Digestion of Algin by Pseudomonas Maltophilia and Pseudomonas Putida V

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1980, p. 92-96 Vol. 39, No. 1 0099-2240/80/01-0092/05$02.00/0

Digestion of Algin by Pseudomonas maltophilia and Pseudomonas putida V. LYLE VON RIESEN Department ofMedical Microbiology, College ofMedicine, University of Nebraska Medical Center, Omaha, Nebraska 68105 Pseudomonas maltophilia and Pseudomonas putida were identified as alginolytic species. Two media used for demonstrating alginolytic activity are described. The applied aspects of the ability of these two species to digest algin are discussed.

In a search for undescribed biochemical activ- Both media contained yeast extract (0.5%), algin (al- ities of nonfastidious, nonfermentative gram- ginic acid, sodium salt, no. A-7128; Sigma Chemical negative bacilli (NFB) which might be used to Co., St. Louis, Mo.) (1%), and bromothymol blue of these taxonomically (0.002%). Agar (0.5%) was used to form an algin-agar aid in the identification medium and animal charcoal (0.1%) to form an algin- troublesome organisms, a survey of a variety of charcoal medium. The media were prepared in 100- to complex polysaccharides as substrates was made 500-ml amounts in the following manner. Calculated by using a few strains of some well-recognized amounts of yeast extract and bromothymol blue (from species of NFB. When it was discovered that a 1% aqueous solution) were added to an appropriate some of the strains ofPseudomonas maltophilia volume of distilled water. This was then warmed on a showed evidence of an ability to hydrolyze algin magnetic stirrer-heater. When warm (40 to 60°C), (sodium alginate), an available collection of ap- algin and charcoal (algin-charcoal medium) or algin proximately 390 strains of NFB was examined and agar (algin-agar medium) were sprinkled into the of individual strains to digest vigorously stirred solution. As heating and stirring for the ability were continued, obvious lumps were mashed, with a algin. Since many of these organisms do not spatula or glass rod being used to aid the magnetic readily produce acids from carbohydrates, it stirrer. By the time the mixture was close to boiling, seemed that some approach other than the con- it was reasonably free of obvious lumps. Heating was ventional method for carbohydrate metabolism discontinued at this point. The algin-charcoal medium (e.g., 1% carbohydrate in OF base) was neces- was then transferred in 4-ml volumes with a hand- sary. This paper describes the methods used to operated Cornwall pipettor to screw-cap tubes (16 by demonstrate alginolytic activity and identifies 125 mm). The algin-agar medium was left in a flask or two species which can digest algin. fleaker. The media were autoclaved at 121°C for not more than five minutes. The short heating period was to avoid the possible breakdown of algin. Immediately MATERUILS AND METHODS upon removal from the autoclave, the algin-agar was Organisms. The organisms used in this study were distributed in petri dishes to at least half volume. The strains of gram-negative bacilli which had been col- lids were left off the petri dishes to allow steam to lected from human, animal (porcine, bovine, and escape. Before use the plates were exposed in an avian), and environmental sources over a period of 25 incubator at 35°C for further drying. The algin-char- years. They had been maintained through this period coal medium was allowed to cool in an upright position in several ways, but generally by culture in cystine- and the tubes were then refrigerated until used. No Trypticase agar (BBL Microbiology Systems, Cock- settling of the charcoal occurred during storage or eysville, Md.) and storage at room temperature in the during use. The pH of the media by the color of the dark. None of the strains produces acid under anaer- indicator was neutral to very slightly alkaline. obic conditions in OF basal medium (14) with 1% The algin-agar plates were inoculated by spotting glucose, and all are therefore designated as NFB. (patching) cultures, grown on Trypticase soy agar