Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.
The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.
Protein glycosylation has been described in both Gram-positive and Gram-negative bacteria and consists of the covalent attachment of a glycan to an amino acid side chain1,2. In prokaryotes, this process usually occurs via two major enzymatic mechanisms: O- and N-glycosylation3. In O-glycosylation, the glycan is attached to the hydroxyl group of a serine (Ser) or threonine (Thr) residue. In N-glycosylation, the glycan is attached to the side chain amide nitrogen of an asparagine (Asn) residue within the tripeptide sequences Asn-X-Ser/Thr, where X could be any amino acid except proline.
Glycans can adopt linear or branched structures and are composed of monosaccharides or polysaccharides covalently linked by glycosidic bonds. In prokaryotes, glycans usually show diversity in sugar composition and structure in comparison to eukaryotic glycans4. Furthermore, two different bacterial glycosylation pathways that differ in how the glycan is assembled and transferred to the acceptor protein have been described: sequential and en bloc glycosylation5,6. For sequential glycosylation, the complex glycan is built up directly on the protein by successive addition of monosaccharides. In en bloc glycosylation, a pre-assembled glycan is transferred to the protein from a lipid-linked oligosaccharide by a specialized oligosaccharyltransferase (OTase). Both pathways have been shown to be involved in N- and O-glycosylation processes7.
Protein glycosylation has a role in modulating the physicochemical and biological properties of proteins. The presence of a glycan can influence how the protein interacts with its ligand, which affects the biological activity of the protein, but can also affect protein stability, solubility, susceptibility to proteolysis, immunogenicity, and microbe-host interactions8,9. However, several glycosylation parameters, such as the number of glycans, glycan composition, position, and attachment mechanism, could also affect protein function and structure.
Glycosyltransferases (GTs) are the key enzymes in the biosynthesis of complex glycans and glycoconjugates. These enzymes catalyze the glycosidic bond formation between a sugar moiety from an activated donor molecule and a specific substrate acceptor. GTs can use both nucleotides and non-nucleotides as donor molecules and target different substrate acceptors, such as proteins, saccharides, nucleic acids, and lipids10. Therefore, understanding GTs at the molecular level is important to identify their mechanisms of action and specificity, and also enables understanding how sugar composition of glycans that modify relevant molecules are related to pathogenicity. The Carbohydrate Active enzyme database (CAZy)11 classifies GTs according to their sequence homology, which provides a predictive tool since, in most of the GT families, the structural fold and mechanisms of action are invariant. However, four reasons make it difficult to predict substrate specificity of many GTs: 1) no clear sequence motif determining substrate specificity has been determined in prokaryotes12, 2) many GTs and OTases show substrate promiscuity13,14, 3) functional GTs are difficult to produce in high yield in recombinant form and 4) the identification of both donor and acceptor substrates is complex. Despite this, recent mutagenesis studies have made it possible to obtain significant advances in the understanding of catalytic mechanisms and subtract binding of GTs.
In bacteria, O-glycosylation seems to be more prevalent than N-glycosylation. The O-glycosylation sites do not show a consensus sequence, and many of the O-glycosylated proteins are secreted or cell-surface proteins, such as flagellins, pili, or autotransporters1. Flagellin glycosylation shows variability in the number of acceptor sites, glycan composition, and structure. For example, Burkholderia spp flagellins have only one acceptor site, while in Campylobacter jejuni, flagellins have as many as 19 acceptor sites15,16. Furthermore, for some bacteria, the glycan is a single monosaccharide, while other bacteria possess heterogeneous glycans compromised of different monosaccharides to form oligosaccharides. This heterogenicity occurs even among strains of the same species. Helicobacter flagellins are only modified by pseudaminic acid (PseAc)17, and Campylobacter flagellins can be modified by PseAc, the acetamidino form of the pseudaminic acid (PseAm) or legionaminic acid (LegAm), and glycans derived from these sugars with acetyl, N-acetylglucosamine, or propionic substitutions18,19. In Aeromonas, flagellins are modified by glycans whose composition ranges from a single PseAc acid derivative to a heteropolysaccharide20, and the attachment of glycans to the flagellin monomers is always via a PseAc derivative.
