Navy, Research Tasks MR005. 05.01-0008B and M4305. 01-1008. 2 Permanent
.... J. 7:567-584. 2. Goldschneider, I., E. C. Gotschlich, and M. S. Artenstein.
INFECTION
AND
Vol. 3, No. 2 Printed in U.S.A.
IMMUNITY, Feb. 1971, p. 274-277
Copyright ©) 1971 American Society for Microbiology
Partial Reassociation Between the Deoxyribonucleic Acids of Neisseria lactamicus and Neisseria meningitidis' SCHRAMEK,2
E. WEISS, N. N. WILSON,
Departmenit of Microbiology, Naval Medical Research Inistitute, Bethesda, Marylanid 20014
The similarity in polynucleotide
sequence
of seven strains of Neisseria lactamicus
to N. meningitidis and to each other was investigated. The per cent reassociation
between single-stranded N. lactamicus deoxyribonucleic acid (DNA), immobilized on membrane filters, with labeled single-stranded DNA fragments derived from strain SD-6 (group C) of N. meningitidis varied from 75 to 56. With strain ATCC 23972 of N. lactamicus furnishing the labeled DNA fragments, reassociation was 72% with N. meningitidis, 31% with N. subflava, and 98 to 72%, with the other strains of N. lactamicus. It is concluded that on the basis of DNA reassociation N. lactamicus can be distinguished from N. meningitidis and N. subflava, but constitutes a relatively heterogeneous species.
Kingsbury (5) tested 11 strains of Neisseria meningitidis for similarity in polynucleotide sequence by the deoxyribonucleic acid (DNA) reassociation technique. Ten of these strains were indistinguishable from each other by this method, suggesting that the considerable variation among them in serological specificity, represented by groups A, B, C, D, X, Y, and 29E, was reflected in small differences in the genome. Only a group Z strain was significantly different from the others. These studies did not extend to the lactose-fermenting strains which, until recently, were considered to be atypical strains of N. meningitidis. Hollis et al. (3) introduced the designation N. lactamicus for these strains. In addition to fermenting lactose, a majority of these strains grow in nutrient agar at 35 to 37 C and cannot be typed because of their tendency to autoagglutinate. Despite their prevalence in the nasopharynx of individuals 12 years or younger, they have not been associated with meningococcal disease (4). This investigation represents a further attempt at differentiation of N. lactamicus from N. meningitidis. MATERIALS AND METHODS Strains ATCC numbers 23970 and 23972 of N.
lactamicus (3) were obtained from the American Type 1 From the Bureau of Medicine and Surgery, Department of the Navy, Research Tasks MR005. 05.01-0008B and M4305. 01-1008. 2 Permanent address: Institute of Virology, Slovak Academy of Sciences, Bratislava, Czechoslovakia.
274
Culture Collection. The other lactose fermenters were obtained from the Naval Biological Laboratory, Oakland, Calif., and are identified by their designations. M-248 and M-714 were isolated by T. D. Keyes and C. Halvorson of the Preventive Medicine Unit No. 6, Pearl Harbor, Hawaii, during a meningococcus carrier survey of ship personnel; M-655 from an adult by the California State Public Health Laboratory, Berkeley, Calif.; and M-921 and M-922 from children by Virginia Dimanche, Cantacuzino Institute, Bucharest, Rumania. All of these strains were isolated from the nasopharynx. The SD-6 strain of N. meninigitidis, group C, a group Z strain also regarded as N. meningitidis (9), and N. subflava were the same as those used in previous studies (5). Since N. lactamicus strains did not grow in Frantz' modified medium (10), labeled DNA was obtained by adding adenine-8-14C to N. lactamicus cultures growing in Trypticase Soy Broth (BBL). The technique of DNA reassociation on membrane filters was identical to that described previously (5). The temperature of incubation during reassociation was 67 C, and the ionic strength of the diluent was 0.6 Na+. The thermal stability of hybrid reassociated DNA was tested by the elution of 14C-DNA fragments from the membrane filters as the temperature was gradually increased (1, 6). The ionic strength was reduced to 0.0065 M Na+ during this portion of the experiment. The results are presented in a different manner than in previous papers (6, 7, 1 1). Instead of selecting a typical elution pattern for each hybrid, regression lines were calculated over the short temperature ranges where elutions were approximately linear. By this method it was possible to combine results obtained in duplicate or triplicate determinations. The temperatures at which half of the
