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Every Base of Synechocystis Genome Now Known

Starting in only 1994, Kazusa DNA Research Institute has completed sequencing the entire genome of Synechocystis PCC 6803, reports Satoshi Tabata. For the record, the strain has precisely 3,573,470 bp, with an average GC content of 48%. Synechocystis has about 3100 likely coding sequences, with two sets of ribosomal RNA gene clusters and 42 potential tRNA genes.

1 Mb of the sequence (from 64% to 92% of the circular map) has already been analyzed and made available electronically through the Kazusa CYANOBASE site (see BULLETIN BOARD). 818 likely coding sequences in this region have been identified based on their lengths or similarity to known genes.

The sequence was reexamined using the program GeneMark, designed to recursively evaluate the probability that an open reading frame (ORF) is indeed a coding sequence, in light of the aggregate of characteristics of other open reading frames in the sequence. The program identified 752 of the 818 previously assigned ORFs, of which 26 were predicted to start at more internal start codons. 50 ORFs not found in the original analysis were predicted by GeneMark to be coding sequences.

Of the 66 ORFs originally identified but not found by GeneMark, 14 showed significant degrees of similarity to known genes, and 10 were found within insertion-sequence-like elements. Perhaps these genes came to Synechocystis by horizontal gene transfer, and therefore possess foreign characteristics that would escape the detection algorithm of the program.

Some of this work has been published [Kotani H et al (1994) DNA Res 1:30-307; Kotani H et al (1995) DNA Res 2:133-142; Kaneko et al (1995) DNA Res 2:153-166. Hirosawa et al (1995) DNA Res 2:239-246], and much more can be found on the CYANOBASE web site.

CONTACT: Satoshi Tabata, Kazusa DNA Research Institute, Laboratory of Gene Structure 2, 1532-3 Yana, Kisarazu, Chiba 292, JAPAN. TEL: 81-438-52-3934, FAX: 81-438-52-3933, E-MAIL: Tabata@Kazusa.Or.Jp


Toxin Suspect in Mass Killing

Tragedy struck this past spring in a Brazilian hospital. 126 patients undergoing hemodialysis in Caruaru, a city in northeastern Brazil, became ill and, over the course of several weeks, 43 of them died of toxic hepatitis. Much evidence has implicated microcystin, a toxin produced by the cyanobacterium Microcystis aeruginosa, as a prime cause of the deaths. If this connection is borne out, it would constitute the first confirmed instances of human fatalities attributed to microcystin poisoning.

Microcystins, potent hepatotoxins that act on protein phosphatases, were found in the liver and serum of affected patients at levels associated with acute or lethal toxicities. Investigation as to the source of the toxin centered on the water source used to feed the hemodialysis unit. Owing to a drought, the hospital had water trucked in from reservoir and treated with chlorine. Chlorination, however, is not effective in destroying microcystin, except with long treatments or at low pH. Indeed, chlorination, by killing any M. aeruginosa present, would be expected to liberate microcystin into the water. Microcystin was detected by high performance liquid chromatography in the water source and in the carbon used to filter the water.

Jose Roberto C. Rocha, the physician who broke the story to the cyanobacterial community, voiced the concern that the case in Caruaru may not be unique. Patients on dialysis often show unexplained alterations in liver enzymes, he said, speculating that this might be caused by low levels of microcystin intoxification. A similar case was reported earlier by Filomena Araujo, Evora, Portugal, in which the deaths of dialysis patients may have been due to microcystin. The water supply was drawn from a river heavily contaminated with Microcystis aeruginosa.

It is important to note that the case against microcystin is not complete. The water may well have contained other toxins, including pesticides, that have not yet come to light. Wayne Carmichael is continuing a study to pin down the cause. The evidence in favor of some wrongdoing is already convincing enough to officials, however, to prompt action. Three physicians involved in the dialysis have been indicted for manslaughter.

Abstracted from material posted to the Toxic Cyanobacteria Web Site (See BULLETIN BOARD).


Cryopreservation of Cyanobacteria

Cryopreservation refers to the storage of a living organism at ultra- low-temperature such that it can be revived and restored to the same living state as before it was stored. Indefinitely long storage times require that the organism be maintained below the glass transformation temperature of aqueous solutions, approximately -130degC, the temperature at which frozen water no longer sublimes and recrystallizes. Although ultra-cold freezers may stabilize some living cells for weeks or even years, liquid nitrogen is required for longer storage times. Cyanobacteria, as is the case for all living cells, suffer severe osmotic stress and/or ice crystal damage during the freezing and thawing processes. The most effective known ways to minimize these potentially lethal effects are to add a cryoprotective compound to the culture prior to its freezing for storage, and to control the transient cooling and warming rates during preservation.

We have been able to cryopreserve virtually all of the approximately 200 strains of cyanobacteria in the UTEX collection of algae [R.C. Starr and J.A. Zeikus (1993) J Phycol 29 (supp)] located in the Department of Botany at the University of Texas at Austin. This includes unicells, branching and unbranching filamentous species, marine and freshwater species, and those with heterocysts and akinetes. We also have successfully stored several photosynthetic mutants of cyanobacterial species provided by Robert Tabita of Ohio State University and John Golbeck of the University of Nebraska. The required procedures are straightforward and inexpensive, but require attention to a few details. One-hundred percent viability is never expected but viabilities over 50% are typical. High viabilities (i.e. >10%) are especially desirable for mutant strains. Here I will describe procedures that cryopreserve nearly all of cyanobacterial strains we have examined.

