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The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be repressed in the best-studied bacterium,Escherichia coliK-12. We show here that theE. coliCRISPR-Cas system is active and serves to inhibit its nine defective (i.e., cryptic) prophages. Specifically, compared to the wild-type strain, reducing the amounts of specific interfering RNAs (crRNA) decreases growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation. Similar results were obtained by inactivating CRISPR-Cas by deleting the entire 13 spacer region (CRISPR array); hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages, most likely through CRISPR array-derived crRNA binding to cryptic prophage mRNA, rather than through cleavage of cryptic prophage DNA; i.e., self-targeting. Consistently, four of the 13E. colispacers contain complementary regions to the mRNA sequences of seven cryptic prophages, and inactivation of CRISPR-Cas increases the level of mRNA for lysis protein YdfD of cryptic prophage Qin and lysis protein RzoD of cryptic prophage DLP-12. Also, lysis is clearly seen via transmission electron microscopy when the whole CRISPR-Cas array is deleted, and eliminating spacer #12, which encodes crRNA with complementary regions for DLP-12 (includingrzoD), Rac, Qin (includingydfD), and CP4-57 cryptic prophages, also results in growth inhibition and cell lysis. Therefore, we report the novel results that (i) CRISPR-Cas is active inE. coliand (ii) CRISPR-Cas is used to tame cryptic prophages, likely through RNAi; i.e., unlike with active lysogens, active CRISPR-Cas and cryptic prophages may stably co-exist.

Digital, Palm Oil Mill Effluent (POME) requires treatment before disposal due to its high organic matter content. In this study, the electrical performance and wastewater treatment efficiency were evaluated for Microbial Fuel Cells (MFC) treating unsterile POME with chemical oxygen demand (COD) from 200 to 10 000 mg/L. Since the inoculum type is a key factor in MFC performance, three types of sludge (methanogenic sludge (MS), facultative sludge (FS), and dry sludge (DS), obtained from the current POME treatment ponds were evaluated as inoculum. Dry sludge (DS) developed a maximum power output of 3.30 W/m3 by oxidizing 71% out of the COD provided by POME (1000 mg/L). Also, raw POME microbiota contributed to an enrichment of the community in DS inoculum along with the operation, in which Geobacter was the predominant genus reaching a current generation of 247 mA/m2 and a power density of 2.36 W/m3. Conversely, pure electrogenic (Shewanella sp.) inoculation led to a diversification process, resulting in a lower current generation of 52 mA/m2 and a power density of 0.10 W/m3. Consequently, microbial community dynamics revealed that MFC inoculation tends to a microbial equilibrium wherein generation of high current density was achieved by gradual microbial enrichment rather than external electrogenic invasion., Ciencias Exacta y Naturales

Anaerobic digestion of sewage sludge (SS) is one of the effective ways to reduce the waste generated from human life activities. To date, there are many reports to improve or repress methane production during the anaerobic digestion of SS. In the anaerobic digestion process, many microorganisms work positively or negatively, and as a result of their microbe-to-microbe interaction and regulation, methane production increases or decreases. In other words, understanding the complex control mechanism among the microorganisms and identifying the strains that are key to increase or decrease methane production are important for promoting the advanced production of bioenergy and beneficial compounds. In this mini-review, the literature on methane production in anaerobic digestion has been summarized based on the results of antibiotic addition, quorum sensing control, and inorganic substance addition. By optimizing the activity of microbial groups in SS, methane or acetate can be highly produced. KEY POINTS: • Bactericidal agents such as an antibiotic alter microbial community for enhanced CH4 production. • Bacterial interaction via quorum sensing is one of the key points for biofilm and methane production. • Anaerobic digestion can be altered in the presence of several inorganic materials.

