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Journal of Bacteriology, December 2001, p. 6841-6851, Vol. 183, No. 23
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6841-6851.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
andMSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824
Received 6 June 2001/Accepted 12 September 2001
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ABSTRACT |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In many filamentous cyanobacteria, vegetative cells can differentiate into heterocysts, cells that are specialized for aerobic fixation of N2. Synthesis of the heterocyst envelope polysaccharide is dependent on the gene hepA in Anabaena sp. strain PCC 7120. In search of genes that are involved in the regulation of hepA, we transposon mutagenized strain DR1069, which bears a chromosomal hepA::luxAB fusion. One resulting mutant, designated HNL3, grows normally in medium with nitrate and shows poor induction of hepA in response to nitrogen deprivation. In HNL3, transposon Tn5-1058 is inserted within gene hcwA, a constitutively expressed open reading frame whose predicted product resembles N-acetylmuramoyl-L-alanine amidases. Reconstruction of the mutation confirmed that the mutant phenotype resulted from the insertion of the transposon. The induction of hepA in HNL3 is partially restored upon recombination of HNL3 with plasmid-borne, wild-type hcwA. Moreover, HcwA expressed in Escherichia coli exhibits wall-lytic activity. These results suggest that the degradation, or possibly reconstruction, of the cell peptidoglycan layer is a prerequisite for heterocyst maturation.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Certain filamentous cyanobacteria, such as Anabaena spp., adapt to deprivation of fixed nitrogen by forming heterocysts, differentiated cells in which nitrogenase reduces atmospheric dinitrogen to ammonia. Because nitrogenase is highly sensitive to oxygen, the interior of the heterocyst must be rendered microaerobic. Microaerobic conditions result from (i) inactivation of the oxygen-producing photosystem II in developing heterocysts; (ii) synthesis, outside the cell wall, of a layer of polysaccharide and, within that layer, a layer of glycolipids that is little permeable to O2; and (iii) reduction of residual permeant O2 to H2O by respiration (37).
A number of genes involved in the biosynthesis and organization of the
polysaccharide and glycolipid layers of the heterocyst envelope have
been identified. Mutation of these genes leaves heterocysts unable to
fix nitrogen in the presence of oxygen (referred to as the
Fox
phenotype [14]) but able to
fix N2 anaerobically. How glycolipids and
polysaccharides are transported and deposited through a preexisting wall remains unclear. Diverse Fox mutants with
abnormalities of envelope deposition have been isolated. For example, a
mutation in hepA prevents the formation of envelope polysaccharides (34). The predicted HepA is a member of
the family of ATP-binding cassette (ABC) inner membrane transport proteins. hepA is first activated 4 to 7 h after
nitrogen stepdown, several hours before morphological differentiation
of heterocysts first becomes apparent by light microscopy (6, 17,
36). By 10 h after nitrogen stepdown, about when
differentiation becomes morphologically evident by transmission light
microscopy, hepA is induced transcriptionally over 10-fold
(6, 36). The induction depends on activation of
hetR several hours earlier (4). Recent studies
have shown that two regions upstream from the transcriptional start
site of hepA are required for the expression of
hepA in nitrogen-free medium; at least the proximal region
is a binding site for particular proteins (39). By
mutagenesis of hepA::luxAB strain
DR1069 with transposon Tn5-1058, additional genes that regulate the expression of hepA in response to nitrogen
stepdown were sought. One such mutant, in which a
hepC::Tn5-1058 fusion led to
constitutive expression of hepA independent of the presence of fixed nitrogen, was earlier described (39). Here we
report the characterization of a similarly identified gene that is,
however, required for the induction of hepA in response to
nitrogen stepdown and whose predicted product shows extensive
similarity to
N-acetylmuramoyl-L-alanine amidases.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Culture conditions and transformation of strains of
Anabaena sp.
