Anabaena sp. strain PCC 7120 gene devH is required for synthesis of the heterocyst glycolipid layer

In response to deprivation for fixed nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 provides a microoxic intracellular environment for nitrogen fixation through the differentiation of semiregularly spaced vegetative cells into specialized cells called heterocysts. The devH gene is induced during heterocyst development and encodes a product with characteristics of a trans-acting regulatory protein. A devH mutant forms morphologically distinguishable heterocysts but is Fox(-), incapable of nitrogen fixation in the presence of oxygen. We demonstrate that rearrangements of nitrogen fixation genes take place normally in the devH mutant and that it is Fix(+), i.e., has nitrogenase activity under anoxic conditions. The Fox(-) phenotype was shown by ultrastructural studies to be associated with the absence of the glycolipid layer of the heterocyst envelope. The expression of glycolipid biosynthetic genes in the mutant is greatly reduced, and heterocyst glycolipids are undetectable.     

Identification of the glycosyl transferase required for synthesis of the principal glycolipid characteristic of heterocysts of Anabaena sp. strain PCC 7120

Nitrogenase is oxygen-labile. Cyanobacterial heterocysts can fix N(2) in an oxic milieu because their interior is micro-oxic, for which the glycolipid layer of the heterocyst envelope is required. ORF all5341 of the Anabaena sp. genome predicts a glycosyl transferase. An insertional mutant of all5341 synthesized only a nonglycosylated form of heterocyst envelope glycolipid, and lacked a glycolipid layer. All5341 appears to be the transferase required to glycosylate the glycolipid aglycone.     

A TolC-like protein is required for heterocyst development in Anabaena sp. strain PCC 7120

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms heterocysts in a semiregular pattern when it is grown on N2 as the sole nitrogen source. The transition from vegetative cells to heterocysts requires marked metabolic and morphological changes. We show that a trimeric pore-forming outer membrane beta-barrel protein belonging to the TolC family, Alr2887, is up-regulated in developing heterocysts and is essential for diazotrophic growth. Mutants defective in Alr2887 did not form the specific glycolipid layer of the heterocyst cell wall, which is necessary to protect nitrogenase from external oxygen. Comparison of the glycolipid contents of wild-type and mutant cells indicated that the protein is not involved in the synthesis of glycolipids but might instead serve as an exporter for the glycolipid moieties or enzymes involved in glycolipid attachment. We propose that Alr2887, together with an ABC transporter like DevBCA, is part of a protein export system essential for assembly of the heterocyst glycolipid layer. We designate the alr2887 gene hgdD (heterocyst glycolipid deposition protein).     

Synthesis of nitrogenase in mutants of the cyanobacterium Anabaena sp. strain PCC 7120 affected in heterocyst development or metabolism.

Mutants of Anabaena sp. strain PCC 7120 that are incapable of sustained growth with air as the sole source of nitrogen were generated by using Tn5-derived transposons. Nitrogenase was expressed only in mutants that showed obvious morphological signs of heterocyst differentiation. Even under rigorously anaerobic conditions, nitrogenase was not synthesized in filaments that were unable to develop heterocysts. These results suggest that competence to synthesize nitrogenase requires a process that leads to an early stage of visible heterocyst development and are consistent with the idea that synthesis of nitrogenase is under developmental control (J. Elhai and C. P. Wolk, EMBO J. 9:3379-3388, 1990). We isolated mutants in which differentiation was arrested at an intermediate stage of heterocyst formation, suggesting that differentiation proceeds in stages; those mutants, as well as mutants with aberrant heterocyst envelopes and a mutant with defective respiration, expressed active nitrogenase under anaerobic conditions only. These results support the idea that the heterocyst envelope and heterocyst respiration are required for protection of nitrogenase from inactivation by oxygen. In the presence of air, such mutants contained less nitrogenase than under anaerobic conditions, and the Fe-protein was present in a posttranslationally modified inactive form. We conclude that internal partial oxygen pressure sufficient to inactivate nitrogenase is insufficient to repress synthesis of the enzyme completely. Among mutants with an apparently intact heterocyst envelope and normal respiration, three had virtually undetectable levels of dinitrogenase reductase under all conditions employed. However, three others expressed oxygen-sensitive nitrogenase activity, suggesting that respiration and barrier to diffusion of gases may not suffice for oxygen protection of nitrogenase in these mutants; two of these mutants reduced acetylene to ethylene and ethane.     

