INTERNATIONALJOURNAL OF SYSTEMATIC BACTERIOLOGY, July 1988, p. 319-320
0020-7713/88/030319-02$02.00/0
Copyright 0 1988, International Union of Microbiological Societies
Vol. 38, No. 3
V-Factor-Dependent Growth of Actinobacillus pleuropneumoniae
Biotype 2 (Bertschinger 2008/76)
D. F. NIVEN" A N D M. LEVESQUE
Department of Microbiology, Macdonald College of McGill University, Ste Anne de Bellevue, Quebec, Canada H9X ICO
A chemically defined medium containing nicotinamide adenine dinucleotide (NAD) supported the growth of
ActinobaciZZus pleuropneumoniae biotype 2 (Bertschinger 2008/76), whereas the same medium lacking NAD did
not. Nicotinamide mononucleotide, nicotinamide riboside, and nicotinamide could substitute for NAD, whereas
nicotinic acid adenine dinucleotide, nicotinic acid mononucleotide, nicotinic acid, and quinolinic acid could not.
Hence, although described as being V factor independent, this organism can exhibit V-factor-dependent
growth.
The organism known previously as Haemophilirs pleuropneumoniae (3) and now referred to as Actinobacillus pleuropneumoniae biotype 1 (7) requires V factor for growth;
whereas nicotinamide adenine dinucleotide (N AD), nicotinamide mononucleotide (NMN), and nicotinamide riboside
(NR) can serve as V factor, nicotinamide (NAm) and the
acid analogs of these compounds cannot (4). A . pleuropneirmoniae biotype 2, on the other hand, is described as being V
factor independent (7). However, to our knowledge, it
remains unknown whether A . pleuropneumoniae biotype 2
can synthesize NAD de novo, unlike biotype 1 (3,or
requires for growth some form of pyridine nucleotide precursor, such as NAm. In this respect, note that most
complex media probably contain NAm or nicotinic acid or
both. Therefore, the purpose of the present study was to
grow A . pleuropneumoniae biotype 2 in a chemically defined
medium (CDM) and to determine the requirement, if any, for
an exogenous pyridine nucleotide precursor.
Cultures of A . pleuropneumoniae biotype 2 (Bertschinger
2008/76) were stored and frozen inocula were prepared as
described previously (6). To obtain organisms for inoculation of CDM, frozen inocula (6) were thawed rapidly, and
0.05-ml volumes were used to inoculate 20-ml volumes of
tryptone-yeast extract medium (6) contained in 125-ml flasks
fitted with Kaput closures (Bellco Glass, Inc., Vineland,
N.J.). These cultures were incubated for 8 h at 37°C with
agitation on a gyratory shaker (200 rpm). The organisms
from these early stationary phase cultures (optical density at
660 nm [OD66,], -2.2; model 240 spectrophotometer; Gilford Instrument Laboratories, Inc., Oberlin, Ohio) were
harvested by centrifugation (21,000 x g , 20 min), washed
with an equal volume of sterile saline, and resuspended
similarly. Portions (0.2 ml) of such suspensions were added
to 125-ml flasks (as described above) containing 20-ml volumes of CDM; this medium was prepared as described by
Herriott et al. (2), except that sodium lactate, hemin, nitrilotriethanol, and NAD were omitted, and glucose and FeCI,
were incorporated to give final concentrations of 10 mM and
10 pM, respectively. Such cultures in CDM were incubated
as described above, but for 15 h. By this time growth had
ceased (OD6607 -0.35), presumably due to pyridine nucleotide source limitation as growth resumed if cultures were
supplemented with 0.5 pM NAD; a similar phenomenon has
been observed with A . pleuropneumoniae biotype 1 (4). The
organisms from these CDM cultures were harvested,
washed, and resuspended as described above, and 0.2-ml
volumes were used to inoculate triplicate 20-ml volumes of
CDM that did or did not contain the filter-sterilized pyridine
nucleotide or precursor (0.5 pM) under study. NR was
produced and NR, NMN, and NAD were purified and
analyzed as described previously ( 3 , except that a step
gradient of HCOOH (increments of 0.1 M) was used to elute
a column (1.5 by 5.5 cm) of anion exchange resin (Dowexformate). Quinolinic acid, nicotinic acid, nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, and NAm
were used without further purification. The latter cultures
were incubated at 37°C with agitation (200 rpm), and growth
was monitored turbidimetrically at 660 nm by using 0.45-ml
samples.