Anabaena sp. strain PCC
7120 and its derivatives (Table 1) were
grown at 30°C in the light (ca. 3,500 ergs
cm2 s1) on a rotary
shaker in AA/8 medium (18) supplemented with 5 mM nitrate
in 125-ml Erlenmeyer flasks. Cultures of derivatives of PCC 7120 were
supplemented with appropriate antibiotics at the concentrations
described by Khudyakov and Wolk (19). Plasmids (Table 1)
were introduced into Anabaena sp. strain PCC 7120 by conjugation (11), and single or double recombinants were
selected as described earlier (4, 5). To induce heterocyst
formation, portions of actively growing cultures were washed three
times with AA/8, suspended in a volume of AA/8 without antibiotics
equal to the original volume, and incubated under growth conditions. Filaments were examined by microscopy 24 to 72 h following
nitrogen stepdown. Samples were prepared for electron microscopy
(2) and micrographed by S. Burns (MSU Center for Electron
Optics).
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DNA manipulation. Recombinant DNA procedures were performed according to established standards (26). Enzymes were purchased from New England BioLabs, Beverly, Mass., or occasionally from other suppliers and used as recommended.
Transposon Tn5-1058 and contiguous chromosomal DNA was excised from HNL3 genomic DNA with ClaI, self-ligated, and transferred to Escherichia coli DH10B (Life Technologies, Inc., Gaithersburg, Md.) by electroporation, yielding plasmid pRL1977. A 0.55-kb fragment, derived from pRL1977, extending from the ClaI site in hcwA to the Psp1406I site ca. 90 bp from the end of IS50R in Tn5-1058, was used as a probe to identify
EMBL3 clones bearing
corresponding wild-type DNA (3). Automated sequencing
(Applied Biosystems Inc., Foster City, Calif.) was performed on both
strands of the DNA by primer walking.
Database comparisons and alignments of the translated sequences were
performed by using the default settings of the algorithm developed by
Altschul et al. (1), using the BLAST network service at
the National Center for Biotechnology Information, and the Genetics
Computer Group Pileup program (Program manual for the Wisconsin
sequence analysis package; Genetics Computer Group, Madison, Wis., 1994).
Transposon mutagenesis and reconstruction of mutant HNL3. Mutagenesis of strain DR1069 with transposon Tn5-1058 was performed as described by Wolk et al. (35). Plasmid pRL1992, a derivative of pRL1977, was transferred to strain DR1069 by conjugation. Several independently isolated neomycin-resistant, erythromycin-sensitive, sucrose-resistant clones were shown by Southern hybridization to be double recombinants.
Construction of a complementing plasmid.
Because the
putative protein encoded by the open reading frame (ORF) intercepted in
mutant HNL3 may be related to the synthesis or turnover of the
heterocyst cell wall (see below), we designated the ORF
hcwA. Our initial attempts to complement the mutation in
HNL3 having foundered on our inability to subclone the entirety of
hcwA in a pUC-based vector, we first synthesized pRL691
(Fig. 1), which bears an interrupted form
of hcwA (long heavy arrow). pRL691 is a derivative of pIC20H
(23) whose insert consists of fragments a, b, and c.
Fragment a, from XhoI (destroyed) to MluI, is
most of a 2.6-kb PCR product of a EMBL3 clone (designated C2) used
as a template, with primers CPW117
(5'-CTCGTCCGAGAATAACGAGTGG-3') from within the right arm of
EMBL3 and CPW118
(5'-GGTACGTCGACAATCCATCGCCTGTAACTCGTA-3'; this is based on a
sequence that is 3' from the MluI site in hcwA). Fragment b, from MluI to ClaI, is derived from
pRL2130, a subclone from a different, 12-kb EMBL3 clone that begins
between the initiation codon of hcwA and its MluI
site and continues beyond its downstream ORF, which we designated
orf1. Within the BsaBI site of fragment b is
inserted a BsaBI-aadA-BsaBI cassette,
derived from the omega interposon (25) that is
bracketed by inverted repeats consisting of portions of the polylinkers
from pJRD184 (15), pRL148 (10) and pRL453
(10). The aadA gene (short heavy arrow) confers
resistance to streptomycin (Sm) and spectinomycin (Sp). In all of the
Smr Spr constructs
described, only these two BsaBI sites can be cut by BsaBI when the constructs are isolated from
dam+ E. coli. Fragment c, from
ClaI of fragment b to the EcoRI site (destroyed)
of pIC20H, is the ClaI-MfeI fragment from pRL2130 that contains the 3' end of hcwA.
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EMBL3; the other PstI site is from the pIC20H polylinker.
pRL669a consists of RSF1010-based plasmid pRL59HE (19)
into the BamHI site of which, downstream from
Ptac, was introduced the
Cmr Emr cassette C.CE3
(Pcat 
cat erm) (Y. Cai and C. P. Wolk, unpublished data). The
PstI-bracketed insert from pRL691 was transferred to the
PstI site of pRL669a, generating plasmids pRL2509a, with
Ptac 5' from the 5' end of hcwA, and
pRL2509b, with hcwA inverted. BAC vector pRL838 is a
Cmr Emr derivative of
pBAC108L (27), with unique NsiI and
NruI sites and a single BsaBI site that can be
cut coming from a dam+ strain. pRL2510 has
the structure of pRL838, but with the two corresponding
BsaBI/NruI fragments inverted (and those sites
destroyed). Insertion of the 5.9-kb, PstI-bracketed fragment
from pRL691 into the NsiI site of pRL2510 generated pRL2430.
pRL2431 was generated from pRL2430 by restriction with BsaBI
and religation, deleting the aadA-bearing fragment. The
ClaI sites in the insert and the vector are separated by 3.6 kb.
Luciferase assays. Luminescence of colonies on filters was assessed as described previously (35). Luciferase activity of suspensions, measured with an ATP photometer (Turner Designs, Sunnyvale, Calif. [12]), was normalized to the concentration of chlorophyll in the sample, which was measured in methanolic extracts (22).
Northern blot hybridizations. The hcwA probe consisted of a digoxigenin (DIG)-labeled antisense RNA generated from pRL2333 with a DIG RNA Labeling Kit (Boehringer Mannheim GmbH, Mannheim, Germany) using T3 RNA polymerase according to the instructions of the manufacturer. Similarly, the orf1 probe consisted of a DIG-labeled single-stranded RNA generated from pRL2332 using T7 RNA polymerase.
Total RNA (5 or 10 µg per lane, as indicated) was extracted from 50-ml cultures of Anabaena sp. strain PCC 7120 that were grown and treated as described under Materials and Methods and then harvested and washed with 10 mM Tris-HCl-0.1 mM EDTA (pH 7.5). To approximately 400 µl of resuspended cells was added an equal volume of phenol-chloroform, 0.2% sodium dodecyl sulfate (SDS) (final concentration) and 150 µl of glass beads (diameters, 212 to 300 µm). The cells were broken by vortexing at the maximum speed of a Vortex-Genie 2 mixer (Fisher Scientific, Pittsburgh, Pa.) for four cycles of 1 min on and 1 min off (on ice). The supernatant solution from a 10-min centrifugation at 14,900 × g and 4°C was ethanol precipitated. The resulting pellet was dissolved in 100 µl of RNase-free water and combined with 350 µl of RLT buffer, a guanidinium isothiocyanate-containing lysis buffer (RNeasy Kit; Qiagen GmbH, Hilden, Germany). Purification then proceeded with the RNeasy kit as described by Qiagen. Total RNA suspended in 20 µl of RNA loading buffer was heated at 65°C for 5 min, chilled on ice for 1 min, and loaded onto gels of 1.2% agarose in 1× MOPS buffer (20 mM 3-N-morpholinepropanesulfonic acid [MOPS], 5 mM sodium acetate, 2 mM EDTA [pH 7.0]) containing 2% formaldehyde. The electrophoresis buffer contained 1× MOPS buffer and 1.33% (final concentration) formaldehyde. After electrophoresis, the gels were washed in 10× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 10 min twice and RNA transferred downwards (8) to a Nytran membrane (Schleicher & Schuell, Dassel, Germany) with 