During the course of this study all strains were more (BBL) for 24 h, onto the surface of the medium, 13 critically examined biochemically to obtain as positive strains per plate. (If spreading occurred, the offending an identication of as many strains as was reasonably organisms were tested separately.) The algin-charcoal possible. The methods used to identify the strains deeps were inoculated in a conventional way. Incuba- were identical to or variations of the methods de- tion of all materials was at 35 ± 1°C for 72 h and then scribed by various workers in this field (10, 13, 24, 29, at room temperature (18 to 22°C) to the end of 10 33, 35, 38). A few of the strains remain unidentified, days. Observations were made at 1, 2, 3, 6, and 10 days. since it did not seem possible to identify them with Over a period of several years, a number of formu- recognized species or alphanumerics. lations of algin media and variations of the procedures Algin media. Two forms of algin media were used. described above were examined. The results shown 92 VOL. 39, 1980 ALGIN DIGESTION BY P. MALTOPHILIA AND P. PUTIDA 93 here are based on the procedures described and the TABLE 1. Biochemical characteristics of alginolytic agar and charcoal media were studied concurrently for Pseudomonas species comparison. Positive strains

RESULTS Test or medium P. maltophilia P. putida (42 strains) (30 strains) By the criteria used in this study (Table 1) for the identification of the 390 strains of NFB the No. % No. % numbers of strains of species and the alphanu- OF glucose 41 98 30 100 merics identified were: Achromobacter xylosox- Oxidase 0 0 30 100 idans, 10; Acinetobacter anitratum, 24; Acine- Motility 38 91 28 93 tobacter Iwoffii, 17; Alcaligenes denitrificans, 3; H2S 0 0 0 0 Alcaligenes faecalis, 3; Alcaligenes odorans, 7; Tryptophan pyrrolase 40 95 2 7 Bordetella bronchiseptica, 3; Bordetella para- Indole 0 0 0 0 pertussis, 1; Flavobacterium meningosepticum, Starch hydrolysis 0 0 0 0 Deoxyribonuclease 42 100 0 0 1; Flavobacterium odoratum, 2; Moraxella spp. Ribonuclease 42 100 0 0 (undifferentiated), 9; Pseudomonas acidovor- Arginine dihydrolase 0 0 29 97 ans, 7; Pseudomonas aeruginosa, 88; Pseudom- NO3-+ N2 0 0 0 0 onas alcaligenes-pseudoalcaligenes group, 18; N03- N02 18 43 0 0 Pseudomonas cepacia, 13; Pseudomonas dimi- Urease 7 17 2 7 nuta, 6; Pseudomonas fluorescens, 2; Pseudom- Gelatinase 42 100 0 0 onas maltophilia, 42; Pseudomonas paucimo- Esculin hydrolysis 40 95 0 0 bilis, 1; Pseudomonasputida, 30; Pseudomonas Acetamide utilization 0 0 0 0 putrefaciens, 7; Pseudomonas stutzeri, 46; Pseu- Algin hydrolysis 23 55 13 43 domonas testosteroni, 3; Vibrio extorquens, 4; CDC (Center for Disease Control) group lIf, 9; strains produced an appearance of a very weakly CDC group IVc-2, 1; CDC group Vd, 7; CDC developed depression during the first 24 to 72 h; group Ve-1, 1; CDC group Ve-2, 3. Twenty-two however, by day 6 this depression was dissi- strains have not yet been matched to any of the pated. Such an appearance with pectate gels was species or groups. Of these strains, 15 are al- attributed by Hildebrand (12) to possible loss of ginolytic. Efforts to identify these are continu- water from the medium to the developing mass ing. of growth, thus causing shrinkage of the me- Species and alphanumerics not identified in dium. this collection were: Agrobacterium radiobac- With the algin-charcoal medium, alginolytic ter, Pseudomonas mallei; Pseudomonas men- activity was observed as a usually sharply de- docina; Pseudomonas pickettii; Pseudomonas marcated settling of the charcoal. With time, pseudomallei; Pseudomonas vesicularis; and and varying with the individual strains, the line CDC groups IIj, IIk-2, IVe, and Va-i. of settled charcoal rapidly or slowly descended. Table 1 shows the characteristics ofthe strains On each day of observation, the amount of set- identified as P. maltophilia and P. putida. thing (depth of clear zone above the line of char- Twenty-three, or 55%, of the 42 strains of P. coal) was measured and recorded. On day 10, all maltophilia and 13, or 