Mid-logarithmic Growth Phase Research Articles

Metabolic engineering of Escherichia coli for seleno-methylselenocysteine production

Selenium (Se) is an essential trace element for life. Seleno-methylselenocysteine (SeMCys) can serve as a Se supplement with anticarcinogenic activity and can improve cognitive deficits. We engineered Escherichia coli for microbial production of SeMCys. The genes involved in the synthesis of SeMCys were divided into three modules–the selenocysteine (SeCys) synthesis, methyl donor synthesis and SMT modules–and expressed in plasmids with different copy numbers. The higher copy number of the SeCys synthesis module facilitated SeMCys production. The major routes for SeCys degradation were then modified. Deletion of the cysteine desulfurase gene csdA or sufS improved SeMCys production the most, and the strain that knocked out both genes doubled SeMCys production. The addition of serine in the mid-logarithmic growth phase significantly improved SeMCys synthesis. When the serine synthetic pathway was enhanced, SeMCys production increased by 12.5 %. Fed-batch culture for sodium selenite supplementation in the early stationary phase improved SeMCys production to 3.715 mg/L. This is the first report of the metabolic engineering of E. coli for the production of SeMCys and provide information on Se metabolism.

An insight into algicidal characteristics of Bacillus altitudinis G3 from dysfunctional photosystem and overproduction of reactive oxygen species

Cyanobacterial blooms negatively affect aquatic ecosystems and human health. Algicidal bacteria can efficiently kill bloom-causing cyanobacteria. Bacillus altitudinis G3 isolated from Dianchi Lake shows high algicidal activity against Microcystis aeruginosa. In this study, we investigated its algicidal characteristics including attack mode, photosynthesis responses, and source and the contribution of reactive oxygen species (ROS). The results showed that G3 efficiently and specifically killed M. aeruginosa mainly by releasing both thermolabile and thermostable algicidal substances, which exhibited the highest algicidal activity (99.8%, 72 h) in bacterial mid-logarithmic growth phase. The algicidal ratio under full-light conditions (99.5%, 60 h) was significantly higher than under dark conditions (<20%, P < 0.001). G3 filtrate caused photosystem dysfunction by decreasing photosynthetic efficiency, as indicated by significantly decreased Fv/Fm and PIABS (P < 0.001) values. It also inhibited photosynthetic electron transfer as indicated by significantly decreased rETR (P < 0.001), especially QA− downstream, as revealed by significantly decreased φEo and ψo, and increased Mo (P < 0.001). These results indicated that the algicidal activity of G3 filtrate is light-dependent, and the cyanobacterial photosystem is an important target. Cyanobacterial ROS and malondialdehyde contents greatly increased by 37.1% and 208% at 36 h, respectively. ROS levels decreased by 49.2% (9 h) when diuron (3-(3-4-dichlorophenyl)-1,1-dimethylurea) partially blocked photosynthetic electron transport from QA to QB. Therefore, excessive ROS were produced from disrupted photosynthesis, especially the inhibited electron transport area in QA− downstream, and caused severe lipid peroxidation with significantly increased MDA content and oxidative stress in cyanobacteria. The ROS scavenger N-acetyl-l-cysteine significantly decreased both cyanobacterial ROS levels (34%) and algicidal ratio (52%, P < 0.05) at 39 h. Thus, excessive ROS production due to G3 filtrate administration significantly contributed to its algicidal effect. G3 could be an excellent algicide to control M. aeruginosa blooms in waters under suitable light conditions.

Microbial metabolomics and network analysis reveal fungistatic effect of basil (Ocimum basilicum) oil on Candida albicans

