HetR-dependent and -independent expression of heterocyst-related genes in an Anabaena strain overproducing the NtcA transcription factor

Abstract

Heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120 depends on both the global nitrogen control transcription factor NtcA and the cell differentiation regulatory protein HetR, with expression of ntcA and hetR being dependent on each other. In this study we constructed strains that constitutively express the ntcA gene leading to high levels of NtcA protein irrespective of the nitrogen source, and we analyzed the effects of such NtcA levels on heterocyst differentiation. In the NtcA-overproducing strain, heterocyst differentiation, induction of NtcA-dependent heterocyst development genes or operons such as devBCA or the cox2 operon, and NtcA-dependent excision of the 11-kb nifD-intervening element only took place under nitrogen deficiency. Although functional heterocysts were produced in response to nitrogen step-down, the NtcA overproducing strain could not grow diazotrophically. Overexpression of ntcA in a hetR background promoted expression of devBCA in response to ammonium withdrawal and excision of the 11-kb element even in the presence of combined nitrogen. Our results show that some NtcA-dependent heterocyst-related genes can be expressed independently of HetR.

FIG. 1.

Structure of the ntcA region in ntcA-overexpressing strains CSEL1 and CSEL2. (A) Sequences from the ntcA gene are shown in gray. DNA regions present in plasmid pCSM1 (see Materials and Methods for details), the ntcA fragment used as probe for Northern hybridization, and the position of oligonucleotide NA1 used for primer extension experiments are indicated. P_ntcA, wild-type ntcA promoter; P_trc, trc promoter. (B) Southern blot analysis of the structure of the ntcA region in strains CSEL1 and CSEL2. Total DNA from wild-type strain PCC 7120 and ntcA-overexpressing strains CSEL1 and CSEL2 was digested with HindIII and hybridized to the probe shown in panel A. Sizes (in kilobases) are indicated on the right. WT, wild-type strain PCC 7120.

FIG. 2.

Expression of ntcA and amount of NtcA protein in Anabaena sp. strain PCC 7120, hetR strain 216, and ntcA-overexpressing strains CSEL1 and CSEL2. (A) Northern blot analysis of expression of the ntcA gene. RNA was isolated from ammonium-grown filaments (lanes 0) or from ammonium-grown filaments incubated in the absence of combined nitrogen for 9 or 24 h. Samples contained 50 μg of RNA. Hybridizations to the ntcA probe (see Fig. 1) (upper panel) or to an rnpB probe (43) used as a loading and transfer control (lower panel) were performed. Note that the ntcA probe used only hybridizes to transcripts that include the second half of the ntcA gene. (B) Primer extension analysis of ntcA transcripts. RNA was isolated from ammonium-grown filaments (lanes 0) or from ammonium-grown filaments incubated in the absence of combined nitrogen for 1 or 9 h. Oligonucleotide NA1 (see position in Fig. 1) was used as a primer in assays containing 25 μg of RNA (wild type) or 5 μg of RNA (CSEL1 and CSEL2). A sequencing ladder was generated with the same oligonucleotide and plasmid pCSM1. (Note that pCSM1 does not contain the wild-type ntcA promoter upstream from ntcA but contains the trc promoter instead. Thus, the sequencing ladder shown corresponds to ntcA only for sequences downstream from the ATG codon.) The po-sitions of the 5′ ends with respect to the ATG codon of the ntcA gene, as well as that corresponding to the transcripts originating from the trc promoter (P_trc), are indicated with arrowheads on the right. Note that the amount of RNA from strains CSEL1 and CSEL2 used in primer extension assays was fivefold lower than for the wild-type samples. (C) Western blot detection of the NtcA protein. Whole-cell extracts were prepared from ammonium-grown filaments (lanes 0) or from ammonium-grown filaments incubated in the absence of combined nitrogen for 9 h. Samples contained extract amounts corresponding to 1.6 μg of Chl. Arrowhead points to the NtcA protein. WT, wild-type strain PCC 7120.

FIG. 3.

Expression of the nir and rbcLXS operons in Anabaena sp. strain PCC 7120 and strain CSEL1. (A) RNA was isolated from ammonium-grown filaments (lanes NH₄⁺) or from ammonium-grown filaments incubated in nitrate-containing medium for 4 h (lanes NO₃⁻) and hybridized to a nir probe. Samples contained 30 μg of RNA. (B) RNA was isolated from ammonium-grown filaments (lanes 0) or from ammonium-grown filaments subjected to nitrogen deficiency for the number of hours indicated in each case and hybridized to a rbcL probe. The lower panels correspond to hybridization to a rnpB probe (43) in both cases. WT, wild-type strain PCC 7120.

FIG. 4.

Expression of devBCA, cox2, and nifHDK in Anabaena sp. strain PCC 7120, the hetR strain 216 and strains CSEL1 and CSEL2. RNA was isolated from ammonium-grown filaments (lanes 0) or from ammonium-grown filaments incubated in the absence of combined nitrogen for the number of hours indicated in each case. Samples were hybridized to devB (A), coxB2 (B), or nifH (C) probes. Samples contained 25 μg of RNA (A and C) or 40 μg of RNA (B). The lower panels correspond to hybridization to a rnpB probe (43) in all cases. Arrowheads point to the transcripts (size in kilobases) previously described for the corresponding genes. WT, wild-type strain PCC 7120.

FIG. 5.

Excision of the 11-kb nifD-intervening element in Anabaena sp. strain PCC 7120, the hetR strain 216 and strains CSEL1 and CSEL2. DNA was isolated from ammonium-grown filaments (lanes 0) or from ammonium-grown filaments subjected to nitrogen deficiency for the number of hours indicated in each case and digested with HindIII. Excision of the 11-kb element was assessed by Southern blot with a fragment of the nifD gene as a probe. The probe hybridizes to a 2.8-kb HindIII fragment in chromosomal DNA prior to rearrangement and to a 1.8-kb HindIII fragment after excision of the nifD element. WT, wild-type strain PCC 7120.