Genetic Cassette Research Articles

DIY series of genetic cassettes useful in construction of versatile vectors specific for Alphaproteobacteria

We have developed a DIY (Do It Yourself) series of genetic cassettes, which facilitate construction of novel versatile vectors for Alphaproteobacteria. All the cassettes are based on defined genetic modules derived from three natural plasmids of Paracoccus aminophilus JCM 7686. We have constructed over 50 DIY cassettes, which differ in structure and specific features. All of them are functional in eight strains representing three orders of Alphaproteobacteria: Rhodobacterales, Rhizobiales and Caulobacterales. Besides various replication and stabilization systems, many of the cassettes also contain selective markers appropriate for Alphaproteobacteria (40 cassettes) and genetic modules responsible for mobilization for conjugal transfer (24 cassettes). All the DIY cassettes are bordered by different types of polylinkers, which facilitate vector construction. Using these DIY cassettes, we have created a set of compatible Escherichia coli-Alphaproteobacteria mobilizable shuttle vectors (high or low copy number in E. coli), which will greatly assist the genetic manipulation of Alphaproteobacteria.

Nasal carriage of methicillin-resistant Staphylococcus aureus among preclinical medical students: epidemiologic and molecular characteristics of methicillin-resistant S. aureus clones

Between May 2008 and October 2009, a total of 2103 interns were randomly tested for nasal colonization of S. aureus and methicillin-resistant Staphylococcus aureus (MRSA). The prevalence of S. aureus among staphylococci specimens was 23.1%, and among the total S. aureus the MRSA prevalence was 9.4%. MRSA isolates were further subtyped using genetic element staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE) band pattern, and multilocus sequence typing. SCCmec type IVa was the most prevalent strain, at 45.4 %. Eleven PFGE patterns were identified in MRSA strains, with 1 predominant (pulsotype A, 45.5%). Eight strains which belonged to clonal complex 78 carried type IVa SCCmec and produced type 3 coagulase. Panton–Valentine leukocidin (PVL) genes (lukS and F-PV) were identified in 10 (45.4%) MRSA strains; these predominately carried ϕSa2958type and ϕSa108PVL-like type PVL phages. After inducing prophages, 8 strains infected other S. aureus isolates and could generate novel PVL-positive strains of S. aureus. The present study demonstrates that interns can carry certain MRSA strains asymptomatically and contribute to the spread of MRSA between the community and hospital.

Engineering of a synthetic electron conduit in living cells

Engineering efficient, directional electronic communication between living and nonliving systems has the potential to combine the unique characteristics of both materials for advanced biotechnological applications. However, the cell membrane is designed by nature to be an insulator, restricting the flow of charged species; therefore, introducing a biocompatible pathway for transferring electrons across the membrane without disrupting the cell is a significant challenge. Here we describe a genetic strategy to move intracellular electrons to an inorganic extracellular acceptor along a molecularly defined route. To do so, we reconstitute a portion of the extracellular electron transfer chain of Shewanella oneidensis MR-1 into the model microbe Escherichia coli. This engineered E. coli can reduce metal ions and solid metal oxides ∼8× and ∼4× faster than its parental strain. We also find that metal oxide reduction is more efficient when the extracellular electron acceptor has nanoscale dimensions. This work demonstrates that a genetic cassette can create a conduit for electronic communication from living cells to inorganic materials, and it highlights the importance of matching the size scale of the protein donors to inorganic acceptors.

Transformation of coffee (Coffea Arabica L. cv. Catimor) with the cry1ac gene by biolistic, without the use of markers

The transformation of coffee plantlets with the cry1ac gene of Bacillus thuringiensis was achieved by biolistic using either the whole pUBC plasmid or only the ubi-cry1ac-nos genetic cassette. The cry1ac gene was inserted into coffee plants in order to confer resistance to the leaf miner Leucoptera coffeella, an insect responsible for considerable losses in coffee crops. Bearing in mind that the genetic cassettes used for this study lack reporter genes and/or selection marker genes, the parameters for the transformation procedure by biolistic were previously standardised with a plasmid carrying the gus reporter gene. The presence of the cry1ac gene in young plantlet tissues was determined by PCR, Southern blot and reverse transcription-PCR. Our results show that the obtainment of viable coffee plantlets, transformed by bombardment with the cry1ac gene and without selection markers nor reporter genes, is feasible.

