Name: replication of the ordered, nonredundant library of pseudomonas aeruginosa strain pa14 transposon insertion mutants
Uploaded: 2018-05-04T13:30:00.000+00:00
Duration: 11 min 35 s
Description: Watch this Scientific Journal Video about Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants at JoVE.com

We would like to thank Lisa Philpotts of the MGH Treadwell Virtual Library for her guidance in the database search. This work was supported by the Cystic Fibrosis Foundation (YONKER16G0 and HURLEY16G0) and NIH NIAID (BPH and ADE: R01 A1095338).

The authors report no financial conflicts of interest. Eliana Drenkard and Frederick Ausubel participated in the creation the PA14 nonredundant transposon mutant library. Bryan Hurley and Lael Yonker currently house and distribute the mutant library as part of the Department of Pediatrics at Massachusetts General Hospital.

The P. aeruginosa PA14NR Set is a valuable resource for the scientific community. According to the March 2017 dataset from Clarivate Analytics&#39; Essential Science Indicators database, Liberati et al. (2006)37, which describes the construction of the PA14NR Set, is ranked in the top 1% of microbiology publications. Google Scholar reports over 600 citations of the Liberati et al. (2006) original manuscript as of August 2017. The library has played an important role in elucidating the mechanisms underlying P. aeruginosa pathogenesis. Importantly, the 96-well plate format of the PA14NR Set facilitates high throughput genetic screens designed to study a variety of P. aeruginosa mutant phenotypes44,45,46. As the PA14NR Set is available for distribution, special precautions must be used to preserve the integrity of this resource.
A number of publications demonstrate the utility of using the PA14NR Set in genetic screens37,38,47,48. For example, as an initial test of the utility of the library, Liberati et al. performed a PVC (polyvinyl chloride) attachment screen, which correlates with the bacteria&#39;s ability to form biofilms37,38,47,48. The library&#39;s format also allows the study of complex genetic traits such as swarming motility, evidenced by the identification of hundreds of genes of interest in a simple screen48. The PA14NR Set was used also in a large-scale MALDI-TOF mass spectrometry-based screen to acquire and analyze intact-cell proteome profile spectra and assess the effectiveness of MALDI-TOF Biotyping47. Applying this technique, researchers were able to determine whether minor genomic differences, such as those that result from transposon insertions, are disruptive to biotyping efforts which are used in clinical and therapeutic environments. Additionally, the PA14NR Set was used to perform a genome-wide screen for attenuation of PA14 virulence in a C. elegans infection model, allowing the identification of previously uncharacterized P. aeruginosa virulence-related genes38, and thereby providing an example of the use of the PA14 library to study host-pathogen interactions.
The PA14NR Set also serves as an important resource for the study of individual mutants. To avoid contamination of the library, best practices for accessing mutants of interest are recommended. Because handling of the library increases the possibility of contamination, it is recommended to store mutants of interest in individual vials to limit access to the PA14NR Set working and master copies. This prevents contamination, improves the longevity of the library, and protects the investment.
In order to preserve the integrity of the important PA14NR Set resource, it is advised that all recipients of the PA14NR Set make copies of the library upon receipt. Despite best efforts, the potential for inadvertent contamination during replication procedures cannot be excluded. Therefore, it is strongly recommended that users complete quality control checks after library replication and after using the library to perform genetic screens. It is also strongly recommended that users make additional copies of the PA14NR Set to perform genetic screens as the probability of interwell contamination increases. In addition, library copies used to perform more than 2-3 genetic screens should be subjected to exhaustive quality control assessments. Unfortunately, there is no proper way to recover libraries that experience loss of clones or interwell contamination. Compromised or contaminated copies must be discarded. As a consequence, it is recommended that users replicate several copies of the library for routine laboratory use, considering that a copy&#39;s useful life is limited. Also it is recommended that users limit access to the &#34;master copy&#34; to preserve the integrity of the PA14NR Set.
The protocols presented here provide a detailed explanation of the replication techniques, including set-up, protocols, and best practices. The replication protocols described for the PA14NR Set can be adapted to generate up to 12 copies of the library and can also be readily adapted to replicate other bacterial mutant libraries. To the best of our knowledge, there are no published protocols available to date with detailed descriptions of the procedures and techniques used for replication, maintenance and validation of bacterial mutant libraries. We hope these protocols will provide users of the PA14NR Set and other bacterial mutant libraries with the information necessary to perform these important tasks.
Whether the library is used for high throughput screens or as a source for individual mutants, it is important to periodically assess the integrity of the copy in use. To do this, adequate training of personnel and re-assessing replication or mutant selection techniques should be done periodically. The use of PA14NR Set control plates and performance of routine PCR amplification and sequencing of random mutants to confirm their identity is highly recommended. Liberati el al. (2006)37 estimated that 2.8% of the total number of mutants in the PA14NR Set was mislabeled, which highlights the need to sequence specific transposon mutants and to generate in-frame deletion mutants before starting any comprehensive study and/or publication of research involving specific genes. Given proper protocols and methods for library replication and selection of individual mutants for further study, the PA14NR Set will contribute to the understanding of P. aeruginosa pathogenicity and the improvement of clinical outcomes for infected patients.

