Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.
18 Related JoVE Articles!
Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis
Institutions: Marshall University.
is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa
is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa
is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa
. To better understand alginate regulation, we describe a protocol using the mini-himar1
transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1
transposon (pFAC) from host E. coli
SM10/λpir into recipient P. aeruginosa
biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas
isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE
(PA4033) and kinB
(PA5484), in strain PAO1 with a wild-type mucA
encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, σ22
). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.
Immunology, Issue 85, Pseudomonas aeruginosa, alginate, mucoidy, mutagenesis, mini-himar1 mariner transposon, pFAC
Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung
Institutions: University of Liverpool , University of Liverpool .
There is growing concern about the relevance of in vitro
antimicrobial susceptibility tests when applied to isolates of P. aeruginosa
from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1
. However, during chronic lung infections in CF, P. aeruginosa
populations exist in biofilms and there is evidence that the environment is largely microaerophilic2
. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3
Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa
growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6
. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa
based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
An ASM assay was developed in a microtitre plate format. P. aeruginosa
biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa
isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a >128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3
. Several in vitro
models have been used previously to study P. aeruginosa
. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9
. In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2
and affect antibiotic susceptibility10
. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.
Immunology, Issue 64, Microbiology, Pseudomonas aeruginosa, antimicrobial susceptibility, artificial sputum media, lung infection, cystic fibrosis, diagnostics, plankton
Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice
Institutions: San Raffaele Scientific Institute, Italian Cystic Fibrosis Research Foundation.
A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa
agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo
. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa
and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa
clinical strains in the agar-beads in vitro
, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa
RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa
colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.
Infection, Issue 85, Opportunistic Infections, Respiratory Tract Infections, Inflammation, Lung Diseases, Cystic Fibrosis, Pseudomonas aeruginosa
Using Coculture to Detect Chemically Mediated Interspecies Interactions
Institutions: University of North Carolina at Chapel Hill .
In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e.
biofilm formation, sporulation, virulence factor production, etc
.) Screening is performed under growth conditions where this phenotype is not
expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis
; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.
Microbiology, Issue 80, High-Throughput Screening Assays, Genes, Reporter, Microbial Interactions, Soil Microbiology, Coculture, microbial interactions, screen, fluorescent transcriptional reporters, Bacillus subtilis
Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection
Institutions: Imperial College London.
, the causative agent of a severe pneumonia named Legionnaires' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella
containing vacuole (LCV). L. pneumophila
infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella
are suitable for investigation of L. pneumophila
infection. G. mellonella
is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae's immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila
is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila
virulence in the G. mellonella
model, including how to grow infectious L. pneumophila
, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila
virulence, describing a new tool to aid our understanding of this complex pathogen.
Infection, Issue 81, Bacterial Infections, Infection, Disease Models, Animal, Bacterial Infections and Mycoses, Galleria mellonella, Legionella pneumophila, insect model, bacterial infection, Legionnaires' disease, haemocytes
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae
Institutions: Rensselaer Polytechnic Institute.
has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae
has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.
Microbiology, Issue 92, Aging, mutations, genome instability, Saccharomyces cerevisiae, fluctuation test, magnetic sorting, mother cell, replicative aging
In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration
Institutions: Harvard Medical School, MGH for Children, Massachusetts General Hospital.
Mucosal surfaces serve as protective barriers against pathogenic organisms. Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils. This process has the potential to be destructive to tissues if excessive or held in an unresolved state. Cocultured in vitro
models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration. This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil. Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface. The in vitro
model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa
(PAO1). Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli
(MC1000). The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls. This in vitro
model system can be manipulated and applied to other mucosal surfaces. Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections. A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro
coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.
Infection, Issue 83, Cellular Biology, Epithelium, Neutrophils, Pseudomonas aeruginosa, Respiratory Tract Diseases, Neutrophils, epithelial barriers, pathogens, transmigration
Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species
Institutions: University of British Columbia .
