Microbiobgy (1994), 140, 3225-3232
Printed in Great Britain
Elements interrupting nitrogen fixation genes
in cyanobacteria: presence and absence of a
nifD element in clones of Nostoc sp. strain Mac
John C. Meeks, Elsie Lin Campbell and Prakash S. Bisent
Author for correspondence: J. C. Meeks. Tel:
Section of Microbiology,
Division of Biological
Sciences, University of
California, Davis, CA 95616,
USA
+ 1 916 752 3346. Fax: + 1 916 752 9014.
Nostoc sp. strain Mac is capable of microaerobic, but not aerobic, nitrogen
fixation (Fox-). Nostoc Mac grows as long, relatively straight, filaments that
are well dispersed in the culture medium. However, spontaneously-arising
revertant strains selected for aerobic nitrogen fixation (Fox+)all grow as coiled
filaments that associate in macroscopic clumps or balls of varying dimensions.
DNA restriction fragment length polymorphism, using nitrogenase (nif)
structural genes as probes, established identity between revertants and the
parental culture. Mapping of the fragments and lack of hybridization to
specific probes indicated the absence of a DNA sequence interrupting the nifD
gene in one Fox+revertant. Such a nifD element is assumed to be present in
essentially all heterocyst-forming cyanobacteria. Only one clone out of 223
Fox- and Fox+Nostoc Mac clones surveyed lacked the nifD element, indicating
that loss of the element is a rare event. The nifD element is present in the
same location in the genome of Nostoc Mac as it is in all other heterocystforming cyanobacteria analysed. No phenotypic differences could be detected
between two Fox+clones containing or lacking the nifD element, including
repression and derepression of nitrogen fixation in response to the presence
or absence of combined nitrogen. We suspect that retention of the nifD
element in vegetative cells of heterocyst-forming cyanobacteria is a
consequence of selective pressure, although such selective conditions in
laboratory cultures have not been identified.
Keywords : diazotrophic cyanobacterium, nif gene organization, nzjD element, nitrogen
fixation, Nostoc sp. Mac
INTRODUCTION
In cyanobacteria, the metabolic process of nitrogen
fixation is expressed under anaerobic, microaerobic or
aerobic growth conditions by unicellular and filamentous
species representative of each of the five major taxonomic
Sections (senszl Rippka e t al., 1979). In the heterocystforming species, nitrogenase (nzj)expression appears to be
confined to the heterocysts (Elhai & Wolk, 1990), whereas
it may be active in any or all cells of the non-heterocystforming species. Moreover, organization of the nif
....,........,,.,..,....... ........,,.,.,,,,,...,,.,..,........,,...............,,..,.,.,,,,.,.,,....,............,..,,,,.,,...,,,...,......,....,......
Microbiology, Barkatullah University, Bhopal, India.
,
t Permanent address: Department of
Abbreviations: ATCC, American Type Culture Collection; Chl a, chlorophyll a; PCC, Pasteur Culture Collection; RFLP, restriction fragment length
polymorphism.
0001-9216 0 1994 SGM
structural genes, n z f l (dinitrogenase reductase), nzjD (asubunit of dinitrogenase) and n$K @-subunit of dinitrogenase), in vegetative cells seems to be distinctly different
between the heterocyst-forming and the non-heterocystforming species (Kallas e t al., 1985; Haselkorn, 1992;
Tandeau de Marsac & Houmard, 1993). In all nonheterocyst-forming, unicellular and filamentous species
examined to date, nzjHDK are contiguously organized in
the genome and transcribed as an operon (Barnum &
Gendel, 1985; Kallas e t al., 1985), similar to the situation
in most proteobacteria (Dean & Jacobson, 1992). While
extensive surveys have not been conducted, in essentially
all heterocyst-forming strains examined, the nzjD gene is
interrupted by a DNA element (Haselkorn, 1992) that
must be removed before the nzfHDK operon can be
transcribed (Golden & Wiest, 1988). Details of the
element and its excision have been described only in
Anabaena sp. strain PCC 7120 (Golden e t al., 1985, 1987,
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3225
1. C. MEEKS, E. L. C A M P B E L L a n d P. S. B I S E N
1991). In Anabaena 7120 the element is 11 kb in size,
bounded by an 11 bp direct repeat sequence, and includes
the genetic sequence (xisA)that encodes the site specific
recombinase (XisA or excisase) responsible for excision
and generation of the transcribed operon (Lammers e t al.,
1986). Excision occurs during latter stages of heterocyst
maturation, but the cellular signal(s) for transcription of
xisA or activation of XisA is unknown (Haselkorn,
1992).
