Anabaena sp. strain PCC 7120 is a filamentous nitrogen-fixing cyanobacterium. When deprived of nitrogen, approximately 10% of vegetative cells can differentiate into heterocysts along filaments in a semi-regular pattern. Key questions about heterocyst differentiation are (1) how the differentiation is initiated and (2) what controls the formation of the semi-regular pattern. Ning and Xu (2004) reported, an Anabaena mutant with alr0099 disrupted by transposon was unable to grow on dinitrogen and showed abnormal heterocyst pattern. Because the gene cluster asr0098-alr0099-asr0100-alr0101-asr0102-alr0103-asr0104 may be transcribed as an operon, the phenotype of the mutant could be due to a polar effect of the transposon within alr0099 on downstream genes. In this study, investigations using gene knock-out and complementation showed that alr0099 and alr0101 excert positive and negative effects on heterocyst differentiation respectively, Northern blot hybridization and GFP reporter gene were employed to analyze the transcription of these genes upon nitrogen stepdown and the regulatory relationship with other genes invoved in heterocyst differentiation, double mutation and bacterial two-hybrid system were employed to investigate the functional connections between these genes.
The researches and results are in four parts:
(1) Insertion mutants of alr0099, asr0100, alr0101, asr0102, alr0103 and asr0104 were generated by double homologous recombination, investigated for heterocyst differentiation and confirmed by complementation. Mutants of alr0099 showed delayed or completely abolished heterocyst differentiation, while a mutant of alr0101 produced multiple contiguous heterocysts. The results indicated that alr0099 and alr0101 were required for positive and negative regulation of heterocyst differentiation respectively.
(2) The transcription of the alr0099 gene cluster was investigated by using Northern blot hybridization and GFP or LuxAB reporter gene. At least two promoters are involved in the transcription of the alr0099 gene cluster. The first is located upstream of alr0099, initiating the transcription of alr0099 that may extends to asr0100 and alr0101; the second is located upstream of asr0100, initiating the transcription of asr0100 and alr0101. These transcripts are upregulated in proheterocysts or heterocysts after nitrogen stepdown.
(3) The regulatory relationships alr0099, alr0101 and other reported genes were investigated using GFP reporter gene. The expression of alr0099 was self inhibited and promoted by alr0101. The transcription of alr0099 and alr0101 were both dependent on hetR but not on hetC. Insertion at different locations within hetR showed different effects on its expression.
(4) Double mutants were constructed for investigations of epistatic interactions between genes, and a bacterial two-hybrid system was employed to investigate the interactions of HetR and Alr0101. Results indicated that a mutation in alr0099 did not influence the phenotypes of hetN or patS mutants, but suppressed Mch phenotype of alr0101, and that a null mutation in alr0101 resumed the formation of intercalary heterocysts ina patA mutant. Alr0101 and HetR had no direct physical interaction, and alr0101, patS and hetN played independent role in pattern formation.
The main conclusions of this study are as follows:
In alr0099 is required for initiating heterocyst differentiation and alr0101 for inhibiting the formation of multiple contiguous heterocysts. These two genes are subject to both independent transcription and co-transcription, and the promoter of alr0099 is under opposite regulation by the two genes. The organization and feedback regulation of the two genes should be part of the mechanism that ensures the formation of heterocysts in proper rate and pattern along filaments.