20× SSC as transfer solution. RNA was cross-linked to the membrane with a UV-Stratalinker (Stratagene, La Jolla, Calif.). Hybridization, washing, and detection of chemiluminescence were based on the instructions in Boehringer Mannheim's manual (The DIG system user's guide for filter hybridizations), modified according to Engler-Blum et al. (13) to achieve a higher sensitivity with the DIG nonradioactive hybridization system. Briefly, prehybridization and hybridization were performed overnight at 68°C in 250 mM sodium phosphate (pH 7.2)-1 mM EDTA-20% SDS-0.5% blocking reagent without and with 2.5 ng of DIG-labeled probe per ml, respectively.Reverse transcription (RT)-PCR. Total RNA was prepared from Anabaena sp. strain PCC 7120 as described in the Northern blot procedure above. Two-microgram aliquots of each RNA preparation were reverse-transcribed by Omniscript Reverse Transcriptase (Qiagen) from random hexamer primers (Promega, Madison, Wis.) in a reaction volume of 20 µl. Two microliters from each cDNA pool was used as a PCR template. Thermal cycling conditions were as follows: 94°C for 2 min, followed by 18 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s. Samples of the same cDNAs were subjected to 18 cycles of PCR with a primer pair specific to rnpB, a constitutively expressed gene from PCC 7120 (30), used as a loading control. The PCR primers used were hepA forward (5'-ACATGATTTTGGCTGCTGATGC-3'), hepA reverse (5'-GCTAAAACAACTTTGATAGCTGC-3'), rnpB forward (5'-GGCGTTGGCGGTTGCAGACC-3'), and rnpB reverse (5'-AGTTGGTGGTAAGCCGGGTTC-3').
Expression of HcwA in E. coli.
Plasmid
pRL2431 or pRL2510 was introduced into E. coli strain BL21
by transformation. Transformants were grown in Luria broth supplemented with 0.2% maltose and 100 µg of ampicillin per ml to an
optical density at 600 nm of 0.6 to 1.0. MgSO4
and CE6 (Novagen, Madison, Wis.) were added to final concentrations
of 10 mM and 2 × 109 to 4 × 109 PFU/ml, respectively. The infected cells were
grown for 3 h and harvested by centrifugation. Cells were
disrupted by cavitation, on ice, with a Vibra cell sonicator (Sonics & Materials, Inc., Danbury, Conn.) equipped with a 2-mm tip, at 40% of
maximum power, for 10 s, and extract was collected by
centrifugation (13,000 × g, 5 min, 4°C).
Detection of lytic activity in SDS-polyacrylamide gel electrophoresis (PAGE) gels. For isolation of cell wall material from PCC 7120, cells were harvested from 500 ml of stationary-phase culture and disrupted with a French press. The crude cell wall material was collected by centrifugation (30,000 × g, 60 min, 4°C) and boiled in 4% SDS for 15 min. The suspension was centrifuged (45,000 × g, 30 min, 20°C), and the sedimented material was washed six times with twice-distilled H2O.
Protein-containing extracts of E. coli were heated at 95°C for 2 min in Laemmli sample buffer and loaded onto an SDS-polyacrylamide gel (10% acrylamide) containing 0.2% (wt/vol) SDS-treated cell walls from PCC 7120. SDS-PAGE was carried out at 4°C. After electrophoresis, the gels were incubated for 12 to 16 h at 37°C in 500 ml of 25 mM Tris-HCl, pH 8.0, containing 1% Triton X-100 to permit protein renaturation. The visibility of transparent bands of lysis in the translucent gel was enhanced by staining with 0.1% methylene blue in 0.01% KOH prior to photography.Nucleotide sequence accession number.