There have been three reports of heterocyst-forming
strains in which vegetative cells lack the nzjD element.
Placement in trans of a constitutively expressed xisA gene
in Anabaena 7120 resulted in excision of the nzfD element
from all cells of a clonal population, a contiguous nzJTrlDK
organization and a viable nitrogen-fixing phenotype
(Brusca e t al., 1990). The moderate thermophile Fischevlla
sp. ATCC 27929 (PCC 71 15) has a contiguous nzjRDK
organization (Saville e t al., 1987), but no other related
species classified in Section V (Rippka e t al., 1979) have
been studied. The dominant symbiont (Anabaena a@lae)
in the water fern Apolla caroliniana has been showri by
restriction mapping to lack a nzfD element (Meeks ei' al.,
1988) and negative results in Southern hybridizations
imply a similar contiguous organization in A . apllae from
four other Apolla species, collected worldwide, that also
represent two divergent evolutionary lines of the symbiotic cyanobacterium (Franche & Cohen-Bazire, 15)87).
In contrast, cyanobacteria cultured from Apolla caroliiziana
contain a nzfD element (Meeks e t al., 1988). Since it is
unlikely that cells would acquire D N A in the transttion
from a symbiotic to a free-living growth state, these
studies defined a negative marker for the dominant
symbiotic cyanobacterium in association with Apolla spp.
and further indicate that this dominant symbiont ha:<not
yet been cultured apart from the fern.
We report here a fourth instance of a heterocyst-forming
species with a contiguous nzfl'lDK organisation in isolation of clonal populations of Nostoc sp. strain Mac (PCC
8009). Nostoc Mac was originally cultured from symbiotic
association within the coralloid roots of the cycad
Macropamia h i d a L. Johnson (Bowyer & Skerman, 1968).
Nostoc Mac is among a relatively small group of photoautotrophic cyanobacteria characterized by rapid heterotrophic growth at the expense of exogenous carbohydrate (Bottomley & van Baalen, 1978; cf. also Smith,
1982). At unknown times in its culture history, Nostoc
Mac accumulated a number of spontaneous mutations,
the most obvious being the losses of hormogonia
formation and aerobic nitrogen fixation (Fox- ;sensz/ Ernst
e t al., 1992). Spontaneous Fox' revertants were isolated
(Enderlin & Meeks, 1983), but these presumptibe revertants have a filament and culture morphology distinct
from the parental Fox- strain, implying the possibility of
a minor Nostoc contaminant in the culture. Therefore, a
DNA restriction fragment length polymorphism (RFLP)
study was initiated using nif structural genes cloned from
Anabaena 7120 as probes to determine identity between
Fox' revertants and the parental culture, as well as to
compare with other Nostoc strains. The results, detailed
here, while establishing such identity, also indicate the
3226
absence of a nzfD element in one revertant, but its
presence in the parental culture and all other Fox'
revertants.
METHODS
Culture conditions, mutant selections and cell cloning. The
Nostoc sp. strain Mac culture was obtained from Dr L. 0.
Ingram, University of Florida, via Dr H. T. Bonnett, University
of Oregon, It is a direct lineage of the culture obtained by D. S.
Hoare (Hoare e t al., 1971) from Bowyer & Skerman (1968) and
is sister to the culture PCC 8009. The basal medium for growth
of all Nostoc cultures was that of Allen & Arnon (1955); it was
used at full strength when solidified with 1 YO (w/v) agar
(purified by the method of Braun & Wood, 1962) or diluted
fourfold in liquid culture. For Fox- cultures, the basal medium
was supplemented with 2.5 mM NH; plus 5.0 mM NO,
(equimolar Na' and K+ salts) and buffered with 5 mM MOPS,
pH 7.5.
