Biol. Mar. Mediterr. (2012), 19 (1): 188-189
V. Pasquale, V. Romano, M. Guida*, V. Mastascusa, M. Greco, R. Sandulli
Department of Environmental Sciences, “Parthenope” University, Naples, Italy.
[email protected]
*Department of Biological Science, Section Physiology and Hygiene, “Federico II” University, Naples, Italy.
HYDROLYTIC EXOENZYME SCREENING OF HETEROTROPHIC
BACTERIA ASSOCIATED WITH CORALLIUM RUBRUM
SCREENING DI ESOENZIMI IDROLITICI IN BATTERI ETEROTROFI
ASSOCIATI A CORALLIUM RUBRUM
Abstract - Corals establish strict ecological associations with several prokaryotic and eukaryotic
microorganisms. Heterotrophic bacterial strains belonging to the genera Actinomyces, Bacillus,
Kocuria, Pseudomonas, Staphylococcus and Vibrio, isolated from the surfaces and the tissues of
Corallium rubrum, have been characterized for the production of hydrolytic exoenzimes (protease,
lipase, fosfolipase, DNase, mucinase, chitinase and amilase).
Key-words: Corallium rubrum, Bacillus, Vibrio, exoenzymes, extracellular hydrolytic enzyme.
Introduction - Recent studies on the ecological relationships between
microorganisms (virus, bacteria and algae) and corals have led to the birth of coral
microbiology (Rosenberg et al., 2007). Several authors have described the interactions
between microorganisms and esacorals, particularly after the spread of diseases
causing the loss of about 30% of the coral population (Rosenberg et al., 2007; Luna
et al., 2007). On the other hand, little is known about the microbiota associated to
Corallium rubrum, a gorgonacea (Anthozoa, Octocorallia) belonging to the family of
Coralliidae. During a survey carried out by Pasquale et al. (2011), Actinomyces sp.,
Bacillus licheniformis, B. megaterium, B. mycoides, B. pumilus, Bacillus sp., Kocuria
palustris, Kocuria sp., Pseudoalteromonas sp., Pseudomonas stutzeri, Pseudomonas
sp., Staphylococcus equorum, S. vitulinus, Vibrio harvey, V. hepatarius and V. shilonii
were reported as the most representative isolates of the culturable heterotrophic
bacterial component associated to colonies of C. rubrum sampled from the sea beds
surrounding the island of Procida (Southern Italy). The aim of this study was to
investigate the production of hydrolythic exoenzymes in bacterial strains isolated
from colonies of C. rubrum.
Materials and methods - Bacterial strains isolated from colonies of C. rubrum
were cultured on R2A (Oxoid, UK) supplemented with 2% NaCl. Subcultures of
the isolates were performed with the agar spot technique on a carbon void medium
(K2HPO4 0.14 g/l, KH2PO4 0.06 g/l, MgSO4∙7H2O 0.1 g/l, FeSO4∙7H2O 0.002 g/l, ZnSO4
0.0002 g/l, MgCl2 0.0002 g/l, NaCl2 20 g/l, agar 1.5%, pH 7.2) and supplemented with
substrates specific for the exoenzymes tested: 1.5% gelatin (gelatinases), 1% skim milk
(proteases), 1.5% mucin type II (mucinases), 1% colloidal chitin (chitinases), 1,5%
soluble starch (amilase), 1% tween 80 (lipase) and 2% egg yolk emulsion (fosfolipase).
The production of DNase was tested using DNase Agar (Merck, Germany). The
exoenzymes production was evaluated observing the presence of hydrolysis haloes
around the bacterial colonies, after up to 7 days incubation at 20 °C.
Results - Gelatinases were the most common enzymes produced by the isolates:
47% of them hydrolyzed gelatin, followed by proteases, with 41% of the isolates able
to hydrolyze skim milk (Tab. 1). The production of mucinase and chitinase was
observed in 38% and 35% of the isolates respectively. Haloes of starch hydrolysis were
observed in 29% of the strains, while only one strain of B. licheniformis, which was
Hydrolytic exoenzyme screening of heterotrophic bacteria associated with C. rubrum
189
the one showing the highest heterogeneity in exoenzymes synthesis, produced DNase.
None of the tested isolates produced lipase or fosfolipase. Bacillus, Pseudoalteromonas
and Vibrio resulted as the genera with the widest pattern of exoenzymes produced.
Among the Vibrionaceae, considered as potential pathogens of corals, V. harvey has
been the species with the broadest profile of exoenzymes produced, while V. shilonii
hydrolyzed only chitin and starch. Vibrio hepatarium, for the first time isolated from
colonies of corals, produced gelatinase, protease and chitinase.
Tab. 1 - Hydrolitic exoenzyme production in bacterial strains associated with C. rubrum.
Produzione di esoenzimi idrolitici in ceppi batterici associati a C. rubrum.
Species identified
B. licheniformis
B. megaterium
B. pumilus
B. mycoides
P. stutzeri
Bacillus sp.
K. palustris
Kocuria sp.
Pseudomonas sp.
Actinomyces sp.
S. equorum
S. vitulinus
Pseudoalteromonas sp.
V. harveyi
V. hepatarium
V. shilonii
% of strains
Strains
10
3
2
1
1
1
3
4
1
1
1
1
2
1
1
1
Gel
6
2
2
1
1
2
1
1
47
SM
5
2
2
1
1
1
1
1
41
Muc
8
1
1
2
1
38
Chi
7
1
2
1
1
35
Sta
2
3
1
1
1
1
1
29
DNase
1
3
Gel: gelatinase; Pro: protease; Muc: mucinase; Amy: amylase; Chi: chitinase.
Gel: gelatinasi; Pro: proteasi; Muc: mucinasi; Amy: amilasi; Chi: chitinasi.
Conclusions - This study demonstrates that the surfaces (mucus and tissue) of C.
rubrum can host a wide diversity of bacterial species able to produce exoenzymes
involved in processes of mineralization of the organic matter in the marine
environment. Further studies, however, are needed to elucidate upon the potential
role of bacterial exoenxymes in coral deseases.
References
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