Lipid Giant Vesicles Engulf Living Bacteria Triggered by Minor Enhancement in Membrane Fluidity.

Antibacterial amphiphiles normally kill bacteria by destroying the bacterial membrane. Whether and how antibacterial amphiphiles alter normal cell membrane and lead to subsequent effects on pathogen invasion into cells have been scarcely promulgated. Herein, by taking four antibacterial gemini amphiphiles with different spacer groups to modulate cell-mimic phospholipid giant unilamellar vesicles (GUVs), bacteria adhesion on the modified GUVs surface and bacteria engulfment process by the GUVs are clearly captured by confocal laser scanning microscopy. Further characterization shows that the enhanced cationic surface charge of GUVs by the amphiphiles determines the bacteria adhesion amount, while the involvement of amphiphile in GUVs results in looser molecular arrangement and concomitant higher fluidity in the bilayer membranes, facilitating the bacteria intruding into GUVs. This study sheds new light on the effect of amphiphiles on membrane bilayer and the concurrent effect on pathogen invasion into cell mimics and broadens the nonprotein-mediated endocytosis pathway for live bacteria.

mucG, mucH, and mucI Modulate Production of Mutanocyclin and Reutericyclins in Streptococcus mutans B04Sm5.

Streptococcus mutans is considered a primary etiologic agent of dental caries, which is the most common chronic infectious disease worldwide. S. mutans B04Sm5 was recently shown to produce reutericyclins and mutanocyclin through the muc biosynthetic gene cluster and to utilize reutericyclins to inhibit the growth of neighboring commensal streptococci. In this study, examination of S. mutans and muc phylogeny suggested evolution of an ancestral S. mutans muc into three lineages within one S. mutans clade and then horizontal transfer of muc to other S. mutans clades. The roles of the mucG and mucH transcriptional regulators and the mucI transporter were also examined. mucH was demonstrated to encode a transcriptional activator of muc. mucH deletion reduced production of mutanocyclin and reutericyclins and eliminated the impaired growth and inhibition of neighboring streptococci phenotypes, which are associated with reutericyclin production. ΔmucG had increased mutanocyclin and reutericyclin production, which impaired growth and increased the ability to inhibit neighboring streptococci. However, deletion of mucG also caused reduced expression of mucD, mucE, and mucI. Deletion of mucI reduced mutanocyclin and reutericylin production but enhanced growth, suggesting that mucI may not transport reutericyclin as its homolog does in Limosilactobacillus reuteri. Further research is needed to determine the roles of mucG and mucI and to identify any cofactors affecting the activity of the mucG and mucH regulators. Overall, this study provided pangenome and phylogenetic analyses that serve as a resource for S. mutans research and began elucidation of the regulation of reutericyclins and mutanocyclin production in S. mutans. IMPORTANCE S. mutans must be able to outcompete neighboring organisms in its ecological niche in order to cause dental caries. S. mutans B04Sm5 inhibited the growth of neighboring commensal streptococci through production of reutericyclins via the muc biosynthetic gene cluster. In this study, an S. mutans pangenome database and updated phylogenetic tree were generated that will serve as valuable resources for the S. mutans research community and that provide insights into the carriage and evolution of S. mutans muc. The MucG and MucH regulators, and the MucI transporter, were shown to modulate production of reutericyclins and mutanocyclin. These genes also affected the ability of S. mutans to inhibit neighboring commensals, suggesting that they may play a role in S. mutans virulence.

Klebsiella and Providencia emerge as lone survivors following long-term starvation of oral microbiota.