To obtain spontaneously-arising Fox+ revertants, NH,NO,grown Nostoc Mac was harvested by centrifugation, washed
once in nitrogen-free medium, suspended in that medium and
10' to 5 x 10' cells spread per plate on medium lacking combined
nitrogen. The plates were wrapped in parafilm and incubated for
3-4 weeks at room temperature (22-25 "C) under approximately
66 pmol mP2s-l of light from 'coolwhite' fluorescent lamps.
Revertant colonies were subcloned and maintained under N,
prototrophic growth condtions.
Clones of Fox- Nostoc Mac for colony hybridizations were
obtained by first fragmenting filaments to an average chain
length of 1-2 cells by sonic cavitation using a microprobe and a
model W 225R sonicator (Heat Systems-Ultra Sonics). The
fragmented cultures were washed by centrifugation and various
dilutions plated on basal medium supplemented with NH,NO,
and 1 % purified agar. The plates were incubated as above.
Individual colonies (205) were picked to 2 ml of liquid culture
and incubated in the light at room temperature until they
reached an average chlorophyll a (Chl a) density of approximately 8 pg ml-'.
Nitrogen fixation. Nitrogenase activity was estimated by
whole-cell reduction of acetylene to ethylene and the rates
normalized to Chl a content as described previously (Meeks e t
al., 1983). T o examine repression of nitrogenase activity, 5 mM
MOPS-buffered (pH 7*5),3 mM NH,f or 5 mM NO, was added
to exponential cultures and the rates of acetylene reduction
monitored at various times for up to 192 h on 2.0 ml subsamples.
In derepression experiments, cultures were transferred from
NH,NO, medium to medium lacking combined nitrogen and
acetylene reduction monitored as before.
In vitro DNA manipulations. All solutions and basic in vitro
DNA manipulations were according to Maniatis e t al. (1982).
Cells grown with N, (all Fox+ clones) or NH,NO, (all Foxclones) were lysed and the DNA purified by the procedure of
Golden e t al. (1985), as modified by Meeks e t al. (1988). The
restriction endonucleases EcoRI, HincII and Hind111 were used
as recommended by the manufacturers (Bethesda Research
Laboratories ; Boehringer Mannheim ; New England Biolabs).
Electrophoretic separation of the fragments in 0.7 % (w/v)
agarose, transfer to nylon membranes (Gene Screen Plus ;
DuPont NEN Products) and hybridization under stringent
conditions were as described by Meeks e t al. (1988).
For colony hybridizations, 1.5 ml of cell suspension from 2.0 ml
cultures was harvested by centrifugation for 1 min at maximum
speed in a microcentrifuge. The cell pellet was washed once with
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nifgene organization in Nostoc sp. strain Mac
R1
1
5.0
2
R2
3 112 113 213
1
2
3
Fox-
112 113 213
1
2
3 112113 U 3 1 2
29133
3 112 113 U 3
-
-
0-5
.......... .. .. ........................................................................................................................................
Fig. 1. Autoradiogram of Anabaena 7120 nifH (pAn154.3) 32P-labelledprobe hybridized t o endonuclease-digested DNA
extracted from Nostoc strain R1 (Rl), strain R2 (RZ), strain Mac parental culture (Fox-), and strain ATCC 29133 (29133).
The DNA was digested singly with the endonucleases EcoRl (l), Hincll (2), Hindlll (3) or in combinations with EcoRl and
Hincll (112), EcoRl and Hindlll (113) and Hincll and Hindlll (U3). The numbers on the side refer t o size in kb of fragments
migrating the distance shown as determined by digestion of lambda phage DNA with BstEll.
10 mM Tris, 1 mM EDTA, pH 8.0 (TE), plus 0.15 M NaCl, the
pellet suspended to a final volume of 50 pl with TE and 10 pl
spotted onto nylon membranes. Cyanobacteria were lysed by
incubation of the membrane for 10 min on top of filter paper
saturated with 0.4 M NaOH. The NaOH in the membrane was
replaced by 10 min incubations on top of filter paper saturated
with 1-0M Tris, pH 8-0, followed by 0-5 M Tris, pH 8.0, plus
1-25M NaC1. The nucleic acids were then fixed by baking at
80 O C for 2 h.
spontaneously-arising mutants, Reversion to Fox' occurred at a frequency of about 2 x 1O-'. Approximately 40
Fox' revertants of Nostuc Mac were accumulated in two
sets of platings separated by a 6 year period. Of the 20
revertants obtained in the initial set of platings, two were
retained and were designated Nostuc Mac strain R1 and
Nostoc Mac strain R2. The parental strain grows as
relatively long filaments of similar-shaped cells and the
filaments are well dispersed in the culture medium. All
The hybridization probes of the n z f l , nzfD and nzjK structural
Fox' revertants display variable vegetative cell sizes and
genes were isolated as Hind111 fragments from subclones of
the coiled filament morphology typical of many Nostuc
Anabaena 7120 DNA (Rice e t al., 1982) as before (Meeks e t al.,
strains
(Rippka e t a/., 1979), although none appeared to
1988). The xisA probe was a 1.65 kb Ba131 derivative of
have regained hormogonia formation. The coiled filapAn207.65,made by W. Buikema. The probes were prepared by
ments also associate in macroscopic clumps or balls of
nick translation (Bethesda Research Laboratories) or random
priming (5 Prime - 3 Prime, Inc.) using [ C G ~ ~ P I ~ C T P varying dimensions and densities whether grown with N,
(Amersham or DuPont NEN) and autoradiograms of the
or NH; as nitrogen source.
hybridization blots were obtained by exposure to X-ray film.
The blots were regenerated as suggested by the manufacturer
Restriction patterns of the nifH gene and relatedness
and used for subsequent hybridizations.
of Nostoc Mac clones
RESULTS A N D DISCUSSION
Isolation and morphological characteristics of Fox+
revertants
Under our liquid culture conditions, Nostuc Mac does not
attain high population densities ; thus, high-volume
cultures must be concentrated and plated to detect
To verify that the distinct morphological differences
between presumptive revertants and the parental culture
were not a consequence of low-level contamination of a
different, but poorly competitive, Nostoc strain, RFLPs
were analysed. The restriction patterns using a n z f l probe
are complex in heterocyst-forming cyanobacteria due to
the existence of extra copies of the n z f l gene; thus this
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3227
J. C. M E E K S , E. L. C A M P B E L L a n d P. S. B I S E N
1
2
3
R1
112 113 U 3
1
2
3
R1
R2
112 113 213
1
2
3
R2
112 113 U 3
1
2
3 112 113 U 3
5.0-
4.8 3.7 -
0.9-
0.5-
Fig. 2. Autoradiogram of Anabaena 7120 nifD (pAn256) 32Plabelled probe hybridized t o endonuclease-digestedDNA from
Nostoc Mac Fox+ revertant strains R1 and R2. The endonuclease
digests and size markers are as in Fig. 1. The membrane in Fig. 1
was stripped of the nifH probe and reprobed with nifD.
probe is highly discriminatory in strain comparisons
(Meeks e t al., 1988). The n z f l restriction pattern was
identical in Nostoc Mac and revertant strains R1 and R2
(Fig. 1). The restriction patterns were also identical in the
three cultures when fragments of nifK,g l n A (glutamine
synthetase) and rbcS (small subunit of ribulose bisphosphatase carboxylase) genes from Anabaena 7120 were used
as probes (data not shown). However, the n z f l pattern
differed from that of Nostoc sp. ATCC 29133 (PCC 73102)
(Fig. l), which was also isolated as a symbiont from
Macroxamia sp. (Rippka e t al., 1979), and from those of
four other Nostoc sp. strains (data not shown). These
results establish clear identity between the Fox- and Fox'
cultures of Nostoc Mac and lack of identity to closely
related strains.
Nostoc Mac is capable of nitrogenase expression under
microaerobic incubation conditions (data not shown).
Thus, the Fox- mutation appears to result in formation of
an altered heterocyst wall and decreased oxygen protection of nitrogenase (Murry & Wolk, 1989). We do not
know the specific nature of the mutation or of the
reversion(s) that corrects the oxygen-sensitive phenotype.