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Received for publication 21 September 1970
VOL. 3, 1971
DNA OF N. LACTAMICUS AND N. MENINGITIDIS
radioactivity had eluted (Em)
were calculated
275
from the
degree of reassociation of SD-6 fragments to strains of N. lactamicus varied from 78 to 56%. There appeared to be no correlation between RESULTS similarity in polynucleotide sequence and geoTwo strains used in this study (ATCC 23970 graphic origin of the strains. Heterogeneity among and 23972) were described by Hollis et al. (3). the N. lactamicus strains is better illustrated by ATCC 23972, but not ATCC 23970, grew on the experiments carried out with labeled strain nutrient agar at 37 C. The characteristics of the 23972. The polynucleotide sequence of 23972 apother strains are shown in Table 1. They all fer- peared to be identical to that of M-922, but reasmented glucose, maltose, and lactose and dis- sociation with the other N. lactamicus strains played f-D-galactosidase activity. Only one strain ranged from 90 to 72%. Reassociation with N. grew well, and one grew slightly on nutrient agar meningitidis was considerably more extensive than at 37 C. Three strains agglutinated in saline and with N. subflava. Experiments with labeled DNA could not be typed. Two strains were agglutinated from strain Z suggest that strain 23972, but not by antisera prepared against N. lactamicus, but 23970, is possibly more closely related to it than not by antimeningococcal sera. The serological to N. meningitidis. The specificity of the reassociation reaction betests were performed by Dannie G. Hollis and Robert E. Weaver of the Center for Disease Con- tween heterologous DNA was studied by comtrol, Atlanta, Ga., who also confirmed our results paring the thermal stability of homologous and indicating that these are typical N. lactamicus heterologous reassociated DNA. The Em values in homologous SD-6 or 23972 were about 65 C. strains. DNA reassociation was studied with labeled (The low Em values are due to the low ionic DNA fragments derived from strains SD-6, Z, strength of the solution used.) The Em values of and 23972. The results are shown in Table 2. The duplexes of labeled SD-6 DNA fragments and
regression lines.
Strain designation
M-655 M-248 M-714 M-921 M-922
Origin
California Hawaii Hawaii
Rumania Rumania
Agglutination by antiserum to
Sugar fermentations
6-D-Galac
Growth
Maltose
activity
agar'
N. lactamicus
-
± +
_
+
Glucose
+ + + + +
Lactose
+ -+ + + + +I + +
+
+
onutin
tosidase
+ + + + +
N.
neningilidis
Autoagglutinable Autoagglutinable Slight Autoagglutinable Autoagglutinable + Autoagglutinable Autoagglutinable
a At 37 C.
TABLE 2. Reassociation of Neisseria DNA to labeled Neisseria DNA fragmentsa Per cent reassociationb of labeled DNA
Source of immobilized DNA
Species
Strain and its origin
SD-6
Neisseria meniingitidis
SD-6, San Diego
(100)
N. lactamicus
23972, CDCC
Group Z, Sweden
M-922, Rumania 23970, CDC M-921, Rumania M-248, Hawaii M-655, California M-714, Hawaii
b c
In ionic concentration of 0.6 M Na+ at 67 C. Mean of two or three experiments, each done in Center for Disease Control, Atlanta, Ga.
23972
72
90
(100)
75 78 78 67 65 70 56
83 77
(100) 98 90 89 87 83 72 31
N. subflava a
Z
fragments
triplicate.
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TABLE 1. Characteristics of Neisseria lactamicus strains
276
WEISS ET AL.
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DISCUSSION The lactose-fermenting strains of Neisseria were placed in a separate species, N. lactamicus, and differentiated from N. nmeningitidis on the basis of phenotypic characteristics (3). It was also shown that the two species play different roles in human infection (4). The studies described here clearly
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TEMPERATURE (C)
FIG. 1. Thermal elution of labeled fragments of DNA derived from Neisseria meningitidis, strain SD-6, reassociated with DNA immobilized on membrane filters from SD-6 (a), N. lactamicus strain 23972 (0) and strain 23970 (a). The regression lines were calculated by the least squares method.
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