Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1998-04-16
2001-04-03
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S909000, C424S093400
Reexamination Certificate
active
06210947
ABSTRACT:
TECHNICAL FIELD
The present invention relates to antimicrobial compounds produced by subcuticular bacteria of a brittlestar, to methods of producing such antimicrobial compounds, and to biologically pure strains of subcuticular bacteria of a brittlestar producing such compounds.
Table of Abbreviations and Symbols
A. gracillima
Amphipholis gracillima
, a species of brittlestar
ATCC
American Type Culture Collection
B. anthracis
Bacillus anthracis
B. megaterium
Bacillus megaterium
B. subtilis
Bacillus subtilis
B. catarrhalis
Branhamella catarrhalis
BE
brittlestar extract
C. diphtheriae
Corynebacterium diphtheriae
DNA
deoxyribonucleic acid
E. aerogenes
Enterobacter aerogenes
E. coli
k-12
Escherichia coli
k-12
HPLC
high performance liquid chromatography
K. pneumoniae
Klebsiella pneumoniae
M. luteus
Micrococcus luteus
M. smegmatis
Mycobacterium smegmatis
%
o
standard symbol indicating salinity by weight
PCR
polymerase chain reaction
P. vulgaris
Proteus vulgaris
P. aeruginosa
Pseudomonas aeruginosa
pSCB
putative subcuticular bacteria
RFLP
restriction fragment length polymorphism
RNA
ribonucleic acid
rRNA
ribosomal ribonucleic acid
SCB
subcuticular bacteria
S. typhimurium
Salmonella typhimurium
S. ureae
Sporosarcina ureae
S. aureus
Staphylococcus aureus
TBE
Tris-borate-EDTA
TEM
Transmission electron microscope
UV
ultraviolet
V. parahemolyticus
Vibrio parahemolyticus
BACKGROUND ART
Associations between marine invertebrates and endosymbiotic bacteria are increasingly recognized as widespread and of biological importance (McKenzie and Kelly 1994). Examples of marine invertebrates utilizing symbiotic bacteria include sponges, annelids, bivalve molluscs, cephalopods, and echinoderms (Kelly et al. 1995). Of the five extant classes of echinoderms, all are known to harbor symbiotic bacteria directly below the cuticle, lying in or within the folds of the dermal lamallae (McKenzie and Kelly 1994; Kelly et al. 1995). Holland and Nealson (1978) first described these bacteria, known as subcuticular bacteria (SCB). Little is known of the biology and role of SCB relative to their hosts, but chemoautotrophic symbionts are found in sea-urchin guts, and some feather stars (criniods) are known to have bacteria enclosed in their pinnules (McKenzie and Kelly 1994).
Many morphological studies of SCB have been done. Transmission electron microscope (TEM) observations of SCB revealed that they are Gram-negative bacilli (McKenzie and Kelly 1994). The SCB found in
Amphipholis
(
Microphiopholis
)
gracillima
(Ophiuroidea) (hereinafter “
A. gracillima
”) were described by McKenzie and Kelly (1994) as single straight rods, with membrane-bound vacuoles. All SCB examined by McKenzie and Kelly (1994) had simple, thin capsules. None of the bacteria observed in
A. gracillima
tissues exhibited evidence for flagella or pili, nor showed any type of mobility (McKenzie and Kelly 1994; Kelly and McKenzie 1995).
SCB could play a role in defense against bacterial infestation during tissue regeneration. Since brittlestars exhibit the ability to autotomize and then regenerate body parts, brittlestars are candidates for pathogenic microbial colonization (Bryan et al. 1994). Bryan et al. (1994) proposed that antimicrobial compounds, produced by echinoderms, would help prevent infection by pathogenic bacteria and thus prevent surface fouling of epithelial tissue that can deplete respiratory capacity, impede elimination of waste products, and reduce tissue elasticity. Lubchenco et al. (1991) suggested SCB and echinoderms may have evolved a mutualistic relationship that provides for protection against colonization of other bacterial species.
