Characterization of a sialyltransferase-deficient mutant of Neisseria gonorrhoeae strain F62: instability of transposon Tn1545 delta3 in gonococci and evidence that multiple genetic loci are essential for lipooligosaccharide sialylation.
Neisseria gonorrhoeae strain JB1 was previously shown to be defective in the sialylation of lipoologosaccharide (LOS) by exogenous CMP-NANA. The LOS components synthesized by the mutant now have been shown by mass spectrometry to be similar to those in the parental strain, F62, and to include the 4.5 kDa widely conserved lacto-N-neotetraose component that can be sialylated. The same two LOS components could be sialylated on the surface of the mutant and parental strains. One major component was sialylatable after chemical extraction of the LOS from either strain. These data confirm that the mutant, JB1, retains the ability to synthesize the LOS target required for the conversion by sialylation of serum-sensitive gonococci to serum resistance. A single base frame-shift mutation was found in the lst gene from the mutant, resulting in the replacement of the final 61 amino acids at the C-terminus of the sialyltransferase by four residues. Seventeen independent clones of the lst gene were isolated from the parental strain, but none of them complemented the sialyltransferase defect of the mutant and no sialyltransferase activity expressed from the clones could be detected in Escherichia coli. Although the data suggest that the mutant might be defective in genes at more than one chromosomal locus and that multiple loci are essential for sialyltransferase synthesis and activity, the alternative possibility, that DNA adjacent to the lst gene encodes a product which is toxic to E. coli, cannot be excluded. The site of insertion of the transposon Tn1545-Delta3 in strain JB1 was cloned and sequenced. The transposon is located in an intergenic region adjacent to genes for a putative ATP-dependent transport protein, but encoding no recognizable function relevant to LOS sialylation. Evidence that transposon Tn1545-Delta3 is unstable in gonococci is presented.
Crooke
H
H
School of Biochemistry, University of Birmingham, Birmingham, B15 2TT, U.K.
Griffiss
J M
JM
John
C M
CM
Lissenden
S
S
Bramley
J
J
Regan
T
T
Smith
H
H
Cole
J
J
D015139:Blotting, Southern; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D004251:DNA Transposable Elements / Q000502:physiology*; D004269:DNA, Bacterial / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D005816:Genetic Complementation Test; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009344:Neisseria gonorrhoeae / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D015342:DNA Probes; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides; D012799:Sialyltransferases; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase