C. Peter Wolk

[83] Conjugal transfer of DNA to cyanobacteria

Publisher Summary This chapter focuses on the conjugal transfer of DNA to Cyanobacteria. Conjugation appears to be a general means to introduce DNA from Escherichia coli into cyanobacteria, using the broad host range conjugal apparatus of an IncP plasmid, such as RP4. RP4, originally isolated from Pseudomonas, has been shown to mediate the transfer of DNA into a wide range of gram-negative bacteria, including such distantly related organisms as myxobacteria, thiobacilli, and unicellular and filamentous cyanobacteria. To obtain stable conjugal transfer it is necessary that (1) conjugal contact be made, (2) transferred DNA escape restriction or degradation, and (3) the DNA replicate autonomously or integrate into one of the replicons of the recipient. The first requirement is probably met in the great majority of gram-negative eubacteria, cyanobacteria included. The task for the experimenter is to find conditions that permit the last two requirements to be met as well. Even with transformable unicellular cyanobacteria, conjugation may be the preferred route of DNA transfer in certain cases. DNA taken up by unicellular cyanobacteria appears to be randomly cut early during the transformation process, so that the efficiency of transfer of a segment of nonhomologous DNA decreases exponentially with its length.

A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers

Several families of positive-selection cloning vectors were constructed, based on the principle of palindrome nonviability first used by Hagan and Warren [Gene 19 (1982) 147-151]. Each vector, derived from either pBR322 or RSF1010 (a broad-host-range plasmid), contains a long inverted repeat (2 x 366 to 2 x 1008 bp) ending in a symmetrical polylinker. Plasmids with long palindromes are not viable in most strains of Escherichia coli and in at least one Gram-positive bacterium. These palindrome-containing vectors therefore transform such strains at a very low frequency unless a DNA fragment is cloned within the polylinker at the center of the palindrome. Transformation by plasmids lacking an insert is reduced by two to four orders of magnitude. Such vectors can be propagated in a palindrome-tolerant strain; however, long symmetrical deletions then occur within the palindrome. To suppress the resulting deletion derivatives, vectors have been constructed so that an extensive deletion would remove the selectable marker. Alternatively, the vectors can be propagated in any strain of E. coli so long as the palindrome is interrupted by a nonpalindromic DNA fragment. We also present several symmetrical polylinkers and drug-resistance cassettes within the vectors. These components can be interchanged to make new positive-selection vectors as needed, and the cassettes are useful in insertional mutagenesis as well. A general method is described to convert virtually any small or medium-sized plasmid into a positive-selection vector.

Heterocyst Metabolism and Development

Heterocysts are differentiated cells that are specialized for fixation of N2 in an aerobic environment. In heterocysts in the light, Photosystem I generates ATP, but no photosynthetic production of O2 takes place. Instead, reductant moves into heterocysts from vegetative cells. In return, fixed nitrogen moves from heterocysts to vegetative cells. In neither case is there certainty about the identity of the traffic molecules. Pathways of electron-donation to N2 have been extensively investigated, but their in-vivo importance remains to be critically tested. Nitrogenase in heterocysts is protected from inactivation by O2 by a variety of means, principally by enhanced respiration and by a barrier, the heterocyst envelope, to entry of O2. However, the respiratory apparatus and the biosynthetic processes that result in synthesis of the barrier have been little studied. The detailed mechanisms underlying metabolic, environmental, and developmental control of nitrogenase are under investigation. Studies of heterocyst development are being greatly facilitated by recent advances in the genetics of Anabaena sp. An autoregulated gene, hetR, that is activated shortly after nitrogen-stepdown is critical for the differentiation of heterocysts. Two enigmas remain to be answered: how is it determined which cells will differentiate; and, after differentiation is initiated, what intercellular interactions and intracellular mechanisms regulate the progression of the differentiation process? An evolutionary and biochemical relationship between the processes leading to the formation of heterocysts and akinetes is suggested.

Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena

The spatially patterned differentiation of hetero‐cysts in the filamentous cyanobacterium Anabaena requires a functional hetR gene. Transcriptional fusions to luxAB show that hetR is transcribed at a low level throughout the filament when Anabaena is grown with combined nitrogen, and that induction of the gene begins within 2 h following nitrogen deprivation. By 3.5 h, induction is localized to spaced foci. By 6h, there is an overall induction of at least threefold in whole cultures, reflecting at least a 20‐fold increase within spatially separated cells. The induction requires the presence of a functional hetR gene, indicating that hetR is autoregulatory. Full induction of a heterocyst structural gene, hepA, also requires a functional hetR locus.