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium present in soil, water, and most human environments, as well as skin microflora. Compared to many bacterial species, P. aeruginosa has a relatively large genome of 5.5-7 Mbp with high G+C content (65-67%). Furthermore, a significant proportion of its genes are involved in metabolic adaptability and are part of regulatory networks, allowing for great flexibility in response to environmental stress1. P. aeruginosa expresses a plethora of virulence factors, exhibits proclivity to form biofilms, possesses the ability to coordinate responses through multiple quorum sensing pathways, and displays a notable capacity to develop antibiotic resistance and tolerance2,3,4,5,6,7,8. These attributes present significant challenges for treating infections caused by P. aeruginosa.
Chronic P. aeruginosa infections can occur in numerous disease states. Cystic fibrosis (CF), a genetic disease caused by mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, results in inspissated, infected secretions within the airway, progressive bronchiectasis and, ultimately, death from respiratory failure9. By adulthood, the majority of patients with CF are chronically infected with P. aeruginosa, which plays a key role in the morbidity and mortality associated with this disease10. Additionally, patients with severe burn injuries11, tracheostomies12, joint replacements13, or indwelling catheters14 are at risk for P. aeruginosa infection related to the bacteria&#39;s ability to form biofilms and escape host inflammatory responses15. Further, colonization occurs without competition after a multi-antibiotic resistant or tolerant population is selected through broad-spectrum, sequential antimicrobial treatment12,16,17,18. Better understanding the pathogenesis of P. aeruginosa will have significant implications for numerous disease states.
Several P. aeruginosa clinical isolates, including strains PAO1, PA103, PA14 and PAK, have been extensively studied to investigate different features of P. aeruginosa pathogenesis. Strain PA14 is a clinical isolate that belongs to one of the most common clonal groups worldwide19,20 and has not been extensively passaged in the laboratory. PA14is highly virulent in vertebrate models of infection, with a notable endotoxin profile21, pili structure22, pathogenicity islands23, type III secretion system (TTSS), cytotoxicity towards mammalian cells24 and profiles in antibiotic resistance and persistence25. Furthermore, PA14 is also highly virulent in numerous host-pathogen model systems, including plant leaf infiltration models26,27,Caenorhabditis elegans infection models28,29, insect models30,31, as well as mouse pneumonia models32,33 and skin burn models34.
Genome-wide mutant libraries are collections of isogenic mutants in nonessential genes that constitute very powerful tools to understand the biology of an organism by allowing analysis of gene function on a genomic scale. Two near-saturation transposon insertion mutant libraries constructed in P. aeruginosa are currently available for distribution. The insertion sites of the transposons have been determined for both libraries. These so-called nonredundant libraries facilitate genome-wide studies of bacterial strains by considerably decreasing the time and cost involved in screening uncharacterized random transposon mutants. The P. aeruginosa PAO1 transposon mutant library, constructed in the MPAO1 isolate of strain PAO1 using transposons ISphoA/hah and ISlacZ/hah35, is curated by the Manoil lab, University of Washington. The library consists of a sequence-verified collection of 9,437 transposon mutants that provides wide genome coverage and includes two mutants for most genes36. Information about the P. aeruginosa PAO1 transposon mutant library is available at the public, internet-accessible Manoil lab website at http://www.gs.washington.edu/labs/manoil/libraryindex.htm. The P. aeruginosa strain PA14 nonredundant transposon insertion mutant library (PA14NR Set) constructed in strain PA14 using transposons MAR2xT7 and TnphoA37 is currently distributed by the Department of Pediatrics at Massachusetts General Hospital. The PA14NR Set comprises a collection of more than 5,800 mutants with single transposon insertions in nonessential genes37. Details on the construction of the PA14NR Set are described in the public, internet-accessible site http://pa14.mgh.harvard.edu/cgi-bin/pa14/home.cgi?section=NR_LIB, which also contains a variety of online search tools to facilitate the use of the PA14NR Set.
The original PA14NR Set comprised 5,459 mutants, selected from a comprehensive library of approximately 34,000 random transposon insertion mutants, that correspond to 4,596 predicted PA14 genes representing 77% of all predicted PA14 genes37. Since the construction of the library in 2006 new mutants were added, and presently the PA14NR Set includes more than 5,800 mutants38 that represent approximately 4,600 PA14 genes. The majority of the PA14 transposon mutants were generated in the wild type background37. Details concerning each member of the mutant library, including genetic background, are available either through searching the online database, or by downloading the Nonredundant Library spreadsheet, both features available on the PA14 website (http://pa14.mgh.harvard.edu/cgi-bin/pa14/home.cgi). The majority of mutants were created using the MAR2xT7 (MrT7) transposon, with a small set created using the TnPhoA (phoA) transposon37. Each transposon has an antibiotic resistance cassette, which allows for mutant selection using gentamicin (MrT7) or kanamycin (phoA). The PA14NR set of mutants is stored in sixty-three 96-well plates and includes two additional 96-well control plates, which consist of wild type PA14 inoculated and uninoculated wells intercalated in a preset pattern. The 96-well plate format paired with the online search tools greatly facilitates the custom development of screening assays that allow users to easily identify genes associated with mutant phenotypes. The online search tools also facilitate the search and selection of additional relevant mutants required for further studies.
The PA14 and PAO1 transposon mutant libraries are very important global resources for the scientific community, and they complement each other in validating the function of unknown genes and pathways of this bacterial pathogen. Coincidentally, since the construction of the PAO1 and PA14 transposon mutation libraries, full-genome DNA sequencing analysis of many P. aeruginosa isolates has shown that PAO1 and PA14 belong to different major subclades of the P. aeruginosa phylogeny7,39,40,41. Because clinical P. aeruginosa isolates are found distributed throughout the phylogeny, the fact that PAO1 and PA14 belong to different P. aeruginosa subgroups enhances the value of the two transposon mutation libraries for comparative studies.
Publications describing the construction and screening of bacterial mutant libraries, including P. aeruginosa libraries35,37,42, are readily available in the literature. However, to the best of our knowledge, no published protocols describing detailed procedures and techniques used for replication, maintenance, and validation of bacterial mutant libraries are available.
The methodology outlined in this publication describes a set of three protocols that facilitate the use and maintenance of the PA14NR Set. The first protocol describes replication of the library as recommended to recipients of the PA14NR Set. The second protocol includes guidelines for streaking, growing, and storing individual mutants identified using the PA14NR Set. The third protocol describes quality control techniques, including PCR amplification of fragments from transposon mutants and subsequent sequencing to confirm mutant identity. This set of protocols may also be adapted for the replication and maintenance of other bacterial mutant libraries or collections. The replication of bacterial mutant libraries or collections is highly advised to preserve the integrity of the &#34;master copy&#34; (original copy received). Replication of several copies of the PA14NR Set for routine laboratory use minimizes the probability of interwell contamination of the master copy.