The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia
species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia
species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.
Immunology, Issue 80, Bacteria, Aerobic, Gram-Negative Bacteria, Immune System Diseases, Respiratory Tract Diseases, Burkholderia, proteins, glass beads, 2-D gel electrophoresis
The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis
Institutions: INRA, Micalis UMR1319, France.
The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1
. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3
. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1
. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella
have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum
, Aspergillus fumigatus
, Candida albicans
) and bacterial pathogens, such as Staphylococcus aureus
, Proteus vulgaris
, Serratia marcescens Pseudomonas aeruginosa
, Listeria monocytogenes
or Enterococcus faecalis4-7
. Regardless of the bacterial species, results obtained with Galleria
larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria
, and strains causing severe human infections are also highly virulent in the Galleria
. Oral infection of Galleria
is much less used and additional compounds, like specific toxins, are needed to reach mortality.
larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 °C to 30 °C) and infection studies can be conducted between 15 °C to above 37 °C12,13
, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014
, measurement of bacterial survival15,16
and examination of the infection process17
. Here, we describe the rearing of the insects, covering all life stages of G. mellonella
. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus
, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19
Infection, Issue 70, Microbiology, Immunology, Molecular Biology, Bacteriology, Entomology, Bacteria, Galleria mellonella, greater wax moth, insect larvae, intra haemocoelic injection, ingestion, animal model, host pathogen interactions
The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms
Institutions: Bar-Ilan University, Bar-Ilan University, Bar-Ilan University, Bar-Ilan University.
Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5
. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7
. There are also reports of biocidal effect of red and near infra red8
as well as green light9
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa
. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa
is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa
strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa
. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa
was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.
Microbiology, Issue 77, Infection, Infectious Diseases, Cellular Biology, Molecular Biology, Biophysics, Chemistry, Biomedical Engineering, Bacteria, Photodynamic therapy, Medical optics, Bacterial viability, Antimicrobial treatment, Laser, Gentamycin, antibiotics, reactive oxygen species, pathogens, microorganisms, cell culture
A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments
Institutions: Delft University of Technology, Delft University of Technology.
This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli
and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9
addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli
to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native β-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2
) of the alkane hydroxylase system from Gordonia
were transformed into E. coli
. For the conversion of long-chain alkanes (C15-C36), theladA
gene from Geobacillus thermodenitrificans
was implemented. For the required further steps of the degradation process, ADH
and ALDH (
originating from G. thermodenitrificans
) were introduced10,11
. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli
K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFDα and β, both from Pyrococcus horikoshii
OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and β improve the protein folding process e.g.
under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n
-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli
to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.
Bioengineering, Issue 68, Microbiology, Biochemistry, Chemistry, Chemical Engineering, Oil remediation, alkane metabolism, alkane hydroxylase system, resting cell assay, prefoldin, Escherichia coli, synthetic biology, homologous interaction mapping, mathematical model, BioBrick, iGEM
Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
Institutions: University of Virginia Health System.
Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2
. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella
species, and Escherichia coli3-5
, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8
. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants.
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9
. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10
, is ideal for LPS extraction.
Immunology, Issue 63, Microbiology, Gram-negative, LPS, extraction, polysaccharide staining, Western immunoblot
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)
Institutions: Cedars-Sinai Medical Center.
MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus
during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.
Infection, Issue 48, Subcutaneous infection, Staphylococcus aureus, MRSA
Microtiter Dish Biofilm Formation Assay
Institutions: Dartmouth Medical School.
Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae
, Escherichia coli
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa
. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.
Immunology, Issue 47, Biofilm, assay, bacteria, fungi, microtiter, static
Pseudomonas aeruginosa Induced Lung Injury Model
Institutions: University of Illinois at Chicago, Emory University, University of Illinois at Chicago.
In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa
or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa
injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.
Immunology, Issue 92, Lung, injury, pseudomonas, pneumonia, mouse model, alveoli