Figure 1 depicts the growth of A . pleuropneumoniae
biotype 2 (Bertschinger 2008/76) in CDM supplemented with
NAD, NMN, NR, or NAm. Mean generation times of 86 min
(CDM containing NAD), 94 min (CDM containing NMN), 98
rnin (CDM containing NR), and 83 rnin (CDM containing
NAm) were calculated from the linear portions of the
plots; consideration of the first three (CDM containing
NAm), four (CDM containing NR or NMN), or five (CDM
containing NAD) data points (i.e., OD660 of <0.55) gave
correlation coefficients of greater than 0.999. On the other
hand, no growth was observed when the organisms were
incubated in nonsupplemented CDM or in CDM supplemented with quinolinic acid, nicotinic acid, nicotinic acid
mononucleotide, or nicotinic acid adenine dinucleotide; the
OD,,, values of these cultures remained constant at approximately 0.003 for at least 36 h. These data demonstrate that
the growth of A . pleuropneumoniae biotype 2 (Bertschinger
2008/76) requires the presence of specific pyridine nucleotides or pyridine nucleotide precursors. Although further
studies will be required to confirm such a suggestion, the
data also indicate that A . pleuropneumoniae biotype 2
(Bertschinger 2008/76), like biotype 1 (3,cannot synthesize
NAD de novo.
The most interesting outcome of these experiments is that
they present us with a significant dilemma; namely, they
demonstrate that A . pleuropneumoniae biotype 2 (Bertschinger 2008/76), although described as being V factor
independent, can exhibit V-factor (NAD, NMN, or NR)dependent growth. On the other hand, NAm, which cannot
support the growth of A . pleuropneumoniae biotype 1 (4),
can satisfy the requirement of the biotype 2 organism for a
pyridine nucleotide precursor. However, as none of several
other compounds involved in procaryotic pyridine nucleo-
* Corresponding author.
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320
NOTES
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INCUBATION TlhE (h)
FIG. 1. Growth of A. pleuropneuinoniar biotype 2 (Bertschinger 2008/76) in CDM supplemented with NAD (O), NMN (0),NR (A),or
‘VAm (A). Culture turbidity values are the logarithms of the mean OD,, values; the error bars represent log (mean OD,, + standard error of the
mean) and log (mean OD,, - standard error of the mean). Where bars are not presented, the symbols encompass the standard error of the mean.
tide metabolism (1)can support the growth of A . pleuropneu,Ynoniae biotype 2 (Bertschinger 2008/76), as is also the case
?withA . pleuropneumoniae biotype 1 (4),one possible soluition to the dilemma is the inclusion of NAm among the
compounds that can serve as V factor. A V-factor-dependent
organism could then be defined as an organism that requires
a pyridine nucleotide source for growth and can use at least
one, but not necessarily all, of the newly defined V-factor
compounds (nicotinamide adenine dinucleotide phosphate,
NAD, NMN, NR, NAm) for this purpose. When this defiinition is used, and provided that other A . pleuropneumoniue
lbiotype 2 strains exhibit a requirement for either NAD,
NMN, NR, or NAm, all A . pleuropneumoniae strains could
Ibe described as being V factor dependent, with biotype 1and
lbiotype 2 reflecting the capacity to utilize NAm. In our
opinion, the only alternative is that V-factor terminology be
abandoned.
This work was supported by the Natural Sciences and Engineering Research Council of Canada.
We thank T. Kouri for contributions in the early stages of this
]project and T. O’Reilly for encouragement and stimulating discus:sions.
LITERATURE CITED
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3. Kilian, M., J. Nicolet, and E. L. Biberstein. 1978. Biochemical
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