It is well-understood that many bacteria have evolved to survive catastrophic events using a variety of mechanisms, which include expression of stress-response genes, quiescence, necrotrophy, and metabolic advantages obtained through mutation. However, the dynamics of individuals leveraging these abilities to gain a competitive advantage in an ecologically complex setting remain unstudied. In this study, we observed the saliva microbiome throughout the ecological perturbation of long-term starvation, allowing only the species best equipped to access and use the limited resources to survive. During the first several days, the community underwent a death phase that resulted in a ∼50-100-fold reduction in the number of viable cells. Interestingly, after this death phase, only three species, Klebsiella pneumoniae, Klebsiella oxytoca, and Providencia alcalifaciens, all members of the family Enterobacteriaceae, appeared to be transcriptionally active and recoverable. Klebsiella are significant human pathogens, frequently resistant to multiple antibiotics, and recently, ectopic colonization of the gut by oral Klebsiella was documented to induce dysbiosis and inflammation. MetaOmics analyses provided several leads for further investigation regarding the ecological success of the Enterobacteriaceae. The isolates accumulated single nucleotide polymorphisms in known growth advantage in stationary phase alleles and produced natural products closely resembling antimicrobial cyclic depsipeptides. The results presented in this study suggest that pathogenic Enterobacteriaceae persist much longer than their more benign neighbors in the salivary microbiome when faced with starvation. This is particularly significant, given that hospital surfaces contaminated with oral fluids, especially sinks and drains, are well-established sources of outbreaks of drug-resistant Enterobacteriaceae.

Transcriptomic and physiological analysis of common duckweed Lemna minor responses to NH4(+) toxicity.

BACKGROUND: Plants can suffer ammonium (NH4 (+)) toxicity, particularly when NH4 (+) is supplied as the sole nitrogen source. However, our knowledge about the underlying mechanisms of NH4 (+) toxicity is still largely unknown. Lemna minor, a model duckweed species, can grow well in high NH4 (+) environment but to some extent can also suffer toxic effects. The transcriptomic and physiological analysis of L. minor responding to high NH4 (+) may provide us some interesting and useful information not only in toxic processes, but also in tolerance mechanisms. RESULTS: The L. minor cultured in the Hoagland solution were used as the control (NC), and in two NH4 (+) concentrations (NH4 (+) was the sole nitrogen source), 84 mg/L (A84) and 840 mg/L (A840) were used as stress treatments. The NH4 (+) toxicity could inhibit the growth of L. minor. Reactive oxygen species (ROS) and cell death were studied using stained fronds under toxic levels of NH4 (+). The malondialdehyde content and the activities of superoxide dismutase and peroxidase increased from NC to A840, rather than catalase and ascorbate peroxidase. A total of 6.62G nucleotides were generated from the three distinct libraries. A total of 14,207 differentially expressed genes (DEGs) among 70,728 unigenes were obtained. All the DEGs could be clustered into 7 profiles. Most DEGs were down-regulated under NH4 (+) toxicity. The genes required for lignin biosynthesis in phenylpropanoid biosynthesis pathway were up-regulated. ROS oxidative-related genes and programmed cell death (PCD)-related genes were also analyzed and indicated oxidative damage and PCD occurring under NH4 (+) toxicity. CONCLUSIONS: The first large transcriptome study in L. minor responses to NH4 (+) toxicity was reported in this work. NH4 (+) toxicity could induce ROS accumulation that causes oxidative damage and thus induce cell death in L. minor. The antioxidant enzyme system was activated under NH4 (+) toxicity for ROS scavenging. The phenylpropanoid pathway was stimulated under NH4 (+) toxicity. The increased lignin biosynthesis might play an important role in NH4 (+) toxicity resistance.

Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors.

Furfural and acetic acid from lignocellulosic hydrolysates are the prevalent inhibitors to Zymomonas mobilis during cellulosic ethanol production. Developing a strain tolerant to furfural or acetic acid inhibitors is difficul by using rational engineering strategies due to poor understanding of their underlying molecular mechanisms. In this study, strategy of adaptive laboratory evolution (ALE) was used for development of a furfural and acetic acid-tolerant strain. After three round evolution, four evolved mutants (ZMA7-2, ZMA7-3, ZMF3-2, and ZMF3-3) that showed higher growth capacity were successfully obtained via ALE method. Based on the results of profiling of cell growth, glucose utilization, ethanol yield, and activity of key enzymes, two desired strains, ZMA7-2 and ZMF3-3, were achieved, which showed higher tolerance under 7 g/l acetic acid and 3 g/l furfural stress condition. Especially, it is the first report of Z. mobilis strain that could tolerate higher furfural. The best strain, Z. mobilis ZMF3-3, has showed 94.84% theoretical ethanol yield under 3-g/l furfural stress condition, and the theoretical ethanol yield of ZM4 is only 9.89%. Our study also demonstrated that ALE method might also be used as a powerful metabolic engineering tool for metabolic engineering in Z. mobilis. Furthermore, the two best strains could be used as novel host for further metabolic engineering in cellulosic ethanol or future biorefinery. Importantly, the two strains may also be used as novel-tolerant model organisms for the genetic mechanism on the "omics" level, which will provide some useful information for inverse metabolic engineering.