Attempts to complement the mutation by conjugation of
cosmid-cloned genomic DNA from revertant strain R2
have been unsuccessful (N. Hommes & J. C. Meeks,
unpublished results). The observation that spontaneous
reversion to a Fox' phenotype also results in a pleio-
3228
Fig. 3. Autoradiogram of Anabaena 7120 xisA 32P-labelled
probe hybridized t o endonuclease-digested DNA from Nostoc
Mac Fox+ revertant strains R1 and R2. The endonuclease digests
and size markers are as in Fig. 1. The membrane in Figs 1 and 2
was stripped of prior probes and reprobed with xisA. The
membrane was stripped after hybridization with xisA and
reprobed with nifD t o verify that the lack of hybridization t o
xisA by fragments from strain R1 was not a consequence of loss
of DNA from the membrane.
morphic phenotype may seem unusual. However, we and
our co-workers (Chapman, 1984; Wallis, 1993) have
noted that selection of a nitrogen-fixation phenotype
often yields colonies with altered morphologies, especially
when it involves mutants that form defective heterocyst
walls with oxygen-sensitive nitrogenase activity. It would
appear that some reactions of the synthetic pathways
resulting in functional heterocyst envelopes are also
involved in determining the morphology of the vegetative
filament. We speculate that this is the situation in Nostoc
Mac and its revertants.
Restriction patterns of the nifD and xisA genes and
map of the nifHDK region in revertant strains R1 and
R2
Hybridization with a nzjD probe resulted in a different
pattern of EcoRI, HincII and HindIII restriction fragments
between the two Fox' revertant strains R1 and R2 (Fig,
2). A faint 0.35 kb band was present in EcoRI digests of
strain R1, but not of strain R2. In single HincII digests,
strain R1 had a single band at 2.3 kb, while strain R2 had
a major band at 4.8 kb and a minor band at 2.3 kb. Digests
with HindIII give identical 3-7 kb bands, but additional
0.9 or 2.2 kb bands were present in strains R1 or R2,
respectively. Identical bands of 1.7, 2.9 (plus 0.4) and
1.3 kb were present in EcoRI HincII, EcoRI HindIII
and HincII HindIII double digests, respectively, of the
+
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+
+
nifgene organization in Nostoc sp. strain Mac
(a) Separated
2
-
231
3
132
1
2
3
1 3
2
2
1
3
1
I
I
1
1
1
1
I
(b) Contiguous
1
/ I
3
2
I
I
2311 3
111 I
I K I
I
D
2
2
1
3
I
1
1
1
I H
(c) Probes from Anabaena sp. 71 20
3
3 3
3
urn
3
1
3
3
I
I
I
I
I
256
I
207.8
+
+
two strains. However, the EcoRI HincII and EcoRI
Hind111 digests gave unique bands of 2.9 and 1.85 kb,
respectively, in strain R2, while strain R1 yielded a 0.35 kb
band characteristic of EcoRI digestion alone. The 0.9 and
2.2 kb bands in HincII HindIII digests of strains R1 and
R2, respectively, are those characteristic of digestion with
HindIII alone. The banding patterns in the parental FoxNostoc Mac cultures were identical to those of revertant
strain R2 (data not shown). As will be detailed below,
fragments in common in the double digests of strains R1
and R2 map to the n z f l end of the nzjD probe, while those
that differ map toward the nifK end.
+
A difference in spacing between nzjD and nifK was implied
by probing the same blots with a fragment of x i s A : this
probe hybridized to genomic DNA from strain R2, but
not to that from strain R1, under high-stringency (Fig. 3)
and low-stringency (data not shown) conditions, Moreover, a similar lack of hybridization to strain R1 DNA,
but hybridization to strain R2, was observed when a
fragment of pAn207.3 was used as a probe (data not
shown). In Anabaena 7120,207.3 maps at the opposite end
from xisA within the nzjD insertion element (Lammers e t
al., 1986). These data indicate that genomic DNA of strain
R1, but not strain R2, lacks two of the genes located
within the nzjD insertion element of Anabaena 7120.
Restriction maps of the nzfTlDK region in the two strains,
based on data from Figs 1 , 2 and 3, plus blots of nzjK (data
not shown), are depicted in Fig. 4. These maps emphasize
that a nzjD element is absent in strain R1, but that one is
present in strain R2 and the parental Fox- culture. Similar
restriction maps of the nzjHDK region, showing the
presence of a nzjD element containing xisA and 207.3,
have been constructed with DNA from Nostoc sp. ATCC
29133 and Nostoc sp. ATCC 27896 (PCC 6310) (J. C.
Meeks, unpublished results). Thus, based on restriction
maps of Nostoc strains Mac R2, ATCC 29133 and ATCC
27896, the nzjD element is present in the same genomic
location as it is found in Anabaena 7120 (Golden e t al.,
1985), A . variabilis (ATCC 29413) (Brusca e t al., 1989),
and Nostoc sp. strains N1 (UCD 120) and UCD 7801
~~
154.3
\
Figrn
4. Physical map of the nif gene regions
in vegetative cells of Nostoc Mac clones. The
maps are based on data in Figs 1, 2 and 3
and an unshown autoradiogram of the
same membrane using nifK (pAn207.8)as
a probe. (a) ‘Separated’ refers to the
restriction map of Fox- Nostoc Mac and Fox+
revertant strain R2. (b) ‘Contiguous’ refers
t o the. restriction map of Fox+ revertant
strain R1 and presumably rearranged DNA in
heterocysts of revertant strain R2. The small
segment of the nifD gene, adjacent to nifK
and defined by the near xisA insertion point
in the Anabaena 7120 nifD element is not
shown, but is assumed to be present in
Nostoc Mac strains. The restriction map of
Anabaena 7120 (c) is from the published
nucleotide sequences and shows the
relationship of the probes to the genes.
Symbols: 1, EcoRl; 2, Hincll; 3, Hindlll.
(Meeks e t al., 1988). Based on incomplete maps, a nzjD
element in the same location was also inferred in Nostoc
sp. strains PCC 7121 and PCC 7906, as well as Calothrix
sp. strain PCC 7601 (Kallas e t al., 1985). These observations support the conclusion that the nzjD element is
widely distributed in heterocyst-forming species, at least
those of Section IV (Rippka e t al., 1979). Absence of the
element in Fischerella sp. ATCC 27929 (Saville e t al., 1987)
renders it unclear whether the element is present in
heterocyst-forming strains classified in Section V since no
other strains have been examined. Although it is not
known when insertion of the nzj5!J element occurred in
species of Section IV, it appears to have been conserved in
its genomic location while strains or species have evolved,
as evidenced by mutations reflected in different restriction
patterns in and around the nifgenes.
The restriction map in Fig. 4 allows identity of the minor
or weakly hybridizing bands to the Anabaena 7120-derived
An256 probe of strain R2 in Fig. 2. For examples, the
weakly hybridizing 2.3 kb band in HincII digests is
probably a consequence of a small fraction of the DNA
having rearranged in heterocysts. Conversely, the faint
bands of 2.9 kb in EcoRI HincII and 1-85kb in EcoRI
HindIII double digests result from weak hybridization to
the region of the nzjD element of An256 between the nzjD
insertion point and 207.3, which also shows low homology to other Nostoc strains (Meeks etal., 1988). This low
homology implies evolutionary divergence of the
sequences within the n z p element.
+
+
nifD restriction patterns and hybridization of xisA to
additional Fox+and Fox- clones
To assess the frequency at which loss of the nzjD element
may occur in the Nostoc Mac population, additional Fox’
revertants were isolated and cloned some 6 years after
isolation of strains R1 and R2. The genomic DNA of
these revertants was digested with HindIII, which yields
distinct banding patterns in strain R1 compared to strain
R2 when probed with nzjD (Fig. 2). The results with 16
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J. C. M E E K S , E. L. CAMPBELL a n d P. S. B I S E N
1 4 6 7 8
9 10 16 17
22 19 27
2
5 20 41 3 18
............................................................................................................