McKenzie and Kelly (1994) showed that regenerating tissue, such as damaged ophiuroid arm tips, are quickly colonized by SCB. When the arms of
Amphipholis gracillima
were surgically removed, SCB colonized the wound closure within 1 hour and almost completely covered the regenerating stump within 24 hours (Dobson 1988). Due to this rapid proliferation, SCB may limit colonization of pathogenic bacteria due to competitive exclusion, production of a compound which exhibits antimicrobial activity, or both.
Despite the aforementioned study of SCB in the prior art, the isolation of putative SCB from intact and regenerating brittlestars and subsequent characterization of those bacteria for ability to chemically inhibit the growth of pathogenic bacteria in culture has not been described. Isolation and characterization of putative SCB are highly desirable given the need to determine whether such bacteria produce antimicrobial compounds and given the broad utility of any such compounds produced by the bacteria as potential antimicrobials.
SUMMARY AND OBJECTS OF THE INVENTION
In accordance with the present invention there are provided biologically pure cultures of subcuticular bacteria isolated from
A. gracillima
and having bacillus morphology, samples of which has been deposited at American Type Tissue Culture (ATCC) on Apr. 14, 1998 under accession numbers 202111, 202112, and 202113, or a mutant or a derivative thereof having the same antimicrobial activity as the culture.
In accordance with the present invention there is also provided a biologically pure culture of a subcuticular bacteria isolated from
A. gracillima
and having bacillus morphology, further characterized as a monoculture selected from monocultures BE 12 through BE 70 as set forth in Table 1 of FIG.
3
and having the anti-microbial activity thereof, or a mutant or a derivative thereof having the same antimicrobial activity as the monoculture. Preferably, the biologically pure culture is further characterized as a monoculture selected from the group consisting of monocultures BE 37, BE 52 and BE 59 as set forth in Table 1 of FIG.
3
and in Examples 1 through 3, displaying Hae III restriction fragment length polymorphism (RFLP) banding patterns of polymerase chain reaction (PCR)-amplified DNA from the 16S rRNA gene as shown in
FIG. 4
, and having the anti-microbial activity thereof, or a mutant or a derivative thereof having the same antimicrobial activity as the respective monoculture.
In accordance with the present invention, there is also provided an antimicrobial composition comprising the biologically pure culture as described above, or a mutant or a derivative of any of such culture, the mutant or the derivative having the same antimicrobial activity as the culture, or antimicrobial material isolated from any of such cultures, together with a carrier or diluent therefor. Further, a method for inhibiting the growth of a microbe comprising the step of contacting said microbe with an inhibiting amount of the antimicrobial composition of the present invention is also provided.
In accordance with the present invention there is also provided an antibiotic or antibiotic fraction derivable from subcuticular bacteria isolated from
A. gracillima
, wherein the isolated bacteria have bacillus morphology. The antibiotic or antibiotic fraction is characterized by (a) antibiotic activity against Gram-positive bacteria, Gram-negative bacteria and combinations thereof; (b) antimicrobial activity over a pH range of about 6.4 to about 8.4; (c) solubility in water and ethanol; (d) insolubility in toluene and chloroform; and (e) precipitability from an aqueous solution using either toluene or chloroform. A process for obtaining such an antibiotic or antibiotic fraction is also provided.
Therefore, it is an object of the present invention to isolate and to characterize putative subcuticular bacteria from
A. gracillima.
It is another object of the present invention to isolate and to characterize compounds produced by such bacteria for use as antimicrobials.
It is a further object of the present invention to provide a biologically pure culture of a subcuticular bacteria isolated from
A. gracillima.
It is yet a further object of the present invention to provide an antibiotic or antibiotic fraction derivable from subcuticular bacteria isolated from
A. gracillima.
It is still a further object of the present invention to provide methods for
Afremova Vera
Appalachain State University
Jenkins & Wilson, P.A.
Marx Irene
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