Transcriptome profiling of Zymomonas mobilis under furfural stress.

Furfural from lignocellulosic hydrolysates is the prevalent inhibitor to microorganisms during cellulosic ethanol production, but the molecular mechanisms of tolerance to this inhibitor in Zymomonas mobilis are still unclear. In this study, genome-wide transcriptional responses to furfural were investigated in Z. mobilis using microarray analysis. We found that 433 genes were differentially expressed in response to furfural. Furfural up- or down-regulated genes related to cell wall/membrane biogenesis, metabolism, and transcription. However, furfural has a subtle negative effect on Entner-Doudoroff pathway mRNAs. Our results revealed that furfural had effects on multiple aspects of cellular metabolism at the transcriptional level and that membrane might play important roles in response to furfural. This research has provided insights into the molecular response to furfural in Z. mobilis, and it will be helpful to construct more furfural-resistant strains for cellulosic ethanol production.

Cariogenic Streptococcus mutans Produces Tetramic Acid Strain-Specific Antibiotics That Impair Commensal Colonization.

Streptococcus mutans is a common constituent of dental plaque and a major etiologic agent of dental caries (tooth decay). In this study, we elucidated the biosynthetic pathway encoded by muc, a hybrid polyketide synthase and nonribosomal peptide synthetase (PKS/NRPS) biosynthetic gene cluster (BGC), present in a number of globally distributed S. mutans strains. The natural products synthesized by muc included three N-acyl tetramic acid compounds (reutericyclin and two novel analogues) and an unacylated tetramic acid (mutanocyclin). Furthermore, the enzyme encoded by mucF was identified as a novel class of membrane-associated aminoacylases and was responsible for the deacylation of reutericyclin to mutanocyclin. A large number of hypothetical proteins across a broad diversity of bacteria were homologous to MucF, suggesting that this may represent a large family of unexplored acylases. Finally, S. mutans utilized the reutericyclin produced by muc to impair the growth of neighboring oral commensal bacteria. Since S. mutans must be able to out-compete these health-associated organisms to persist in the oral microbiota and cause disease, the competitive advantage conferred by muc suggests that this BGC is likely to be involved in S. mutans ecology and therefore dental plaque dysbiosis and the resulting caries pathogenesis.

Identification of the Bacterial Biosynthetic Gene Clusters of the Oral Microbiome Illuminates the Unexplored Social Language of Bacteria during Health and Disease.

