Cytological and biochemical requirements for the establishment of a polar cell.

Our research is aimed at understanding the biochemical and cytological basis of cell polarity in zygotes of the brown alga, Fucus distichus L. Powell. One manifestation of this polar cell is the localization of a sulphated fucan polysaccharide (F2) in only one region of the zygote cell surface, the rhizoid cell wall. The focus of this paper is centered around the mechanism responsible for the directional transport of Golgi vesicles containing F2 and the biochemical properties of F2 that might specify its localized fate. Recent findings indicate that the various sulphated polysaccharides in the brown algae are complexes resulting from linkages of two basic polymers: an alpha-(1----2)-linked fucan that contains high levels of ester sulphate (F3), and a uronic acid-rich polymer (F1). The fucan complex F2, which is localized in the rhizoid wall, is composed of a fucan sulphate core (F3) to which uronic acid polymers (similar to F1) are attached. Our results, using a purified endoguluronate lyase, indicate that guluronate bridges link these subunits of F2. The carbon backbone of F2 is not synthesized de novo after fertilization. However, F2 is sulphated, and possibly assembled, beginning 10 h after fertilization, after which it is locally inserted into the rhizoid wall, and held in the wall structure only by calcium ionic bonds. Although sulphation is required for localization of F2, it is not known if the uronic acid side-chains are also assembled at the time of sulphation, and/or required for localization. The fact that F3 (F2 without the side-chains) is secreted uniformly into the zygote wall suggests that the uronic acid chains of F2 may play a critical role in its localization. A sulphated F2 alone is not sufficient for its localization since in the presence of cytochalasin, vesicles containing F2 are not transported to the rhizoid. Recent studies point to a central role for a cytoskeletal element, possibly microfilaments, in the directional transport of these vesicles. We have used the techniques of isoelectric focusing and electrophoretic mobility to study surface charge of these Golgi vesicles to determine if charge might be one factor that specifies their localization. Vesicles that contain the sulphated fucan F3 are secreted randomly and have the same surface charge as those containing F2 that are directionally transported. However, there is no stable endogenous electrical current at the time when F3 vesicles are randomly secreted, whereas a current is detectable when F2 vesicles are localized.(ABSTRACT TRUNCATED AT 400 WORDS)

Alginase enzyme production by Bacillus circulans.

Stream and soil samples were screened for microorganisms that would use alginate from mucoid Pseudomonas aeruginosa as the sole carbon and energy source. A pure culture containing large aerobic rods was isolated. The cells were about 0.8 by 2.5 microns in size, had lateral or peritrichous flagella, had a negative Gram stain reaction, and produced spores on sporulation medium. Purified DNA was approximately 46 mol% G+C as measured by thermal denaturation. From these and other biochemical tests, the organism was identified as Bacillus circulans. The enzyme activity that degraded alginate appeared in the culture medium. Upon gel filtration, alginase activity eluted as a single peak at a position corresponding to a protein of 40,000 daltons. Activity recovered from this one-step, partial purification showed apparent endomannuronidase specificity. Like other alginases previously reported, the enzyme appeared likely to be a lyase (or eliminase). However, no Bacillus species or other gram-positive bacteria have heretofore been reported to produce extracellular enzymes with alginase activity. Several other B. circulans strains from the American Type Culture Collection also appeared to have inducible extracellular alginase activity.

Properties of alginate lyases from marine bacteria.

Alginate lyases (EC 4.2.2.3) from two marine bacteria were isolated and partially characterized. A cell-bound lyase from isolate A3 had a molecular weight of approximately 100,000 and cleaved mannuronate blocks, apparently in an exo manner. A lyase recovered from the culture medium of isolate W3 was soluble in saturated ammonium sulfate, cleaved guluronate blocks, apparently in an endo manner, and had a molecular weight of 35,000. The thiobarbiturate test and urea-polyacrylamide gel electrophoresis were used to determine substrate specificity and mode of substrate cleavage by the enzymes.

Isolation of marine bacteria capable of producing specific lyases for alginate degradation.

Alginate lyases (EC 4.2.2.3) were isolated from cultures of several marine bacterial isolates. The lyases were induced by native alginate and had activity toward both the mannuronic acid and the guluronic acid blocks of the alginate polymer. The guluronic acid-specific lyase was recovered from the medium, whereas the mannuronic acid-specific lyase was retained with the bacteria.