Markerless gene editing in Neisseria gonorrhoeae Jones, Rebekah A.; Yee, Wearn Xin; Mader, Kahlio ...
Microbiology (Society for General Microbiology),
06/2022, Volume:
168, Issue:
6
Journal Article
Peer reviewed
Neisseria gonorrhoeae
, the gonococcus, is a pathogen of major public health concern, but sophisticated approaches to gene manipulation are limited for this species. For example, there are few ...methods for generating markerless mutations, which allow the generation of precise point mutations and deletions without introducing additional DNA sequence. Markerless mutations are central to studying pathogenesis, the spread of antimicrobial resistance (AMR) and for vaccine development. Here we describe the use of
galK
as a counter-selectable marker that can be used for markerless mutagenesis in
N. gonorrhoeae
.
galK
encodes galactokinase, an enzyme that metabolizes galactose in bacteria that can utilize it as a sole carbon source. GalK can also phosphorylate a galactose analogue, 2-deoxy-galactose (2-DOG), into a toxic, non-metabolisable intermediate, 2-deoxy-galactose-1-phosphate. We utilized this property of GalK to develop a markerless approach for mutagenesis in
N. gonorrhoeae
. We successfully deleted both chromosomally and plasmid-encoded genes, that are important for gonococcal vaccine development and studies of AMR spread. We designed a positive-negative selection cassette, based on an antibiotic resistance marker and
galK
, that efficiently rendered
N. gonorrhoeae
susceptible to growth on 2-DOG. We then adapted the
galK
-based counter-selection and the use of 2-DOG for markerless mutagenesis, and applied biochemical and phenotypic analyses to confirm the absence of target genes. We show that our markerless mutagenesis method for
N. gonorrhoeae
has a high success rate, and should be a valuable gene editing tool in the future.
•An infectious bacterial artificial chromosome (BAC) clone for an emerging pseudorabies virus variant was developed.•The BAC clone, pBAC-JS2012, contains the full-length genome of the PRV variant ...strain JS-2012 without any gene deletion.•The BAC clone, pBAC-JS2012, contains the full-length genome of the PRV variant strain JS-2012 without any gene deletion.
Since late 2011, a pseudorabies virus (PRV) variant with increased virulence in old pigs had caused major disease outbreaks and great economic losses to the pig industry in China. The gene mutations that contributed to the increased virulence were unclear. To study the basis of the enhanced pathogenicity, an infectious bacterial artificial chromosome (BAC) clone consisting of the complete genome of the PRV variant was developed. Using homologous recombination and Cre/LoxP recombination, the recombinant virus rJS-2012-BAC carrying a BAC insertion downstream of the open reading frame (ORF) of gG was constructed. The circular genome of rJS-2012-BAC was extracted from infected Vero cells and transformed into Escherichia coli DH10B, generating the BAC clone pBAC-JS2012. The loxP sites flanking the BAC vector were used to excise the BAC sequences using Cre recombinase. The reconstituted BAC-excision virus, vJS2012 L, from pBAC-JS2012 exhibited similar biological properties to the wild-type virulent strain JS-2012. To manipulate the BAC clone pBAC-JS2012, the galK selection system was adopted to delete the gI/gE gene from pBAC-JS2012 in E. coli and to generate the gI/gE-deleted virus vJS2012-ΔgE/gI. The BAC clone, pBAC-JS2012, retained the same level of virulence as its parent strain and was easily manipulated using a galK system which would facilitate the study of the enhanced pathogenicity of the PRV variant as well as other studies on PRV.
Streptococcus thermophilus
is one of the most important homo-fermentative thermophilic bacteria, which is widely used as a starter culture in dairy industry. Both wild-type galactose-negative (Gal
−
...)
S. thermophilus
AR333 and galactose-positive (Gal
+
)
S. thermophilus
S-3 in this study were isolated from Chinese traditional dairy products. Here, to access the mechanism of the difference of galactose utilization between strains AR333 and S-3, the expression of
gal
–
lac
operons was examined using real-time qPCR in the presence of different sugars, and the gene organization of
gal
–
lac
operons was characterized using comparative genomics analysis. As compared with medium containing glucose, the expression of
gal
–
lac
operons in AR333 and S-3 was significantly activated (> 5-fold) in the presence of galactose or lactose in the medium. More importantly, the expression of
gal
operon in S-3 was higher than that of AR333, suggesting that the strength of
gal
promoter in AR333 and S-3 may be different. The genomes of AR333 and S-3 were the first time sequenced to provide insight into the difference of
gal
–
lac
operons in these two strains. Comparative genomics analysis showed that gene order and individual gene size of
gal
–
lac
operons are conserved in AR333 and S-3. The DNA sequence of
gal
operon responsible for galactose utilization between AR333 and S-3 is almost identical except that
galK
promoter of S-3 possesses single base pair mutation (G to A substitution) at -9 box
galK
region. Moreover, the expression of red fluorescent protein can be activated by
galK
promoter of S-3, but cannot by
galK
promoter of AR333 in galactose medium, suggesting that
gal
operon is silent in AR333 and active in S-3 under galactose-containing medium. Overall, our results indicated that single point mutation at -9 box in the
galK
promoter can significantly affect the expression of
gal
operon and is largely responsible for the Gal
+
phenotype of
S. thermophilus
.