3.7 -
Fig. 5, Autoradiogram of Anabaena 7120
nifD (pAn256) 32P-labelledprobe hybridized
t o Hindlll-digested DNA from Nostoc Mac
Fox+ revertant strains R1 (1) and R2 (2) plus
16 additional Fox+ revertant strains. The size
markers of the dominant bands are in kb
and were determined as in Fig. 1. The faint
band near the bottom of the blot in lanes
other than (1) reflects the 0-9kb fragment
that arises when a fraction of the DNA
rearranges in heterocysts of the N,-grown
cultures. The faint band at about 3-0 kb in
all lanes has not been identified.
2.3 -
0.9 -
15 -10 --
............................................................................................................
5 --
20
40
60
80
Time (h)
20
such randomly selected revertants, plus strains R1 and R2,
are shown in Fig. 5. In all cases the restriction pattern of
these revertantswas characteristic of strain R2,-implying
the presence of a n z p element.
To increase the sample size and uncouple two potentially
different alterations, excision of the nzj9 element and
reversion of the Fox- phenotype, in the genomic D N A of
Nostoc Mac, 205 clones were isolated on NH,NO,supplemented medium. These clones, plus strain R1 as the
negative control, were subjected to colony lysis and
probed with xisA, yielding a positive result in all cases
except strain R1 (data not shown). These results support
the idea that loss of the n z p element in vegetative cells is
a rare event.
Nitrogen repression phenotype of revertant strains
R1 and R2
Heterocyst differentiation and subsequent nitrogenase
expression are regulated by the presence or absence of
combined nitrogen in the medium (Wolk, 1982). To
determine if the nzj9 element is directly involved in
nitrogen control, repression and derepression of nitrogenase activity was examined in strains R1 and R2 (Fig. 6).
3230
-
40
60
80
Fig. 6. Effect of added NO; or NH; to
dinitrogen-growing cultures (a), or of
removal from the presence of NH,NO,.(b),
on nitrogenase activity (acetylene reduction)
in Nostoc Mac revertant strains R1 and R2, as
a function of time. Open symbols in (a) refer
t o NH; addition, filled symbols refer t o NO;
addition; triangles, strain R1; circles, strain
R2.
The kinetics of repression by NH; or NO, of nitrogenfixing cultures were identical in the two strains, as were
the kinetics of derepression when cultures were transferred from NH,NO, to combined-nitrogen-free medium. These results indicate that genetic information
within the nzj9 element does not influence other genes in
response to combined nitrogen availability. It is noteworthy that NO, was as effective as NH; in repression of
nitrogenase expression in the Nostoc Mac clones ; in many
diazotrophic cyanobacteria, NO, is a less effective repressor (van Baalen, 1987).
We have superficially surveyed other phenotypic characteristics in comparative experiments with strains R1 and
R2. These include heterotrophic growth with glucose or
sucrose as carbon sources, complementary chromatic
adaptation and viability after desiccation, with no discernible phenotypic differences. Phage susceptibility has
not been examined.
Significance of the "ifD element
The results of our limited screening of Fox- and Foxf
Nostoc Mac clones indicate that loss of the nzJD element is
a rare event; nevertheless, the isolation of strain R1
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nifgene organization in Nostoc sp. strain Mac
indicates that it does occur on a random basis. Since the
excision is a deletion event, and there is no experimental
evidence for reintegration of the element, cells that have
excised the element should accumulate in the population,
unless there is some selective pressure for its retention. If
the frequency of random excision is similar to that of
spontaneous mutation (about lo-') and 1-10 ml of about
lo' cells ml-' are transferred at each subculturing, it is
possible that cells lacking the nzjD element are rarely
carried in the subcultures. Thus, the 223 clones examined
would be an insufficient population size from which to
draw meaningful conclusions. However, the lack of
accumulation of such cells seems unlikely given the length
of time Nostoc Mac has been maintained in culture
collections and subcultured (6 years of subculturing by us
prior to establishing -80 OC stocks).
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conditions are obscure. We have seen no phenotypic
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under our benign culture conditions, but we have not
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This work was supported by the US National Science Foundation (grant IBN 9206130) and the US Department of
Agriculture Competitive Research Grants Office (grant 9037120-5602). We thank R. Haselkorn and W. Buikema for
advice and biological materials.
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Received 18 May 1994; revised 15 August 1994; accepted 17 August
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