Two genes encoding protein kinases of the HstK family are involved in synthesis of the minor heterocyst-specific glycolipid in the cyanobacterium Anabaena sp. strain PCC 7120

Abstract

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 can fix N(2) under oxic conditions, and the activity of nitrogen fixation occurs exclusively in heterocysts, cells differentiated from vegetative cells in response to a limitation of a combined-nitrogen source in the growth medium. At the late stages of heterocyst differentiation, an envelope layer composed of two glycolipids is formed to limit the entry of oxygen so that the oxygen-sensitive nitrogenase can function. The genome of Anabaena sp. strain PCC 7120 possesses a family of 13 genes (the hstK family), all encoding proteins with a putative Ser/Thr kinase domain at their N termini and a His-kinase domain at their C termini. In this study, we showed that the double mutant D4.3 strain, in which two members of this gene family, pkn44 (all1625) and pkn30 (all3691), were both inactivated, failed to fix N(2) in the presence of oxygen (Fox(-)). In an environment without oxygen, a low level of nitrogenase activity was detectable (Fix(+)). Heterocyst development in the mutant D4.3 was delayed by 24 h and arrested at a relatively early stage without the formation of the glycolipid layer (Hgl(-)). Only the minor species of the two heterocyst-specific glycolipids (HGLs) was missing in the mutant. We propose that DevH, a putative transcription factor, coordinates the synthesis of both HGLs, while Pkn44/Pkn30 and the previously characterized PrpJ may represent two distinct regulatory pathways involved in the synthesis of the minor HGL and the major HGL, respectively.

FIG. 1.

Schematic presentation of pkn44 and pkn30. The strain D4.3 was constructed by interrupting pkn44 by the insertion of the Ω element (Sp^r Sm^r) into an AccI site and by interrupting pkn30 by insertion of a neomycin resistance gene cassette into a BamHI site. Relevant domains of Pkn44 and Pkn30 are also shown.

FIG. 2.

Growth curves of the wild type and D4.3 in the presence of fixed nitrogen (A) or under diazotrophic conditions (B). Growth was followed by measuring the optical density at 700 nm (OD₇₀₀). Diamond, wild type; square, D4.3. Both strains were adjusted to an OD₇₀₀ of 0.05 at the beginning of the growth measurement. Experiments were repeated twice, and a representative result is shown.

FIG. 3.

Expression of nifH in the wild type and D4.3. (A) Rearrangement of the nifD element in the wild type (WT) and the strain D4.3. Primers A1 and A3 (A1A3) were used to amplify the DNA fragment corresponding to the nonrearranged DNA, and primers A1 and A2 (A1A2) were used to amplify the DNA fragment corresponding to the rearranged DNA. Cells were cultured for 3 days in the presence of ammonium (NH₄⁺) and then incubated for 18 h in the absence of combined nitrogen (N₂). DNA was extracted and used as template for PCR amplification. PCR products were loaded on an 0.8% agarose gel and separated by electrophoresis. (B) Immunodetection of NifH in the wild type (WT) and D4.3. Filaments were cultured in the absence of fixed nitrogen for 24 or 48 h and then incubated under four conditions: lanes 1 and 2, in the air; lanes 3 and 4, in the air and the presence of DCMU; lanes 5 and 6, in argon (Ar) instead of air; lanes 7 and 8, in argon instead of air and in the presence of DCMU. Fifty micrograms of total protein was separated on a 15% sodium dodecyl sulfate-polyacrylamide gel, transferred onto a nitrocellulose membrane, and incubated with antibodies against NifH. (C) The relative abundance of nifH transcripts determined by real-time quantitative PCR. The amounts of nifH transcripts were normalized to those of rnpB as an internal standard. The amount of the nifH transcripts measured under oxic conditions with wild-type cells at 24 h after the deprivation of combined nitrogen was set at 1, and all the other data were calculated relative to this value.

FIG. 4.

Heterocyst development after starvation for combined nitrogen in the wild type (A) and D4.3 (B). Strains were cultured for 48 h under diazotrophic conditions. Red fluorescent images reflecting photosynthetic pigments (right panels) or bright-field images (left panels) were pictured with the same filaments under a microscope. Arrows indicate the positions of heterocysts.

FIG. 5.

The ultrastructure of heterocysts (A) and components of heterocyst glycolipids (B). (A) Electron microscopic images of the wild type (WT) and strain D4.3. Filaments were cultured in the absence of a fixed-nitrogen source. Boxed areas in the upper panels were enlarged with higher magnification in the lower panels. P, polysaccharide layer; GL, glycolipid layer. (B) Thin-layer chromatographic analysis of glycolipids in the wild type (WT) and D4.3. Cells were cultured in the presence of ammonium (NH₄⁺) or in the absence of combined nitrogen (N₂). The heterocyst-specific glycolipids are indicated by arrows; the upper one (the minor HGL) corresponds to 1-(O-α-d-glucopyranosyl)-3-keto-25-hexacosanol, and the lower one (the major HGL) corresponds to 1-(O-α-d-glucopyranosyl)-3,25-hexacosanediol.

FIG. 6.

Analysis of transcripts of genes related to HGL synthesis or deposition. (A) Analyses by RT-PCR in the wild type (WT) and the mutant D4.3 strain. Total RNA was extracted from samples grown for 24 h and 48 h after deprivation of combined nitrogen. After cDNA synthesis, PCR was carried out for 20, 22, 24, and 26 cycles for rnpB; 24, 26, and 28 cycles for hglE_ACDK and devABC; 21, 23, and 25 cycles for devH; and 23, 25, and 27 cycles for asr5349 and asr5350. PCR products were loaded on a 1.2% agarose gel and separated by electrophoresis. For hglDE_AK and devABC, the results are shown after 24 cycles; for rnpB, hglC, and devH, the results are shown after 22, 26, and 21 cycles, respectively; and for asr5349 and asr5350, results of PCR are shown after 27 cycles. The rnpB gene served as a control for the amount of RNA template. (B) Transcriptional analysis of asr5349 and asr5350 by real-time quantitative PCR in the wild type (gray bars) and D4.3 (open bars). The transcript levels were normalized to those of rnpB; the transcript level of wild-type cells at 24 h after the deprivation of combined nitrogen was set to 1, and other data were calculated according to this value.