SAMPLE PREPARATION: Transfer cyanobacterial liquid culture into a cryovial[1] in preparation for cryopreservation. Alternatively the culture can be grown directly as a lawn on a tiny agar slant prepared within the cryovial [K. Bodas, K.R. Diller, and J.J. Brand (1995) Cryo-Letters 16:267-274]. If the cryovial contains liquid culture, pellet the cells by centrifugation in a clinical centrifuge and discard the supernatant [2]. Add cryoprotective solution containing 1.0 ml of half-strength growth medium (BG-11 works well for virtually all fresh-water cyanobacteria) containing 5% methanol or 8% DMSO to the pelleted cells [3]. Alternatively, if the culture is growing on an agar slant, transfer 1 ml of cryoprotective solution above the slant[4,5].

[1] Two-ml or 1.8-ml polyethylene or polypropylene cryovials are especially convenient for handling and storage efficiency, although 1-ml and 5-ml cryovials also work well.

[2] We have constructed acrylic sleeves that fit into the tube holders of the rotor, positioning the cryovials securely in place within a clinical centrifuge rotor, flush with the top of the tube holders. The cryovials can also be inserted into unmodified tube holders for centrifugation in a clinical centrifuge.

[3] Although glycerol is an effective cryoprotective agent for many bacteria, it is not effective for most cyanobacteria. Methanol at approximately 5% (v/v) is suitable for most strains. However, we have been successful with concentrations of methanol ranging from 2% to 12.5%, and DMSO ranging from 4 to 15 %, depending on the culture. A small fraction of some cultures survive with no added cryoprotective agent.

[4] When the cryoprotective agent is added directly above the culture on an agar slant, the tube is shaken gently prior to freezing, to dislodge some of the cells and ensure that the liquid penetrates through the culture. Cells pelleted from liquid suspension are fully suspended in the cryoprotective solution.

[5] Cells are killed by exposure to bright light when in cryoprotective solution. Keep the culture in subdued room light while handling, and in complete darkness at other times.

FREEZING: The cryovial containing the culture in cryoprotective agent at room temperature is inserted into a special "freezing container"[6] which has been pre-chilled to refrigerator temperature. The freezing container is then placed into a -70degC freezer for 2 hours. Then the cryovial is quickly removed from the freezing container, placed into a storage container, and plunged into liquid nitrogen for indefinite storage.[7]

[6] The "Mr. Frosty" freezing container (Nalgene) is satisfactory for nearly all cyanobacteria. It is inexpensive to purchase and holds eighteen 2-ml cryovials simultaneously. Its contents cool at slightly less than 1degC per minute when it is placed into a -70degC freezer.

[7] Sterility is a problem when storing plastic cryovials in liquid nitrogen. Vials equipped with gaskets and those with inside threads seal most tightly, but liquid nitrogen always creeps into some cryovials. This provides a conduit for entry of bacteria, some of which remain viable in bulk liquid nitrogen. Several manufacturers sell heat-shrink tubing that serves as a tight-fitting sleeve around the entire cryovial and lid, thereby eliminating liquid nitrogen leakage. Bacterial contamination can be eliminated also by storage in sealed glass ampoules or by storing plastic cryovials in the nitrogen vapor just above the liquid, although these procedures introduce additional safety and convenience considerations.

THAWING AND RECOVERY: Cultures to be revived are removed from liquid nitrogen storage and warmed rapidly to room temperature[8]. Cells are immediately pelleted by centrifugation of the cryovial[9,10], and the supernatant is discarded. One ml of fresh growth medium is placed into the vial to suspend the pellet. The cryovial lid is slightly loosened to allow gas exchange, and the contents of the vial are kept in complete darkness for 1-2 days. The culture can then be placed on agar or in liquid growth media under normal growth conditions. The viable cells should begin normal growth within 1 - 2 days in light, although they are especially susceptible to damage by excessive light intensity for the first day or two of illumination.

[8] Warm rapidly by plunging the tightly sealed, still-frozen cryovials into a dish of water at 35degC. An appropriately selected volume of water will cool to approximately 25degC as the cryovial contents are warmed to that same temperature.

[9] Centrifugation of a thawed culture in a cryovial containing an agar slant is best done in an angle rotor that pellets the cells on the agar surface without appreciably altering the position of the agar in the tube.

[10] Cultures of eukaryotic algae especially, and cyanobacteria to some extent, are susceptible to mechanical damage during recovery from storage at low temperature. Cells should be pelleted at the minimum R.C.F. that facilitates pelleting. Excessive agitation should be avoided when suspending the pellet.

Comments welcome!

Jerry J. Brand, Botany Dept., Univ. of Texas at Austin E-MAIL: JBrand@UTxsvs.Cc.UTexas.Edu


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