43%, of the 30 strains of tubes showing no evidence of settling, i.e., com- P. putida were alginolytic. All alginolytic strains, parable to the uninoculated control tube, were including the 15 unidentified strains, were posi- recorded as negative. Most of the alginolytic tive on both algin media. strains of both P. maltophilia and P. putida Alginolytic activity was observed with the al- were positive within 24 h of inoculation. A few gin-agar medium, as previously described for strains of both species required up to 72 h to alginolytic activity (9, 42) and for pectinolytic become positive. Table 2 shows the number of activity (36, 37), as a defmite break in the plane strains of P. maltophilia and P. putida produc- of the surface of the medium. With the majority ing 100% settling of the charcoal by days 1, 3, 6, of the positive strains, this was a distinctly dif- and 10. As a further evaluation of the alginolytic ferentiated, bowl-shaped depression containing activity, when the settling of the charcoal was the patch of growth. With some strains a shallow considered 100%, the tubes were manually depression was formed, and in this reaction the shaken to resuspend the charcoal and to obtain growth was frequently contiguous with the pe- an impression of the viscosity of the medium. rimeter of the depression. Most ofthe alginolytic Generally, with P. maltophilia the viscosity was strains of P. maltophilia were positive within 24 as for water (algin presumably completely di- h of inoculation; most strains of P. putida re- gested); with P. putida the viscosity was as for quired 48 h to become positive. Some negative a thin syrup (algin not completely digested). 94 VON RIESEN APPL. ENVIRON. MICROBIOL. TABLE 2. Rate oflO0% settling of charcoal for growth, the absence ofthat amino acid in the No. (%) showing 100% settling by day: algin-utilization basal media, as in the work of Organism Waksman et al. (39) and Kooiman (19), would 1 3 6 10 have precluded isolation of that species. Stanier P. maltophilia (23 0 16 (73%) 19 (86%) 22 (97%) and his co-workers (30) in their study on the strains) taxonomy of the pseudomonads stated, "Many P. putida 0 4 (31%) 8 (62%) 10 (77%) bacteria that seem to conform to our definition (13 strains) of aerobic pseudomonads have been isolated from soil, fresh water, and sea water by selection for the ability to grow at the expense of complex DISCUSSION polysaccharides (cellulose, agar, chitin, alginic Aerobacter (16), Agarbacterium (41), Algi- acid) and a number of specific, physiologically nobacter (18, 34), Alginomonas (4, 5, 6, 9, 18, defined genera (e.g., Cellulomonas, Cellvibrio, 34), Alginovibrio (22, 31, 32, 34), Alteromonas Alginomonas) have been proposed for them. We (1), Bacterium (39), Bacteroides (28), Beneckea shall not attempt to cite here the voluminous (2, 25), Klebsiella (3), Pseudomonas (4, 5, 6, 15, literature on these organisms; but it should be 42), and Vibrio (2) are genera of bacteria said to kept in mind that they may well constitute a contain alginolytic strains. Of these, Pseudomo- large and varied group ofaerobic pseudomonads, nas and one species ofAlginomonas (A. nonfer- which has so far never been subjected to system- mentans) fit the description of NFB. Most of atic comparative study." Interestingly, perhaps the species of Alginomonas (4) were originally even surprisingly, this group (30) did not include isolated from seawater and described by Waks- algin among the many substrates studied for man et al. (39) in 1934 as species of Bacterium. utilization by pseudomonads, including P. mal- In 1945, Thjotta and Kass (34) described the tophilia and P. putida. genus Alginomonas, and Kass et al. (18) Although this study was initiated to find new changed the Waksman Bacterium species to substrates which would aid in the identification species of Alginomonas, adding A. nonfermen- of NFB, the incidence of positive strains in the tans. None of these species (4) nor the uniden- two clearly defined species is not sufficiently tified strains isolated by Kooiman (19) and Qua- high to be of value for identification