In general, glycosylation of flagellins is essential for flagellar filament assembly, motility, virulence, and host specificity. However, while flagellins of C. jejuni16, H. pylori17, and Aeromonas sp.21 cannot assemble into filament unless the protein monomers are glycosylated, Pseudomonas spp. and Burkholderia spp.15 do not require glycosylation for flagella assembly. Furthermore, in some C. jejuni strains, changes in sugar composition of the flagella glycan affect bacterial-host interaction and may play a role in evading certain immune responses16. Autoagglutination is another phenotypic characteristic affected by modifications in the composition of glycans associated with flagellins. A lower autoagglutination leads to a reduction in the ability to form microcolonies and biofilm22. In some bacteria, the ability of flagella to trigger a pro-inflammatory response was linked to flagellin glycosylation. Thus, in P. aeruginosa, glycosylated flagellin induces a higher pro-inflammatory response than unglycosylated23.
Aeromonas are Gram-negative bacteria ubiquitous in the environment, which allows them to be at the interface of all One Health components24. Mesophilic Aeromonas have a single polar flagellum, which is constitutively produced. More than half of clinical isolates also express lateral flagellin, inducible in high viscosity media or plates. Different studies have related both flagella types with the early stages of bacterial pathogenesis25. While polar flagellins reported to date are O-glycosylated at 5-8 Ser or Thr residues of its central immunogenic domains, lateral flagellins are not O-glycosylated in all the strains. Although polar flagella glycans from different strains show diversity in their carbohydrate composition and chain length20, the linking sugar has been shown to be a pseudaminic acid derivative.
The goal of this manuscript is to describe a method to obtain null mutants in specific GTs or chromosomal regions containing GTs to analyze their involvement in the biosynthesis of relevant polysaccharides and in bacterial pathogenicity, as well as the role of the glycan itself. As an example, we identify and delete a chromosomal region containing GTs of Aeromonas to establish its involvement in polar flagellin glycosylation and analyze the role of the flagellin glycan. We show how to delete a specific GT to establish its function in the biosynthesis of this glycan and the role of modified glycan. Although using Aeromonas as an example, the principle can be used to identify and study flagella glycosylation islands of other Gram-negative bacteria and analyze the function of GTs involved in the biosynthesis of other glycans such as the O-antigen lipopolysaccharide.
The schematic representation of the procedure is shown in Figure 1.
1. Bioinformatic identification of flagella glycosylation island (FGIs) in Aeromonas
2. Generation of null mutants in flagella glycosylation island genes
NOTE: This method of mutagenesis is based on the allelic exchange of polymerase chain reaction (PCR) in-frame deletion products using the suicide vector pDM428 (GenBank: KC795686.1). Replication of pDM4 vector is lambda pir dependent, and the complete allelic exchange is coerced by utilizing the sacB gene located on the vector.
3. Motility assays
NOTE: In some bacterial species with glycosylated flagella, modifications in the levels of glycosylation or glycan composition affect the assembly of flagellins, which is usually reflected as a motility reduction or absence of motility. Therefore, two motility assays were performed with the null mutants.
4. Flagella purification
This methodology provides an effective system to generate null mutants in genes or chromosomal regions of Aeromonas that can affect flagella glycosylation and the role of flagella filament (Figure 1).
The protocol starts with the bioinformatic identification of putative FGIs and the genes encoding GTs presents in this region. In Aeromonas, the chromosomal location of FGIs is based on the detection of three types of genes: genes involved in the biosynthesis of the pseudaminic acid (pseI and pseC), polar flagellin genes, and luxC. Strains whose polar flagella are glycosylated by a single pseudaminic acid derivative, such as A. hydrophila AH-130, do not have genes encoding GTs between the pse genes located in FGIs group I27. Most of the strains belonging to this FGI group show polar flagellin genes adjacent to this region. In contrast, strains whose polar flagella is glycosylated with a heteropolysaccharide glycan, such as A. piscicola AH-319, show different genes encoding GTs downstream of luxC, localized between pse genes of FGIs group II27, and they are not always adjacent to polar flagellin genes (Figure 2A).