<table><tbody><tr><td>&lt;em&gt;Materials for Library Replication&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Sterile 96-well Tissue-culture treated, case of 50</td><td>Corning Life Sciences</td><td>353072</td><td>via Fisher Scientific</td></tr><tr><td>Sterile 96 Well Clear V-Bottom 2000&amp;mu;L Deep Well Plates, case of 25</td><td>Corning Life Sciences</td><td>3960</td><td>via Fisher Scientific</td></tr><tr><td>Nunc OmniTray (rectangular plates), case of 60</td><td>Thermo Scientific Rochester</td><td>242811</td><td>via Fisher Scientific</td></tr><tr><td>Rectangular Ice Pan, Midi (4L)</td><td>Corning Life Sciences</td><td>432104</td><td>via Fisher Scientific</td></tr><tr><td>Secure-Gard Cone Mask, case of 300</td><td>Cardinal Health</td><td>AT7509</td><td>via Fisher Scientific</td></tr><tr><td>AluminaSeal, pack of 100</td><td>Diversified Biotech</td><td>ALUM-100</td><td>via Fisher Scientific</td></tr><tr><td>Breathe-Easy membrane, pack of 100</td><td>Diversified Biotech</td><td>BEM-1</td><td>via Sigma-Aldrich</td></tr><tr><td>Sterile, individually wrapped, 50mL Solution Trough/Reagent Reservoir, case of 100</td><td>Sorenson</td><td>S50100</td><td>via Westnet Incorporated</td></tr><tr><td>Plate roller</td><td>VWR</td><td>60941-118</td><td>via VWR</td></tr><tr><td>Cryo Laser Labels - CRYOLAZRTAG 2.64&quot; x 0.277&quot;, pack of 16 sheets</td><td>GA International</td><td>RCL-11T1-WH</td><td>via Labtag.com (template for printing also available from Labtag.com)</td></tr><tr><td>96-well replicator</td><td>V &amp;amp; P Scientific, Inc.</td><td>Custom 407C, 3.18mm pin diameter, 57mm long</td><td>via V &amp;amp; P Scientific, Inc.</td></tr><tr><td>Multitron Pro, 3mm Shaking incubator</td><td>Infors HT</td><td>l10003P</td><td>via Infors HT</td></tr><tr><td>Picus 12 Channel 50-1200&amp;mu;L Electronic Pipette</td><td>Sartorius</td><td>735491PR</td><td>via Sartorius</td></tr><tr><td>Filter Tips 50-1200&amp;mu;L, pack of 960</td><td>Biohit</td><td>14-559-512</td><td>via Fisher Scientific; use electronic multichannel-compatible tips</td></tr><tr><td>Dry Ice</td><td>&lt;em&gt;User-specific vendor&lt;/em&gt;</td><td></td><td></td></tr><tr><td>&lt;em&gt;Materials for Individual Mutant Storage&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td><td>Fisher Scientific</td><td>05-408-130</td><td>via Fisher Scientific</td></tr><tr><td>Pipettes (P1000, P200, P20, P2)</td><td>Gilson</td><td>F167370</td><td>via Gilson</td></tr><tr><td>&lt;em&gt;Materials for Quality Control PCR&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td><td>Fisher Scientific</td><td>05-408-130</td><td>via Fisher Scientific</td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td>ND-2000</td><td>via ThermoFisher</td></tr><tr><td>PCR Thermocycler</td><td></td><td></td><td></td></tr><tr><td>Omnistrips PCR Tubes with domed lids</td><td>Thermo Scientific</td><td>AB0404</td><td>via Fisher Scientific</td></tr><tr><td>ART Barrier low-retention pipette tips (10 uL, 100 uL, 1000 uL)</td><td>Molecular BioProducts, Inc.