Ethnopharmacological relevanceFungal infections remain a serious problem worldwide that require effective therapeutic strategies. Essential oil of basil (Ocimum basilicum L., BEO) being traditionally used extensively for the treatment of bacterial and fungal infection has a long history. However, the potential mechanism of action was still obscure, especially from the metabolic perspective. Materials and methodsThe fungistatic effect of BEO on Candida albicans (C. albicans) was evaluated by measurement of minimum inhibitory concentration (MIC) and morphological analysis. A high-coverage microbial metabolomics approach was utilized to identify the alterations of intracellular metabolites of C. albicans at mid-logarithmic growth phase in response to the subinhibitory concentration of BEO, by using gas chromatography coupled to time-of-fight mass spectrometry (GC-TOFMS). Following the metabolic fingerprinting, systematic network analysis was performed to illustrate the potential mechanism of BEO involved in the suppression of C. albicans. ResultsThe damage in cellular membranes of C. albicans treated by BEO above MIC was observed on the scanning electron microscope (SEM) micrographs. Metabolomics results showed that, among 140 intracellular metabolites identified by comparison with reference standards, thirty-four had significantly changed abundances under 0.2 MIC of BEO treatment, mainly involving in central carbon metabolism (glycolysis/gluconeogenesis, pentose phosphate pathway and TCA cycle), amino acids, polyamines and lipids metabolism. Pathway and network analyses further found that fifteen ingredients of BEO mainly terpenoids and phenyl-propanoids, potentially participated in the metabolic regulation and may be responsible for the suppression of C. albicans. ConclusionsThe findings highlighted that integrated microbial metabolomics and network analyses could provide a methodological support in understanding the functional mechanisms of natural antimicrobial agents and contribute to drug discovery.

Pan-Proteomic Analysis and Elucidation of Protein Abundance among the Closely Related Brucella Species, Brucella abortus and Brucella melitensis.

Brucellosis is a zoonotic infection caused by bacteria of the genus Brucella. The species, B. abortus and B. melitensis, major causative agents of human brucellosis, share remarkably similar genomes, but they differ in their natural hosts, phenotype, antigenic, immunogenic, proteomic and metabolomic properties. In the present study, label-free quantitative proteomic analysis was applied to investigate protein expression level differences. Type strains and field strains were each cultured six times, cells were harvested at a midlogarithmic growth phase and proteins were extracted. Following trypsin digestion, the peptides were desalted, separated by reverse-phase nanoLC, ionized using electrospray ionization and transferred into an linear trap quadrapole (LTQ) Orbitrap Velos mass spectrometer to record full scan MS spectra (m/z 300–1700) and tandem mass spectrometry (MS/MS) spectra of the 20 most intense ions. Database matching with the reference proteomes resulted in the identification of 826 proteins. The Cluster of Gene Ontologies of the identified proteins revealed differences in bimolecular transport and protein synthesis mechanisms between these two strains. Among several other proteins, antifreeze proteins, Omp10, superoxide dismutase and 30S ribosomal protein S14 were predicted as potential virulence factors among the proteins differentially expressed. All mass spectrometry data are available via ProteomeXchange with identifier PXD006348.

Direct Binding and Regulation by Fur and HapR of the Intermediate Regulator and Virulence Factor Genes Within the ToxR Virulence Regulon in Vibrio cholerae.

Cholera toxin (CT) and toxin coregulated pilus (TCP, TcpA is the major subunit) are two major virulence factors of Vibrio cholerae, both of which play critical roles in developing severe diarrhea in human. Expression of CT and TCP is under the tight control of the regulatory cascade known as the ToxR virulence regulon, which is composed of three regulators ToxR, TcpP, and ToxT. Besides, their expression is also regulated by the quorum sensing (QS) master regulator HapR and the regulatory protein Fur. Though transcription of tcpP, toxT, and/or tcpA are reported to be regulated by HapR and Fur, to date there are no studies to verify their direct regulations. In the present study, we showed that HapR directly repress the transcription of tcpP and tcpA by binding to their promoter regions, and possibly repress toxT transcription in an indirect manner. Fur directly activated the transcription of tcpP, toxT, and tcpA by binding to their promoters. Taking account of the sequential expression of hapR, fur, tcpP, toxT, and tcpA in the different growth phases of V. cholerae, we deduce that at the early mid-logarithmic growth phase, Fur binds to the promoters of tcpP, toxT, and tcpA to activate their transcription; while at the later mid-logarithmic growth phase, HapR can bind to the promoters of tcpP and tcpA to repress their transcription. Our study reveals the new recognition in the virulence regulatory pathways in V. cholerae and suggests the complicated and subtle regulation network with the growth density dependence.