Bacterial artificial chromosomes (BACs) are genomic tools that can carry several hundred kilobases of exogenous genomic material. This allows to incorporate sufficiently large DNA stretches to ...include most if not all upstream and downstream cis-regulatory elements of a gene in order to mimic and analyze its endogenous regulation of expression using a reporter protein in vivo. Here, we illustrate the generation of a BAC:LIF-EGFP transgenic mouse line to describe a simplified version of BAC transgenesis using galK-based recombineering.
Deficiencies of galactokinase (GALK) and UDP-epimerase (GALE) are implicated with galactose metabolic disorders. The aim of the study was the identification of mutations in GALK and GALE genes and ...clinical evaluation of patients.
Five patients with GALK and five with GALE deficiency were picked up via the Neonatal Screening Program. Additionally, two females, 4 years old, were referred with late diagnosed galactosemia, as rare cases. Mutational analysis was conducted via Sanger sequencing, while in silico analysis tools were utilized for the novel mutation. Psychomotor and speech development tests were performed, as well.
The mutation p.Pro28Thr was identified in both alleles in GALK-deficient patients of Roma (gypsy) origin, whereas the novel p.Asn39Ser was detected in two non-Roma patients. In GALE-deficient patients benign and/or likely benign mutations were found. Psychomotor and speech delay were determined in the Roma GALK patients. In each of the late diagnosed females, four mutations were identified in all galactosemia-related genes.
The mutational spectrums of GALE- and GALK-deficient patients in Greece are presented for the first time along with a clinical evaluation. Mutational analysis in all galactosemia-related genes of symptomatic patients is highly recommended for future cases.
•Using galK positive and negative screening methods, we managed to replace gene sequences in the BAC precisely.•We deleted the first 100kb at the 5’regulatory region carried by the BAC vectors via ...homologous recombination.•Pronuclear injection efficiency of BAC transgenic mice reached 8.5% using the Tol2 transposon system.
Bacterial artificial chromosomes (BACs) are vectors that are capable of carrying gene fragments of up to 300kb in size, and in theory, harbor cis-regulatory elements that are necessary for the expression of specific genes. Therefore, BACs can effectively alleviate or even eliminate the position effect induced by gene-integration, rendering these as ideal expression vectors of exogenous genes. However, the number of relevant studies involving BACs as vectors of exogenous genes are limited. In the present study, we converted the BAC regulatory region of the Mus musculus Wap gene into a mammary gland-specific expression vector. Using the galK-based positive-negative selection method, we seamlessly replaced the Wap gene in a BAC with Homo sapiens GPX3, MT2, and Luc genes while keeping the original mammary gland-specific regulatory sequence intact, without introducing any extra sequences (Loxp/Frt). To improve the efficiency of creating BAC transgenic mice, we used a Tol2 transposon system optimized for mammalian codons and eliminated 100kb of sequence from the BAC 5′ end (173kb), which resulted in an 8.5% rate of successful gene transmission via pronuclear injection. The results of the present study indicate that seamlessly constructed BAC expression vectors can be used for the transmission of the GPX3 gene.
► We developed a galK-UTR BAC recombineering method to mutate the diploid IR2 gene of EHV-1. ► The key idea is to block one of the target diploid genes by galK untranslated region (UTR). ► The IR2 ...mutant virus was successfully generated. ► The galK-UTR method will provide a useful tool in studies of herpesviruses.
Bacterial artificial chromosome (BAC) recombineering using galK selection allows DNA cloned in Escherichia coli to be modified without introducing an unwanted selectable marker at the modification site. Genomes of some herpesviruses have a pair of inverted repeat sequences that makes it very difficult to introduce mutations into diploid (duplicate) genes using the galK selection method. To mutate diploid genes, we developed a galK-UTR BAC recombineering procedure that blocks one copy of the target diploid gene by insertion of a galK untranslated region (UTR), which enables the simple mutation of the other copy. The blocked copy can then be replaced with an UTR-specific primer pair. The IR2 gene of equine herpesvirus 1 (EHV-1) maps within both the internal (IR) and terminal repeat (TR) of the genomic short region and is expressed at low levels because its promoter is TATA-less. Both IR2 promoters in EHV-1 BAC were replaced with a mutant IR2 promoter containing three Sp1-binding motifs and a consensus TATA box by galK-UTR BAC recombineering. The expression level of the IR2 protein controlled by the modified promoter increased approximately 4-fold as compared to that of wild-type EHV-1. The galK-UTR method will provide a useful tool in studies of herpesviruses.
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