The sequence of
hcwA and orf1 reported in this paper has been
submitted to GenBank under accession no. AF216288. The presence of
orf2, shown in Fig. 2, the
sequence of Anorf2, shown in Fig. 3, and
predicted lengths of restriction fragments that extend beyond those
reported in AF216288 are based in whole or in part on sequence data
that subsequently became available at the website
http://www.kazusa.or.jp/cyano /anabaena/distribute.html (Kazusa
DNA Research Institute, Chiba, Japan).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolation of mutant HNL3.
Mutant HNL3 was isolated as a
transposon Tn5-1058-derivative of
hepA::luxAB strain DR1069, which
upon nitrogen stepdown showed greatly decreased luminescence
(35) compared with strain DR1069 (Table
2). Tn5-1058 mutant HNL3 grew
normally in nitrate-containing liquid medium. As shown by electron
microscopy, the immature-appearing heterocysts that differentiated upon
nitrogen stepdown of HNL3, like the heterocysts of its parental strain
DR1069 (32), formed a laminated layer of glycolipids but
no envelope polysaccharide layer (data not shown).
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Cloning of hcwA and orf1
A
10.5-kb ClaI fragment containing the transposon and
contiguous Anabaena sp. strain DNA was recovered, as
pRL1977, from HNL3. The flanking DNA, including a nearby
SpeI site, was sequenced using primers extending outward
from the transposon. Clones of fragments of wild-type DNA were isolated
from a EMBL3 phage library of Anabaena sp. strain PCC
7120 DNA (3) using the flanking DNA as probe. Sequence
analysis of both strands of a region surrounding the
SpeI site showed the presence of three parallel,
neighboring ORFs (Fig. 2). The central ORF, into which
Tn5-1058 had inserted, predicts a 68.0-kDa protein of
627 amino acids that shares 70.2 and 36.8% identity with presumptive
N-acetylmuramoyl-L-alanine amidases of
Nostoc punctiforme (JGI website
http://spider.jgi-psf.org/JGI_microbial/html /nostoc_homepage.html)
and Synechocystis sp. strain PCC 6803, respectively, and 16.7% identity with CwlB of Bacillus
subtilis, as well as 59% identity and 73% similarity with the
627-amino acid protein predicted by the upstream ORF, which we
designated orf2 (Fig. 2 and 3). (Despite this extensive
amino acid similarity, there is extensive nucleotide disparity, so that
the Northern blot hcwA probe that shares 578 bp of
nucleotide identity with hcwA has a longest string of 20 nucleotides identical with orf2.)
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Reconstruction of the mutation in mutant HNL3.
To determine
whether the phenotype of HNL3 was the result of insertion of the
transposon, pRL1992 was introduced into strain DR1069, resulting in
replacement of hcwA with
hcwA::Tn5-1058. The structure of the
double recombinants was confirmed by Southern hybridization to
ClaI-digested DNA from strains DR1069, HNL3, and CPB7084
(Fig. 5). Heterocysts in these strains
have the same appearance in the microscope, and the expression of
hepA is greatly reduced in CPB7084, as in HNL3 (Table 2).
When the DR1992 mutation was constructed in wild-type PCC 7120, the
resulting strain grew normally in medium with fixed nitrogen. In
response to nitrogen deprivation, it formed what appeared to be normal
heterocysts with only slight defects in envelopes sometimes seen by
bright-field microscopy, but it was nonetheless
Fox (data not shown).
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Expression of hcwA and orf1
Total RNA was isolated from PCC 7120 cultures grown with fixed nitrogen
or deprived of fixed nitrogen for 6, 12, or 18 h. Using DNA
fragments specific for hcwA and orf1 as
probes, Northern hybridization (Fig. 6)
indicated that both genes had basal expression in medium with fixed
nitrogen. After nitrogen stepdown, transcription of hcwA
was not significantly altered. However, transcription of
orf1 increased substantially. The hcwA
and orf1 probes hybridized to RNA fragments of ca. 2.3 and 1.0 kb, respectively, lengths which are ca. 15 to 20% greater than
those of the coding regions of the corresponding genes.
