Clustered genes required for the synthesis of heterocyst envelope polysaccharide in Anabaena sp. strain PCC 7120

Abstract

As demonstrated with alr2835 (hepA) and alr2834 (hepC) mutants, heterocysts of Anabaena sp. strain PCC 7120, a filamentous cyanobacterium, must have an envelope polysaccharide layer (the Hep+ phenotype) to fix dinitrogen in an oxygen-containing milieu (the Fox+ phenotype). Transpositions presumptively responsible for a Fox- phenotype were localized in open reading frames (ORFs) near hepA and hepC. A mutation in each of nine of these ORFs was complemented by a clone bearing only that single, intact ORF. Heterocysts of the nine mutants were found to lack an envelope polysaccharide layer. Complementation of mutations in alr2832 and alr2840 may have resulted from recombination. However, alr2825, alr2827, alr2831, alr2833, alr2837, alr2839, and alr2841, like hepA and hepC, are required for a Hep+ Fox+ phenotype.

FIG. 1.

Map (20) of genes in an “expression island” (12) of the genome of Anabaena sp. strain PCC 7120. The positions of transposons in mutants that were successfully used for complementation are indicated by triangles. The sole insertion found in alr2834 was reported earlier (44). The marker extends 1.5 kb.

FIG. 2.

Wild-type and mutant strains of Anabaena sp. strain PCC 7120 unstained (A), stained with Alcian blue (B), and lipid extracted with chloroform and methanol (2:1, vol/vol) (C). Only the wild-typestrain retained heterocyst envelope polysaccharide, as assessed by staining with Alcian blue and retention of an envelope layer evident after lipid extraction. Aberrant overall coloration resulting from various intensities of illumination was partially corrected with Photoshop 7.0. Arrowheads point to heterocysts.

FIG. 3.

Tests of complementation by clones bearing, intact, only the gene mutated in the corresponding mutant: FQ885 (alr2825) plus pRL2875, FQ57 (alr2827) plus pRL2864, FQ1631 (alr2831) plus pRL2862, FQ773 (alr2832) plus pRL2863, FQ470 (alr2833) plus pRL2865a, FQ428 (alr2837) plus pRL2877, FQ794 (alr2839) plus pRL2876, FQ1630 (alr2840) plus pRL2873, and FQ344 (alr2841) plus pRL2866. Spots were grown from cells of wild-type Anabaena sp. strain PCC 7120 (lanes 1), a particular FQ mutant (lanes 2), and four independent, exconjugant clones of the particular FQ mutant bearing the corresponding pRL plasmid (lanes 3 to 6). The left, center, and right panels present, respectively, the results observed (for any one mutant, simultaneously) 2 to 3 weeks after spotting cells on agar-solidified media AA, AA+N, and AA+N plus 200 μg of Nm ml⁻¹ and 10 μg of Sp ml⁻¹. Cells of the wild-type strain grew in the presence or absence of nitrate but failed to grow in the presence of antibiotic; cells of each mutant grew in the presence of nitrate unless counterselected by antibiotics, but failed to grow in the absence of nitrate; addition of the corresponding cloned genes permitted the mutants to grow with only N₂ as nitrogen source or in the presence of antibiotics.

FIG.4.

Use of PCR to analyze possible recombination products in complemented mutants. (A) A plasmid (RSF1010 derivative) bears a fragment (in gray) that contains a single, intact (wild-type) copy of an ORF (shown as a white arrow) that has not recombined with the transposon (Tn)-interrupted copy of the same ORF in the genome. The ORF in the plasmid may be expressed from the glnA promoter or, perhaps, from an intervening, native promoter. PCR primers P3 and P4 are genomic sequences, up- and downstream from the cloned region, respectively, and primers P1 and P2 are vector sequences present, respectively, up- and downstream from the cloning region of the plasmid (P1 is also upstream from the glnA promoter). (B) Single, homologous recombination upstream from the transposon (at X in diagram A) would give rise to structure B, in which the uninterrupted ORF is positioned downstream from its natural upstream sequence. PCR would be expected to yield a product of predictable size with primers P2 and P3. (C) Single, homologous recombination downstream from the transposon (at Y in diagram A) would give rise to structure C, in which the uninterrupted ORF and any 3′ cotranscribed sequences would be placed under the influence of the glnA promoter and/or an intervening native promoter. PCR would be expected to yield a product of predictable size with primers P1 and P4. (D) Double, homologous recombination at both sides of the transposon would give rise to structure D, identical with that of the wild-type strain. With DNA from the wild-type strain or a double recombinant as template, PCR would be expected to yield a product of predictable size with primers P3 and P4. Such a band should be obtained with DNA from the mutant strain as template only if segregation of the mutation were incomplete, because the extension period was too short for the polymerase to traverse the transposon. If DNA from the mutant yielded no band, a band of the same size as from the wild-type strain, using DNA from the complemented mutant as template, would indicate that a double recombination event had cured the transposon from one or more copies of the genome. Lane numbers in inset tables E and F refer to lane numbers in Fig. 5, below. Inset table F shows PCR band sizes predicted (lanes 1 and 6) or predicted conditionally upon incomplete segregation (lane 2) or single (lanes 4 and 5) or double (lane 3) recombination.

FIG. 5.

PCR analyses of N₂-grown cultures derived from complemented mutants. Panels correspond to analysis of total DNA, as template, from wild-type Anabaena sp. strain PCC 7120 (lanes 1), or (in lanes 2) the following mutants: FQ885 (alr2825) (A); FQ57 (alr2827) (B); FQ1631 (alr2831) (C); FQ773 (alr2832) (D); FQ470 (alr2833) (E); FQ428 (alr2837) (F); FQ794 (alr2839) (G); FQ1630 (alr2840) (H); FQ344 (alr2841) (I). Lanes 3 to 6: complemented strains FQ885 plus pRL2875, FQ57 plus pRL2864, FQ1631 plus pRL2862, FQ773 plus pRL2863, FQ470 plus pRL2865a, FQ428 plus pRL2877, FQ794 plus pRL2876, FQ1630 plus pRL2873, and FQ344 plus pRL2866. Lanes M are New England BioLabs 2-log DNA size-standard markers (10, 8, 6, 5, 4, and 3 kb [bright]; 2, 1.5, 1.2, and 1.0 kb [bright]; 0.9, 0.8, 0.7, 0.6, and 0.5 kb [bright]; and 0.4, 0.3, 0.2, and 0.1 kb). Primers were ORF-specific P3 and P4 (lanes 1 to 3), P2 plus P3 (lanes 4), P1 plus P4 (lanes 5), and P1 and P2 (lanes 6), specific for the ends of the cloning region of pRL1383a and its derivatives pRL2831a and pRL2831b. In the panels (see inset table F of Fig. 4), the band in lane 1 shows the size expected for wild-type DNA, indicating the efficacy of P3 and P4 to prime the expected PCR with genomic DNA; lanes 2 lack any band other than from primer dimerization, indicating that segregation of the mutations was complete; and lanes 3 show in several instances (panels C, E, H, and I) the presence of a band in the complemented mutant, indicative of double recombination having taken place in these recombination-proficient cells. Single recombination shown by bands in lanes 4 and 5 is discussed in the text. The band in each lane 6 shows the size expected for the plasmid added to the corresponding strain, indicating the efficacy of P1 and P2 to prime the expected PCR with the plasmid present in total DNA from the complemented strain.