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Escherichia coli (E. coli) cells are present in fecal materials that can be the main source for disease‐causing agents in water. As a result, E. coli is recommended as a water quality indicator. We have developed an innovative platform to detect E. coli for monitoring water quality on-site by integrating paper-based sample preparation with nucleic acid isothermal amplification. The platform carries out bacterial lysis and DNA enrichment onto a paper pad through ball-based valves for fluid control, with no need of laboratory equipment, followed by loop-mediated isothermal amplification (LAMP) in a battery-operated coffee mug, and colorimetric detection. We have used the platform to detect E. coli in environmental water samples in about 1 h, with a limit of quantitation of 0.2 CFU/mL, and 3 copies per reaction. The platform was confirmed for detecting multiple E. coli strains, and for water samples of different salt concentrations. We validated the functions of the platform by analyzing recreational water samples collected near the Atlantic Ocean that contain different concentrations of salt and bacteria. [ABSTRACT FROM AUTHOR]

The Pseudomonas aeruginosa RsaL is a negative regulator of the quorum sensing signal synthesis gene lasI. The expression of RsaL is directly activated by the LasI cognate regulator LasR. Thus, RsaL and LasI-LasR (LasI/R) form a regulatory loop. Further studies revealed that RsaL is a global regulator which controls the expression of numerous genes through quorum sensing system dependent and independent pathways. However, whether RsaL is involved in antibiotic tolerance remains elusive. In this study, we found that the mutation of rsaL increased bacterial tolerance to ciprofloxacin and carbenicillin. Through motif search, gene expression analyses and electrophoretic mobility shift assays, we found that RsaL directly represses the expression of the narK1K2GHJI operon, which is involved in the tolerance to ciprofloxacin. We further demonstrated that the narK1K2GHJI operon is directly regulated by LasR. In combination, our study revealed a novel operon under the control of the RsaL, LasI/R regulatory loop. [ABSTRACT FROM AUTHOR]

Expression of a putative acyltransferase encoded by NCgl- 0350 of Corynebacterium glutamicum is induced by cell-free culture fluids obtained from stationary-phase growth of both C. glutamicum and Pseudomonas aeruginosa, providing evidence for interspecies communication. Here, we further confirmed that such communication occurs by showing that acyltransferase expression is induced by culture fluid obtained from diverse Gram-negative and -positive bacterial strains, including Escherichia coli, Salmonella Typhimurium, Bacillus subtilis, Staphylococcus aureus, Mycobacterium sp. strain JC1, and Mycobacterium smegmatis. A homologous acyltransferase encoded by PA5238 of P. aeruginosa was also induced by fluids obtained from P. aeruginosa as well as other bacterial strains, as observed for NCgl0350 of C. glutamicum. Because C. glutamicum is difficult to study using molecular approaches, the homologous gene PA5238 of P. aeruginosa was used to identify PA5309 as an upstream regulator of expression. A homologous D-amino acid dehydrogenase encoded by NCgl- 2909 of C. glutamicum was cloned based on amino acid similarity to PA5309, and its role in the regulation of NCgl0350 expression was confirmed. Moreover, NCgl2909 played positive roles in growth of C. glutamicum. Thus, we identified a D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in C. glutamicum. [ABSTRACT FROM AUTHOR]

Ribosome is responsible for protein synthesis and is able to monitor the sequence and structure of the nascent peptide. Such ability plays an important role in determining overall gene expression profile of the bacteria through ribosome stalling and rescuing. In this review, we briefly summarize our current understanding of the regulation of gene expression through ribosome stalling and rescuing in bacteria, as well as mechanisms that modulate ribosome activity. Understanding the mechanisms of how bacteria modulate ribosome activity will provide not only fundamental insights into bacterial gene regulation, but also new candidate targets for the development of novel antimicrobial agents. [ABSTRACT FROM AUTHOR]