Origin and Genetic Evolution of the Vertebrate Skeleton

The current understanding of the origin and evolution of the genetic cassette for the vertebrate skeletal system is reviewed. Molecular phylogenetic analyses of fibrillar collagen genes, which encode the main component of both cartilage and mineralized bone, suggest that genome duplications in vertebrate ancestors were essential for producing distinct collagen fibers for cartilage and mineralized bone. Several data Indicate co-expression of the ancestral copy of fibrillar collagen with the SoxE and Runx transcription factors. Therefore, the genetic cassette may have already existed in protochordate ancestors, and may operate in the development of the pharyngeal gill skeleton. Accompanied by genome duplications in vertebrate ancestors, this genetic cassette may have also been duplicated and co-opted for cartilage and bone. Subsequently, the genetic cassette for cartilage recruited novel genetic material via domain shuffling. Aggrecan, acquired by means of domain shuffling, performs an essential role in cartilage as a shock absorber. In contrast, the cassette for bone recruited new genetic material produced by tandem duplication of the SPARC/osteonectin genes. Some of the duplicated copies of SPARC/osteonectin became secretory Cabinding phosphoproteins (SCPPs) performing a central role in mineralization by regulating the calcium phosphate concentration. Comparative genome analysis revealed similar molecular evolutionary histories for the genetic cassettes for cartilage and bone, namely duplication of the ancestral genetic cassette and recruitment of novel genetic material.

Isolation, characterization, and utilization of a temperature-sensitive allele of a Pseudomonas replicon

In order to facilitate genetic study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, we isolated a conditional, temperature-sensitive plasmid origin of replication. We mutagenized the popular Pseudomonas stabilizing fragment from pRO1610 in vitro using the Taq thermostable DNA polymerase in a polymerase chain reaction (PCR). Out of approximately 23,000 potential clones, 48 temperature-sensitive mutants were isolated. One mutant was further characterized and the origin of replication was designated as mSF ts1 . The mutations that resulted in a temperature-sensitive phenotype in mSF ts1 were localized to the 1.2 kb of minimum sequence required for replication in P. aeruginosa. The DNA sequence analysis revealed two mutations within the coding sequence of the Replication control (Rep) protein. Growth of P. aeruginosa carrying the temperature-sensitive plasmid at the non-permissive temperature of 42 °C resulted in loss of the plasmid by greater than 99.9999% of the cells after 16 h of growth. In order to facilitate its utilization, the mSF ts1 was converted into a genetic cassette flanked by mirrored restriction endonuclease digestion sites of a pUC1918 derivative. We demonstrate utilization of the mSF ts1 for genetic studies involving complementation and regeneration of a mutant in P. aeruginosa research.

Gene synthesis demystified

DNA fabrication of genetic cassettes at base-level precision is transforming genetic engineering from a laborious art to an information-driven discipline. Although substantial advances have been made in the development of DNA fabrication, the methods employed vary widely based on the length of the DNA. All of these methods are available commercially, but can also be performed at the molecular biology bench using typical reagents and procedures. Because the technology is not mature and is still evolving rapidly, it is helpful to gain some understanding of the different steps in this process and the associated technical challenges to successfully take advantage of DNA fabrication in a research project.

Novel Proteins That Modulate Type IV Pilus Retraction Dynamics in Pseudomonas aeruginosa

