Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-11-19
2002-08-20
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S041000, C435S072000, C435S822000, C435S909000, C435S252100
Reexamination Certificate
active
06436680
ABSTRACT:
The present invention relates to a new bacterial strain of the genus Vibrio, to the exopolysaccharides produced by the said strain and to their uses.
Some microorganisms obtained from the deep submarine hydrothermal medium produce biomolecules whose particular structure and composition confer on them properties of great potential industrial interest; among these biomolecules are a wide variety of polysaccharides some of which have already been the subject of studies intended to determine their structures and their properties.
The studies mentioned below have related more specifically to the exopolysaccharides (EPS) excreted by bacteria of the genus Alteromonas which are cultured under laboratory conditions, and in particular on glucose-enriched medium.
VINCENT et al. [Appl. Environ. Microbiol., 60, 4134-4141 (1994)] have thus described an exopolysaccharide, called EPS-1545, excreted by a strain, designated by the reference HYD-1545, of a bacteria of the genus Alteromonas. The EPS obtained by ethanol precipitation comprises between 49 and 55% of neutral monosaccharides, and between 32.5 and 39% of uronic acids.
GUEZENNEC et al., [Carbohydrate Polymers, 24, 287-294 (1994)] have classified the EPSs produced by various bacteria of the genus Alteromonas into 5 different groups on the basis of their composition: group 1 consists of EPS comprising between about 50 and 60% of neutral monosaccharides, about 10% of uronic acids, between 0.5 and 3.7% of osamines, and between 11.5 and 21.5% of sulphates; group 2 consists of EPS comprising about 50% of neutral monosaccharides, possessing a low content of uronic acids (8%), comprising between 1 and 3.2% of osamines, and whose content of sulphates is between less than 10% and 13%; the group 3 EPSs comprise between 40 and 50% of neutral monosaccharides, have a very low content of uronic acids (between S and 7%), comprise between 1.2 and 1.7% of osamines, and have a content of sulphates varying between 8.9 and 17.2; group 4 consists of EPS comprising 46 to 49% of neutral monosaccharides, possessing a high content of uronic acids (between 34 and 40%), comprising between 0.2 and 1.6% of osamines, and having a content of sulphates of between 9.7 and 13%; group 5 consists of EPS having a relatively low content of neutral monosaccharides (38 to 47%), a high content of uronic acids (26 to 32%), comprising between 1 and 1.6% of osamines, and between 5.2 and 10.1% of sulphates; one of the EPSs of this group comprises, in addition, one hexuronic acid carrying a lactate substituent.
A number of the strains and polymers described in the publications by VINCENT et al. and GUEZENNEC et al. cited above are the subject of application PCT FR 94/00169 published under WO 94/18340.
Recently, RAGUENES et al. [Appl. Environ. Microbiol, 62, 67-73 (1996)] have described an EPS, different from the preceding ones, which is obtained from a bacterium of the genus Alteromonas (
Alteromonas macleodii
subsp. fijiensis). This EPS comprises about 38% of neutral monosaccharides, 38% of uronic acids, 2.3% of osamines and about 5% of sulphates.
The inventors, continuing their research studies on bacteria obtained from the deep submarine hydrothermal medium have now isolated from Pompei worm (
Alvinella pompejana
) a new strain, called HE800, belonging to the genus Vibrio. This strain, which was deposited on Oct. 17, 1995 at the CNCM (Collection Nationale de Cultures de Microorganismes, 28, rue du Docteur Roux, 75724 PARIS, FRANCE) under number I-1629, represents a new species of Vibrio, for which the name
Vibrio diabolicus
is proposed.
Vibrio diabolicus
is a Gram-negative bacillus, about 0.8 &mgr;wide and 2.2 &mgr;m long; it is mobile with the aid of a polar flagellum in liquid medium and of peritrichous flagella in solid medium; it is a non-encapsulated, non-pigmented and non-luminescent bacterium.
It is a catalase+, cytochrome oxidase+, chitinase+, faculative anaerobic bacterium. It reduces nitrates to nitrites. It is sensitive to the vibriostatic agent 0/129 (2, 4-diamino-6, 7-diisopropylpteridine).