In-situ electrochemical analysis of microbial activity

Microbes have a wide range of metabolic capabilities available that makes them industrially useful organisms. Monitoring these metabolic processes is a crucial component in efficient industrial application. Unfortunately, monitoring these metabolic processes can often be invasive and time consuming and expensive, especially within an anaerobic environment. Electrochemical techniques, such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) offer a non-invasive approach to monitor microbial activity and growth. EIS and CV were used to monitor Clostridium phytofermentans, an anaerobic and endospore-forming bacterium. C. phytofermentans ferments a wide range of sugars into hydrogen, acetate, and ethanol as fermentation by-products. For this study, both traditional microbiological and electrochemical techniques were used to monitor the growth of C. phytofermentans and the formation of fermentation products. An irreversible reduction peak was observed using CV beginning at mid-logarithmic phase of growth. This peak was associated with C. phytofermentans and not the spent medium and was indicative of a decrease in carbon and energy sources to the cells. Additionally, EIS analysis during growth provided information related to increased charge transfer resistance of the culture also as a function of carbon and energy source depletion. Results demonstrate that CV and EIS are useful tools in the monitoring the physiological status of bioprocesses.

Expression of Hemolysin Is Regulated Under the Collective Actions of HapR, Fur, and HlyU in Vibrio cholerae El Tor Serogroup O1.

The biotype El Tor of serogroup O1 and most of the non-O1/non-O139 strains of Vibrio cholerae can produce an extracellular pore-forming toxin known as cholera hemolysin (HlyA). Expression of HlyA has been previously reported to be regulated by the quorum sensing (QS) and the regulatory proteins HlyU and Fur, but lacks the direct evidence for their binding to the promoter of hlyA. In the present work, we showed that the QS regulator HapR, along with Fur and HlyU, regulates the transcription of hlyA in V. cholerae El Tor biotype. At the late mid-logarithmic growth phase, HapR binds to the three promoters of fur, hlyU, and hlyA to repress their transcription. At the early mid-logarithmic growth phase, Fur binds to the promoters of hlyU and hlyA to repress their transcription; meanwhile, HlyU binds to the promoter of hlyA to activate its transcription, but it manifests direct inhibition of its own gene. The highest transcriptional level of hlyA occurs at an OD600 value of around 0.6–0.7, which may be due to the subtle regulation of HapR, Fur, and HlyU. The complex regulation of HapR, Fur, and HlyU on hlyA would be beneficial to the invasion and pathogenesis of V. cholerae during the different infection stages.

CRP-cAMP mediates silencing of Salmonella virulence at the post-transcriptional level.

Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3’UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3’UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3’UTR as a hub for post-transcriptional control of Salmonella invasion gene expression.

Comparative exoproteome analyses of Lactobacillus spp. reveals species- and strain-specific proteins involved in their extracellular interaction and probiotic potential

Due to their health-promoting effects, the probiotic applications of lactobacilli have been investigated. Aside from their basic cellular functions, the extracellular activities of probiotic lactobacilli influence their establishment and interaction with the host. Many extracellular proteins play important roles in bacterial colonization and survival in their host's gastrointestinal environment. In this study, we compared the exoproteome of three lactobacillus strains isolated from the gastrointestinal tracts of pigs and chickens, namely, Lactobacillus mucosae LM1, L. johnsonii PF01, and L. johnsonii C1–10. Extracellular proteins collected in the mid-logarithmic growth phase were identified and quantified using a Q Exactive™ Orbitrap mass spectrometer (MS). Of 99 total extracellular proteins, 83% belonged to L. mucosae LM1; L. johnsonii PF01 strains had fewer extracellular proteins. Enolase, which is involved in the glycolysis pathway and has moonlighting functions in the adhesion of probiotic bacteria, was found in the core exoproteomes of the three strains. The most abundant proteins of each strain were phosphoketolase in L. mucosae LM1, aggregation-promoting factor in L. johnsonii PF01, and levansucrase in L. johnsonii C1–10. The observed differences between these three exoproteomes in terms of cellular and molecular function elucidate the extracellular activities of these isolates.

Transcriptional Regulation of the Type VI Secretion System 1 Genes by Quorum Sensing and ToxR in Vibrio parahaemolyticus.

Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis, harbors two separate T6SSs on chromosomes 1 and 2, i.e., T6SS1 (VP1386-1420) and T6SS2 (VPA1025-1046). T6SS1 contains at least 7 putative operons: VP1386-1387, VP1388-1390, VP1392-1391, VP1393-1406, VP1400-1406, VP1409-1407, and VP1410-1420. V. parahaemolyticus AphA and OpaR are the two master regulators of quorum sensing (QS) system that are highly expressed at low cell density and high cell density, respectively. ToxR is a membrane-bound virulence regulatory protein conserved across the Vibrio family. In the present work, we show that ToxR coordinates with AphA and OpaR to repress T6SS1 expression in V. parahaemolyticus. OpaR binds to the promoters of VP1388-1390, VP1400-1406, and VP1409-1407 to repress their transcription, but it appears to negatively regulate VP1393-1406 transcription in an indirect manner. By contrast, AphA negatively regulated the above four T6SS1 operons in an indirect manner. In addition, ToxR binds to the promoters of VP1400-1406 and VP1409-1407 to inhibit their transcription, but it presents an indirect interaction with VP1388-1390 and VP1393-1406 promoters. Notably, the expression of ToxR also manifested in a QS-dependent manner and the highest expression occurred at LCD. Meanwhile, the highest expression of T6SS1 occurred at an OD600 value of 0.6 to 0.8 due to the tight regulation of ToxR and QS, suggesting T6SS1 functions only during the mid-logarithmic growth phase. These observations provide significant insight into the molecular mechanism of T6SS1 gene regulation by QS and ToxR in V. parahaemolyticus.

Nanoparticles surface treatment on cemented materials for inhibition of bacterial growth

Bacterial growth on cemented materials such as concrete can cause degradation and early ageing. This paper explores the bactericidal characteristics of cementitious materials surface treated with zinc oxide (ZnO) and silver (Ag) nanoparticles. The growth of gram positive Bacillus cereus and gram negative Escherichia coli has been monitored. The growth inhibitory performance of nanoparticles against two model bacteria was further examined at different surface pH of mortar samples and different bacterial cell densities. After the seven-day experimental period, more than 60% reduction in the number of viable cells of both model bacteria was obtained at all tested pH values when nanoparticles with a concentration of 250mg/l were added at the beginning of culturing time. However, delayed addition of nanoparticles at the mid-logarithmic growth phase, i.e., higher cell densities, resulted in less growth inhibition. Ag nanoparticles showed the highest inhibition due to the high degree of nanoparticle stability and uniform particle size. On the other hand, the stability of ZnO nanoparticles in the experimental mixture was significantly influenced by the pH change and aggregation, which resulted in lower inhibition efficiencies. Gram-positive Bacillus cereus was found to be less sensitive to both tested nanoparticles, compared with Gram-negative Escherichia coli. The experiment demonstrates the efficacy of nanoparticles in inhibiting bacterial growth on cementitious materials.

Micafungin alters the amino acid, nucleic acid and central carbon metabolism of Candida albicans at subinhibitory concentrations: novel insights into mechanisms of action.

Echinocandins are an important class of antifungal agents in the treatment of invasive candidiasis. However, little is known about the metabolomic effects of echinocandins on Candida . We therefore performed LC-high-resolution MS (LC-HRMS)-based metabolomics profiling of the response of Candida albicans cells to increasing concentrations of micafungin to determine the metabolic response of Candida to micafungin subinhibitory injury. Isolates of C. albicans were cultured on nitrocellulose filters to mid-logarithmic phase of growth and micafungin (0-0.25 mg/L) was added. At mid-logarithmic phase, replicates were metabolically quenched. Intracellular metabolites were analysed by LC-HRMS. Changes in pool sizes of individual metabolites were analysed by Student's t -test adjusted for multiple hypothesis testing by Benjamini-Hochberg correction. Metabolites were ascribed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways database. Among 3446 detected metabolites, 204 were identified by comparison against pure standard or comparison against a library of mass-retention-time pairs. Fifty had significantly altered abundances in response to increasing micafungin concentrations. Pool sizes of amino acids, nucleic acids and polyamine metabolism were significantly increased at subinhibitory concentrations, while exposure to inhibitory concentrations resulted in a precipitous decrease consistent with fungicidal activity. Micafungin induces a re-routing of metabolic pathways inhibiting protein synthesis and cell replication. These results shed light on new mechanisms of action of echinocandins.