Pseudomonas aeruginosa uses type IV pili to colonize various materials and for surface-associated twitching motility. We previously identified five phylogenetically distinct alleles of pilA in P. aeruginosa, four of which occur in genetic cassettes with specific accessory genes (J. V. Kus, E. Tullis, D. G. Cvitkovitch, and L. L. Burrows, Microbiology 150:1315-1326, 2004). Each of the five pilin alleles, with and without its associated pilin accessory gene, was used to complement a group II PAO1 pilA mutant. Expression of group I or IV pilA genes restored twitching motility to the same extent as the PAO1 group II pilin. In contrast, poor twitching resulted from complementation with group III or group V pilA genes but increased significantly when the cognate tfpY or tfpZ accessory genes were cointroduced. The enhanced motility was linked to an increase in recoverable surface pili and not to alterations in total pilin pools. Expression of the group III or V pilins in a PAO1 pilA-pilT double mutant yielded large amounts of surface pili, regardless of the presence of the accessory genes. Therefore, poor piliation in the absence of the TfpY and TfpZ accessory proteins results from a net increase in PilT-mediated retraction. Similar phenotypes were observed for tfpY single and tfpY-pilT double knockout mutants of group III strain PA14. A PilAV-TfpY chimera produced few surface pili, showing that the accessory proteins are specific for their cognate pilin. The genetic linkage between specific pilin and accessory genes may be evolutionarily conserved because the accessory proteins increase pilus expression on the cell surface, thereby enhancing function.

Tight Modulation of Escherichia coli Bacterial Biofilm Formation through Controlled Expression of Adhesion Factors

Despite the economic and sanitary problems caused by harmful biofilms, biofilms are nonetheless used empirically in industrial environmental and bioremediation processes and may be of potential use in medical settings for interfering with pathogen development. Escherichia coli is one of the bacteria with which biofilm formation has been studied in great detail, and it is especially appreciated for biotechnology applications because of its genetic amenability. Here we describe the development of two new genetic tools enabling the constitutive and inducible expression of any gene or operon of interest at its native locus. In addition to providing valuable tools for complementation and overexpression experiments, these two compact genetic cassettes were used to modulate the biofilm formation capacities of E. coli by taking control of two biofilm-promoting factors, autotransported antigen 43 adhesin and the bscABZC cellulose operon. The modulation of the biofilm formation capacities of E. coli or those of other bacteria capable of being genetically manipulated may be of use both for reducing and for improving the impact of biofilms in a number of industrial and medical applications.

A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine

Pyrrolysine has entered natural genetic codes by the translation of UAG, a canonical stop codon. UAG translation as pyrrolysine requires the pylT gene product, an amber-decoding tRNA(Pyl) that is aminoacylated with pyrrolysine by the pyrrolysyl-tRNA synthetase produced from the pylS gene. The pylTS genes form a gene cluster with pylBCD, whose functions have not been investigated. The pylTSBCD gene order is maintained not only in methanogenic Archaea but also in a distantly related Gram-positive Bacterium, indicating past horizontal gene transfer of all five genes. Here we show that lateral transfer of pylTSBCD introduces biosynthesis and genetic encoding of pyrrolysine into a naïve organism. PylS-based assays demonstrated that pyrrolysine was biosynthesized in Escherichia coli expressing pylBCD from Methanosarcina acetivorans. Production of pyrrolysine did not require tRNA(Pyl) or PylS. However, when pylTSBCD were coexpressed with mtmB1, encoding the methanogen monomethylamine methyltransferase, UAG was translated as pyrrolysine to produce recombinant monomethylamine methyltransferase. Expression of pylTSBCD also suppressed an amber codon introduced into the E. coli uidA gene. Strains lacking one of the pylBCD genes did not produce pyrrolysine or translate UAG as pyrrolysine. These results indicated that pylBCD gene products biosynthesize pyrrolysine using metabolites common to Bacteria and Archaea and, furthermore, that the pyl gene cluster represents a "genetic code expansion cassette," previously unprecedented in natural organisms, whose transfer allows an existing codon to be translated as a novel endogenously synthesized free amino acid. Analogous cassettes may have served similar functions for other amino acids during the evolutionary expansion of the canonical genetic code.