</td><td>Z676543 (10 uL), Z676713 (100 uL), Z676802 (1000 uL)</td><td>via Sigma-Aldrich</td></tr><tr><td>Pipettes (P1000, P200, P20, P2)</td><td>Gilson</td><td>F167370</td><td>via Gilson</td></tr><tr><td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td><td>Fisher Scientific</td><td>05-408-130</td><td>via Fisher Scientific</td></tr><tr><td>MasterPure DNA Purification Kit</td><td>Epicentre</td><td>MCD85201</td><td>via Epicentre Technologies Corp</td></tr><tr><td>GeneRuler 1 kb Plus DNA Ladder, ready-to-use</td><td>Thermo Scientific</td><td>SM1333</td><td>via ThermoFisher</td></tr><tr><td>RediLoad Loading Buffer</td><td>Invitrogen</td><td>750026</td><td>via ThermoFisher</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Chemicals for Library and Individual Mutant Storage&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Glycerol MB Grade, 1L</td><td>Sigma Aldrich</td><td>G5516</td><td>via Sigma-Aldrich</td></tr><tr><td>LB Broth</td><td></td><td></td><td>Per 1L dH&lt;sub&gt;2&lt;/sub&gt;O: 10g tryptone, 5g yeast extract, 5g NaCl, 1ml 1N NaOH (Current Protocols in Molecular Biology.&amp;nbsp; Wiley, 1994.)</td></tr><tr><td>Tryptone</td><td>Sigma Aldrich</td><td>T7293</td><td>via Sigma-Aldrich</td></tr><tr><td>Yeast Extract</td><td>Sigma Aldrich</td><td>Y1625</td><td>via Sigma-Aldrich</td></tr><tr><td>Sodium Chloride</td><td>Sigma Aldrich</td><td>S7653</td><td>via Sigma-Aldrich</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma Aldrich</td><td>S8045</td><td>via Sigma-Aldrich</td></tr><tr><td>LB&amp;nbsp; agar</td><td></td><td></td><td>See preparation above, add 15g Bacto Agar</td></tr><tr><td>Bacto Agar</td><td>Sigma Aldrich</td><td>A5306</td><td>via Sigma-Aldrich</td></tr><tr><td>Gentamicin sulfate, 10g</td><td>BioReagent</td><td>1405-41-0</td><td>via Sigma-Aldrich</td></tr><tr><td>Kanamycin sulfate</td><td>Gibco</td><td>11815024</td><td>via ThermoFisher</td></tr><tr><td>Ethanol, 190 proof</td><td>Decon</td><td>04-355-221</td><td>via Fisher Scientific</td></tr><tr><td>&lt;em&gt;Chemicals for Quality Control PCR&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Primers</td><td>&lt;em&gt;User-preferred vendor&lt;/em&gt;</td><td></td><td>See primers listed in Table 3</td></tr><tr><td>Corning cellgro Molecular Biology Grade Water</td><td>Corning</td><td>46000CV</td><td>via Fisher Scientific</td></tr><tr><td>Taq Polymerase Buffer</td><td>Invitrogen</td><td>10342020</td><td>via ThermoFisher</td></tr><tr><td>Taq DNA Polymerase, recombinant</td><td>Invitrogen</td><td>10342020</td><td>via ThermoFisher</td></tr><tr><td>dNTPs</td><td>Invitrogen</td><td>10297018</td><td>via ThermoFisher</td></tr><tr><td>Agarose</td><td>Sigma</td><td>A9539</td><td>via Sigma-Aldrich</td></tr></tbody></table>