Infections are often not caused by a colonization of Pseudomonas aeruginosa alone but by a consortium of other bacteria. Little is known about the impact of P. aeruginosa on the growth of other bacteria upon coinfection. Here, cellree culture supernatants obtained from P. aeruginosa suppressed the growth of a number of bacterial strains such as Corynebacterium glutamicum, Bacillus subtilis, Staphylococcus aureus, and Agrobacterium tumefaciens, but had little effect on the growth of Escherichia coli and Salmonella Typhimurium. The growth suppression effect was obvious when P. aeruginosa was cultivated in M9 minimal media, and the suppression was not due to pyocyanin, a well-known antimicrobial toxin secreted by P. aeruginosa. By performing transposon mutagenesis, PA5070 encoding TatC was identified, and the culture supernatant of its mutant did not suppress the growth. HPLC analysis of supernatants showed that pyoverdine was a secondary metabolite present in culture supernatants of the wild-type strain, but not in those of the PA5070 mutant. Supplementation of FeCl as a source of iron compromised the growth suppression effect of supernatants and also recovered biofilm formation of S. aureus, indicating that pyoverdine-mediated iron acquisition is responsible for the growth suppression. Thus, this study provides the action of TatC-dependent pyoverdine translocation for the growth suppression of other bacteria, and it might aid understanding of the impact of P. aeruginosa in the complex community of bacterial species upon coinfection. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor-II (IGF2) is a growth factor for the control of cell proliferation and apoptosis. To explore the clinical use of human IGF2, an efficient method for production of a large amount of active recombinant hIGF2 is necessary. Human IGF2 cDNA was cloned into pET32 vector where it is under the control of an IPTGinducible T7 promoter. High level soluble thioredoxin (Trx)-hIGF2 fusion protein was produced at room temperature following IPTG induction, amounting up to 20% of the total soluble bacterial proteins. The recombinant Trx-hIGF2 fusion protein was purified to an approximate 95% purity using Ni-NTA affinity chromatography with an overall yield of 120 mg protein per liter of bacterial culture. After cleavage of the Trx fusion fragment by recombinant enterokinase, the tag-free recombinant hIGF2 protein (rhIGF2) was purified by passage through the Ni+-NTA affinity column again. Biological activity of the purified hIGF2 was determined by its ability to support NIH/3T3 cells proliferation and to activate AKT signaling pathways. Our results demonstrate that tag-free active rhIGF2 can easily be obtained for various applications from E. coli using the procedure described in this report. [ABSTRACT FROM AUTHOR]

The proinflammatory cytokine interleukin-1β plays an important role in protecting the host against airway infection; however, it can also trigger a massive influx of neutrophils into the airways, causing tissue damage. Anti-inflammatory treatments are particularly in demand for patients suffering from chronic inflammatory diseases. Sophora flavescens is a traditional herbal medicine used to reduce inflammation, but no study has examined its ability to block IL-1β production. Here, we show that S. flavescens reduced the Pseudomonas aeruginosa-induced expression of IL-1β by lung epithelial cells and macrophages. S. flavescens was also effective at reducing IL-1β production induced by either Staphylococcus aureus or phorbol 12-myristate 13-acetate, indicating that the effect is generalizable to diverse inflammatory stimuli. In addition, S. flavescens blocked the phosphorylation of IKKα/β, key upstream kinases involved in the degradation of IκBα, and the cleavage of caspase-1, a key component of the inflammasome. Thus, this study demonstrates that S. flavescens exerts its anti-inflammatory effects by blocking P. aeruginosa-mediated NF-κB/inflammasome activation and the subsequent production of IL-1β. [ABSTRACT FROM AUTHOR]

genetic locus, encoding putative acyltransferase, was induced by autoinducers in Corynebacterium glutamicum. The autoinducers were maximally produced by the bacterium after 24 h culture. Those molecules are resistant to proteinase K treatment (300 μg ml) for 30 min at 37°C or at 121°C for 15 min, and remained stable after extensive storage at 4°C. Autoinducers in the cell-free culture fluids from Corynebacterium ammoniagenes and Pseudomonas aeruginosa also induced the expression of acyltransferase in C. glutamicum, suggesting possible cross-recognition of the autoinducers by C. glutamicum. C. glutamicum thus possesses an autoinduction system which secretes autoinducers during growth, triggering the expression of downstream genes, exemplified by the putative acyltransferase gene. [ABSTRACT FROM AUTHOR]

Epithelial cells act as the first line of host defense against microbes by producing a range of different molecules for clearance. Chemokines facilitate the clearance of invaders through the recruitment of leukocytes. Thus, upregulation of chemokine expression represents an important innate host defense response against invading microbes such as Streptococcus pneumoniae. In this study, we report that the expression of Monocyte Chemotactic Protein 1 (MCP1) was highly induced in response to S. pneumoniae in vitro and in vivo. Among numerous virulence factors, pneumococcal pneumolysin was found to be the major factor responsible for this induction. Furthermore, MCP1 induction was mediated by the p38 mitogen-activated protein kinase (MAPK) whose activation was controlled by MAPK phosphatase 1 (MKP1). Therefore, this study reveals novel roles of pneumolysin in mediating MKP1 expression for the regulation of MCP1 expression in human epithelial cells. [ABSTRACT FROM AUTHOR]