Community-acquired methicillin-resistant Staphylococcus aureus: the role of Panton–Valentine leukocidin

Community-acquired (CA) methicillin-resistant Staphylococcus aureus (MRSA) infection among individuals without healthcare-associated (HA) risk factors was first recognized about a decade ago. It has now emerged as an epidemic that is responsible for rapidly progressive, fatal diseases including necrotizing pneumonia, severe sepsis and necrotizing fasciitis. Unlike HA-MRSA, CA-MRSA are usually pan-susceptible to non-β-lactam antimicrobials. In addition to novel methicillin resistance genetic cassettes, many CA-MRSA harbor a phage harboring Panton–Valentine Leukocidin (PVL) genes and some data support the idea that PVL is responsible at least in part for the increased virulence of CA-MRSA. The tight association between the novel methicillin resistance cassettes and PVL phage cannot be explained, as they integrate into distinct sites on the S. aureus chromosome. This paper presents the evidence that CA-MRSA isolates are distinct strains emerging de novo from CA-methicillin susceptible isolates rather than from HA-MRSA isolates that have escaped from the hospital setting and that these novel CA-MRSA isolates may be more virulent than HA-MRSA. The second aim is to outline the progress in understanding the role of PVL in CA-MRSA pathogenesis.

Predominance of staphylococcal cassette chromosome mec (SCCmec) type IV among methicillin-resistant Staphylococcus aureus (MRSA) in a Swedish county and presence of unknown SCCmec types with Panton-Valentine leukocidin genes

Methicillin-resistant Staphylococcus aureus (MRSA) is an established nosocomial pathogen, but has recently begun to appear in the community. The clones in the community may not have originated in the hospital setting, and are referred to as community-acquired MRSA (CA-MRSA). Resistance to methicillin is mediated by the gene mecA, which is carried by the mobile genetic element staphylococcal cassette chromosome mec (SCCmec). SCCmec typing (I-IV) of all clinical isolates of MRSA (n = 92) from 1987 to 2004 in Orebro County, Sweden, was performed by real-time LightCycler PCR to detect the essential genetic components mecA, mecR1, IS1272, ccrA and ccrB. Forty-one isolates harboured type IV SCCmec, of which ten could be classified further as subtype IVa, and 27 as subtype IVc. No isolates belonged to subtype IVb, but four isolates could not be subtyped, and may be examples of novel type IV SCCmec subtypes. Thirty-five MRSA isolates, assigned to six different pulsotypes by pulsed-field gel electrophoresis, did not belong to SCCmec types I-IV. The Panton-Valentine leukocidin (PVL) genes were identified in two of these pulsotypes. Only SCCmec type IV has been associated previously with the PVL toxin, but the results suggest that new PVL-positive clones with novel SCCmec types may be arising and disseminating in the community.

The Neisseria meningitidis Serogroup A Capsular Polysaccharide O-3 and O-4 Acetyltransferase

Neisseria meningitidis serogroup A capsular polysaccharide (CPS) is composed of a homopolymer of O-acetylated, alpha1-->6-linked ManNAc 1-phosphate that is distinct from the capsule structures of the other meningococcal disease-causing serogroups, B, C, Y, and W-135. The serogroup A capsule biosynthetic genetic cassette consists of four open reading frames, mynA-D (sacA-D), that are specific to serogroup A, but the functions of these genes have not been well characterized. mynC was found to encode an inner membrane-associated acetyltransferase that is responsible for the O-acetylation of the CPS of serogroup A. The wild-type CPS as revealed by 1H NMR had 60-70% O-acetylated ManNAc residues that contained acetyl groups at O-3, with some species acetylated at O-4 and at both O-3 and O-4. A non-polar mynC mutant generated by introducing an aphA-3 kanamycin resistance cassette produced CPS with no O-acetylation. A serogroup A capsule-specific monoclonal antibody was shown to recognize the wild-type O-acetylated CPS, but not the CPS of the mynC mutant, which lacked O-acetylation. MynC was C-terminally His-tagged and overexpressed in Escherichia coli to obtain the predicted approximately 26-kDa protein. The acetyltransferase activity of purified MynC was demonstrated in vitro using [14C]acetyl-CoA. MynC O-acetylated the O-acetylated CPS of the mynC mutant and further acetylated the wild-type CPS of serogroup A meningococci, but not the CPS of serogroup B or C meningococci. Genetic complementation of the mynC mutant confirmed the function of MynC as the serogroup A CPS O-3 and O-4 acetyltransferase. MynC represents a new subclass of O-acetyltransferases that utilize acetyl-CoA to decorate the D-mannosamine capsule of N. meningitidis serogroup A.

Local variants of Staphylococcal cassette chromosome mec in sporadic methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative Staphylococci: evidence of horizontal gene transfer?