Waterborne diseases due to pathogen contamination in water are a serious problem all over the world. Accurate and simultaneous detection of pathogens in water is important to protect public health. In this study, we developed a method to simultaneously detect various pathogenic Escherichia coli by sequencing the amplicons of multiplex PCR. Our newly designed multiplex PCR amplified five genes for pathogenic E. coli (uidA, stx1, stx2, STh gene, and LT gene). Additional two PCR assays (for aggRand eae) were also designed and included in the amplicon sequencing analysis. The same assays were also used for digital PCR (dPCR). Strong positive correlations were observed between the sequence read count and the dPCR results for most of the genes targeted, suggesting that our multiplex PCR-amplicon sequencing approach could provide quantitative information. The method was also successfully applied to monitor the level of pathogenic E. coli in river water and wastewater samples. The approach shown here could be expanded by targeting genes for other pathogens.

Alcaligenaceae and Chromatiaceae were previously reported as the specific pollution bioindicators in the receiving river water contaminated by palm oil mill effluent (POME) final discharge. Considering the inevitable sensitivity of bacteria under environmental stresses, it is crucial to assess the survivability of both bacteria in the fluctuated environmental factors, proving their credibility as POME pollution bioindicators in the environment. In this study, the survivability of Alcaligenaceae and Chromatiaceae from facultative pond, algae (aerobic) pond and final discharge were evaluated under varying sets of temperature (25-40°C), pH (pH 7-9) and low/high total suspended solid (TSS) contents of POME collected during low/high crop seasons of oil palm, respectively. Following treatment, the viability status and compositions of the bacterial community were assessed using flow cytometry-based assay and high-throughput Illumina MiSeq, respectively, in correlation with the changes of physicochemical properties. The changes in temperature, pH and TSS indeed changed the physicochemical properties of POME. The functionality of bacterial cells was also shifted where the viable cells and high nucleic acid contents reduced at elevated levels of temperature and pH but increased at high TSS content. Interestingly, the Alcaligenaceae and Chromatiaceae continuously detected in the samples which accounted for more than 0.5% of relative abundance, with a positive correlation with biological oxygen demand (BOD5) concentration. Therefore, either Alcaligenaceae or Chromatiaceae or both could be regarded as the reliable and specific bacterial indicators to indicate the pollution in river water due to POME final discharge despite the fluctuations in temperature, pH and TSS.

Because colony formation is essential to seek bacterial functions by the direct observation of phenotype, the diversification of colony formation for culturable bacteria is a big challenge in the research field of Environmental Biotechnology. In this study, the biodiversity of cultivable bacteria (colony or liquid culture) was compared by using Luria-Bertani (LB) medium and waste sewage sludge (WSS) under different dilutions and temperatures. When WSS was used as a bacterial source, whereas the highest number of colonies was found at the concentration of WSS (5%), a particular concentration of LB (10%) or WSS (1%) as a growth medium showed the best number of the operational taxonomic units (OTUs) of colonies. The results of bacterial community structure indicated that there are 1, 8, and 12 bacterial genera found uniquely in the agar plates of LB, 10% LB, and 5% WSS. By contrast, when palm oil mill effluent sludge was used as a bacterial source, the effect of dilution was different with WSS. When comparing the biodiversity between colonies and liquid culture, a high OTU value was observed in the colonies on the plate. In addition, 30°C showed the highest number of colonies in LB, 10% LB, and 5% WSS whereas the best OTUs were observed at 37°C for LB and 10% LB, and at 25°C for 5% WSS. This study demonstrates the diversification of cultivable bacteria through the number of OTUs in diluted LB medium and WSS, which is beneficial to isolate a unique bacterial strain. Key points • Impacts of diluted LB medium and WSS for colony formation were determined. • Difference of concentration of LB and WSS made different effects on colony formation. • Temperature change affected on diluted LB and WSS as media.

Pseudomonas aeruginosa is the main bacterial model to study cooperative behaviors, since it yields exoproducts such as exoproteases and siderophores that act as public goods and can be exploited by selfish non-producers that behave as social cheaters. Non-producers of the siderophore pyoverdine are typically isolated in media with low free iron, mainly casamino acids medium supplemented with transferrin. Nevertheless, using a protein as the iron chelator could additionally select mutants unable to produce exoproteases that degrade the transferrin to facilitate iron release. Here, we investigated the dynamics of pyoverdine and exoprotease production in media in which iron was limited by using either transferrin or a cation chelating resin. Our experiments show that concomitant loss of pyoverdine and exoprotease production readily develops in media with transferrin whereas only lack of pyoverdine emerges in medium treated with the resin. Genomic characterization of the exoprotease- and pyoverdine-less mutants revealed large deletions (13 to 33 Kb) including Quorum Sensing (lasR, rsal and lasl) and flagellar genes. Complementation experiments, PCR and motility tests confirmed the deletions. Our work shows that using transferrin as an iron chelator imposes simultaneous selective pressure for the loss of pyoverdine and exoprotease production. The unintended effect of transferrin observed in our experiment settings can help revisiting or informing the design of similar studies.