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Complementation of the HNL3 mutation. To determine whether the phenotype of the HNL3 mutation is due to a polar effect on the expression of the orf1 gene, we attempted complementation of the original mutation. Removal of the aadA-bearing BsaBI fragment from within hcwA (Fig. 1) by restriction (which appeared complete) and religation of high-copy-number plasmid pRL691 or lower-copy-number, RSF1010-based plasmids pRL2509a and pRL2509b, produced no spectinomycin-sensitive transformants. When BAC-based plasmid pRL2430 was digested with BsaBI and religated, Cmr Sms Sps transformants (bearing pRL2431) of E. coli were obtained only at 30°C, not at 37°C, whereas E. coli bearing pRL2430 grows well at 37°C. Thus, cloning of intact hcwA proved possible only in a very-low-copy-number, F-plasmid-based vector, and then only at 30°C, not 37°C, implying that hcwA is expressed in E. coli and that its product is toxic to the bacterial host.
Rather than try to complement the mutation in trans by adding a replicon functional in Anabaena sp. to plasmid pRL2431, pRL2431 was introduced into strain HNL3 through single recombination. As confirmed by Southern hybridization (Fig. 7B), recombination took place either 5' (strain CPB8089) or 3' (strain CPB8090) from the site of insertion of the transposon within strain HNL3, producing one intact copy and one interrupted copy of hcwA. Only in CPB8090 was orf1 downstream from an intact copy of hcwA. However, both tested recombinants partially restored the induction of hepA::luxAB (Table 3). Because, in addition, the transcripts appeared independent (Fig. 6), it is unlikely that the blocking of hepA expression is due to a polar effect, on orf1, of the insertion of the transposon.
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Expression of hepA in hcwA
mutant.
To test the possibility that the blocked induction of
hepA::luxAB in strain HNL3 was
influenced by the loss of hepA itself, the effect of
hcwA on hepA was explored in a genetic background with an intact hepA gene. Plasmid pRL1992 was introduced
into PCC 7120, generating hcwA-knockout mutant DR1992. The
expression of hepA in response to nitrogen stepdown was
detected by semiquantitative RT-PCR. In wild-type PCC 7120, induction
of hepA was detected as early as 6 h after nitrogen
deprivation, and the hepA transcript continued to
accumulate. In the hcwA mutant, in contrast, expression of
hepA did not exceed wild-type background levels at 6 and
12 h after stepdown but became comparable with that in the wild
type at 18 h (Fig. 8). This
experiment indicates that the loss of hcwA decreased
activation of hepA early in heterocyst development, corroborating the result of Table 2 without reliance on a
lux reporter.
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Expression of HcwA in E. coli and characterization
of its lytic activity.
In pRL2431, gene hcwA is
preceded by a T7 promoter. Expression of HcwA was induced in BL21 by
infecting the cells with CE6, a source of T7 RNA polymerase. Plasmid
pRL2510, the parental BAC vector without an hcwA insert, was
used as a negative control. The lytic activity of HcwA was visualized
in a zymogram. Protein extracts of BL21 (pRL2431) were separated in an
SDS-PAGE gel containing cell walls from PCC 7120. The zymogram showed
that the recombinant E. coli produces a lytic enzyme with an
apparent molecular mass of ca. 74 kDa (Fig.
9). E. coli harboring pRL2510
expressed no detectable lytic activity.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The cell walls of cyanobacteria, outside of the cytoplasmic membrane, consist of a peptidoglycan layer, an outer membrane, and often a sheath (16, 33). These cell walls are qualitatively similar to those of gram-negative bacteria. However, the thickness of the peptidoglycan layer, together with its degree of cross-linking and the presence of covalently linked polysaccharide, is more characteristic of the walls of gram-positive cells (31).
