose degradation. Here we show the giant pandas' gut microbiota does not significantly contribute to cellulose and lignin degradation. We found that no operational taxonomic unit had a nearest neighbor identified as a cellulolytic species or strain with a significant higher abundance in juvenile than cubs, a very low abundance of putative lignin and cellulose genes existed in part of analyzing samples but a significant higher abundance of genes involved in starch and hemicellulose degradation in juveniles than cubs. Moreover, a significant lower abundance of putative cellulolytic genes and a significant higher abundance of putative α-amylase and hemicellulase gene families were present in giant pandas than in omnivores or herbivores.
... However, to the best of our knowledge, there are no commercial probiotic strain originating from pandas. In our previous study, we found a bacterial model B. subtilis strain HH2 isolated from the feces of a healthy giant panda; this strain showed a good adaptation to the herbivore intestinal cellulose environment and exhibited several probiotic functions based on transcriptional regulation [7]. However, the secretion and antibacterial effects of surfactin from this probiotic candidate in the presence of high-fiber conditions remain unclear. ...
... Glucose medium was slightly modified from previous studies [7,10] and contained 70 mmol K 2 HPO 4 , 30 mmol KH 2 PO 4 , 25 mmol (NH 4 ) 2 SO 4 , 0.5 mmol MgSO 4 , 10 μmol MnSO 4 , 22 mg ferric ammonium citrate, 8 g potassium glutamate, 6 g potassium succinate, 1% glucose, 0.5 mmol CaCl 2 , 5 μmol MnCl 2 , and 1000 mL ddH 2 O at pH 7.2. The three other media used in this study included mixed-1, mixed-2, and cellulose media which were formulated in the same manner as the glucose medium, except that the main carbon sources were 0.33% sodium carboxymethylcellulose plus 0.67% glucose (mixed-1), 0.67% sodium carboxymethylcellulose plus 0.33% glucose (mixed-2), or 1% sodium carboxymethylcellulose (cellulose) instead of 1% glucose. ...
... Generally, glucose culture condition can be beneficial for the expression of most genes and can promote bacterial growth and division. In a previous study, we showed that cellulose was not an ideal carbon source for B. subtilis HH2; most nonessential genes (e.g., genes involved in chemotaxis and motility) in the cellulose group were down-regulated compared with that in the glucose group in order to conserve energy [7]. However, in this study, we found that B. subtilis HH2 showed optimal growth in a glucose medium, accompanied by low surfactin expression. ...
Surfactin secreted by Bacillus subtilis can confer strong, diverse antipathogenic effects, thereby benefitting the host. Carbon source is an important factor for surfactin production. However, the mechanism that bacteria utilize cellulose, the most abundant substance in the intestines of herbivores, to produce surfactin remains unclear. Here, we used B. subtilis HH2, isolated from the feces of a giant panda, as a model to determine changes in surfactin expression in the presence of different concentrations of cellulose by quantitative polymerase chain reaction and high-performance liquid chromatography. We further investigated the antimicrobial effects of surfactin against three common intestinal pathogens (Escherichia coli, Staphylococcus aureus, and Salmonella enterica) and its resistance to high temperature (60–121°C), pH (1–12), trypsin (100–300 μg/mL, pH 8), and pepsin (100–300 μg/mL, pH 2). The results showed that the surfactin expressed lowest in bacteria cultured in the presence of 1% glucose medium as the carbon source, whereas increased in an appropriate cellulose concentration (0.67% glucose and 0.33% cellulose). The surfactin could inhibit E. coli and Staphylococcus aureus, but did not affect efficiently for Salmonella enterica. The antibacterial ability of surfactin did not differ according to temperature (60–100°C), pH (2–11), trypsin (100–300 μg/mL), and pepsin (100–300 μg/mL; P > 0.05), but decreased significantly at extreme environments (121°C, pH 1 or 12; P < 0.05) compared with that in the control group (37°C, pH = 7, without any protease). In conclusion, our findings indicated that B. subtilis HH2 could increase surfactin expression in an appropriate cellulose environment and thus provide benefits to improve the intestinal health of herbivores.