Methane metabolism in wetlands involves diverse groups of bacteria and archaea, which are responsible for the biological decomposition of organic matter under certain anoxic conditions. Recent advances in environmental omics revealed the phylogenetic diversity of novel microbial lineages, which have not been previously placed in the traditional tree of life. The present study aimed to verify the key players in methane production, either well-known archaeal members or recently identified lineages, in peat soils collected from wetland areas in Japan. Based on an ana-lysis of microbial communities using 16S rRNA gene sequencing and the mole-cular cloning of the functional gene, mcrA, a marker gene for methanogenesis, methanogenic archaea belonging to Methanomicrobiales, Methanosarcinales, Methanobacteriales, and Methanomassiliicoccales were detected in anoxic peat soils, suggesting the potential of CH

The four hydrogenase isozymes; hydrogenase 1 (Hyd-1), hydrogenase 2 (Hyd-2), hydrogenase 3 (Hyd-3) and hydrogenase 4 (Hyd-4) of Escherichia coli have been reported for their crucial functions in the hydrogen metabolism; however, their distinctive roles could not be completely understood. In this study, four ideal hydrogenase operon mutants, Δhyb hyc hyf, Δhya hyc hyf, Δhya hyb hyf, and Δhya hyb hyc, in which only a single hydrogenase is intact in the genome, were constructed as well as one quadruple mutant (Δhya hyb hyc hyf) that all four hydrogenase operons were deleted. First, single operon mutants and single-gene mutants for each hydrogenase showed different hydrogen productivity and growth in the anaerobic fermentation, indicating that bacterial phenotype regarding the hydrogen metabolism via the deletion of each operon is different with that of each single gene. Then, 4 triple hydrogenase operon mutants and one quadruple mutant were investigated to evaluate the hydrogen metabolism (hydrogen production and uptake) using glucose or glycerol as a substrate of hydrogen fermentation. With both the carbon sources, only Hyd-2 and Hyd-3 were able to produce hydrogen. Furthermore, all the hydrogenases showed hydrogen uptake activity. In addition, no hydrogen production and hydrogen uptake were detected in the quadruple mutant which does not have all 4 hydrogenases. Hydrogen production from Hyd-2 and Hyd-3 was further confirmed by complementing their operons in the cloning vector pBR322.

Biochar has proven to be a feasible additive for mitigating nitrogen loss during the composting process. This study aims to evaluate the influence of biochar addition on bacterial community and physicochemical properties changes, including ammonium (NH4+), nitrite (NO2−) and nitrate (NO3−) contents during the composting of poultry manure. The composting was carried out by adding 20% (w/w) of biochar into the mixture of poultry manure and rice straw with a ratio of 2:1, and the same treatment without biochar was prepared as a control. The finished product of control compost recorded the high contents of NO2− and NO3− (366 mg/kg and 600 mg/kg) with reduced the total NH4+ content to 10 mg/kg. Meanwhile, biochar compost recorded a higher amount of total NH4+ content (110 mg/kg) with low NO2− and NO3− (161 mg/kg and 137 mg/kg) content in the final composting material. The principal component analysis showed that the dynamics of dominant genera related to Halomonas, Pusillimonas, and Pseudofulvimonas, all of which were known as nitrifying and denitrifying bacteria, was significantly correlated with the dynamic of NO2− and NO3− content throughout the composting process. The genera related to Pusillimonas, and Pseudofulvimonas appeared as the dominant communities as the NO2− and NO3− increased. In contrast, as the NO2− and NO3− concentration decreased, the Halomonas genus were notably enriched in biochar compost. This study revealed the bacterial community shifts corresponded with the change of physicochemical properties, which provides essential information for a better understanding of monitoring and improving the composting process.