Bioemulsifier (BE-AM1) produced by Solibacillus silvestris AM1 is a functional amyloid that modulates bacterial cell-surface properties

A novel estuarine bacterial strain, Solibacillus silvestris AM1, produces an extracellular, thermostable and fibrous, glycoprotein bioemulsifier (BE-AM1). The amyloid nature of the bioemulsifier (BE-AM1) was confirmed by biophysical techniques (Congo red based polarization microscopy, ThioflavinS based fluorescent microscopy, fibrous arrangement in transmission electron microscopy and secondary structure measurement by FTIR and CD spectrum analysis). Cell-bound BE-AM1 production by S. silvestris AM1 during the mid-logarithmic phase of growth coincided with a decrease in cell surface hydrophobicity, and an increase in cell autoaggregation and biofilm formation. It was observed that the total interfacial interaction energy () for the surface of the bioemulsifier producing S. silvestris AM1 and different derivatized surfaces of polystyrene (silanized and sulfonated) was found to support biofilm formation. This study has revealed that the BE-AM1, a bacterial bioemulsifier, is a functional amyloid and has a role in biofilm formation and cell surface modulation in S. silvestris AM1.

Effects of fluconazole on the metabolomic profile of Candida albicans.

Little is known about the effects of fluconazole on the metabolism of Candida albicans. We performed LC/MS-based metabolomic profiling of the response of C. albicans cells to increasing doses of fluconazole. C. albicans cells were cultured to mid-logarithmic growth phase in liquid medium and then inoculated in replicate on to nitrocellulose filters under vacuum filtration. Organisms were cultured to mid-logarithmic growth phase and treated with 0-4 mg/L fluconazole. Following metabolic quenching at mid-logarithmic growth phase, intracellular metabolites were extracted and analysed by LC/MS. Changes in pool sizes of individual metabolites were verified by Student's t-test, adjusted for multiple hypothesis testing by Benjamini-Hochberg correction. Distribution of metabolites was analysed by the Kyoto Encyclopedia of Genes and Genomes metabolic pathways database. We reproducibly detected 64 metabolites whose identities were confirmed by comparison against a pure standard and a library of accurate mass-retention time pairs. These 64 metabolites were broadly representative of eukaryotic central metabolic pathways. Among them 12 had their mean abundance significantly altered in response to increasing fluconazole concentrations. Pool sizes of four intermediates of central carbon metabolism (α-ketoglutarate, glucose-6-phosphate, phenylpyruvate and ribose-5-phosphate) and mevalonate were increased by 0.5-1.5-fold (P ≤ 0.05). Five amino acids (glycine, proline, tryptophan, aminoisobutanoate and asparagine) and guanine were decreased by 0.5-0.75-fold (P ≤ 0.05). Fluconazole treatment of C. albicans resulted in increased central carbon and decreased amino acid synthesis intermediates, suggesting a rerouting of metabolic pathways. The function of these metabolomic changes remains to be elucidated; however, they may represent previously unrecognized mechanisms of metabolic injury induced by fluconazole against C. albicans.

Screening and Characterization of Bacteriocin-Like Inhibitory Substances Produced by Bangladeshi Strains of Bacillus thuringiensis

Bacteriocins are ribosomally synthesized antibacterial peptides which have the potential to be used as natural food preservatives as well as alternative to antibiotics. Here, we report the production of bacteriocin-like inhibitory substances (BLIS) from the indigenous strains of Bacillus thuringiensis. Deferred antagonism bacteriocin assay and agar well diffusion methods suggested that several of the tested strains have high levels of bacteriocin-like activity against the common food-borne pathogens, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus. These bacteriocins are generally produced at the mid-logarithmic phase of growth with optimum temperature of 37 ºC, pH-7.0 and 24 h of incubation. Heat stability assay demonstrated that the bacteriocins produced from the strains are highly heat stable and can retain activity up to 100 ºC. Our study suggests that these bacteriocins may be potential candidates for use as biodegradable natural food preservatives and alternative antimicrobial agents to solve the increasing trends of problems of antibiotic resistance.Bangladesh Pharmaceutical Journal 18(2): 149-156, 2015

Enhanced intracellular survival and epithelial cell adherence abilities of Burkholderia pseudomallei morphotypes are dependent on differential expression of virulence-associated proteins during mid-logarithmic growth phase.