purposes. trano and Caldwell (27) from marine algae can Identification of strains ofP. maltophilia and P. be identified with P. maltophilia or P. putida. putida as alginolytic may be warranted, how- Many of the alginolytic species of Pseudomo- ever, in other situations. Since algin is used in nas described earlier by Humm (15) have since foods as a thickening and a stabilizing agent (21), been relegated to lists of "uncertain species" in identification of NFB which have the ability to Bergey's Manual (5). Other reports of algino- digest this food additive may have some further lytic pseudomonads have referred to P. atlan- significance beyond whatever value this infor- tica (42), P. carrageenovora (40, 42), Pseudom- mation might provide from a diagnostic or tax- onas sp. (17, 23), or a pseudomonad (7, 9, 26). onomic standpoint. Since P. maltophilia and P. None of the descriptions of the pseudomonads putida are commonly present in the environ- which have been examined would identify P. ment, a coming together ofalgin-containing food maltophilia or P. putida. and the specific bacteria might lead to some Alginolytic activity in NFB was observed in interesting results. this study in two well-defined species, P. mal- Still another way in which the alginolytic ac- tophilia and P. putida, and in several less well- tivity of positive strains of these species may be defined species or groups. Alginolytic activity put to use is in the determination ofthe structure was demonstrated in 55% of the strains of P. of algin. The algin molecule is described as con- maltophilia and in 43% of the strains of P. taining three fractions: a mannuronate polymer, putida. It seems somewhat surprising, therefore, a guluronate polymer, and a polymer comprised that these two species ofPseudomonas have not of alternating units of mannuronate and gulu- previously been identified as alginolytic. This ronate (21). Recent work (7, 17, 23) has shown would not seem to have been due to a lack of that the alginases of marine pseudomonads are interest in the bacterial degradation of algin. A specific for the guluronide linkages. In this study, possible explanation is that many searches for the differences observed in the viscosity of 10- alginolytic bacteria have focused, for the most day-old cultures might suggest that P. malto- part, on marine organisms (1, 2, 7, 15, 18, 19, 22, philia may be able to act upon more of these 27, 34, 39). P. maltophilia and P. putida appar- linkages (guluronide and mannuronide) than ently are not commonly present in the marine does P. putida. The alginases of these two spe- environment (1, 2, 30). Another point to consider cies, therefore, may be of some value to those is that, since P. maltophilia requires methionine who are attempting to establish the mannuron- VOL. 39, 1980 ALGIN DIGESTION BY P. MALTOPHILIA AND P. PUTIDA 95 ate-guluronate configuration in the algin mole- 3. Boyd, J., and J. R. Turvey. 1977. Isolation of a poly-a- L-guluronate lyase from Klebsiella aerogenes. Carbo- cule (3, 7, 8, 17, 23, 32). hydr. Res. 57:163-171. Each of the media used in this study to eval- 4. Breed, R. S., E. G. D. Murray, and N. R. Smith (ed.). uate alginolytic activity has its own merits. The 1957. Bergey's manual of determinative bacteriology, agar medium can be used to test as many as 13 7th ed. The Williams & Wilkins Co., Baltimore. It can also 5. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Ber- strains simultaneously. be used to gey's manual of determinative bacteriology, 8th ed. The obtain isolates from the environment (some iso- Williams & Wilkins Co., Baltimore. lates from soil have been obtained this way). A 6. Buchanan, R. E., J. G. Holt, and E. F. Lessel, Jr. (ed.). disadvantage of the agar medium is that some 1966. Index Bergeyana. The Williams & Wilkins Co., is encountered in a Baltimore. difficulty obtaining relatively 7. Davidson, L. W., L. W. Sutherland, and C. J. Lawson. dry surface. This problem was previously noted 1976. Purification and properties of an alginate lyase by Yaphe (42). 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