The region involved in the biosynthesis of the flagella heteropolysaccharide glycan and the function of putative GTs contained there was confirmed by the construction of null mutants. The generation of each mutant requires four primers: A, B, C, and D (Table 1). Primer B anneals 5-6 codons downstream of the start of the gene (fgi-4) or first gene of the cluster (fgi-1) to be deleted and primer A anneals 600-800 bp upstream from the start of this gene (fgi-4 or fgi-1). Primer C anneals upstream of the last 5-6 codons of the gene (fgi-4) or last gene of the cluster (fgi-12) to be deleted and primer D anneals 600-800 bp downstream from the stop of this gene (fgi-4 or fgi-12) (Figure 2B). Primers A and D for the deletion of fgi cluster and fgi-4 of A. piscicola AH-3 contain a restriction site for the endonuclease BamHI at their 5' ends (Table 1). This endonuclease has no internal targets in the AB nor CD PCR fragments. One important step is the design of B and C primers. Both have to be in-frame to not break the open reading frame of gene or fragment to be deleted. The 21 bp complementary sequences at the 5' end of B and C primers allow the generation of AB and CD amplicons with 3' end complementary sequences, which allow the joining and extending of these amplicons. The PCR program to join and extend the amplicons consists of an initial denaturalization step and five cycles with an annealing temperature of 54 °C (Supplementary Material). Then, the addition of A and D primers allows the amplification of the extended fragment (Figure 3). The enzymatic action of BamHI gives rise to an amplicon with sticky ends compatible for ligation with the suicide vector pDM4 digested with BglII.
Given that pDM4 is a λpir dependent plasmid, the ligation product was electroporated into the E. coli strain MC1061λpir (thi thr1 leu6 proA2 his4 argE2 lacY1 galK2 ara14 xyl5 supE44 λ pir) and selected in chloramphenicol plates. pDM4 does not have a direct system to select the recombinant clones, and colonies were analyzed by PCR with a pDM4 primers pair (pDM4for and pDM4rev) (Table 1 and Supplementary Material) that flank the cloning region. The E. coli strain MC1061λpir without pDM4 was used as the negative control in the PCR reaction.
To perform the allelic exchange, the recombinant pDM4 plasmid was transferred to the recipient strain A. piscicola AH-3. Given that many Aeromonas are not transformable by electroporation, the recombinant pDM4 was transferred by conjugation using triparental mating (Figure 4). To select the transconjugant colonies, we use a rifampicin-resistant recipient strain, which allows selecting the Aeromonas strain from the conjugation mating. Furthermore, the selected colonies were submitted to the oxidase test because while Aeromonas is oxidase-positive, E. coli is oxidase negative. The first and second recombinations were confirmed by PCR with external primers (E and F primers) of amplified regions (AB and CD fragments) (Table 1, Supplementary Material, and Figure 5A).
In Aeromonas, the glycosylation of polar flagellins is essential for the assembly of the flagellar filament. The presence of functional polar flagella was analyzed using motility assays of the bacterial colonies with deleted GTs genes or region. Light microscopy assays showed that swimming motility in liquid medium was reduced in both mutants in comparison to the wild-type strain. Furthermore, both showed a decreased radial expansion in relation to the wild-type strain when motility was analyzed on soft agar (Figure 5B). Given that both mutants were able to swim but with reduced motility in relation to the wild-type strain, their polar flagella were purified, and the flagellin molecular weight was analyzed in a 12% SDS-polyacrylamide gel. This analysis showed that both mutants have polar flagellins with lower molecular weight than the wild-type strain (Figure 5C), which suggests alterations in the flagella glycan. Flagella purified by CsCl gradients (Figure 5C) will be used in mass-spectrometry assays to identify glycan composition and null mutants used in biofilm, adhesion, or other assays to identify the role of the glycan and glycan composition.
Figure 1: Overview of the steps used in this procedure. Scheme of the process described in the protocol to identify flagella glycosylation island of Aeromonas, and GTs involved in glycan biosynthesis. Please click here to view a larger version of this figure.
Figure 2: Bioinformatic detection of chromosomal regions and primer design. (A) Scheme of chromosomal regions identified in A. hydrophila AH-1 and A. piscicola AH-3. A. hydrophila AH-1 flagella glycosylation island is representative of strains whose flagella glycan only have a pseudaminic acid derivative, and A. piscicola AH-3 flagella glycosylation island is representative of strains whose flagella glycan is a heteropolysaccharide. Genes denoted in black are involved in the biosynthesis of pseudaminic acid, and those denoted yellow are putative GTs. (B) Scheme of the chromosomal location of primer pairs designed for the PCR in-frame deletions. Blue boxes in A and D primers contain the restriction binding site for an endonuclease. Red boxes in B and C primers contain 21 bp complementary sequences. Please click here to view a larger version of this figure.