Evidence that the barrier to the penetration of oxygen into heterocysts depends upon two layers of the cell envelope

Mutants of Anabaena sp. PCC 7120 with O2-sensitive acetylene-reducing activity were studied to identify envelope components that contribute to the barrier limiting diffusion of oxygen into the heterocyst. Mutant strain EF114, deficient in a heterocyst-specific glycolipid, reduced acetylene only under strictly anaerobic conditions. Analysis of in vivo O2 uptake as a function of dissolved pO2 showed that EF114 has lost the low affinity, diffusion-limited respiratory component associated with heterocysts in wild-type filaments. The low affinity respiratory activity was also lost in EF116, a mutant in which the cohesiveness of the outer polysaccharide layer was reduced. Restoration of aerobic nitrogen fixation in a spontaneous revertant of EF116 and in a strain complemented with cosmid 41E11 was associated with restoration of the low affinity component of respiratory activity. The results provide evidence that the barrier to diffusion of gas into heterocysts depends upon both the glycolipid layer and the polysaccharide layer of the heterocyst envelope.

FISCHERELLIN, A NEW ALLELOCHEMICAL FROM THE FRESHWATER CYANOBACTERIUM FISCHERELLA MUSCICOLA1

The benthic cyanobacterium Fischerella muscicola (Thur.) Gom. UTEX 1829 produces a secondary metabolite, fischerellin, that strongly inhibits other cyanobacteria and to a lesser extent members of the Chlorophyceae. Eubacteria are not affected. The major active compound is lipophilic and exhibits a molecular ion at m/z 408. It is heat‐ and acid‐stable but decomposes in 1 M sodium hydroxide (80° C. 1 h). Fischerellin inhibits the photosynthetic but not the respiratory electron transport of cyanobacteria and chlorophytes. Its site of action is located in PS II. Two other species of Fischerella also produce fischerellin, indicating that the synthesis of such allelochemicals might be characteristic of the genus.

Production, by filamentous, nitrogen-fixing cyanobacteria, of a bacteriocin and of other antibiotics that kill related strains

Colonies of sixty-five filamentous cyanobacteria were screened for the production of temperate phages and/or antibiotics on solid medium. None of them was observed to release phages. However, seven N2-fixing strains were found to produce antibiotics very active against other cyanobacteria. The antibiotic produced by Nostoc sp. 78-11 A-E represents a bacteriocin of low molecular weight. Nostoc sp. ATCC 29132 appears to secrete, together with an antibiotic, a protein that inhibits its action.

New Anabaena and Nostoc cyanophages from sewage settling ponds

We have isolated, from sewage settling ponds, 16 cyanophages for heterocyst forming, filamentous cyanobacteria of the genera Anabaena and Nostoc. These phages fall into three groups based on morphology, host range, one-step growth curves, and restriction digests. On the basis of these criteria they can be distinguished from cyanophages A-1(L), A-4(L), N-1, and AN-10 which we received from other laboratories. Certain of the newly described phages are similar in morphology to the short-tailed LPP cyanophages, and others to the long-tailed AS cyanophages.

Clustered genes required for synthesis and deposition of envelope glycolipids in Anabaena sp. strain PCC 7120

Photoreduction of dinitrogen by heterocyst‐forming cyanobacteria is of great importance ecologically and for subsistence rice agriculture. Their heterocysts must have a glycolipid envelope layer that limits the entry of oxygen if nitrogenase is to remain active to fix dinitrogen in an oxygen‐containing milieu (the Fox+ phenotype). Genes alr5354 (hglD), alr5355 (hglC) and alr5357 (hglB) of the filamentous cyanobacterium, Anabaena sp. strain PCC 7120, and hglE of Nostoc punctiforme are required for synthesis of heterocyst envelope glycolipids. Newly identified Fox– mutants bear transposons in nearby open reading frames (orfs) all5343, all5345–asr5349 and alr5351–alr5358. Complementation and other analysis provide evidence that at least orfs all5343 (or a co‐transcribed gene), all5345, all5347, alr5348, asr5350–alr5353 and alr5356, but not asr5349, are also required for a Fox+ phenotype. Lipid and sequence analyses suggest that alr5351–alr5357 encode the enzymes that biosynthesize the glycolipid aglycones. Electron microscopy indicates a role of all5345 through all5347 in the normal deposition of the envelope glycolipids.

Lipids of membranes and of the cell envelope in heterocysts of a blue-green alga

SummaryHeterocysts of Anabaena cylindrica, isolated rapidly in the cold, were found—in contrast to earlier reports—to contain all of the same lipids and lipophilic pigments, and in about the same proportions, as vegetative cells. In broken filaments and in heterocysts damaged during isolation, the membrane lipids and certain pigments (myxoxanthophyll and an unidentified red pigment) break down rapidly. The glycolipids specific to heterocyst-forming blue-green algae are localized in the laminated layer of the heterocyst envelope. A possible role of the laminated layer is discussed.