Small molecules are the primary communication media of the microbial world. Recent bioinformatic studies, exploring the biosynthetic gene clusters (BGCs) which produce many small molecules, have highlighted the incredible biochemical potential of the signaling molecules encoded by the human microbiome. Thus far, most research efforts have focused on understanding the social language of the gut microbiome, leaving crucial signaling molecules produced by oral bacteria and their connection to health versus disease in need of investigation. In this study, a total of 4,915 BGCs were identified across 461 genomes representing a broad taxonomic diversity of oral bacteria. Sequence similarity networking provided a putative product class for more than 100 unclassified novel BGCs. The newly identified BGCs were cross-referenced against 254 metagenomes and metatranscriptomes derived from individuals either with good oral health or with dental caries or periodontitis. This analysis revealed 2,473 BGCs, which were differentially represented across the oral microbiomes associated with health versus disease. Coabundance network analysis identified numerous inverse correlations between BGCs and specific oral taxa. These correlations were present in healthy individuals but greatly reduced in individuals with dental caries, which may suggest a defect in colonization resistance. Finally, corroborating mass spectrometry identified several compounds with homology to products of the predicted BGC classes. Together, these findings greatly expand the number of known biosynthetic pathways present in the oral microbiome and provide an atlas for experimental characterization of these abundant, yet poorly understood, molecules and socio-chemical relationships, which impact the development of caries and periodontitis, two of the world's most common chronic diseases.IMPORTANCE The healthy oral microbiome is symbiotic with the human host, importantly providing colonization resistance against potential pathogens. Dental caries and periodontitis are two of the world's most common and costly chronic infectious diseases and are caused by a localized dysbiosis of the oral microbiome. Bacterially produced small molecules, often encoded by BGCs, are the primary communication media of bacterial communities and play a crucial, yet largely unknown, role in the transition from health to dysbiosis. This study provides a comprehensive mapping of the BGC repertoire of the human oral microbiome and identifies major differences in health compared to disease. Furthermore, BGC representation and expression is linked to the abundance of particular oral bacterial taxa in health versus dental caries and periodontitis. Overall, this study provides a significant insight into the chemical communication network of the healthy oral microbiome and how it devolves in the case of two prominent diseases.

Kinetic model of continuous ethanol fermentation in closed-circulating process with pervaporation membrane bioreactor by Saccharomyces cerevisiae.

Unstructured kinetic models were proposed to describe the principal kinetics involved in ethanol fermentation in a continuous and closed-circulating fermentation (CCCF) process with a pervaporation membrane bioreactor. After ethanol was removed in situ from the broth by the membrane pervaporation, the secondary metabolites accumulated in the broth became the inhibitors to cell growth. The cell death rate related to the deterioration of the culture environment was described as a function of the cell concentration and fermentation time. In CCCF process, 609.8 g L(-1) and 750.1 g L(-1) of ethanol production were obtained in the first run and second run, respectively. The modified Gompertz model, correlating the ethanol production with the fermentation period, could be used to describe the ethanol production during CCCF process. The fitting results by the models showed good agreement with the experimental data. These models could be employed for the CCCF process technology development for ethanol fermentation.

Inhibition effect of secondary metabolites accumulated in a pervaporation membrane bioreactor on ethanol fermentation of Saccharomyces cerevisiae.

The secondary metabolites accumulated in a pervaporation membrane bioreactor during ethanol fermentation were mostly composed of acetic acid, lactic acid, propionic acid, citric acid, succinic acid and glycerol. The inhibition effect of these compounds at a broad concentration range was studied through ethanol fermentation by Saccharomyces cerevisiae. An increasing concentration of the secondary metabolites led to longer lag time and a reduction of cell growth. The specific cell growth rate, cell yield, ethanol productivity were only 0.061 h(-1), 0.024, 0.47 g L(-1) h(-1) respectively, when the medium contained 3.12 g of acetic acid, 10.23 g of lactic acid, 2.72 g of propionic acid, 1.35 g of citric acid, 2.26 g of succinic acid and 49.25 g of glycerol per liter (a concentration level in pervaporation membrane bioreactor at later fermentation period). By increasing pH of the medium to 6.0-8.0, the inhibition of these secondary metabolites could be greatly relieved.

Enhanced ethanol fermentation in a pervaporation membrane bioreactor with the convenient permeate vapor recovery.

A continuous and closed-circulating fermentation (CCCF) system with a pervaporation membrane bioreactor was built for ethanol fermentation without a refrigeration unit to condense the permeate vapor. Two runs of experiment with a feature of complete and continuous coupling of fermentation and pervaporation were carried out, lasting for 192h and 264h, respectively. The experimental measurement indicated that the enhanced fermentation could be achieved with additional advantages of convenient permeate recovery and energy saving of the process. During the second experiment, the average cell concentration, glucose consumption rate, ethanol productivity, ethanol yield and total ethanol amount produced reached 19.8gL(-1), 6.06gL(-1)h(-1), 2.31gL(-1)h(-1), 0.38, and 609.8gL(-1), respectively. During the continuous fermentation process, ethanol removal in situ promoted the cell second growth obviously, but the accumulation of the secondary metabolites in the broth became the main inhibitor against the cell growth and fermentation.