Escherichia coli K-12, being one of the best understood and thoroughly analyzed organisms, is the preferred platform for genetic and biochemical research. Among all genetic engineering approaches applied on E. coli, the homologous recombination approach is versatile and precise, which allows engineering genes or large segments of the chromosome directly by using polymerase chain reaction (PCR) products or synthetic oligonucleotides. The previously explained approaches for random insertion and deletions were reported as technically not easy and laborious. This study, first, finds the minimum length of homology extension that is efficient and accurate for homologous recombination, as 30 nt. Second, proposes an approach utilizing PCR products flanking ambiguous NNN-sequence (30-nt) extensions, which facilitate the homologous recombination to recombine them at multiple regions on the genome and generate insertion-deletion mutations. Further analysis found that these mutations were varying in number, that is, multiple genomic regions were deleted. Moreover, evaluation of the phenotype of all the multiple random insertion-deletion mutants demonstrated no significant changes in the normal metabolism of bacteria. This study not only presents the efficiency of ambiguous sequences in making random deletion mutations, but also demonstrates their further applicability in genomics.

Although Escherichia coli has four hydrogenases, their definite roles in fermentation are still not clear. In this study, all the operon deletion mutants of E.coli hydrogenases (∆hya, ∆hyb, ∆hyc, or ∆hyf) were constructed to evaluate the hydrogen metabolism in comparison to their respective single-gene deletion mutants of large subunits (∆hyaB, ∆hybC, ∆hycE, and ∆hyfG). Besides the hyc operon mutant that expectedly showed no hydrogen synthesis, the hyb operon mutant showed low hydrogen production and demonstrated significantly reduced growth under anaerobic conditions. The present work also provided first-hand data where deleterious effects of operon deletion were compared with single-gene deletion mutations and the results showed that the former type of deletion was found to cause more prominent phenotypic effects than the latter one. Interestingly, hyb operon mutant was remarkably distinct from other operon mutants, specifically in its inability to utilize glucose under both aerobic and anaerobic conditions. Further studies on this mutant revealed a significant reduction of the total intracellular ATP and NADH concentrations, which could explain its impaired glucose metabolism. In this way, Hyd-2 was verified as crucial not only in glucose metabolism but also in energy balance and redox homeostasis of the cells. Furthermore, a decreased expression of glucose metabolism-associated genes, particularly ppc and pykA, indicated their regulation by hyb operon, and thereby, glucose consumption. Moreover, the transcriptional changes in this mutant indicated the wide genomic connectivity of hyb operon to other metabolisms.

The ubiquitous signaling molecule autoinducer 2 (AI-2) is involved in intra- and interspecies communication, most notably between Gram-negative and Gram-positive bacteria. AI-2 accumulates during the exponential phase of the Escherichia coli (E. coli) monoculture and then rapidly decreases upon entry into the stationary phase. However, deleting both the genes encoding AI-2 synthase (LuxS) and the lsr operon regulator (LsrR) in the E. coli genome causes impaired AI-2 production and continuous AI-2 scavenging from the environment. This genetically-engineered E. coli mutant capable of quenching AI-2 quorum sensing (QS) system was utilized to evaluate the effect of AI-2 quenching on the anaerobic digestion of waste activated sludge (WAS) because the role of QS system via AI-2 in the process remains obscure. In this study, E. coli ∆luxS lsrR mutant cells were microencapsulated in sodium alginate beads and incubated with WAS anaerobically. After 15 days of anaerobic fermentation, the WAS containing double mutant cells produced significantly more methane than that of the parent E. coli cells. AI-2 quenching occurred concurrently with a shift of microbial communities that contribute to increasing acetate consumption by the Methanosarcina spp. resulting in an increase in methane production. KEY POINTS: • Impact of autoinducer 2 quenching in complex bacterial populations were determined. • Key microorganisms contributing to the increase of methane in WAS anaerobic digestion were found. • The AI-2 quenching is a potential regulatory in wastewater treatment and bioenergy research.