replication of the ordered, nonredundant library of pseudomonas aeruginosa strain pa14 transposon insertion mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Twelve new copies of the PA14NR Set were replicated using Protocol I, and a quality control assessment of the new copies generated was conducted using Protocol III.
PA14NR Set mutant plates along with Control Plates, which consist of wild type PA14 inoculated and uninoculated wells intercalated in a preset pattern (Figure 4A), were replicated following methology described in Protocol I. Control plates are included in the PA14NR Set to evaluate possible interwell contamination when performing plate replication. Additionally, control plates can be used to practice replication techniques prior to accessing plates containing transposon insertion mutants. Growth of control plates was visually inspected after replication on LB agar plates and overnight incubation to ensure the presence of expected growth patterns (Figure 4B). Presence or absence of bacterial growth in deep well blocks inoculated with Control Plate 1 was assessed after overnight incubation by reading OD600 in spectrophotometer. Results showed substantial growth in the wild type PA14 inoculated wells and complete absence of growth in the uninoculated wells (Figure 4C). Although there was some variability in the growth of wild type PA14 cultures, there was complete absence of growth in the uninoculated wells (Figure 4D).
Thirty-eight mutant strains randomly selected from one of the newly generated copies of the PA14NR Set were analyzed by sequencing DNA fragments generated by arbitrary PCR. Arbitrary PCR reactions were carried out to amplify DNA fragments from regions surrounding transposon insertions of the 38 mutants selected, and the PCR fragments obtained were subsequently sequenced. An example of PCR fragments obtained after PCR amplification of nine distinct transposon insertion mutants is shown in Figure 5. As annealing of arbitrary primers occurs at random, fragment length cannot be predicted. When no bands were visible, PCR reactions were repeated. The identity of the transposon insertions contained in the 38 mutants analyzed was found using the PA14 Transposon Insertion Mutant Library link (http://pa14.mgh.harvard.edu/cgi-bin/pa14/search.cgi) to select the primers necessary to perform arbitrary PCR reactions.
Sequencing results obtained from the 38 mutant strains were BLASTed against the complete genome of strain PA14 using the blast link provided on the PA14NR Library website (http://pa14.mgh.harvard.edu/cgi-bin/pa14/blast.cgi). Sequencing results from randomly selected mutants were also aligned against the gene sequences that corresponded to each individual mutant (obtained using the PA14NR Set plate position search tool found on the PA14NR Library website http://pa14.mgh.harvard.edu/cgi-bin/pa14/search.cgi?searchType=SEARCH_PLATE_POSITIONS). Sequence alignments were performed using the Align Sequences Nucleotide BLAST tool provided by NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=MegaBlast&#38;PROGRAM=blastn&#38;BLAST_PROGRAMS=megaBlast&#38;PAGE_TYPE=BlastSearch&#38;BLAST_SPEC=blast2seq&#38;DATABASE=n/a&#38;QUERY=&#38;SUBJECTS=).
Two of the 38 mutants selected generated low quality sequence results, probably due to high GC content in the region containing the transposon insertion, and could not be analyzed further. Sequence results from 35 of the 36 succesfully sequenced mutants matched the sequences of the genes corresponding to the PA14NR Set selected mutants. Only one out of the 36 sequences failed to match the sequence of the gene that corresponds to the mutant selected. However, it was not established if this discrepancy resulted from a problem that occurred during replication of the new copies of the PA14NR Set or could be attributed to the 2.8% mislabeling error estimated earlier for the PA14NR Set37.