It uses a wide variety of carbon substrates; it can use in particular, as sole carbon source, any one of the following substrates: glycerol, ribose, galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamine, maltose, sucrose, trehalose, starch, glycogen, gluconate, caprate, citrate and malate.
Its growth is optimum at a temperature of between 30 and 45° C., a pH of between 7 and 8, and a salinity of between 20 and 50 g/l of NaCl; its generation time under these conditions is between 18 and 28 minutes.
The G+C content of its DNA is 49.6%. The phylogenic analysis of the 16S rRNA gene (according to the protocol established by RUIMY et al. [Int. J. Syst. Bacteriol 44, 416-426 (1994)] have made it possible to establish that it belongs to a well-defined taxon which also includes
Vibrio mytili, Vibrio nereis
and
Vibrio tubiashii
. The results of this analysis are illustrated by FIG.
1
. The percentage homology of the DNA of the HE800 strain with that of the 3 most closely related Vibrio mentioned above is 27%, 15% and 5%, respectively.
Table I below illustrates the metabolic properties of the HE800 strain (only the positive responses are included in this table)
TABLE I
Assimilation of glycerol
+
Assimilation of ribose
+
Assimilation of galactose
+
Assimilation of glucose
+
Assimilation of fructose
+
Assimilation of mannose
+
Assimilation of mannitol
+
Assimilation of N-acetylglucosamine
+
Assimilation of maltose
+
Assimilation of sucrose
+
Assimilation of trehalose
+
Assimilation of starch
+
Assimilation of glycogen
+
Assimilation of gluconate
+
Assimilation of caprate
+
Assimilation of citrate
+
Assimilation of malate
+
Reduction of nitrates to nitrites
+
Production of indole
+
Acidification of glucose
+
Hydrolysis of esculin (&bgr;-glucosidase)
+
Hydrolysis of gelatin (protease)
+
b-galactosidase (PNPG)
+
Lysine decarboxylase
+
Ornithine decarboxylase
+
Tryptophan deaminase
+
Alkaline phosphatase
++
Esterase (C4)
+
Esterase lipase (C8)
+++
Leucine arylamidase
+++
Trypsin
+
Chymotrypsin
+
Acid phosphatase
+++
The above morphological, biochemical and phylogenetic characteristics make it possible to include the HE800 strain in the genus Vibrio [BAUMANN et al.: Genus Vibrio, Bergey's Manual of Systematic Bacteriology, Vol. 1, 342-352. (1984) KRIEG, and HOIT (ed.); The Williams and Wilkins Co., Baltimore].
Because the DNA/DNA percentage homology with the three subspecies of the abovementioned genus Vibrio is less than 30%, the HE800 strain may be considered as representing a new species of Vibrio [WAYNE et al., Report of the Ad Hoc Committee on reconciliation of approaches to bacterial systematics, Int. J. Syst. Bacteriol. 463-464, (1987)], for which the name:
Vibrio diabolicus
is proposed.
The subject of the present invention is strains of bacteria possessing the above-defined characteristics of the species
Vibrio diabolicus
, and in particular the HE800 strain; this includes in particular bacteria whose DNA has a percentage homology greater than 28%, preferably greater than 30%, and advantageously greater than 50%, with the DNA of the HE800 strain. This subject also includes the bacteria obtained from bacteria of the species
Vibrio diabolicus
, and in particular of the strain HE800, by mutation, or by genetic recombination, such as for example bacteria of the species
Vibrio diabolicus
harbouring a plasmid carrying a heterologous gene.
The present invention also includes the various products which may be obtained from the said bacteria, which comprises in particular their cellular fractions, the enzymes as well as the nucleic acid preparations which may be extracted therefrom, as well as the products excreted or secreted by these bacteria.
This comprises polysaccharides capable of be
Guezennec Jean
Pignet Patricia
Raguenes Gérard
Rougeaux Hélène
Instit Francais de Recherche pour l'Exploitation de la Mer
Prats Francisco
Srivastava Kailash C.
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