The mecA gene in Staphylococcus aureus is located on the genetic element staphylococcal cassette chromosome (SCC). Different SCCmecs have been classified according to their putative recombinase genes (ccrA and ccrB) and overall genetic composition. Clinical isolates of coagulase-negative staphylococci (CoNS; n = 39) and S. aureus (n = 20) from Norway, India, Italy, Finland, the United States, and the United Kingdom were analyzed by pulsed-field gel electrophoresis, which showed that most isolates were genetically unrelated. Cluster analyses of 16S rRNA gene and pta sequences confirmed the traditional biochemical species identification. The mecI, mecR1, mecA, and ccrAB genes were detected by PCRs, identifying 19 out of 20 S. aureus and 17 out of 39 CoNS isolates as carriers of one of the three published ccrAB pairs. New variants of SCCmec were identified, as well as CoNS isolates containing ccrAB genes without the mec locus. ccrAB and mec PCRs were verified by hybridization. Sequence alignments of ccrAB genes showed a high level of diversity between the ccrAB alleles from different isolates, i.e., 94 to 100% and 95 to 100% homology for ccrAB1 and ccrAB2, respectively. All of the ccrAB3 genes identified were identical. Genetically unique and sporadic methicillin-resistant S. aureus (MRSA) contained local variants of ccrAB gene pairs identical to those found in MR-CoNS but different from those in MRSA from other regions. Allelic variants of ccrAB in isolates from the same geographic region showed sequence conservation independent of species. The species-independent sequence conservation found suggests that there is a closer genetic relationship between ccrAB2 in Norwegian staphylococci than between ccrAB2 sequences in international MRSA and Norwegian MRSA. This might indicate that different staphylococcal species acquire these genes locally by horizontal gene transfer.

Construction of new unencapsulated (rough) strains of Streptococcus pneumoniae

To construct rough strains of Streptococcus pneumoniae in which the capsule locus was completely deleted, a genetic cassette to be used as a donor DNA in transformation was developed. The cassette contained an aphIII gene, conferring kanamycin resistance, flanked by segments of dexB and aliA. Since, in all strains of S. pneumoniae the capsule locus is between dexB and aliA, the DNA segments of these two genes allow insertion of a 1354-bp DNA fragment containing aphIII into the pneumococcal chromosome, determining the deletion of the whole capsule locus. The capsule locus was deleted from the classic type 2 and type 3 Avery's strains, from R6 (whose complete genome sequence is released) and Rx1 (the two most commonly used transformation recipients), from a type 3 clinical strain and type 19F clinical isolate G54 (whose draft genome sequence is annotated). The effect of capsule removal was tested in 4 isogenic pairs. In unencapsulated strains, growth rate increased up to 56% and transformation frequency increased up to 1075-fold. A correlation was observed between the increase in growth rate and an increase in transformation frequency.

Homology, limbs, and genitalia.

Similarities in genetic control between the main body axis and its appendages have been generally explained in terms of genetic co-option. In particular, arthropod and vertebrate appendages have been explained to invoke a common ancestor already provided with patterned body outgrowths or independent recruitment in limb patterning of genes or genetic cassettes originally used for purposes other than axis patterning. An alternative explanation is that body appendages, including genitalia, are evolutionarily divergent duplicates (paramorphs) of the main body axis. However, are all metazoan limbs and genitalia homologous? The concept of body appendages as paramorphs of the main body axis eliminates the requirement for the last common ancestor of limb-bearing animals to have been provided with limbs. Moreover, the possibility for an animal to express complex organs ectopically demonstrates that positional and special homology may be ontogenetically and evolutionarily uncoupled. To assess the homology of animal genitalia, we need to take into account three different sets of mechanisms, all contributing to their positional and/or special homology and respectively involved (1) in the patterning of themain body axis, (2) in axis duplication, followed by limb patterning mechanisms diverging away from those still patterning the main body axis (axis paramorphism), and (3) in controlling the specification of sexual/genital features, which often, but not necessarily, come into play by modifying already developed and patterned body appendages. This analysis demonstrates that a combinatorial approach to homology helps disentangling phylogenetic and ontogenetic layers of homology.