Heterocysts exhibit structural differences from vegetative cells.
Outside a wall that appears similar to that of vegetative cells,
heterocysts are enveloped, except where they are attached to vegetative
cells, by a layer of glycolipids (32) and a layer of
polysaccharide (7). The interpretation that the underlying cell wall of heterocysts is similar to that of vegetative cells is
based in part on low-resolution electron micrographs and in part on
evidence that suggests that hexosamine is incorporated into the wall
early during the differentiation process and that slow synthesis or
turnover of N-acetylglucosamine, a constituent of
peptidoglycan, occurs throughout the process (9).
Defective synthesis of lipopolysaccharide, a constituent of the wall,
leads to apparently defective deposition of the heterocyst envelope and
to a Fox phenotype (38).
Heterocyst envelope glycolipids and polysaccharides synthesized within
and at the plasmalemma must traverse the cell wall before they are
deposited at the outside surface of differentiating heterocysts.
Therefore, rearrangement, perforation, or partial degradation of the
peptidoglycan layer may be required for assembly of the glycolipid or
polysaccharide layer of the heterocyst envelope. It is germane that 1 of at least 12 putative penicillin-binding proteins encoded by
Anabaena sp. strain PCC 7120 is required for aerobic
nitrogen fixation, although its encoding gene appears to be expressed
equally strongly in the presence of nitrate (20).
While searching for genes whose products control the expression of hepA, we found that a mutation in hcwA, which putatively encodes an N-acetylmuramoyl-L-alanine amidase, extensively reduces the induction of hepA upon nitrogen deprivation. The expression of hepA is restored, albeit incompletely, by recombination with an intact copy of hcwA. Incompleteness of restoration may be due to competition between intact and truncated copies of HcwA for binding to substrate or to other proteins. Alternatively, the increased copy number of sequences 5' from hcwA might indirectly affect the expression of hepA, perhaps as a result of titration of effectors by increased binding to upstream regulatory sequences. The transcript of a neighboring gene, orf1, differs in size from that of hcwA and appears to be an independent transcript, suggesting that the effect of the mutation in hcwA is not a polar effect on the transcription of orf1. hcwA appears, both by similarity (Fig. 3) and by its lytic activity on walls of Anabaena sp. strain PCC 7120 (Fig. 9), to encode a metabolic enzyme similar to an autolysin. It therefore seems unlikely that HcwA directly regulates the expression of hepA.
In wild-type PCC 7120, the expression of hcwA remains relatively constant upon nitrogen deprivation, and hcwA mutants grow well in medium with fixed nitrogen. Similarly, B. subtilis genes that encode autolysins that function during spore germination are transcribed at an earlier stage of development (28).
We conjecture that HcwA increases the permeability of the peptidoglycan layer of the cell wall of the developing heterocyst, thereby facilitating the penetration of glycolipids and polysaccharides. We suggest that in mutant HNL3, an external signal that regulates hepA may fail to reach the inside of developing heterocysts. Alternatively, components of the heterocyst envelope may accumulate inside those cells and negatively regulate the expression of hepA.
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ACKNOWLEDGMENTS |
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We thank Xiaoqiong Qin for a sample of tobacco rRNA.
This work was supported by the U.S. Department of Energy under grant DOE-FG02-91ER20021 and by NSF grant MCB 9723193.
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FOOTNOTES |
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* Corresponding author. Mailing address: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824. Phone: (517) 353-2049. Fax: (517) 353-9168. E-mail: wolk{at}msu.edu.
Present address: Department of Entomology, Michigan State
University, East Lansing, MI 48824.
Present address: Schering-Plough Research Institute, Kenilworth,
NJ 07033-0539.
§ Present address: Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824.
Permanent address: N. Vavilov Institute of General Genetics,
Moscow 117809, Russia.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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