MSX-resistant mutants of Anabaena 7120 with derepressed heterocyst development and nitrogen fixation

To investigate the role of ammonium-assimilating enzyme in heterocyst differentiation, pattern formation and nitrogen fixation, MSX-resistant and GS-impaired mutants of Anabaena 7120 were isolated using transposon (Tn5-1063) mutagenesis. Mutant Gs1 and Gs2 (impaired in GS activity) exhibited a similar rate of nitrogenase activity compared to that of the wild type under dinitrogen aerobic conditions in the presence and absence of MSX. Filaments of Gs1 and Gs2 produced heterocysts with an evenly spaced pattern in N₂-grown conditions, while addition of MSX altered the interheterocyst spacing pattern in wild type as well as in mutant strains. The wild type showed complete repression of heterocyst development and nitrogen fixation in the presence of NO₃ ^– or NH₄ ⁺, whereas the mutants Gs1 and Gs2 formed heterocysts and fixed nitrogen in the presence of NO₃ ^– and NH₄ ⁺. Addition of MSX caused complete inhibition of glutamine synthetase activity in wild type but Gs1 and Gs2 remained unaffected. These results suggest that glutamine but not ammonium is directly involved in regulation of heterocyst differentiation, interheterocyst spacing pattern and nitrogen fixation in Anabaena.     

Heterocyst development and diazotrophic metabolism in terminal respiratory oxidase mutants of the cyanobacterium Anabaena sp. strain PCC 7120

Heterocyst development was analyzed in mutants of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bearing inactivated cox2 and/or cox3 genes, encoding heterocyst-specific terminal respiratory oxidases. At the morphological level, the cox2 cox3 double mutant (strain CSAV141) was impaired in membrane reorganization involving the so-called honeycomb system that in the wild-type strain is largely or exclusively devoted to respiration, accumulated glycogen granules at conspicuously higher levels than the wild type (in both vegetative cells and heterocysts), and showed a delay in carboxysome degradation upon combined nitrogen deprivation. Consistently, chemical analysis confirmed higher accumulation of glycogen in strain CSAV141 than in the wild type. No impairment was observed in the formation of the glycolipid or polysaccharide layers of the heterocyst envelope, consistent with the chemical detection of heterocyst-specific glycolipids, or in the expression of the heterocyst-specific genes nifHDK and fdxH. However, nitrogenase activity under oxic conditions was impaired in strain CSAV135 (cox3) and undetectable in strain CSAV141 (cox2 cox3). These results show that these dedicated oxidases are required for normal development and performance of the heterocysts and indicate a central role of Cox2 and, especially, of Cox3 in the respiratory activity of the heterocysts, decisively contributing to protection of the N(2) fixation machinery against oxygen. However, in contrast to the case for other diazotrophic bacteria, expression of nif genes in Anabaena seems not to be affected by oxygen.     

Biosynthesis of heterocyst glycolipids of Anabaena cylindrica

The incorporation of sodium acetate-[1-¹⁴C] into the heterocyst glycolipids of Anabaena cylindrica cultures from 60–234 hr old is reported. Incorporation of radioactivity was maximal in 88 hr old cultures. In 60 hr and 88 hr cultures about 90 % of the radioactivity of the heterocyst glycolipids was found in the non-saponifiable glycolipid fraction, whereas in older cultures this fraction contained only 75 % of the radioactivity. Acid hydrolysis of non-saponifiable heterocyst glycolipid fractions showed that in 60 hr cultures, 81 % of the radioactivity occurs in the lipid moiety, whereas in older cultures a greater proportion (40–53 %) of the radioactivity was found in the sugar residue. The lipid fraction obtained by acid hydrolysis contained a mixture of labelled long chain mono-, di- and trihydric alcohols. In young (60 hr) cultures the primary alcohol fraction was most heavily labelled (57.3 % of the radioactivity in the non-saponifiable glycosides) with much smaller amounts in the diol and triol (8.4 and 15.1 % respectively), whereas in older cultures (234 hr) the primary alcohol (23.6 %) diol (22.5 %) and triol (18.9 %) fractions contained ca equal amounts of radioactivity.     