Persistence enables a subpopulation of tolerant bacteria to survive in the presence of a bactericidal antibiotic. Reports indicate the non-inherited nature of persistence; however, it also seems to be a genetically influenced phenomenon that has evolved to allow the organisms to survive in sudden environmental insults. In contrary to some reports, lowered available pool of ATP, NAD, and ROS leads to increased survival via slowing down the metabolism and resulting in an increased persister fraction. In this study, the hydrogenases of Escherichia coli were analyzed for their role in persistence, where four hydrogenase operon deletion mutants were evaluated before and after antibiotic treatment. As result, the expressions of hydrogenases and cellular viability (specifically hyd-1, 2, 3) were found elevated in antibiotic-treated cells, indicating their influential roles in persistence. Further work elucidated the affected ATP, NAD, and metabolism in the antibiotic-treated mutant cells. The transcriptomic analysis further revealed a wide genomic connectivity of hydrogenases to influence persistence in E. coli. Hydrogenases were mainly reported for their essential roles in bacterial hydrogen metabolism, this study further demonstrates their probable role in bacterial survival against antibiotic stress.

Zero-emission technology for palm oil mill effluent (POME) has led to a breakthrough in the palm oil industry in relation to the goal of sustainable development. However, there are limited resources on how this technology has affected the bacterial community in the receiving river that has previously been polluted with POME final discharge. Thus, the current study assessed the recoverability of the unexplored bacterial community in the receiving water of a constructed river water system post-zero emission of POME final discharge. An artificial river water system was constructed in this study, where the viability status and the composition of the bacterial community were assessed for 15 days using a flow cytometry-based assay and high-throughput sequencing by Illumina MiSeq, respectively. The zero-emission of POME final discharge reduced not only the physicochemical properties and nutrient contents of the receiving water, but also the bacterial cells’ viability from 40.3% to 24.5% and shifted the high nucleic acid (HNA) to low nucleic acid (LNA) content (38.7% to 34.5%). The proposed POME bacterial indicators, Alcaligenaceae and Chromatiaceae were not detectable in the rainwater (control) but were detected in the artificial river water system after the introduction of POME final discharge at the compositions of 1.0–1.3% and 2.2–5.1%, respectively. The implementation of a zero-emission system decreased the composition of Chromatiaceae from 2.2% on day 8 until it was undetectable on day 15, while Alcaligenaceae was continuously reduced from 1.2% to 0.9% within that similar time frame. As indicated by principal coordinate (PCO) analysis, the reductions in biological oxygen demand (BOD5) would further diminish the compositions of these bioindicators. The zero-emission of POME final discharge has demonstrated its efficacy, not only in reducing the polluting properties, but also in the bacterial biodiversity rebound in the affected water system.

Escherichia coli has approximately 4300 open reading frames and about 178 of them are annotated as pseudogenes. The existence of the pseudogenes in E. coli had raised the question of whether they contribute to any cell function. Recently, several pseudogenes have been studied; however, these genes are still considered rare and tend to be ignored as so-called junk. Our recent study has shown several pseudogenes in E. coli are essential in hydrogen metabolism. ydfW is one of the pseudogenes associated with hydrogen metabolism. Here, we found carbon dioxide and nitrate in the fermentation medium of mutant ydfW suggesting that this strain may induce nitrate pathways. The Fdh-N complex is comprised of FdnG, FdnH, and FdnI subunits which correspond to the nitrate-nitrite pathway. This finding is supported by transcription analysis that shows formate dehydrogenase (fdnG) and nitrate reductase (narG) are significantly induced (25- and 18-fold, respectively). FdnG and NarG play a significant role in the reduction of nitrate into nitrite; hence, their genes are induced by the build-up of nitrate. Based on in silico docking analysis, we predict that YdfW is covalently bound to the Fdh-N complex in the cytoplasm. Hence, deletion of ydfW is expected has influenced the nitrate-nitrite pathway. Subsequently, the situation will affect the hydrogen production in E. coli.

