Summary
DNA sequence analysis, Tnpho and Tntac-1, mutagenesis, deletion analysis, expression under bacteriophage T7 gene 10 promoter control, subcellular fractionation and complementation tests were used to study the function of DNA located in the centre of thepulC-O operon fromKlebsiella oxytoca strain UNF5023. The characterized region of the operon includes five genes (pulG,pulH,pulI,pulJ andpulK) coding for apparently integral inner membrane proteins which are required for pullulanase secretion. The results presented here and previously show that thepulC-O operon contains at least 11 pullulanase secretion genes.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Baneyx F, Georgiou G (1990) In vivo degradation of secreted fusion proteins by theEscherichia coli outer membrane protease OmpT. J Bacteriol 173:491–494
Chow WY, Berg DE (1988) Tn5tacI, a derivative of transposon Tn5 that generates conditional mutations. Proc Natl Acad Sci USA 85:6468–6472
Dale RM, McClure BA, Houchins JP (1985) A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18S rDNA. Plasmid 13:31–40
d'Enfert C, Pugsley AP (1987) A gene fusion approach to the study of pullulanase export and secretion inEscherichia coli. Mol Microbiol 1:159–168
d'Enfert C, Pugsley AP (1989)Klebsiella pneumoniae pulS gene encodes an outer membrane lipoprotein required for pullulanase secretion. J Bacteriol 171:3673–3679
d'Enfert C, Ryter A, Pugsley AP (1987) Cloning and expression inEscherichia coli of theKlebsiella pneumoniae genes for production, surface localization and secretion of the lipoprotein pullulanase. EMBO J 6:3531–3538
d'Enfert C, Reyss I, Wandersman C, Pugsley AP (1989) Protein secretion by Gram-negative bacteria. Characterization of two membrane proteins required for pullulanase secretion byEscherichia coli K-12. J Biol Chem 264:17462–17468
Dreyfus M, Jacques N (1990) Translation initiation inEscherichia coli: old and new questions. Mol Microbiol, in press
Manoil CC, Beckwith J (1985) TnphoA, a transposon probe for protein export signals. Proc Natl Acad Sci USA 82:8129–8133
Manoil C, Mekalaonos J, Beckwith J (1990) Alkaline phosphatase fusions: sensors of subcellular location. J Bacteriol 172:515–518
Michaelis S, Chapon C, d'Enfert C, Pugsley AP, Schwartz M (1985) Characterization and expression of the structural gene for pullulanase, a maltose-inducible secreted protein ofKlebsiella pneumoniae. J Bacteriol 164:633–638
Miller JH (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p 433
Perbal B (1988) A Practical Guide to Molecular Cloning. Wiley, New York
Pugsley AP (1988) The immunity and lysis genes of ColN plasmid pCHAP4. Mol Gen Genet 211:335–341
Pugsley AP (1989) Protein Targeting. Academic Press, San Diego, USA
Pugsley AP, Chapon C, Schwartz M (1986) Extracellular pullulanase ofKlebsiella pneumoniae is a lipoprotein. J Bacteriol 166:1083–1088
Pugsley AP, Kornacker MG, Ryter A (1990) Analysis of the subcellular location of pullulanase produced byEscherichia coli carrying thepulA gene fromKlebsiella penumoniae strain UNF5023. Mol Microbiol 4:59–72
Pugsley AP, Oudega B (1987) Methods for studying colicins and their plasmids. In: Hardy K (ed) Plasmids, a Practical Approach. IRL Press, Oxford, UK, pp 105–161
Pugsley AP, Reyss I (1990) Five genes at the 3′ end of theKlebsiella pneumoniae pulC operon are required for pullulanase secretion. Mol Microbiol, 365–379
Schnaitman CA (1981) Cell fractionation. In: Gehardt P, Murray RSE, Costilow RN, Nester E, Woody WA, Krieg NR, Phillips GB (eds) Manual of Methods for General Bacteriology. ASM Publications, Washington DC, USA, pp 52–61
Spratt BG, Hedge PJ, te Heesen S, Edelman A, Broome-Smith JK (1986) Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9. Gene 41:337–342
Tabor S, Richardson CC (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82:1074–1078
Takeshita S, Sato M, Toba M, Masahashi W, Hashimoto-Goto T (1987) High copy number and low copy number plasmid vectors forlacZ α-complementation and chloramphenicol or kanamycin-resistance selection. Gene 61:63–74
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
von Heijne G (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683–4690
Author information
Authors and Affiliations
Additional information
Communicated by W. Goebel
Rights and permissions
About this article
Cite this article
Reyss, I., Pugsley, A.P. Five additional genes in thepulC-O operon of the gram-negative bacteriumKlebslella oxytoca UNF5023 which are required for pullulanase secretion. Mol Gen Genet 222, 176–184 (1990). https://doi.org/10.1007/BF00633815
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00633815