Evidence that the barrier to the penetration of oxygen into heterocysts depends upon two layers of the cell envelope

Mutants of Anabaena sp. PCC 7120 with O₂-sensitive acetylene-reducing activity were studied to identify envelope components that contribute to the barrier limiting diffusion of oxygen into the heterocyst. Mutant strain EF114, deficient in a heterocyst-specific glycolipid, reduced acetylene only under strictly anaerobic conditions. Analysis of in vivo O₂ uptake as a function of dissolved pO₂ showed that EF114 has lost the low affinity, diffusion-limited respiratory component associated with heterocysts in wild-type filaments. The low affinity respiratory activity was also lost in EF116, a mutant in which the cohesiveness of the outer polysaccharide layer was reduced. Restoration of aerobic nitrogen fixation in a spontaneous revertant of EF116 and in a strain complemented with cosmid 41E11 was associated with restoration of the low affinity component of respiratory activity. The results provide evidence that the barrier to diffusion of gas into heterocysts depends upon both the glycolipid layer and the polysaccharide layer of the heterocyst envelope.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Induction of increase in the heterocyst frequency of Anabaena sp. strain ATCC33047. Effect on ammonium photoproduction

Abstract Several approaches have been followed to increase the nitrogenase level in filaments of Anabaena ATCC33047. In a nitrogen-free medium lacking added molybdate and supplemented with 10 mM tungstate, growth was impaired as a result of decreased nitrogenase activity level. Under these conditions, the filaments exhibited nitrogen starvation symptoms and a high heterocyst frequency, with heterocysts being up to 28% of the total number of cells in the filaments, while a regular pattern of heterocyst distribution was maintained. Normal nitrogenase level and nitrogen status were recovered upon molybdate addition, with resumption of growth and decrease of the heterocyst frequency with time until reaching a value of about 10%. The yield of ammonium photoproduction from N₂ by filaments displaying different heterocyst frequencies and treated with l -methionine- d,l -sulfoximine (MSX) was determined. Maximal rates were obtained with filaments containing 16% of the cells differentiated as heterocysts. Results indicate that appropriate manipulation of the heterocyst frequency leads to an improvement in the efficiency of conversion of light energy into chemical energy through photoreduction of N₂ to ammonium.     

PrpJ, a PP2C-type protein phosphatase located on the plasma membrane, is involved in heterocyst maturation in the cyanobacterium Anabaena sp. PCC 7120

Protein phosphatases play important roles in the regulation of cell growth, division and differentiation. The cyanobacterium Anabaena PCC 7120 is able to differentiate heterocysts specialized in nitrogen fixation. To protect the nitrogenase from inactivation by oxygen, heterocyst envelope possesses a layer of polysaccharide and a layer of glycolipids. In the present study, we characterized All1731 (PrpJ), a protein phosphatase from Anabaena PCC 7120. prpJ was constitutively expressed in both vegetative cells and heterocysts. Under diazotrophic conditions, the mutant DeltaprpJ (S20) did not grow, lacked only one of the two heterocyst glycolipids, and fragmented extensively at the junctions between developing cells and vegetative cells. No heterocyst glycolipid layer could be observed in the mutant by electron microscopy. The inactivation of prpJ affected the expression of hglE(A) and nifH, two genes necessary for the formation of the glycolipid layer of heterocysts and the nitrogenase respectively. PrpJ displayed a phosphatase activity characteristic of PP2C-type protein phosphatases, and was localized on the plasma membrane. The function of prpJ establishes a new control point for heterocyst maturation because it regulates the synthesis of only one of the two heterocyst glycolipids while all other genes so far analysed regulate the synthesis of both heterocyst glycolipids.     

Metabolic Adaptations of Azospirillum brasilense to Oxygen Stress by Cell-to-Cell Clumping and Flocculation

The ability of bacteria to monitor their metabolism and adjust their behavior accordingly is critical to maintain competitiveness in the environment. The motile microaerophilic bacterium Azospirillum brasilense navigates oxygen gradients by aerotaxis in order to locate low oxygen concentrations that can support metabolism. When cells are exposed to elevated levels of oxygen in their surroundings, motile A. brasilense cells implement an alternative response to aerotaxis and form transient clumps by cell-to-cell interactions. Clumping was suggested to represent a behavior protecting motile cells from transiently elevated levels of aeration. Using the proteomics of wild-type and mutant strains affected in the extent of their clumping abilities, we show that cell-to-cell clumping represents a metabolic scavenging strategy that likely prepares the cells for further metabolic stresses. Analysis of mutants affected in carbon or nitrogen metabolism confirmed this assumption. The metabolic changes experienced as clumping progresses prime cells for flocculation, a morphological and metabolic shift of cells triggered under elevated-aeration conditions and nitrogen limitation. The analysis of various mutants during clumping and flocculation characterized an ordered set of changes in cell envelope properties accompanying the metabolic changes. These data also identify clumping and early flocculation to be behaviors compatible with the expression of nitrogen fixation genes, despite the elevated-aeration conditions. Cell-to-cell clumping may thus license diazotrophy to microaerophilic A. brasilense cells under elevated oxygen conditions and prime them for long-term survival via flocculation if metabolic stress persists.     