Pseudomonas aeruginosa RW9 is a promising candidate for the bioremediation of chromium hexavalent (Cr(VI)) pollution, as it resists a high concentration of up to 60 mg/L of Cr(VI). Leaving cells exposed to Cr(VI) has large bioreduction potential, implying its capacity to extract the ions from the contaminated medium. In this study, the tolerance for and distribution of Cr(VI) were investigated to identify the cells’ adaptation and removal strategies. Micro-characterization analysis was conducted to assess the effect of Cr(VI) on the cells. The cells’ elongation was observed at higher Cr(VI) concentrations, signifying their adaptation to DNA damage caused by Cr(VI) toxicity. Cr(VI) distribution analysis showed that the strain developed a complex mechanism to adapt to Cr(VI), based on surface-bound (0.46 mg/L), intracellularly accumulated (1.24 mg/L) and extracellular sequestration (6.74 mg/L), which accounted for 85% of the removal efficiency. The extracellular sequestration might be attributable to the production of metabolites, in accordance with the fourier-transform infrared spectroscopy (FTIR) spectra and orcinol analysis that confirmed the presence of a glycolipid biosurfactant, rhamnolipid. Remarkably, the rhamnolipid was slightly induced in the presence of Cr(VI). From the data obtained, it was confirmed that this local strain is well equipped to survive high doses of Cr(VI) and has great potential for application in Cr(VI) bioremediation.

Photocatalysis has gained attention as a viable wastewater remediation technique. However, the difficulty of recovering powder-based photocatalyst has often become a major limitation for their on-site practical application. Herein, we report on the successful in-situ preparation of a novel three-dimensional (3D) photocatalyst consisting of Cu

Aim As options to treat recalcitrant bacterial infections which are increasingly limited due to multidrug-resistant strains, searching for new, effective antibacterial compounds is necessary. One strategy is to generate treatment alternatives by drug repurposing. Methods and results In this work, phenotypic microarrays were used for the screening of miscellaneous compounds against the growth and biofilm formation of Acinetobacter baumannii, an important emergent multidrug-resistant opportunistic pathogen. The results showed that the phenothiazine derivatives, such as promethazine, trifluoperazine, thioridazine, and chlorpromazine, inhibited the growth of antibiotic-sensitive and multidrug-resistant strains (showing minimal inhibitory concentrations ranging from 0·05 to 0·6 g l-1 and minimal bactericidal concentrations ranging from 0·1 to 2·5 g l-1 ). All phenothiazine derivatives were active against biofilm cells (with minimal biofilm eradication concentrations ranging from 0·5 to >3 g l-1 ). Chlorpromazine promoted reactive oxigen species (ROS) production, and cell membrane and DNA damage. Chlorpromazine showed synergy with antibiotics such as ceftazidime, meropenem, and colistin and was an effective treatment for experimentally infected Galleria mellonella when combined with ceftazidime. Conclusions It was demonstrated that phenothiazine derivatives, especially chlorpromazine, are drugs with attractive antibacterial properties against nosocomial MDR strains of A. baumannii, by generating ROS and cell membrane and DNA damage. Significance and impact of the study The present study indicates that repurposing phenothiazine derivatives for treating recalcitrant infections by A. baumannii could be promising.

Chitosan nanofiber has a highly uniform structure of 20–50 nm in diameter and shows high dispersibility in water due to its submicron size and high surface-to-volume ratio. The stacked nanofibers film is useful for breathability because it has a gap with a size of several tens of nm or more. However, the chemical bonds between the nanofibers cannot be broken during use. In this study, the thin films were obtained by filtration of chitosan nanofibers and 3-glycidoxypropyltrimethoxysilane (GPTMS) mixture. The addition of GPTMS changed the wettability, mechanical property and stability in water of the thin films. Bacitracin zinc salt (BZ) has been used for the localized dermatological medicines and loaded in the films. BZ interacted electrostatically with the thin films matrix and the release of BZ was controlled by the amount of GPTMS. A higher released amount of BZ showed higher antibacterial effects toward S. aureus. The film was also tested their toxicity by L929 fibroblasts. The release of less than 11.9 μg of BZ showed antibacterial effects, but were not toxic for fibroblast cells.