Bamboo: a new source of carbohydrate for biorefinery.

Bamboo is perennial woody grass, which distributed widely in the world and belonged to the Gramineae family and Bambuseae subfamily. It may be consider as a candidate lignocellulosic substrate for bio-ethanol production for its environmental benefits and higher annual biomass yield. The conversion of bamboo into bio-ethanol, bio-methane, natural food, flavonoids, and functional xylo-oligosaccharides production were reviewed in this paper. Future prospects for research include pretreatment, enzymatic hydrolysis and fermentation will also be performed to improve the whole process of ethanol production more economical. And revealing the molecular regulation mechanism of the fast growth of bamboo will provide chance for improving bamboo or other energy plants biomass yield through genetic engineering.

Acetone-butanol-ethanol fermentation in a continuous and closed-circulating fermentation system with PDMS membrane bioreactor.

Acetone-butanol-ethanol (ABE) fermentation by combining a PDMS membrane bioreactor and Clostridium acetobutylicum was studied, and a long continuous and closed-circulating fermentation (CCCF) system has been achieved. Two cycles of experiment were conducted, lasting for 274 h and 300 h, respectively. The operation mode of the first cycle was of fermentation intermittent coupling with pervaporation, and the second cycle was of continuous coupling. The average cell weight, glucose consumption rate, butanol productivity and butanol production of the first cycle were 1.59 g L(-1), 0.63 g L(-1)h(-1), 0.105 g L(-1)h(-1) and 28.03 g L(-1), respectively. Correspondingly, the four parameters of the second cycle were 1.68 g L(-1), 1.12 g L(-1)h(-1), 0.205 g L(-1)h(-1) and 61.43 g L(-1), respectively. The results indicate the fermentation behaviors under continuous coupling mode were superior to that under intermittent coupling mode. Besides, two peak values were observed in the time course profiles, which means the microorganism could adapt the long CCCF membrane bioreactor system.

Adaptive evolution of Saccharomyces cerevisiae in a continuous and closed circulating fermentation (CCCF) system coupled with PDMS membrane pervaporation.

As an efficient means of strain improvement, adaptive evolution is a technique with great potential. Long-term cultivation of Saccharomyces cerevisiae was performed in a polydimethylsiloxane membrane bioreactor system which was constructed by coupling the fermentation with pervaporation. A parent strain was subjected to three rounds of fermentation-screening-transfer procedure lasting 1,500 h in a continuous and closed circulating fermentation (CCCF) system, and its 600-generation descendant S33 was screened. In shaking flask culture test, the selected strain S33 from the third round showed great superiority over the parent strain in the residual broth medium, with the ethanol yield and specific ethanol productivity increasing by 34.5 and 34.7 %, respectively. In the long-term CCCF test, the fermentation performance of the descendant strain in the third round was higher than that of its parent strain in the second round. These results show the potential of this novel adaptive evolution approach in optimization of yeast strains.

Ethanol fermentation kinetics in a continuous and closed-circulating fermentation system with a pervaporation membrane bioreactor.

The kinetics of ethanol fermentation by Saccharomyces cerevisiae was studied in a continuous and closed-circulating fermentation (CCCF) system with a polydimethylsiloxane (PDMS) pervaporation membrane bioreactor. Three sequential 500-h cycles of CCCF experiments were carried out. A glucose volumetric consumption of 3.8 g L(-1) h(-1) and ethanol volumetric productivity of 1.39 g L(-1) h(-1) were obtained in the third cycle, with a specific glucose utilization rate of 0.32 h(-1) and ethanol yield rate of 0.13 h(-1). The prolonged fermentation time and good fermentation performance indicate that the CCCF would be a feasible and promising fermentation process technology.