Predicted Glycosyl Transferase Genes Located outside the HEP Island Are Required for Formation of Heterocyst Envelope Polysaccharide in Anabaena sp. Strain PCC 7120

Abstract

During maturation, heterocysts form an envelope layer of polysaccharide, called heterocyst envelope polysaccharide (HEP), whose synthesis depends on a cluster of genes, the HEP island, and on an additional, distant gene, hepB, or a gene immediately downstream from hepB. We show that HEP formation depends upon the predicted glycosyl transferase genes all4160 at a third locus and alr3699, which is adjacent to hepB and is cotranscribed with it. Mutations in the histidine kinase genes hepN and hepK appear to silence the promoter of hepB and incompletely down-regulate all4160.The filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 produces terminally differentiated cells, called heterocysts, when it is deprived of fixed nitrogen. These specialized cells are internally microoxic as a result of (i) cessation of oxygen production; (ii) intensification of respiration; and (iii) deposition of a thick envelope, comprising an outer polysaccharide layer and an inner glycolipid layer, that impedes the entry of oxygen (35). The glycolipid layer is the principal barrier to the entry of oxygen, whereas the polysaccharide layer is evidently required for the integrity of the glycolipid layer. The homogeneous layer of heterocyst envelope polysaccharide (HEP) has been chemically analyzed in two other Anabaena sp. strains and in a Cylindrospermum sp. strain. In those strains, HEP is composed of oligosaccharide repeating units whose mannose-(glucose) 3 backbone is decorated with side chains that are different in the different strains. In the Anabaena sp. strains, a highly similar polysaccharide was found in the envelopes of akinetes, a kind of spore. It was crudely estimated that, exclusive of regulatory genes, on the order of 20 genes are required for synthesis of the concatenated subunits (33).Through DNA microarray analysis and near-saturation transposon mutagenesis, numerous Anabaena sp. strain PCC 7120 open reading frames in the HEP island (genes alr2825 through alr2841) of the chromosome were found to be required specifically for formation of the envelope polysaccharide layer (7, 17). Mutants with mutations in the presumptively glycosyl transferase-encoding genes hepB (alr3698) (23, 36), alr3699, and all4160 (this study) that are distant from the HEP island in the genome also lack the polysaccharide layer. Regulation of HEP deposition depends upon signal transduction systems that comprise, at least, the histidine kinase HepN (11,26), the response regulator HenR (11), the serine/threonine kinase HepS (11), and the interacting two-component regulatory elements HepK and DevR A (39, 40). In addition, proteins Abp2 and Abp3 bind to the DNA sequence upstream of hepC, and inactivation of abp2 and abp3 greatly reduces the expression of hepA and hepC (20). abp2 and abp3 mutants, however, lack the glycolipid layer of the heterocyst envelope rather than the polysaccharide layer.In the 1,076 sites of insertion mapped in a transposon mutagenesis project screening for Fox Ϫ mutants (i.e., mutants unable to fix dinitrogen i...

The sigE Gene Is Required for Normal Expression of Heterocyst-Specific Genes in Anabaena sp. Strain PCC 7120

“…The hglE2 gene is necessary for the heterocyst glycolipid layer and is developmentally upregulated during the middle stage of heterocyst differentiation (1,15,55). In the sigE mutant strain, the hglE2 expression levels at 12 h were similar to those in the wild-type strain; however, by 36 h after nitrogen step-down, the transcript levels of hglE2 were only half of the wild-type levels.…”

Identification of Ten Anabaena sp. Genes That under Aerobic Conditions Are Required for Growth on Dinitrogen but Not for Growth on Fixed Nitrogen

“…These results support the proposition (22) that all4388 is a Fox gene. Expression of all4388 was upregulated 3, 8, and 24 h after nitrogen deprivation, whereas many of the genes required for deposition of HEP (17,37) are first significantly upregulated at 8 h (data in reference 7 analyzed by the method in reference 41).…”

“…Additionally, if the estimate is correct that Anabaena sp. has approximately 100 to 140 Fox genes (38), the majority of Fox genes have been discovered, many of them by our transposon mutagenesis screen (10,11,13,17,19,37,40).…”

A Major Facilitator Superfamily Protein, HepP, Is Involved in Formation of the Heterocyst Envelope Polysaccharide in the Cyanobacterium Anabaena sp. Strain PCC 7120

“…strain PCC 7120 (sometimes called a Nostoc sp. ), genes involved in the formation of both of these layers are clustered (2,14,17,28), although functionally related hep genes are also present elsewhere in the chromosome (39,59).…”

“…Extensive mutagenic analysis has, however, been performed in Anabaena sp. strain PCC 7120 (see, e.g., references 17,28,34,39,59, and 65) but not in A. variabilis. We here characterize a gene, open reading frame (ORF) all1711, that encodes a major facilitator superfamily (MFS) protein that is needed for diazotrophic growth and production of Hep and which, when overexpressed, elicits production of extracellular (presumed) polysaccharide in vegetative cells.…”