Effect on heterocyst differentiation of nitrogen fixation in vegetative cells of the cyanobacterium Anabaena variabilis ATCC 29413

Heterocysts are terminally differentiated cells of some filamentous cyanobacteria that fix nitrogen for the entire filament under oxic growth conditions. Anabaena variabilis ATCC 29413 is unusual in that it has two Mo-dependent nitrogenases; one, called Nif1, functions in heterocysts, while the second, Nif2, functions under anoxic conditions in vegetative cells. Both nitrogenases depended on expression of the global regulatory protein NtcA. It has long been thought that a product of nitrogen fixation in heterocysts plays a role in maintenance of the spaced pattern of heterocyst differentiation. This model assumes that each cell in a filament senses its own environment in terms of nitrogen sufficiency and responds accordingly in terms of differentiation. Expression of the Nif2 nitrogenase under anoxic conditions in vegetative cells was sufficient to support long-term growth of a nif1 mutant; however, that expression did not prevent differentiation of heterocysts and expression of the nif1 nitrogenase in either the nif1 mutant or the wild-type strain. This suggested that the nitrogen sufficiency of individual cells in the filament did not affect the signal that induces heterocyst differentiation. Perhaps there is a global mechanism by which the filament senses nitrogen sufficiency or insufficiency based on the external availability of fixed nitrogen. The filament would then respond by producing heterocyst differentiation signals that affect the entire filament. This does not preclude cell-to-cell signaling in the maintenance of heterocyst pattern but suggests that overall control of the process is not controlled by nitrogen insufficiency of individual cells.     

PROTEIN TURNOVER AND HETEROCYST DIFFERENTIATION IN THE CYANOBACTERIUM ANABAENA VARIABILIS1

Under conditions of starvation for fixed nitrogen, cells of the filamentous cyanobacterium Anabaena variabilis Kütz, degrade much of their protein prior to heterocyst differentiation. Cells starved for a source of fixed nitrogen initially degraded about 2% of their protein per hour; by 24 h after nitrogen stepdown about 40% of the protein was degraded. Most of the acid-soluble radiolabeled material was excreted into the medium. Proteolysis was completely inhibited by chloramphenicol, by cyanide, or in the dark, hut was only partially inhibited in the presence of dichlorophenyl dimethylurea. Methionine sulfoximine (MSX) (an inhibitor of glutamine synthetase) in the presence of ammonia caused heterocysts to form. MSX treated cells degraded protein; however, the amount of protein degraded was much less than in cells starved for ammonia. Glutamine, which can serve as a nitrogen source for this strain, did not prevent starvation-induced proteolysis and did not prevent the differentiation of heterocysts.     

Action of 2,4-dichlorophenoxyacetic acid and rifampicin on heterocyst differentiation in the blue-green alga,Nostoc linckia

2,4-Dichlorophenoxyacetic acid, a commonly used herbicide, increased the growth of the filamentous blue-green alga,Nostoc linckia at doses upto 100 μg /ml. The herbicidetreated N₂-cultures showed enhanced heterocyst frequency and N₂-growth. Thus, the herbicide stimulated algal growth at the expense of molecular nitrogen under aerobic growth conditions. Rifampicin caused chain formation of heterocysts. This was effectively counteracted by 2,4-dichlorophenoxyacetic acid, suggesting a biological interaction between them at the level of the heterocyst spacing control mechanism.     

Analysis of Cyanothece sp. BH68K mutants defective in aerobic nitrogen fixation

The unicellular cyanobacterium, Cyanothece sp. BH68K, is capable of performing both oxygen-sensitive nitrogen fixation and oxygenic photosynthesis within a single cell. To understand the oxygen protection mechanisms of nitrogenase, mutants defective in nitrogen fixation (Nif^-) were isolated by use of diethyl sulfate as a mutagen. Out of 24 mutants screened, 6 mutants could not express nitrogenase activity under aerobic conditions, but expressed activity under anaerobic conditions (Fox^-); 4 mutants showed no activity under both aerobic and anaerobic conditions (Fix^-); and the remaining mutants were impaired in both aerobic and anaerobic nitrogenase activity (Imp). Respiratory oxygen consumption and photosynthetic oxygen evolution were analyzed in the wild-type and in two Fox^- mutants. In the wild-type the appearance of high aerobic nitrogenase activity was correlated with an increase in dark respiration, whereas no such increase was seen in the Fox^- mutants. We propose that in Fox^- mutants, respiratory oxygen consumption plays an important role in maintaining aerobic nitrogenase activity.