Figure 3: Scheme depicting the method to construct PCR in-frame deletions and ligation to the suicide plasmid pDM4. MCS: multi cloning site, CmR: chloramphenicol resistant genes, sacB: encodes the Bacillus subtilis levansucrase, whose expression is induced by sucrose and is lethal for Gram-negative bacteria, and mob: mobilization genes. Please click here to view a larger version of this figure.
Figure 4: Triparental conjugation and procedure used for the allelic exchange. First recombination occurs due to the absence of λpir into the Aeromonas strains and gives rise to the integration of the recombinant plasmid into the selected gene. Second recombination is induced by growth in LB with 10% sucrose, which leads to the expression of sacB gene, and the plasmid is excised from the chromosome. After the cross-over, the wild-type or the mutated gene can remain on the bacterial chromosome. Null mutants are selected by PCR with external primers. RifR: rifampicin-resistant; CmR: chloramphenicol resistant; SpcR: spectinomycin resistant. Please click here to view a larger version of this figure.
Figure 5: Confirmation of null mutants and phenotypic analysis of polar flagella. (A) PCR with fgi-4 external primers (EF primers) of A. piscicola AH-3 to confirm the allelic exchange after second recombination in chloramphenicol sensitive colonies. Lane WT: A. piscicola AH-3; lanes 1-8: chloramphenicol-sensitive colonies of the second recombination; St: HyperLadder 1 Kb marker. Lanes 1-3 and 5-8 show the colonies with wild-type fgi-4 gene as lane WT. Lane 4 shows a colony with the deleted fgi-4 gene. (B) Motility on soft agar of A. piscicola AH-3 and null mutants in the fgi region (AH-3ΔFgi) and fgi-4 gene (AH-3ΔFgi-4) at 25 °C. (C) Polar flagellum from AH-3 (lane1) and the null mutants in the fgi region (lane 2) and fgi-4 gene (lane 3), isolated, purified in CsCl gradients, analyzed in 12% SDS-PAGE, and stained using Coomassie blue. Size standard (St). Please click here to view a larger version of this figure.
Primer name | Sequence in 5’ to 3’ direction | Used for | ||
A. piscicola AH-3 | ||||
A-Flgi1 | CGCGGATCCGACTGTACCCGTTTCAATCA | fgi mutant | ||
B-Flgi1 | CCCATCCACTAAACTTAAACAGATCACCTCGAACTCGAAA | |||
C-Flgi12 | TGTTTAAGTTTAGTGGATGGGGGAACCTTAAATGCCATGA | |||
D-Flgi12 | CGCGGATCCCAGTCTTCAGCTTCCATCC | |||
E-Flgi1 | ACCCGCTTCATTCGCTAT | |||
F-Flgi12 | TCCGATTTTCTGACTCAGGG | |||
A-Fgi4 | CGCGGATCCGATGCGTACGCTAATATGAA | fgi-4 mutant | ||
B-Fgi4 | CCCATCCACTAAACTTAAACACATATTATCTTGCCCCTGAT | |||
C-Fgi4 | TGTTTAAGTTTAGTGGATGGGATGGAGCTAATCACTCGTTT | |||
D-Fgi4 | CGCGGATCCACATATCAACCCCCAAC | |||
E-Fgi4 | ATTTCCCTGCCAAATACG | |||
F-Fgi4 | CCTGCCAACAGGATGTAAG | |||
pDM4 vector | ||||
pDM4for | AGTGATCTTCCGTCACAGG | Insertions into the pDM4 vector | ||
pDM4rev | AAGGTTTAACGG TTGTGGA |
Table 1: Primers used for the construction of null mutants. Overlapping regions in primers B and C are underlined. BamHI site is bolded in primers A and D.
Supplementary Material. Reagents and reactions used in asymmetric PCRs to obtain the AB and CD amplicons, Pre-AD and AD PCRs to extend and obtain the AD amplicons, pDM4 PCRs to verify the insertion of deleted construct in pDM4 vector, and EF PCRs to verify the first and second recombination. Please click here to download this File.
