Abstract
Streptococcus zooepidemicus is a bacterial pathogen used for production of hyaluronan in industry. Intensive research has significantly contributed to our understanding of S. zooepidemicus biology and pathogenesis. However, the lack of an effective targeted gene inactivation system in S. zooepidemicus has notably prevented the functional genomics analysis of this gram-positive bacterium. Here, we report the development of a markerless gene deletion system in S. zooepidemicus. We constructed a sacB expression cassette on the thermosensitive suicide vector pSET4s and demonstrated its use as a counterselection marker in S. zooepidemicus. We validated the efficiency of this system by deletion of hasA, which synthesizes the important virulence factor hyaluronic acid (HA) capsule. The genotype of the resultant hasA mutant was confirmed by polymerase chain reaction and sequencing. Deletion of hasA resulted in non-mucoid morphology, loss of HA capsule formation, and HA production. These defects can be rescued by introduction of a plasmid containing wild-type hasA expression cassette. Moreover, compared with wild type, hasA mutant showed no significant difference in expressions of other members of the hasABCDE operon, further suggesting that the loss of hasA contributed to the defects observed with ΔhasA mutant. Our results describe the first establishment of a sacB-based counterselection system in S. zooepidemicus, along with the first demonstration of hasA that is the only gene encoding a functional hyaluronan synthase in this bacterium.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Barnham M, Cole G, Efstratiou A, Tagg JR, Skjold SA, Barnham M, Cole G, Efstratiou A, Tagg J, Skjold S (1987) Characterization of Streptococcus zooepidemicus (Lancefield group C) from human and selected animal infections. Epidemiol Infect 98(2):171–182
Beare PA, Larson CL, Gilk SD, Heinzen RA (2012) Two systems for targeted gene deletion in Coxiella burnetii. Appl Environ Microbiol 78(13):4580–4589
Beres SB, Sesso R, Pinto SWL, Hoe NP, Porcella SF, DeLeo FR, Musser JM (2008) Genome sequence of a Lancefield group C Streptococcus zooepidemicus strain causing epidemic nephritis: new information about an old disease. PLoS One 3(8):e3026
Biswas I, Gruss A, Ehrlich SD, Maguin E (1993) High-efficiency gene inactivation and replacement system for gram-positive bacteria. J Bacteriol 175(11):3628–3635
Blank LM, Hugenholtz P, Nielsen LK (2008) Evolution of the hyaluronic acid synthesis (has) operon in Streptococcus zooepidemicus and other pathogenic streptococci. J Mol Evol 67(1):13–22
Bochner BR, Huang H-C, Schieven GL, Ames BN (1980) Positive selection for loss of tetracycline resistance. J Bacteriol 143(2):926–933
Cherepanov PP, Wackernagel W (1995) Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158(1):9–14
Chong BF, Blank LM, Mclaughlin R, Nielsen LK (2005) Microbial hyaluronic acid production. Appl Microbiol Biotechnol 66(4):341–351
Crater DL, Dougherty BA, Rijn I (1995) Molecular characterization of hasC from an operon required for hyaluronic acid synthesis in group A streptococci. J Biol Chem 270:28676–28680
DeAngelis PL, Papaconstantinou J, Weigel PH (1993) Isolation of a Streptococcus pyogenes gene locus that directs hyaluronan biosynthesis in acapsular mutants and in heterologous bacteria. J Biol Chem 268(20):14568–14571
Gay P, Le Coq D, Steinmetz M, Ferrari E, Hoch J (1983) Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expressionof the gene in Escherichia coli. J Bacteriol 153(3):1424–1431
Hamilton CM, Aldea M, Washburn B, Babitzke P, Kushner S (1989) New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol 171(9):4617–4622
Hirayama Y, Sakanaka M, Fukuma H, Murayama H, Kano Y, Fukiya S, Yokota A (2012) Development of a double-crossover markerless gene deletion system in Bifidobacterium longum: functional analysis of the α-galactosidase gene for raffinose assimilation. Appl Environ Microbiol 78(14):4984–4994
Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77(1):51–59
Leenhouts K, Buist G, Bolhuis A, Ten Berge A, Kiel J, Mierau I, Dabrowska M, Venema G, Kok J (1996) A general system for generating unlabelled gene replacements in bacterial chromosomes. Mol Gen Genet 253(1–2):217–224
Liu L, Liu Y, Li J, Du G, Chen J (2011) Microbial production of hyaluronic acid: current state, challenges, and perspectives. Microb Cell Fact 10(1):1–9
Ma Z, Geng J, Zhang H, Yu H, Yi L, Lei M, C-p L, Fan H-j HS (2011) Complete genome sequence of Streptococcus equi subsp. zooepidemicus strain ATCC 35246. J Bacteriol 193(19):5583–5584
Mangan J, Sole K, Mitchison D, Butcher P (1997) An effective method of RNA extraction from bacteria refractory to disruption, including mycobacteria. Nucleic Acids Res 25(3):675–676
Mollerach M, López R, García E (1998) Characterization of the galU gene of Streptococcus pneumoniae encoding a uridine diphosphoglucose pyrophosphorylase: a gene essential for capsular polysaccharide biosynthesis. J Exp Med 188(11):2047–2056
Pelicic V, Reyrat J-M, Gicquel B (1996) Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria. J Bacteriol 178(4):1197–1199
Reyrat J-M, Pelicic V, Gicquel B, Rappuoli R (1998) Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infect Immun 66(9):4011–4017
Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145(1):69–73
Stragier P, Bonamy C, Karmazyn-Campelli C (1988) Processing of a sporulation sigma factor in Bacillus subtilis: how morphological structure could control gene expression. Cell 52(5):697
Takamatsu D, Osaki M, Sekizaki T (2001a) Construction and characterization of Streptococcus suis–Escherichia coli shuttle cloning vectors. Plasmid 45(2):01–113
Takamatsu D, Osaki M, Sekizaki T (2001b) Thermosensitive suicide vectors for gene replacement in Streptococcus suis. Plasmid 46(2):140–148
Tan Y, Xu D, Li Y, Wang X (2012) Construction of a novel sacB-based system for marker-free gene deletion in Corynebacterium glutamicum. Plasmid 67(1):44–52
Wei Z, Fu Q, Chen Y, Cong P, Xiao S, Mo D, He Z, Liu X (2012) The capsule of Streptococcus equi ssp. zooepidemicus is a target for attenuation in vaccine development. Vaccine 30(31):4670–4675
Weigel PH, DeAngelis PL (2007) Hyaluronan synthases: a decade-plus of novelglycosyltransferases. J Biol Chem 282(51):36777–36781
Wessels MR, Bronze MS (1994) Critical role of the group A streptococcal capsule in pharyngeal colonization and infection in mice. Proc Natl Acad Sci USA 91:12238–12242
Acknowledgments
This work was supported by grants from seventh Singapore–China JRP (2011DFA31280), National Natural Science Foundation of China (31270125), the 863 project (2012AA020403), and the Cheung Kong Scholars Program of China (IRT1166).
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(PDF 169 kb)
Rights and permissions
About this article
Cite this article
Sun, X., Yang, D., Wang, Y. et al. Development of a markerless gene deletion system for Streptococcus zooepidemicus: functional characterization of hyaluronan synthase gene. Appl Microbiol Biotechnol 97, 8629–8636 (2013). https://doi.org/10.1007/s00253-013-5058-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-013-5058-8