Comparative analyses were performed on the ability of wild-type and small colony variant of B. pseudomallei to adhere, invade and form plaques on human epithelial cells. In addition, proteomic analysis of these different colony morphotypes was also carried out. This research provides insights into the virulence and pathogenesis attributes of B. pseudomallei and contributes to better understand the pathogenesis of melioidosis.

A ΔdinB mutation that sensitizes Escherichia coli to the lethal effects of UV- and X-radiation

The DinB (PolIV) protein of Escherichia coli participates in several cellular functions. We investigated a dinB mutation, Δ(dinB-yafN)883(::kan) [referred to as ΔdinB883], which strongly sensitized E. coli cells to both UV- and X-radiation killing. Earlier reports indicated dinB mutations had no obvious effect on UV radiation sensitivity which we confirmed by showing that normal UV radiation sensitivity is conferred by the ΔdinB749 allele. Compared to a wild-type strain, the ΔdinB883 mutant was most sensitive (160-fold) in early to mid-logarithmic growth phase and much less sensitive (twofold) in late log or stationary phases, thus showing a growth phase-dependence for UV radiation sensitivity. This sensitizing effect of ΔdinB883 is assumed to be completely dependent upon the presence of UmuDC protein; since the ΔdinB883 mutation did not sensitize the ΔumuDC strain to UV radiation killing throughout log phase and early stationary phase growth. The DNA damage checkpoint activity of UmuDC was clearly affected by ΔdinB883 as shown by testing a umuC104 ΔdinB883 double-mutant. The sensitivities of the ΔumuDC strain and the ΔdinB883 ΔumuDC double-mutant strain were significantly greater than for the ΔdinB883 strain, suggesting that the ΔdinB883 allele only partially suppresses UmuDC activity. The ΔdinB883 mutation partially sensitized (fivefold) uvrA and uvrB strains to UV radiation, but did not sensitize a ΔrecA strain. A comparison of the DNA sequences of the ΔdinB883 allele with the sequences of the Δ(dinB-yafN)882(::kan) and ΔdinB749 alleles, which do not sensitize cells to UV radiation, revealed ΔdinB883 is likely a “gain-of-function” mutation. The ΔdinB883 allele encodes the first 54 amino acids of wild-type DinB followed by 29 predicted residues resulting from the continuation of the dinB reading frame into an adjacent insertion fragment. The resulting polypeptide is proposed to interfere directly or indirectly with UmuDC function(s) involved in protecting cells against the lethal effects of radiation.

Rapid Protocol for Preparation of Electrocompetent &lt;em&gt;Escherichia coli&lt;/em&gt; and &lt;em&gt;Vibrio cholerae&lt;/em&gt;

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent &lt;em&gt;Escherichia coli&lt;/em&gt; and &lt;em&gt;Vibrio cholerae&lt;/em&gt;

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Automated SPME–GC–MS monitoring of headspace metabolomic responses of E. coli to biologically active components extracted by the coating

Monitoring extracellular metabolites of bacteria is very useful for not only metabolomics research but also for assessment of the effects of various chemicals, including antimicrobial agents and drugs. Herein, we describe the automated headspace solid-phase microextraction (HS-SPME) method coupled with gas chromatography–mass spectrometry (GC–MS) for the qualitative as well as semi-quantitative determination of metabolic responses of Escherichia coli to an antimicrobial agent, cinnamaldehyde. The minimum inhibitory concentration of cinnamaldehyde was calculated to be 2gL−1. We found that cinnamaldehyde was an important factor influencing the metabolic profile and growth process. A higher number of metabolites were observed during the mid-logarithmic growth phase. The metabolite variations (types and concentrations) induced by cinnamaldehyde were dependent on both cell density and the dose of cinnamaldehyde. Simultaneously, 25 different metabolites were separated and detected (e.g., indole, alkane, alcohol, organic acids, esters, etc.) in headspace of complex biological samples due to intermittent addition of high dose of cinnamaldehyde. The study was done using an automated system, thereby minimizing manual workup and indicating the potential of the method for high-throughput analysis. These findings enhanced the understanding of the metabolic responses of E. coli to cinnamaldehyde shock effect and demonstrated the effectiveness of the SPME–GC–MS based metabolomics approach to study such a complex biological system.