The critical early step of this method is the identification of regions involved in the glycosylation of flagella and putative GTs because these enzymes show high homology and are involved in many processes. Bioinformatic analysis of Aeromonas genomes in public databases shows that this region is adjacent to the polar flagella region 2, which contains the flagellin genes in many strains and contains genes involved in the biosynthesis of pseudaminic acid27. This has made it possible to develop a guideline for detecting flagella glycosylation islands in Aeromonas that could be used to identify this region in other bacteria whose flagellar glycan contain pseudaminic acid derivatives. In addition, although this method describes how to analyze a gene cluster involved in flagella glycosylation, it can also be used to study genes encoding proteins that might be involved in flagella formation, rotation, or regulation in different Gram-negative bacteria.
Also important in the generation of PCR in-frame deletions is the design of B and C primers. These primers should be localized 5-6 codons downstream of the start (B primer) and upstream of the stop (C primer) of the selected gene or region to be deleted and should not break the open reading frame. Furthermore, an important factor to consider is the length of the homology region amplified to generate the AB and CD amplicons. This protocol recommends that both amplicons contain a homolog region of 600-800 bp each. Shorter homolog regions make the first recombination more challenging.
Aeromonas strains do not carry the lambda pir gene, which is required to replicate the pDM4 suicide plasmid. Therefore, the pDM4 recombinant plasmid must integrate into the chromosomal DNA. After the triparental mating, it is important to identify the bacterial colonies in which the first recombination has occurred. These colonies contain the recombinant pDM4 inserted into the homologous chromosomal region. Identification of recombinant colonies is performed using a PCR reaction with primers (E and F primers) external to the region targeted to be deleted. If a standard DNA polymerase is used, the E-F primers will only lead to the generation of a PCR product in the wild-type. This primer pair will not produce any PCR product in bacterial colonies in which the pDM4 recombinant plasmid has been inserted into the chromosomal DNA. The lack of PCR product is due to the distance between the annealing sequences of E and F primers. This distance cannot be amplified by standard polymerases. However, other factors can also prevent the formation of PCR products. Therefore, first recombinant colonies can be identified by DNA polymerases for long fragment amplification or by PCR reactions using pairs of primers consisting of a pDM4 and an external primer. When large genetic clusters are deleted, amplification in the wild-type strain requires the use of DNA polymerases for long fragment amplification. Bacterial colonies with the first recombination can be identified by PCR with pDM4-external pairs of primers. However, first and second recombinations are usually produced at the same time, leaving the pDM4 with the wild-type or with the deleted gene after recombination. This leads to chloramphenicol resistant colonies whose PCR using external primers give amplicons with an identical size of the wild-type gene or small amplicons, which correlate to the deleted gene or fragment. Colonies shown to have the correct recombinant insert were incubated with sucrose to remove the non-inserted pDM4. Sucrose induces the expression of the sacB gene located on the pDM4, which encodes a lethal enzyme for gram-negative bacteria. Therefore, only colonies lacking the suicide plasmid will grow in LB with sucrose. To support the identity of colonies containing the deleted gene, the fragment amplified with the external primers pair can then be sequenced.
Implementing this in-frame mutation method in Aeromonas allows the generation of more stable null mutants without modifications in the expression levels of downstream genes. Other methods, such as inserting an antibiotic cassette, assure the transcription of downstream genes, but the expression level could be modified. Furthermore, this method could be extrapolated to other target genes and regions, including other Gram-negative bacteria29,31,32,33.
In some bacterial strains, the loss or modification of glycan linked to flagellins can lead to the inability of flagella assembly or the instability of flagella filament, which leads to a reduction of polar flagella motility. While it is easy to distinguish a non-motile phenotype from a motile one using motility assays in liquid media, differences in the degree of motility can be difficult to quantify. Motility plate assays are required to measure differences in the degree of motility. However, some bacterial species, including many mesophilic Aeromonas strains, express inducible lateral flagella when growing in high viscous media or plates, in addition to the constitutive polar flagella. Both flagella types contribute to the bacterial motility in semi-solid plates. Therefore, modifications of polar or lateral flagella only lead to reductions in the migration diameters. Thus, AH-3ΔFgi and AH-3ΔFgi-4 mutants show only reduced motility in relation to the wild-type strain. To improve the evaluation of motility produced by the rotation of polar flagella, the expansion diameter of null mutants should be compared in relation to not only the wild-type strain but also a mutant lacking the polar flagellum. Furthermore, a reduction in the number of glycan residues or changes in the glycan composition influences the electrophoretic motility of glycosylated flagellins. For example, the molecular weight of glycosylated flagellins observed using SDS-PAGE gels is higher than predicted from flagellin amino acid sequence, and disruptions to the glycan are observed as reductions in their molecular weight. In some cases, the changes in glycan modification to the flagellin protein may not be detectable using SDS-PAGE. In these cases, mass spectrometry analysis of either intact protein or flagellin peptides may be required to confirm the presence and mass of glycan.