<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57298/57298fig4.jpg" /> 
Figure 4: PA14NR set control plates. A) Layout of PA14NR Set Control Plates 1 and 2. B) Picture of PA14 NR Set Control Plates 1 and 2 replicated on LB Agar (view from top of agar plate). C) Average bacterial growth (+/-SD) measured in inoculated and uninoculated wells of Control Plate 1 after overnight incubation in Deep Well Block. Readings were performed at OD600. D) Bacterial growth measured in inoculated and uninoculated wells of Control Plate 1 after overnight incubation in Deep Well Block. Readings were performed at OD600 in individual wells. Even columns correspond to measurements performed in uninoculated wells; odd columns correspond to measurements performed in PA14-inoculated wells. <a href="/files/ftp_upload/57298/57298fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57298/57298fig5.jpg" /> 
Figure 5: PCR1 and PCR2 fragments obtained from amplification reactions. Example of fragments obtained using Arbitrary PCR amplification reactions. Fragments in lanes 1 - 9 correspond to different mutants selected at random to perform quality controls of PA14NR Set replicated plates. MWM: DNA Molecular weight marker. DNA MWM sizes are in base pairs. 
PA14NR Set mutant strains tested: Lane 1: 07_4 A10, lane 2: 07_4 B4, lane 3: 08_1 C3, lane 4: 08_1 H6, lane 5: 08_3 G10, lane 6: 08_4 B8, lane 7: 08_4 H3, lane 8: 09_2 D12, lane 9: 09_2 G5. <a href="/files/ftp_upload/57298/57298fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants at JoVE.com

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

replication-ordered-nonredundant-library-pseudomonas-aeruginosa

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.0K Views.  Massachusetts General Hospital. Pseudomonas aeruginosa infectie veroorzaakt significante morbiditeit in kwetsbare hosts. De nonredundant transposon invoeging mutant bibliotheek van P. aeruginosa stam PA14, uitgeroepen tot PA14NR ingesteld, vergemakkelijkt de analyse van gene functionaliteit in tal van processen. Hier gepresenteerd is een protocol voor het genereren van hoge kwaliteit kopieën van de PA14NR ingesteld mutant bibliotheek.

Video: Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

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.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Genetics

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa 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/&#955;pir into recipient P. aeruginosa PAO1 via 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, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

Biology

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The &#8220;last-line&#8221; antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.

We describe a method to generate saturating transposon mutant libraries in Gram-negative bacteria and subsequent preparation of DNA amplicon libraries for high-throughput sequencing. As an example, we focus on the ESKAPE pathogen, Acinetobacter baumannii, but this protocol is amenable to a wide range of Gram-negative organisms.

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

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