Limbs and tail as evolutionarily diverging duplicates of the main body axis.

Contrasting hypotheses have been proposed to explain the pervasive parallels in the patterning of arthropod and vertebrate appendages. These hypotheses either call for a common ancestor already provided with patterned appendages or body outgrowths, or for the recruitment in limb patterning of single genes or genetic cassettes originally used for purposes other than axis patterning. I suggest instead that body appendages such as arthropod and vertebrate limbs and chordate tails are evolutionarily divergent duplicates (paramorphs) of the main body axis, that is, its duplicates, albeit devoid of endodermal component. Thus, vertebrate limbs and arthropod limbs are not historical homologs, but homoplastic features only transitively related to real historical homologs. Thus, the main body axis and the axis of the appendages have distinct but not independent evolutionary histories and may be involved in processes of homeotic co-option producing effects of morphological assimilation. For instance, chordate segmentation may have originated in the posterior appendage (tail) and subsequently extended to the trunk.

Engineering of a stable whole-cell biocatalyst capable of (S)-styrene oxide formation for continuous two-liquid-phase applications.

Recombinant strains of Pseudomonas putida KT2440 carrying genetic expression cassettes with xylene oxygenase- and styrene monooxygenase-encoding genes on their chromosomes could be induced in shaking-flask experiments to specific activities that rivaled those of multicopy-plasmid-based Escherichia coli recombinants. Such strains maintained the introduced styrene oxidation activity in continuous two-liquid-phase cultures for at least 100 generations, although at a lower level than in the shaking-flask experiments. The data suggest that placement of target genes on the chromosome might be a suitable route for the construction of segregationally stable and highly active whole-cell biocatalysts.

An alkane-responsive expression system for the production of fine chemicals

Membrane-located monooxygenase systems, such as the Pseudomonas putida mt-2-derived xylene oxygenase, are attractive for challenging transformations of apolar compounds, including enantiospecific epoxidations, but are difficult to synthesize at levels that are useful for application to biotechnological processes. In order to construct efficient biocatalysis strains, we utilized the alkane-responsive regulatory system of the OCT plasmid-located alk genes of Pseudomonas oleovorans GPo1, a very attractive system for recombinant biotransformation processes. Determination of the nucleotide sequence of alkS, whose activated gene product positively regulates the transcription of the structural genes alkBFGHJKL, on a 3.7-kb SalI-HpaI OCT plasmid fragment was completed, and the N-terminal amino acid sequence of an AlkS-LacZ fusion protein was found to be consistent with the predicted DNA sequence. The alkS gene and the alkBp promoter were assembled into a convenient alkane-responsive genetic expression cassette which allowed expression of the xylene oxygenase genes in a recombinant Escherichia coli strain at a specific activity of 91 U per g (dry weight) of cells when styrene was the substrate. This biocatalyst was used to produce (S)-styrene oxide in two-liquid-phase cultures. Volumetric productivities of more than 2 g of styrene oxide per h per liter of aqueous phase were obtained; these values represented a fivefold improvement compared with previous results.

Construction of attenuated HIV-1 accessory gene immunization cassettes

Delivery of genetic expression cassettes into animals can effectively induce both humoral and cellular immunity to the expressed gene product. Previously, we used this strategy to immunize against HIV-1 structural and enzymatic proteins in mice, non-human primates and in humans. In contrast, the use of the accessory genes including vif, vpr, vpu and nef as immunotherapeutic vaccine targets has not been well characterized. Our goal is to design an effective genetic HIV vaccine, which includes the accessory genes as part of a multi-component immunogen. In order to develop accessory genes as genetic vaccines, we have molecularly cloned and analysed the sequence variation and immunogenic potential present in these genes derived from viral isolates obtained from HIV-1 infected patients and laboratory isolates. Prototype genetic variants were selected and their ability to induce humoral and cellular immune responses was studied in animal models. We observed that attenuated accessory genes can effectively induce both humoral and cellular responses in mice and the resulting immune response is directly correlated with DNA concentrations delivered and the number of boosts. This strategy can be used generally to develop an effective, safe DNA vaccine for any pathogen.