Pathogenicity of Aeromonas, as well as other Gram-negative bacteria, can be affected by the absence of flagellum but also by changes in the composition of flagella glycans. These changes can affect bacterial interaction to biotic and abiotic surfaces, autoagglutination, play a role in evading immune response and/or induction of pro-inflammatory response. Therefore, adherence, biofilm formation, and pro-inflammatory response assays are required to evaluate the relation between flagella glycosylation and Aeromonas pathogenicity.
The authors have nothing to disclose.
This work was supported by the National Research Council Canada, for the Plan Nacional de I + D (Ministerio de Economía y Competitividad, Spain) and for the Generalitat de Catalunya (Centre de Referència en Biotecnologia).
ABI PRISM Big Dye Terminator v. 3.1 Cycle Sequencing Ready Reaction Kit | Applied Biosystems | 4337455 | Used for sequencing |
AccuPrime Taq DNA Polymerase, high fidelity | Invitrogen | 12346-086 | Used for amplification of AB, CD and AD fragments |
Agarose | Conda-Pronadise | 8008 | Used for DNA electrophoresis |
Alkaline phosphatase, calf intestinal (CIAP) | Promega | M1821 | Used to remove phosphate at the 5’ end |
Bacto agar | Becton Dickinson | 214010 | Use for motility analysis |
BamHI | Promega | R6021 | Used for endonuclease restriction |
BglII | Promega | R6081 | Used for endonuclease restriction |
BioDoc-It Imagin System | UVP | Bio-imaging station used for DNA visualization | |
Biotaq polymerase | Bioline | BIO-21040 | Used for colony screening |
Cesium chloride | Applichem | A1126,0100 | Used for flagella purification |
Chloramphenicol | Applichem | A1806,0025 | Used for triparental mating |
Cytiva illustra GFX PCR DNA and Gel Band Purification Kit | Cytivia | 28-9034-71 | Used for purification of PCR amplicons and DNA fragments. |
EDTA | Applichem | 131026.1211 | Used for DNA electrophoresis |
Electroporation cuvettes 2 mm gap | VWR | 732-1133 | Used for transformation |
Ethidium bromide | Applichem | A1152,0025 | Use for DNA visualization |
HyperLadder 1 Kb marker | Bioline | BIO-33053 | DNA marker |
Invitrogen Easy-DNA gDNA Purification Kit | Invitrogen | 10750204 | Used for bacterial chromosomal DNA purification |
Luria-Bertani (LB) Miller agar | Condalab | 996 | Used for Escherichia coli culture |
Luria-Bertani (LB) Miller broth | Condalab | 1551 | Used for Escherichia coli culture |
Nanodrop ND-1000 | NanoDrop Techonologies Inc | Spectrophotometer used for DNA quantification | |
Rifampicin | Applichem | A2220,0005 | Used for triparental mating |
SOC Medium | Invitrogen | 15544034 | Used for electroporation recovery |
Spectinomycin | Applichem | A3834,0005 | Used for triparental mating |
SW 41 Ti Swinging-Bucket Rotor | Beckman | 331362 | Used for flagella purification |
T4 DNA ligase | Invitrogen | 15224017 | Used for ligation reaction |
Trypticasein soy agar | Condalab | 1068 | Used for Aeromonas grown |
Trypticasein soy broth | Condalab | 1224 | Used for Aeromonas grown |
Tryptone | Condalab | 1612 | Use for motility analysis |
Tris | Applichem | A2264,0500 | Used for DNA electrophoresis and flagella purification |
Triton X-100 | Applichem | A4975,0100 | Used for bacterial lysis |
Ultra Clear tubes (14 mm x 89 mm) | Beckman | 344059 | Used for flagella purification |
Veriti 96 well Thermal Cycler | Applied Biosystems | Used for PCR reactions | |
Zyppy Plasmid Miniprep II Kit | Zymmo research | D4020 | Used for isolation of plasmid DNA |